TY  - CONF
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Nikolić, Stanko N
AU  - Rabasović, Mihailo D
AU  - Clayton, Andrew HA
AU  - Krmpot, Aleksandar J
PY  - 2023
UR  - https://www.eventclass.org/contxt_emim2023/online-program/session?s=PW36#e609
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6288
AB  - Introduction
Studies of lipid droplet (LD) physiology in fungi are still in their infancy but their quantitation
has relevance to issues in biomedicine, agriculture and industrial waste. Third Harmonic
Generation (THG) microscopy is non-invasive, produces inherently confocal images and
doesn’t require fixation or external labeling, which make it suitable for in vivo LD imaging [1,
2]. We present in vivo and label-free imaging of LD in individual fungal cells by THG
microscopy to assess the effects of nitrogen starvation. The LD quantification was performed
by two image analysis techniques.
Methods
THG microscopy was applied for the first time to a filamentous fungus and our choice was the
oleaginous fungus Phycomyces blakesleeanus. To observe the changes in LD number, the
22h old hyphae culture was divided into control and nitrogen starved groups (N-starved). A
home built nonlienar microscope with Yb:KGW laser at 1040 nm (200 fs pulses, 83 MHz
repetition rate) was used for THG imaging of live unstained hyphae [3]. THG signal was
detected by PMT in the transmission arm after passing through a Hoya glass UV filter with the
peak at 340 nm. 2D THG images of LDs (Fig. 1a) were analyzed by Image Correlation
Spectroscopy (ICS) measuring spatially-correlated fluctuations [4] and software particle
counting – Particle Size Analysis (PSA).
Results/Discussion
The small volume of hyphae suspension was placed between two coverslips of 170 μm
thickness in order to meet the criteria for the best numerical aperture of the objective lens and
for better transmission of THG signal. The high resolution of the microscopic system, the
hyphae thickness (ca 10 μm) and medium transparency made it possible for the whole
hyphae to be optically sectioned and a 3D model to be reconstructed (Fig. 1b and video).
Since ICS was primarily developed for fluorescent images and was not used to analyze THG
images, we have tested it by comparing the results to the PSA. Nitrogen starvation as
expected [5] increased LD number compared to control which was confirmed by both methods and obtained results are in good agreement. The overall increase of LDs during
growth without available nitrogen is found to be between 3 and 4.5 h time point, followed with
the loss of population of larger-than-average LDs during prolonged starvation.
Conclusions
THG microscopy is suitable for imaging and quantification of changes in lipid droplet number,
brought upon by complete removal of nitrogen, from such low density/diameter baseline. In
addition, we demonstrate that the ICA is suitable for THG images, although it is primarily
developed and have been mostly used for fluorescence signals so far.
PB  - European Society for Molecular Imaging
C3  - European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria
T1  - Third harmonic generation imaging of live fungal cells – quantifying lipid droplets dynamics during nitrogen starvation
SP  - 1093
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6288
ER  - 

@conference{
author = "Pajić, Tanja and Todorović, Nataša and Živić, Miroslav and Nikolić, Stanko N and Rabasović, Mihailo D and Clayton, Andrew HA and Krmpot, Aleksandar J",
year = "2023",
abstract = "Introduction
Studies of lipid droplet (LD) physiology in fungi are still in their infancy but their quantitation
has relevance to issues in biomedicine, agriculture and industrial waste. Third Harmonic
Generation (THG) microscopy is non-invasive, produces inherently confocal images and
doesn’t require fixation or external labeling, which make it suitable for in vivo LD imaging [1,
2]. We present in vivo and label-free imaging of LD in individual fungal cells by THG
microscopy to assess the effects of nitrogen starvation. The LD quantification was performed
by two image analysis techniques.
Methods
THG microscopy was applied for the first time to a filamentous fungus and our choice was the
oleaginous fungus Phycomyces blakesleeanus. To observe the changes in LD number, the
22h old hyphae culture was divided into control and nitrogen starved groups (N-starved). A
home built nonlienar microscope with Yb:KGW laser at 1040 nm (200 fs pulses, 83 MHz
repetition rate) was used for THG imaging of live unstained hyphae [3]. THG signal was
detected by PMT in the transmission arm after passing through a Hoya glass UV filter with the
peak at 340 nm. 2D THG images of LDs (Fig. 1a) were analyzed by Image Correlation
Spectroscopy (ICS) measuring spatially-correlated fluctuations [4] and software particle
counting – Particle Size Analysis (PSA).
Results/Discussion
The small volume of hyphae suspension was placed between two coverslips of 170 μm
thickness in order to meet the criteria for the best numerical aperture of the objective lens and
for better transmission of THG signal. The high resolution of the microscopic system, the
hyphae thickness (ca 10 μm) and medium transparency made it possible for the whole
hyphae to be optically sectioned and a 3D model to be reconstructed (Fig. 1b and video).
Since ICS was primarily developed for fluorescent images and was not used to analyze THG
images, we have tested it by comparing the results to the PSA. Nitrogen starvation as
expected [5] increased LD number compared to control which was confirmed by both methods and obtained results are in good agreement. The overall increase of LDs during
growth without available nitrogen is found to be between 3 and 4.5 h time point, followed with
the loss of population of larger-than-average LDs during prolonged starvation.
Conclusions
THG microscopy is suitable for imaging and quantification of changes in lipid droplet number,
brought upon by complete removal of nitrogen, from such low density/diameter baseline. In
addition, we demonstrate that the ICA is suitable for THG images, although it is primarily
developed and have been mostly used for fluorescence signals so far.",
publisher = "European Society for Molecular Imaging",
journal = "European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria",
title = "Third harmonic generation imaging of live fungal cells – quantifying lipid droplets dynamics during nitrogen starvation",
pages = "1093",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6288"
}

Pajić, T., Todorović, N., Živić, M., Nikolić, S. N., Rabasović, M. D., Clayton, A. H.,& Krmpot, A. J.. (2023). Third harmonic generation imaging of live fungal cells – quantifying lipid droplets dynamics during nitrogen starvation. in European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria
European Society for Molecular Imaging., 1093.
https://hdl.handle.net/21.15107/rcub_ibiss_6288

Pajić T, Todorović N, Živić M, Nikolić SN, Rabasović MD, Clayton AH, Krmpot AJ. Third harmonic generation imaging of live fungal cells – quantifying lipid droplets dynamics during nitrogen starvation. in European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria. 2023;:1093.
https://hdl.handle.net/21.15107/rcub_ibiss_6288 .

Pajić, Tanja, Todorović, Nataša, Živić, Miroslav, Nikolić, Stanko N, Rabasović, Mihailo D, Clayton, Andrew HA, Krmpot, Aleksandar J, "Third harmonic generation imaging of live fungal cells – quantifying lipid droplets dynamics during nitrogen starvation" in European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria (2023):1093,
https://hdl.handle.net/21.15107/rcub_ibiss_6288 .

TY  - CONF
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Stevanović, Katarina
AU  - Pajić, Tanja
AU  - Cvetić Antić, Tijana
AU  - Atlagić, Kristina
AU  - Krmpot, Aleksandar
PY  - 2023
UR  - https://microbiology.bg.ac.rs/workshop2023/
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6248
AB  - The use of chemical fungicides is detrimental for soil and aquatic ecosystems.
Although highly effective, they raise concerns about impact on the environment.
Therefore, the need to reduce the use of chemical fungicides and protect
the soil and water, pose the challenge to find novel, eco-friendly, but also
more efficient, means of crop protection from phytopathogenic fungi. We are
aiming to utilize the native Trichoderma species, as biological control agent,
for controlling or eliminating the phytopathogenic fungi. Our research will be
based on cell nano-surgery of fungal cell wall using ultrashort laser pulses and
subsequent patch clamping on the released protoplast membrane. This will
enable the studies of activity and potency of specific class of antimicrobial short
peptides, peptaibols, released by Trichoderma species, which act by forming
ion channels in pathogen membrane as one of the main mechanisms of their
fungicide action. The result of this project will be the developed instrumentation
and unique method based on nano-surgery and patch clamp for studies of ionic
channels in native fungal plasma membrane. Testing a range of autochthonous
Trichoderma sp. isolates will enable formulation of their combinations that work
best. The selected Trichoderma strains with the highest standardized peptaibol
activity in specific combination of conditions, can be used as a starting point for
development of new, more potent biocontrol agents.
PB  - Belgrade: University of Belgrade, Faculty of Biology
C3  - Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia
T1  - Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent
SP  - 84
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6248
ER  - 

@conference{
author = "Todorović, Nataša and Živić, Miroslav and Stevanović, Katarina and Pajić, Tanja and Cvetić Antić, Tijana and Atlagić, Kristina and Krmpot, Aleksandar",
year = "2023",
abstract = "The use of chemical fungicides is detrimental for soil and aquatic ecosystems.
Although highly effective, they raise concerns about impact on the environment.
Therefore, the need to reduce the use of chemical fungicides and protect
the soil and water, pose the challenge to find novel, eco-friendly, but also
more efficient, means of crop protection from phytopathogenic fungi. We are
aiming to utilize the native Trichoderma species, as biological control agent,
for controlling or eliminating the phytopathogenic fungi. Our research will be
based on cell nano-surgery of fungal cell wall using ultrashort laser pulses and
subsequent patch clamping on the released protoplast membrane. This will
enable the studies of activity and potency of specific class of antimicrobial short
peptides, peptaibols, released by Trichoderma species, which act by forming
ion channels in pathogen membrane as one of the main mechanisms of their
fungicide action. The result of this project will be the developed instrumentation
and unique method based on nano-surgery and patch clamp for studies of ionic
channels in native fungal plasma membrane. Testing a range of autochthonous
Trichoderma sp. isolates will enable formulation of their combinations that work
best. The selected Trichoderma strains with the highest standardized peptaibol
activity in specific combination of conditions, can be used as a starting point for
development of new, more potent biocontrol agents.",
publisher = "Belgrade: University of Belgrade, Faculty of Biology",
journal = "Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia",
title = "Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent",
pages = "84",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6248"
}

Todorović, N., Živić, M., Stevanović, K., Pajić, T., Cvetić Antić, T., Atlagić, K.,& Krmpot, A.. (2023). Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent. in Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia
Belgrade: University of Belgrade, Faculty of Biology., 84.
https://hdl.handle.net/21.15107/rcub_ibiss_6248

Todorović N, Živić M, Stevanović K, Pajić T, Cvetić Antić T, Atlagić K, Krmpot A. Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent. in Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia. 2023;:84.
https://hdl.handle.net/21.15107/rcub_ibiss_6248 .

Todorović, Nataša, Živić, Miroslav, Stevanović, Katarina, Pajić, Tanja, Cvetić Antić, Tijana, Atlagić, Kristina, Krmpot, Aleksandar, "Novel research project – BioPhysFun for advancement of characterization of Trichoderma as biological control agent" in Book of Abstracts: ICGEB Workshop: Trends in microbial solutions for sustainable agriculture; 2023 Sep13-15; Belgrade, Serbia (2023):84,
https://hdl.handle.net/21.15107/rcub_ibiss_6248 .

TY  - CONF
AU  - Stevanović, Katarina
AU  - Pajić, Tanja
AU  - Krmpot, Aleksandar
AU  - Rabasović, Mihailo
AU  - Žižić, Milan
AU  - Živić, Miroslav
AU  - Todorović, Nataša
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6246
AB  - The success of patch-clamp giga-ohm seal formation on filamentous fungi Phycomyces blakesleeanus protoplasts was investigated to evaluate their usefulness in ion channel studies on filamentous fungi. Protoplasts were obtained by laser-mediated nanosurgery of the cell wall stained with Calcofluor White. To enable a successful seal formation, it is critical to prevent cell wall regeneration. Since wall integrity responses in fungi involve kinase-dependent pathways, we aimed to sufficiently reduce intracellular ATP availability using the respiratory inhibitor azide. The effect of azide on phosphate metabolites of Phycomyces blakesleeanus, as determined by 31P NMR spectroscopy, was a reduction in intracellular ATP accompanied by a decrease in long chain polyphosphates. Subsequently, all seal formation measurements were performed in the presence of azide, and protoplast viability was confirmed by cytoplasmic streaming. The success of seal formation depends on the size of the protoplasts, as larger protoplasts are more prone to successful seal formation. It was also found that the laser power used in nanosurgery could influence the success of seal formation, as higher values were associated with a lower success rate. The protoplasts released by laser nanosurgery produced by our newly developed method are a good model system for patch clamp on filamentous fungal membranes because their plasma membrane can easily form high-quality seals with the patch pipette.
PB  - Kragujevac: Institute for Information Technologies, University of Belgrade
C3  - Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia
T1  - Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts
DO  - 10.46793/ICCBI23.221S
SP  - 221
EP  - 224
ER  - 

@conference{
author = "Stevanović, Katarina and Pajić, Tanja and Krmpot, Aleksandar and Rabasović, Mihailo and Žižić, Milan and Živić, Miroslav and Todorović, Nataša",
year = "2023",
abstract = "The success of patch-clamp giga-ohm seal formation on filamentous fungi Phycomyces blakesleeanus protoplasts was investigated to evaluate their usefulness in ion channel studies on filamentous fungi. Protoplasts were obtained by laser-mediated nanosurgery of the cell wall stained with Calcofluor White. To enable a successful seal formation, it is critical to prevent cell wall regeneration. Since wall integrity responses in fungi involve kinase-dependent pathways, we aimed to sufficiently reduce intracellular ATP availability using the respiratory inhibitor azide. The effect of azide on phosphate metabolites of Phycomyces blakesleeanus, as determined by 31P NMR spectroscopy, was a reduction in intracellular ATP accompanied by a decrease in long chain polyphosphates. Subsequently, all seal formation measurements were performed in the presence of azide, and protoplast viability was confirmed by cytoplasmic streaming. The success of seal formation depends on the size of the protoplasts, as larger protoplasts are more prone to successful seal formation. It was also found that the laser power used in nanosurgery could influence the success of seal formation, as higher values were associated with a lower success rate. The protoplasts released by laser nanosurgery produced by our newly developed method are a good model system for patch clamp on filamentous fungal membranes because their plasma membrane can easily form high-quality seals with the patch pipette.",
publisher = "Kragujevac: Institute for Information Technologies, University of Belgrade",
journal = "Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia",
title = "Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts",
doi = "10.46793/ICCBI23.221S",
pages = "221-224"
}

Stevanović, K., Pajić, T., Krmpot, A., Rabasović, M., Žižić, M., Živić, M.,& Todorović, N.. (2023). Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts. in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia
Kragujevac: Institute for Information Technologies, University of Belgrade., 221-224.
https://doi.org/10.46793/ICCBI23.221S

Stevanović K, Pajić T, Krmpot A, Rabasović M, Žižić M, Živić M, Todorović N. Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts. in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia. 2023;:221-224.
doi:10.46793/ICCBI23.221S .

Stevanović, Katarina, Pajić, Tanja, Krmpot, Aleksandar, Rabasović, Mihailo, Žižić, Milan, Živić, Miroslav, Todorović, Nataša, "Patch clamp pipette giga seal forming success on the nanosurgery-obtained filamentous fungi protoplasts" in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia (2023):221-224,
https://doi.org/10.46793/ICCBI23.221S . .

TY  - CONF
AU  - Vitkovac, Aleksandra
AU  - Pajić, Tanja
AU  - Bukumira, Marta
AU  - Stanić, Marina
AU  - Rabasović, Mihailo
AU  - Todorović, Nataša
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6247
AB  - Adaptive changes in mitochondrial morphology are associated with changes in the mitochondrial function and metabolic fitness of eukaryotic cells. We previously described in young hyphae of the filamentous fungus Phycomyces blakesleeanus a dramatic effect of an increase in ambient temperature during growth: a 3°C warmer environment compared with a control temperature of 22°C resulted in the appearance of long elongated (“tubular”) mitochondria accompanied by an increase in lipid droplet density. Here, we examined how cooler ambient temperature (18°C) during growth affects mitochondrial morphology in P. blakesleeanus compared with the control grown at 22°C. We used two-photon fluorescence imaging (TPEF) of live hyphae stained with the vital mitochondrial dye rhodamine 123. Extraction of relevant parameters (number, size, and shape of mitochondria) from TPEF images was performed using the Ilastik machine learning-based software. The suitability of the Ilastik analysis was compared with the Particle Analysis (ImageJ). Cold treatment resulted in the appearance of tubular mitochondrial morphology that was absent in the control group. Tubular mitochondrial morphology appears to be an adaptive feature that occurs in both warmer and colder conditions and is likely part of the stress response.
PB  - Kragujevac: Institute for Information Technologies, University of Belgrade
C3  - Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia
T1  - Slight cooling during growth induced changes in filamentous fungi hypha mitochondrial morphology
DO  - 10.46793/ICCBI23.334V
SP  - 334
EP  - 337
ER  - 

@conference{
author = "Vitkovac, Aleksandra and Pajić, Tanja and Bukumira, Marta and Stanić, Marina and Rabasović, Mihailo and Todorović, Nataša",
year = "2023",
abstract = "Adaptive changes in mitochondrial morphology are associated with changes in the mitochondrial function and metabolic fitness of eukaryotic cells. We previously described in young hyphae of the filamentous fungus Phycomyces blakesleeanus a dramatic effect of an increase in ambient temperature during growth: a 3°C warmer environment compared with a control temperature of 22°C resulted in the appearance of long elongated (“tubular”) mitochondria accompanied by an increase in lipid droplet density. Here, we examined how cooler ambient temperature (18°C) during growth affects mitochondrial morphology in P. blakesleeanus compared with the control grown at 22°C. We used two-photon fluorescence imaging (TPEF) of live hyphae stained with the vital mitochondrial dye rhodamine 123. Extraction of relevant parameters (number, size, and shape of mitochondria) from TPEF images was performed using the Ilastik machine learning-based software. The suitability of the Ilastik analysis was compared with the Particle Analysis (ImageJ). Cold treatment resulted in the appearance of tubular mitochondrial morphology that was absent in the control group. Tubular mitochondrial morphology appears to be an adaptive feature that occurs in both warmer and colder conditions and is likely part of the stress response.",
publisher = "Kragujevac: Institute for Information Technologies, University of Belgrade",
journal = "Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia",
title = "Slight cooling during growth induced changes in filamentous fungi hypha mitochondrial morphology",
doi = "10.46793/ICCBI23.334V",
pages = "334-337"
}

Vitkovac, A., Pajić, T., Bukumira, M., Stanić, M., Rabasović, M.,& Todorović, N.. (2023). Slight cooling during growth induced changes in filamentous fungi hypha mitochondrial morphology. in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia
Kragujevac: Institute for Information Technologies, University of Belgrade., 334-337.
https://doi.org/10.46793/ICCBI23.334V

Vitkovac A, Pajić T, Bukumira M, Stanić M, Rabasović M, Todorović N. Slight cooling during growth induced changes in filamentous fungi hypha mitochondrial morphology. in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia. 2023;:334-337.
doi:10.46793/ICCBI23.334V .

Vitkovac, Aleksandra, Pajić, Tanja, Bukumira, Marta, Stanić, Marina, Rabasović, Mihailo, Todorović, Nataša, "Slight cooling during growth induced changes in filamentous fungi hypha mitochondrial morphology" in Book of Proceedings: 2nd International Conference on Chemo and Bioinformatics ICCBIKG 2023; 2023 Sep 28-29; Kragujevac, Serbia (2023):334-337,
https://doi.org/10.46793/ICCBI23.334V . .

TY  - CONF
AU  - Pajić, Tanja
AU  - Kozakijević, Suzana
AU  - Krmpot, Aleksandar J.
AU  - Živić, Miroslav
AU  - Todorović, Nataša
AU  - Rabasović, Mihailo D.
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6249
AB  - Mitochondrial function, and consequently cellular metabolic status and fitness of a cell, is tightly linked
to the dynamic changes of mitochondrial morphology, including mitochondrial fusion, fission and
mitophagy [1]. Lipid droplets (LDs) can be in close contact with mitochondria, and accumulate
autophagy or mitophagy generated material during the reparatory processes [2]. The effect of increased
ambient temperature on mitochondrial morphology and LDs density in living cells of the filamentous
fungus Phycomyces blakesleeanus was investigated. For in vivo imaging of mitochondria and LDs
multiphoton microscopy was used. Multiphoton microscopy enables 3D imaging in high resolution and
reduced photodamage and photobleaching of the sample using IR ultrafast pulsed lasers. Mitochondria
were stained with the vital dye Rhodamine123 (Rh123) which enters these organelles based on their
membrane potential - mitochondria must be healthy/active to stain. A wavelength of 800 nm from Ti:Sa
laser (160 fs pulse duration, 76 MHz repetition rate) was used for two-photon excitation of Rh123. The
laser beam was focused by the Zeiss Plan Neofluar 40x1.3 objective lens and the signal was detected
through a bandpass interference filter MF530/43 (ThorLabs, USA). For LDs staining a Nile Red dye
was used and excited by Yb: KGW laser at 1040 nm [3]. Six morphological types of mitochondria were
observed in the hyphae of this fungus: intermediate type - normal, intermediate with small semicircular
tubules, tubular, elongated tubular, fragmented (small ellipsoid tubule) and fragmented with exclusively
spheroid-shaped mitochondria. Changes in mitochondrial morphology were induced by a small
temperature change. A 3°C increase in ambient temperature, from 22°C, had a dramatic effect on
mitochondrial morphology, inducing the appearance of a predominantly tubular mitochondrial
morphology. The total area percentage of mitochondria showed an increasing trend when grown at
25°C. Increasing the ambient temperature to 25°C induced a statistically significant increase in the
percentage of hyphal area occupied by LDs from 2.9 ± 1.6 to 4.7 ± 2.2 (mean value and SD in percentage
of hyphal area). The observed response to the small temperature increase points to the physiological
adaptation of hyphal metabolism.
PB  - Belgrade: Vinča Institute of Nuclear Sciences – National Institute of the Republic of Serbia, University of Belgrade
C3  - Book of abstracts: 9th International School and Conference on Photonics PHOTONICA 2023 with joint events: Understanding interaction light - biological surfaces possibility for new electronic materials and devices and Biological and bioinspired structures for multispectral surveillance and Quantum sensing integration within microfluidic Lab-on-a Chips for biomedical applications; 2023 Aug 28 - Sep 01; Belgrade, Serbia
T1  - In vivo multiphoton imaging of a filamentous fungus Phycomyces blakesleeanus: the effect of small ambient temperature increase on mitochondrial morphology and lipid droplets density
SP  - 84
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6249
ER  - 

@conference{
author = "Pajić, Tanja and Kozakijević, Suzana and Krmpot, Aleksandar J. and Živić, Miroslav and Todorović, Nataša and Rabasović, Mihailo D.",
year = "2023",
abstract = "Mitochondrial function, and consequently cellular metabolic status and fitness of a cell, is tightly linked
to the dynamic changes of mitochondrial morphology, including mitochondrial fusion, fission and
mitophagy [1]. Lipid droplets (LDs) can be in close contact with mitochondria, and accumulate
autophagy or mitophagy generated material during the reparatory processes [2]. The effect of increased
ambient temperature on mitochondrial morphology and LDs density in living cells of the filamentous
fungus Phycomyces blakesleeanus was investigated. For in vivo imaging of mitochondria and LDs
multiphoton microscopy was used. Multiphoton microscopy enables 3D imaging in high resolution and
reduced photodamage and photobleaching of the sample using IR ultrafast pulsed lasers. Mitochondria
were stained with the vital dye Rhodamine123 (Rh123) which enters these organelles based on their
membrane potential - mitochondria must be healthy/active to stain. A wavelength of 800 nm from Ti:Sa
laser (160 fs pulse duration, 76 MHz repetition rate) was used for two-photon excitation of Rh123. The
laser beam was focused by the Zeiss Plan Neofluar 40x1.3 objective lens and the signal was detected
through a bandpass interference filter MF530/43 (ThorLabs, USA). For LDs staining a Nile Red dye
was used and excited by Yb: KGW laser at 1040 nm [3]. Six morphological types of mitochondria were
observed in the hyphae of this fungus: intermediate type - normal, intermediate with small semicircular
tubules, tubular, elongated tubular, fragmented (small ellipsoid tubule) and fragmented with exclusively
spheroid-shaped mitochondria. Changes in mitochondrial morphology were induced by a small
temperature change. A 3°C increase in ambient temperature, from 22°C, had a dramatic effect on
mitochondrial morphology, inducing the appearance of a predominantly tubular mitochondrial
morphology. The total area percentage of mitochondria showed an increasing trend when grown at
25°C. Increasing the ambient temperature to 25°C induced a statistically significant increase in the
percentage of hyphal area occupied by LDs from 2.9 ± 1.6 to 4.7 ± 2.2 (mean value and SD in percentage
of hyphal area). The observed response to the small temperature increase points to the physiological
adaptation of hyphal metabolism.",
publisher = "Belgrade: Vinča Institute of Nuclear Sciences – National Institute of the Republic of Serbia, University of Belgrade",
journal = "Book of abstracts: 9th International School and Conference on Photonics PHOTONICA 2023 with joint events: Understanding interaction light - biological surfaces possibility for new electronic materials and devices and Biological and bioinspired structures for multispectral surveillance and Quantum sensing integration within microfluidic Lab-on-a Chips for biomedical applications; 2023 Aug 28 - Sep 01; Belgrade, Serbia",
title = "In vivo multiphoton imaging of a filamentous fungus Phycomyces blakesleeanus: the effect of small ambient temperature increase on mitochondrial morphology and lipid droplets density",
pages = "84",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6249"
}

Pajić, T., Kozakijević, S., Krmpot, A. J., Živić, M., Todorović, N.,& Rabasović, M. D.. (2023). In vivo multiphoton imaging of a filamentous fungus Phycomyces blakesleeanus: the effect of small ambient temperature increase on mitochondrial morphology and lipid droplets density. in Book of abstracts: 9th International School and Conference on Photonics PHOTONICA 2023 with joint events: Understanding interaction light - biological surfaces possibility for new electronic materials and devices and Biological and bioinspired structures for multispectral surveillance and Quantum sensing integration within microfluidic Lab-on-a Chips for biomedical applications; 2023 Aug 28 - Sep 01; Belgrade, Serbia
Belgrade: Vinča Institute of Nuclear Sciences – National Institute of the Republic of Serbia, University of Belgrade., 84.
https://hdl.handle.net/21.15107/rcub_ibiss_6249

Pajić T, Kozakijević S, Krmpot AJ, Živić M, Todorović N, Rabasović MD. In vivo multiphoton imaging of a filamentous fungus Phycomyces blakesleeanus: the effect of small ambient temperature increase on mitochondrial morphology and lipid droplets density. in Book of abstracts: 9th International School and Conference on Photonics PHOTONICA 2023 with joint events: Understanding interaction light - biological surfaces possibility for new electronic materials and devices and Biological and bioinspired structures for multispectral surveillance and Quantum sensing integration within microfluidic Lab-on-a Chips for biomedical applications; 2023 Aug 28 - Sep 01; Belgrade, Serbia. 2023;:84.
https://hdl.handle.net/21.15107/rcub_ibiss_6249 .

Pajić, Tanja, Kozakijević, Suzana, Krmpot, Aleksandar J., Živić, Miroslav, Todorović, Nataša, Rabasović, Mihailo D., "In vivo multiphoton imaging of a filamentous fungus Phycomyces blakesleeanus: the effect of small ambient temperature increase on mitochondrial morphology and lipid droplets density" in Book of abstracts: 9th International School and Conference on Photonics PHOTONICA 2023 with joint events: Understanding interaction light - biological surfaces possibility for new electronic materials and devices and Biological and bioinspired structures for multispectral surveillance and Quantum sensing integration within microfluidic Lab-on-a Chips for biomedical applications; 2023 Aug 28 - Sep 01; Belgrade, Serbia (2023):84,
https://hdl.handle.net/21.15107/rcub_ibiss_6249 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Stevanović, Katarina
AU  - Todorović, Nataša
AU  - Lević, Steva
AU  - Savić Šević, Svetlana
AU  - Pantelić, Dejan
AU  - Živić, Miroslav
AU  - Rabasović, Mihailo D
AU  - Krmpot, Aleksandar J
PY  - 2023
UR  - https://www.eventclass.org/contxt_emim2023/online-program/session?s=PW36#e609
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6289
AB  - Introduction
Electrophysiology studies of ion channels, in live filamentous fungi by patch clamp method are notpossible due to presence of rigid chitinous cell wall that prevents patch clamp pipette to access theplasma membrane. We present laser nano-surgery of the fungal cell wall that enables patch clampelectrophysiology studies. Similar approaches as one-time reports utilizing nanosecond laser pulseslong time ago were not pursued further [1,2]. Here, we demonstrate reproducible method usingfemtosecond lasers accompanied by two-photon excitation fluorescence (TPEF) imaging of hyphae.
Methods
A wild-type strain of filamentous fungus
Phycomyces blakesleeanus
(Burgeff ) [NRRL 1555(-)] were grownon glass coverslips with hand-etched grid, coated with a thin layer of 50% collagen type I as animmobilizer. Home built nonlinear laser scanning microscope [3,4] utilizing Ti:Sa tunable fs laser wasused for TPEF imaging of hyphae and the cell surgery. The latter is enabled with the custom made add-on in soft ware. Coverslip with hyphae is transferred to another microscope setup for patch clamp,consisting of micromanipulators and precise electronics for pA current measurements. The surgicalincisions and released protoplasts were additionally imaged by scanning electron microscopy for whichtreated hyphae had to undergo critical point drying procedure.
Results/Discussion
Hyphae were stained by Calcofluor White and treated with an exocytosis inhibitor (brefeldin A) and arespiration inhibitor (sodium azide) to prevent cell wall regeneration. Since the cell wall and the plasmamembrane are in the close contact [4] hyphae were kept in hyperosmotic solution to retract thecytoplasm from the cell wall. Surgical spot-wise pattern was precisely positioned at TPEF image ofselected hypha at the place where the plasma membrane was retracted. The dwell time (1s) and thelaser power (4-15mW) were set with fixed repetition rate (76MHz), pulse duration (160fs) and laserwavelength (730nm). Upon the surgery, hyphae were gently deplasmolysed. A protoplast with plasmamembrane accessible for the patch clamp pipette was released through the surgical incision (Fig 1). The>GΩ seal resistance was achieved. Numerous ion channels are recorded in diff erent configurations (oncell, inside-out, whole cell and out-out) (Fig 2).
Conclusions
The whole process (cell surgery + patch clamping) is rather complex and specific steps have to be strictlyfollowed for high success rate and reproducibility. Also, chemicals concentrations, solutions osmolarity,timing and cutting parameters have to be kept in the specified narrow range. Obtained currentrecordings provide valuable information on fungal cell membrane ionic channels.
PB  - European Society for Molecular Imaging
C3  - European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria
T1  - Laser nano-surgery of fungal cell wall to enable patch clamping
SP  - 1095
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6289
ER  - 

@conference{
author = "Pajić, Tanja and Stevanović, Katarina and Todorović, Nataša and Lević, Steva and Savić Šević, Svetlana and Pantelić, Dejan and Živić, Miroslav and Rabasović, Mihailo D and Krmpot, Aleksandar J",
year = "2023",
abstract = "Introduction
Electrophysiology studies of ion channels, in live filamentous fungi by patch clamp method are notpossible due to presence of rigid chitinous cell wall that prevents patch clamp pipette to access theplasma membrane. We present laser nano-surgery of the fungal cell wall that enables patch clampelectrophysiology studies. Similar approaches as one-time reports utilizing nanosecond laser pulseslong time ago were not pursued further [1,2]. Here, we demonstrate reproducible method usingfemtosecond lasers accompanied by two-photon excitation fluorescence (TPEF) imaging of hyphae.
Methods
A wild-type strain of filamentous fungus
Phycomyces blakesleeanus
(Burgeff ) [NRRL 1555(-)] were grownon glass coverslips with hand-etched grid, coated with a thin layer of 50% collagen type I as animmobilizer. Home built nonlinear laser scanning microscope [3,4] utilizing Ti:Sa tunable fs laser wasused for TPEF imaging of hyphae and the cell surgery. The latter is enabled with the custom made add-on in soft ware. Coverslip with hyphae is transferred to another microscope setup for patch clamp,consisting of micromanipulators and precise electronics for pA current measurements. The surgicalincisions and released protoplasts were additionally imaged by scanning electron microscopy for whichtreated hyphae had to undergo critical point drying procedure.
Results/Discussion
Hyphae were stained by Calcofluor White and treated with an exocytosis inhibitor (brefeldin A) and arespiration inhibitor (sodium azide) to prevent cell wall regeneration. Since the cell wall and the plasmamembrane are in the close contact [4] hyphae were kept in hyperosmotic solution to retract thecytoplasm from the cell wall. Surgical spot-wise pattern was precisely positioned at TPEF image ofselected hypha at the place where the plasma membrane was retracted. The dwell time (1s) and thelaser power (4-15mW) were set with fixed repetition rate (76MHz), pulse duration (160fs) and laserwavelength (730nm). Upon the surgery, hyphae were gently deplasmolysed. A protoplast with plasmamembrane accessible for the patch clamp pipette was released through the surgical incision (Fig 1). The>GΩ seal resistance was achieved. Numerous ion channels are recorded in diff erent configurations (oncell, inside-out, whole cell and out-out) (Fig 2).
Conclusions
The whole process (cell surgery + patch clamping) is rather complex and specific steps have to be strictlyfollowed for high success rate and reproducibility. Also, chemicals concentrations, solutions osmolarity,timing and cutting parameters have to be kept in the specified narrow range. Obtained currentrecordings provide valuable information on fungal cell membrane ionic channels.",
publisher = "European Society for Molecular Imaging",
journal = "European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria",
title = "Laser nano-surgery of fungal cell wall to enable patch clamping",
pages = "1095",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6289"
}

Pajić, T., Stevanović, K., Todorović, N., Lević, S., Savić Šević, S., Pantelić, D., Živić, M., Rabasović, M. D.,& Krmpot, A. J.. (2023). Laser nano-surgery of fungal cell wall to enable patch clamping. in European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria
European Society for Molecular Imaging., 1095.
https://hdl.handle.net/21.15107/rcub_ibiss_6289

Pajić T, Stevanović K, Todorović N, Lević S, Savić Šević S, Pantelić D, Živić M, Rabasović MD, Krmpot AJ. Laser nano-surgery of fungal cell wall to enable patch clamping. in European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria. 2023;:1095.
https://hdl.handle.net/21.15107/rcub_ibiss_6289 .

Pajić, Tanja, Stevanović, Katarina, Todorović, Nataša, Lević, Steva, Savić Šević, Svetlana, Pantelić, Dejan, Živić, Miroslav, Rabasović, Mihailo D, Krmpot, Aleksandar J, "Laser nano-surgery of fungal cell wall to enable patch clamping" in European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria (2023):1095,
https://hdl.handle.net/21.15107/rcub_ibiss_6289 .

TY  - JOUR
AU  - Stevanović, Katarina S
AU  - Čepkenović, Bogdana
AU  - Križak, Strahinja
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Živić, Miroslav Ž
PY  - 2023
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6088
AB  - Ion channels are vital components of filamentous fungi signaling in communication with their environment. We exploited the ability of the apical region of growing sporangiophores of Phycomyces blakesleeanus to form membrane-enveloped cytoplasmic droplets (CDs), to examine ion currents in the filamentous fungi native plasma membrane. In hypoosmotic conditions, the dominant current in the CDs is ORIC, an osmotically activated, anionic, outwardly rectified, fast inactivating instantaneous current that we have previously characterized. Here, we examined the effect of ATP on ORIC. We show that CDs contain active mitochondria, and that respiration inhibition by azide accelerates ORIC inactivation. ATP, added intracellularly, reduced ORIC run-down and shifted the voltage dependence of inactivation toward depolarized potentials, in a manner that did not require hydrolysis. Notably, ATP led to slowing down of ORIC inactivation, as evidenced by an increased time constant of inactivation, τin, and slower decline of τin during prolonged recordings. Flavonoids (genistein and quercetin) had the effect on ORIC opposite to ATP, acting as current inhibitors, possibly by disrupting the stabilizing effect of ATP on ORIC. The integration of osmotic sensing with ATP dependence of the anionic current, typical of vertebrate cells, is described here for the first time in filamentous fungi.
PB  - Springer Nature
T2  - Scientific Reports
T1  - ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore
IS  - 1
VL  - 13
DO  - 10.1038/s41598-023-39021-9
SP  - 11897
ER  - 

@article{
author = "Stevanović, Katarina S and Čepkenović, Bogdana and Križak, Strahinja and Pajić, Tanja and Todorović, Nataša and Živić, Miroslav Ž",
year = "2023",
abstract = "Ion channels are vital components of filamentous fungi signaling in communication with their environment. We exploited the ability of the apical region of growing sporangiophores of Phycomyces blakesleeanus to form membrane-enveloped cytoplasmic droplets (CDs), to examine ion currents in the filamentous fungi native plasma membrane. In hypoosmotic conditions, the dominant current in the CDs is ORIC, an osmotically activated, anionic, outwardly rectified, fast inactivating instantaneous current that we have previously characterized. Here, we examined the effect of ATP on ORIC. We show that CDs contain active mitochondria, and that respiration inhibition by azide accelerates ORIC inactivation. ATP, added intracellularly, reduced ORIC run-down and shifted the voltage dependence of inactivation toward depolarized potentials, in a manner that did not require hydrolysis. Notably, ATP led to slowing down of ORIC inactivation, as evidenced by an increased time constant of inactivation, τin, and slower decline of τin during prolonged recordings. Flavonoids (genistein and quercetin) had the effect on ORIC opposite to ATP, acting as current inhibitors, possibly by disrupting the stabilizing effect of ATP on ORIC. The integration of osmotic sensing with ATP dependence of the anionic current, typical of vertebrate cells, is described here for the first time in filamentous fungi.",
publisher = "Springer Nature",
journal = "Scientific Reports",
title = "ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore",
number = "1",
volume = "13",
doi = "10.1038/s41598-023-39021-9",
pages = "11897"
}

Stevanović, K. S., Čepkenović, B., Križak, S., Pajić, T., Todorović, N.,& Živić, M. Ž.. (2023). ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore. in Scientific Reports
Springer Nature., 13(1), 11897.
https://doi.org/10.1038/s41598-023-39021-9

Stevanović KS, Čepkenović B, Križak S, Pajić T, Todorović N, Živić MŽ. ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore. in Scientific Reports. 2023;13(1):11897.
doi:10.1038/s41598-023-39021-9 .

Stevanović, Katarina S, Čepkenović, Bogdana, Križak, Strahinja, Pajić, Tanja, Todorović, Nataša, Živić, Miroslav Ž, "ATP modulation of osmotically activated anionic current in the membrane of Phycomyces blakesleeanus sporangiophore" in Scientific Reports, 13, no. 1 (2023):11897,
https://doi.org/10.1038/s41598-023-39021-9 . .

TY  - JOUR
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Nikolić, Stanko N
AU  - Rabasović, Mihailo D
AU  - Clayton, Andrew H A
AU  - Krmpot, Aleksandar J
PY  - 2022
UR  - http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC9637149
UR  - https://www.nature.com/articles/s41598-022-23502-4
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5235
AB  - We report the utilization of Third-Harmonic Generation microscopy for label-free live cell imaging of lipid droplets in the hypha of filamentous fungus Phycomyces blakesleeanus . THG microscopy images showed bright spherical features dispersed throughout the hypha cytoplasm in control conditions and a transient increase in the number of bright features after complete nitrogen starvation. Colocalization analysis of THG and lipid-counterstained images disclosed that the cytoplasmic particles were lipid droplets. Particle Size Analysis and Image Correlation Spectroscopy were used to quantify the number density and size of lipid droplets. The two analysis methods both revealed an increase from 16 × 10 −3 to 23 × 10 −3 lipid droplets/µm 2 after nitrogen starvation and a decrease in the average size of the droplets (range: 0.5–0.8 µm diameter). In conclusion, THG imaging, followed by PSA and ICS, can be reliably used for filamentous fungi for the in vivo quantification of lipid droplets without the need for labeling and/or fixation. In addition, it has been demonstrated that ICS is suitable for THG microscopy.
PB  - Berlin: Nature Portfolio
T2  - Scientific Reports
T1  - Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi
IS  - 1
VL  - 12
DO  - 10.1038/s41598-022-23502-4
SP  - 18760
ER  - 

@article{
author = "Pajić, Tanja and Todorović, Nataša and Živić, Miroslav and Nikolić, Stanko N and Rabasović, Mihailo D and Clayton, Andrew H A and Krmpot, Aleksandar J",
year = "2022",
abstract = "We report the utilization of Third-Harmonic Generation microscopy for label-free live cell imaging of lipid droplets in the hypha of filamentous fungus Phycomyces blakesleeanus . THG microscopy images showed bright spherical features dispersed throughout the hypha cytoplasm in control conditions and a transient increase in the number of bright features after complete nitrogen starvation. Colocalization analysis of THG and lipid-counterstained images disclosed that the cytoplasmic particles were lipid droplets. Particle Size Analysis and Image Correlation Spectroscopy were used to quantify the number density and size of lipid droplets. The two analysis methods both revealed an increase from 16 × 10 −3 to 23 × 10 −3 lipid droplets/µm 2 after nitrogen starvation and a decrease in the average size of the droplets (range: 0.5–0.8 µm diameter). In conclusion, THG imaging, followed by PSA and ICS, can be reliably used for filamentous fungi for the in vivo quantification of lipid droplets without the need for labeling and/or fixation. In addition, it has been demonstrated that ICS is suitable for THG microscopy.",
publisher = "Berlin: Nature Portfolio",
journal = "Scientific Reports",
title = "Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi",
number = "1",
volume = "12",
doi = "10.1038/s41598-022-23502-4",
pages = "18760"
}

Pajić, T., Todorović, N., Živić, M., Nikolić, S. N., Rabasović, M. D., Clayton, A. H. A.,& Krmpot, A. J.. (2022). Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi. in Scientific Reports
Berlin: Nature Portfolio., 12(1), 18760.
https://doi.org/10.1038/s41598-022-23502-4

Pajić T, Todorović N, Živić M, Nikolić SN, Rabasović MD, Clayton AHA, Krmpot AJ. Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi. in Scientific Reports. 2022;12(1):18760.
doi:10.1038/s41598-022-23502-4 .

Pajić, Tanja, Todorović, Nataša, Živić, Miroslav, Nikolić, Stanko N, Rabasović, Mihailo D, Clayton, Andrew H A, Krmpot, Aleksandar J, "Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi" in Scientific Reports, 12, no. 1 (2022):18760,
https://doi.org/10.1038/s41598-022-23502-4 . .

TY  - CONF
AU  - Stevanović, Katarina
AU  - Pajić, Tanja
AU  - Živić, Miroslav
AU  - Krmpot, Aleksandar
AU  - Rabasović, Mihailo
AU  - Todorović, Nataša
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5742
AB  - Prisustvo i aktivni karakter ćelijskog zida koji prekriva plazma membranu filamentoznih gljiva su do sad bili nesavladive prepreke da joj se pristupi elektrofiziološkim metodama na način koji bi omogućio kvalitetno registrovanje jonskih struja. Nedavno, mi smo razvili i optimizovali protokol koji omogućava kvalitetno snimanje struja, metodom nametnute voltaže na deliću membrane, sa plazma membrane protoplasta gljive oslobođenih od zida subćelijskom mikrohirurgijom korišćenjem femtosekundnog Ti:Sa lasera.1,2 Među registrovanim plazmamembranskim strujama protoplasta gljive dominantne su struje nošene različitim anjonskim vrstama. Na osnovu potencijala reverzije u asimetričnim jonskim uslovima identifikovani tipovi jonskih struja su, u najvećem broju slučajeva, bili slabo (42%) ili izrazito (35%) selektivni za hlor u prisustvu K, NO3, glutamatnog jona i visokih koncentracija Ca+2. Među nekoliko različitih struja slabije selektivnosti za hlor, daleko najzastupljenija je struja, konduktivnosti 21 ± 1 pS, prisutna u 42% svih kontakata registracije. Analiza aktivnosti pojedinačnih kanala ove, najzastupljenije, struje pokazuje da verovatnoća otvorenosti kanala slabo raste sa depolarizacijom membrane (Po(-80) = 0,3; Po(50) = 0,45) i opada na više depolarisanim vrednostima (Po(80) = 0,2). Učestalost paketića aktivnosti je najveća na hiperpolarisanim potencijalima (f(-80 mV) > 200 Hz) i dvostruko manja na depolarisanim (f(80 mV) < 100 Hz).
AB  - Присуство и активни карактер ћелијског зида који прекрива плазма мембрану
филаментозних гљива су до сад били несавладиве препреке да јој се приступи
електрофизиолошким методама на начин који би омогућио квалитетно
регистровање јонских струја. Недавно, ми смо развили и оптимизовали протокол
који омогућава квалитетно снимање струја, методом наметнуте волтаже на делићу
мембране, са плазма мембране протопласта гљиве ослобођених од зида
субћелијском микрохирургијом коришћењем фемтосекундног Ti:Sa ласера.1,2 Међу
регистрованим плазмамембранским струјама протопласта гљиве доминантне су
струје ношене различитим анјонским врстама. На основу потенцијала реверзије у
асиметричним јонским условима идентификовани типови јонских струја су, у
највећем броју случајева, били слабо (42%) или изразито (35%) селективни за хлор
у присуству K, NO3, глутаматног јона и високих концентрација Ca+2. Међу
неколико различитих струја слабије селективности за хлор, далеко најзаступљенија
је струја, кондуктивности 21 ± 1 pS, присутна у 42% свих контаката регистрације.
Анализа активности појединачних канала ове, најзаступљеније, струје показује да
вероватноћа отворености канала слабо расте са деполаризацијом мембране (Po(-80) =
0,3; Po(50) = 0,45) и опада на више деполарисаним вредностима (Po(80) = 0,2).
Учесталост пакетића активности је највећа на хиперполарисаним потенцијалима
(f(-80 mV) > 200 Hz) и двоструко мања на деполарисаним (f(80 mV) < 100 Hz).
PB  - Belgrade: Serbian Biological Society
C3  - Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
T1  - Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom
T1  - Доминантне струје плазма мембране гљиве Phycomyces blakesleeanus регистроване са протопласта добијених микрохирургијом ћелијског зида фемтосекундним ласером
SP  - 24
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5742
ER  - 

@conference{
author = "Stevanović, Katarina and Pajić, Tanja and Živić, Miroslav and Krmpot, Aleksandar and Rabasović, Mihailo and Todorović, Nataša",
year = "2022",
abstract = "Prisustvo i aktivni karakter ćelijskog zida koji prekriva plazma membranu filamentoznih gljiva su do sad bili nesavladive prepreke da joj se pristupi elektrofiziološkim metodama na način koji bi omogućio kvalitetno registrovanje jonskih struja. Nedavno, mi smo razvili i optimizovali protokol koji omogućava kvalitetno snimanje struja, metodom nametnute voltaže na deliću membrane, sa plazma membrane protoplasta gljive oslobođenih od zida subćelijskom mikrohirurgijom korišćenjem femtosekundnog Ti:Sa lasera.1,2 Među registrovanim plazmamembranskim strujama protoplasta gljive dominantne su struje nošene različitim anjonskim vrstama. Na osnovu potencijala reverzije u asimetričnim jonskim uslovima identifikovani tipovi jonskih struja su, u najvećem broju slučajeva, bili slabo (42%) ili izrazito (35%) selektivni za hlor u prisustvu K, NO3, glutamatnog jona i visokih koncentracija Ca+2. Među nekoliko različitih struja slabije selektivnosti za hlor, daleko najzastupljenija je struja, konduktivnosti 21 ± 1 pS, prisutna u 42% svih kontakata registracije. Analiza aktivnosti pojedinačnih kanala ove, najzastupljenije, struje pokazuje da verovatnoća otvorenosti kanala slabo raste sa depolarizacijom membrane (Po(-80) = 0,3; Po(50) = 0,45) i opada na više depolarisanim vrednostima (Po(80) = 0,2). Učestalost paketića aktivnosti je najveća na hiperpolarisanim potencijalima (f(-80 mV) > 200 Hz) i dvostruko manja na depolarisanim (f(80 mV) < 100 Hz)., Присуство и активни карактер ћелијског зида који прекрива плазма мембрану
филаментозних гљива су до сад били несавладиве препреке да јој се приступи
електрофизиолошким методама на начин који би омогућио квалитетно
регистровање јонских струја. Недавно, ми смо развили и оптимизовали протокол
који омогућава квалитетно снимање струја, методом наметнуте волтаже на делићу
мембране, са плазма мембране протопласта гљиве ослобођених од зида
субћелијском микрохирургијом коришћењем фемтосекундног Ti:Sa ласера.1,2 Међу
регистрованим плазмамембранским струјама протопласта гљиве доминантне су
струје ношене различитим анјонским врстама. На основу потенцијала реверзије у
асиметричним јонским условима идентификовани типови јонских струја су, у
највећем броју случајева, били слабо (42%) или изразито (35%) селективни за хлор
у присуству K, NO3, глутаматног јона и високих концентрација Ca+2. Међу
неколико различитих струја слабије селективности за хлор, далеко најзаступљенија
је струја, кондуктивности 21 ± 1 pS, присутна у 42% свих контаката регистрације.
Анализа активности појединачних канала ове, најзаступљеније, струје показује да
вероватноћа отворености канала слабо расте са деполаризацијом мембране (Po(-80) =
0,3; Po(50) = 0,45) и опада на више деполарисаним вредностима (Po(80) = 0,2).
Учесталост пакетића активности је највећа на хиперполарисаним потенцијалима
(f(-80 mV) > 200 Hz) и двоструко мања на деполарисаним (f(80 mV) < 100 Hz).",
publisher = "Belgrade: Serbian Biological Society",
journal = "Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia",
title = "Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom, Доминантне струје плазма мембране гљиве Phycomyces blakesleeanus регистроване са протопласта добијених микрохирургијом ћелијског зида фемтосекундним ласером",
pages = "24",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5742"
}

Stevanović, K., Pajić, T., Živić, M., Krmpot, A., Rabasović, M.,& Todorović, N.. (2022). Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
Belgrade: Serbian Biological Society., 24.
https://hdl.handle.net/21.15107/rcub_ibiss_5742

Stevanović K, Pajić T, Živić M, Krmpot A, Rabasović M, Todorović N. Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia. 2022;:24.
https://hdl.handle.net/21.15107/rcub_ibiss_5742 .

Stevanović, Katarina, Pajić, Tanja, Živić, Miroslav, Krmpot, Aleksandar, Rabasović, Mihailo, Todorović, Nataša, "Dominantne struje plazma membrane gljive Phycomyces blakesleeanus registrovane sa protoplasta dobijenih mikrohirurgijom ćelijskog zida femtosekundnim laserom" in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia (2022):24,
https://hdl.handle.net/21.15107/rcub_ibiss_5742 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Rabasović, Mihailo
AU  - Krmpot, Aleksandar
PY  - 2022
UR  - http://www.photonicsworkshop.ipb.ac.rs/15/images/RFotonika2022KnjigaApstrakata-B5-CIP.pdf
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5735
AB  - Lipids in oleaginous filamentous fungi are considered to be a valuable alternative resource for various biotechnological applications. In vivo label-free imaging enables monitoring of fungi lipid droplets (LD) accumulation with the minimal unwanted effects on the metabolism. LDs are the main source of contrast [1] in the Third Harmonic Generation (THG) microscopy method [2] due to their optical properties (high refractive index). The THG phenomenon is utilized in nonlinear laser scanning microscopy that employs ultra-short laser pulses for imaging. We present in vivo and label-free THG imaging of the individual hyphae of Phycomyces blakesleeanus (Figure 1), an oleaginous filamentous fungus with very rapid growth rate. The THG signal was detected in the forward direction (transmission arm) by PMT through Hoya glass UV filter with peak transmission at 340 nm. The Yb KGW laser, wavelength 1040 nm, pulse duration 200 fs, and repetition rate 83 MHz, has been the source  of the infrared femtosecond pulses. The LDs from THG images were quantified by two image analysis techniques: Image Correlation Spectroscopy (ICS) and software particle counting Particle Size Analysis (PSA). We used hyphae that undergo nitrogen starvation, which is known to cause alterations in lipid metabolism and increase of cellular lipid reserve. The two analysis methods gave similar results. The applicability of the described procedure can be easily extended to other unicellular organisms for the in vivo quantification of LDs since there is no need for sample labeling, fixation or any other specific preparation. In addition, we demonstrate that the ICA is suitable for THG images, although it is primarily developed and have been mostly used for fluorescence signals so far. REFERENCES [1] D. Débarre, W. Supatto et al., Nat. Methods, 3(1), (2006), 47-53. [2] R. W. Boyd, Nonlinear Optics, Academic Press (2008).
PB  - Belgrade: Institute of Physics
C3  - Book of Abstracts: 15th Photonics Workshop: Conference; 2022 Mar 13-16; Kopaonik, Serbia
T1  - Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi
SP  - 52
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5735
ER  - 

@conference{
author = "Pajić, Tanja and Todorović, Nataša and Živić, Miroslav and Rabasović, Mihailo and Krmpot, Aleksandar",
year = "2022",
abstract = "Lipids in oleaginous filamentous fungi are considered to be a valuable alternative resource for various biotechnological applications. In vivo label-free imaging enables monitoring of fungi lipid droplets (LD) accumulation with the minimal unwanted effects on the metabolism. LDs are the main source of contrast [1] in the Third Harmonic Generation (THG) microscopy method [2] due to their optical properties (high refractive index). The THG phenomenon is utilized in nonlinear laser scanning microscopy that employs ultra-short laser pulses for imaging. We present in vivo and label-free THG imaging of the individual hyphae of Phycomyces blakesleeanus (Figure 1), an oleaginous filamentous fungus with very rapid growth rate. The THG signal was detected in the forward direction (transmission arm) by PMT through Hoya glass UV filter with peak transmission at 340 nm. The Yb KGW laser, wavelength 1040 nm, pulse duration 200 fs, and repetition rate 83 MHz, has been the source  of the infrared femtosecond pulses. The LDs from THG images were quantified by two image analysis techniques: Image Correlation Spectroscopy (ICS) and software particle counting Particle Size Analysis (PSA). We used hyphae that undergo nitrogen starvation, which is known to cause alterations in lipid metabolism and increase of cellular lipid reserve. The two analysis methods gave similar results. The applicability of the described procedure can be easily extended to other unicellular organisms for the in vivo quantification of LDs since there is no need for sample labeling, fixation or any other specific preparation. In addition, we demonstrate that the ICA is suitable for THG images, although it is primarily developed and have been mostly used for fluorescence signals so far. REFERENCES [1] D. Débarre, W. Supatto et al., Nat. Methods, 3(1), (2006), 47-53. [2] R. W. Boyd, Nonlinear Optics, Academic Press (2008).",
publisher = "Belgrade: Institute of Physics",
journal = "Book of Abstracts: 15th Photonics Workshop: Conference; 2022 Mar 13-16; Kopaonik, Serbia",
title = "Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi",
pages = "52",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5735"
}

Pajić, T., Todorović, N., Živić, M., Rabasović, M.,& Krmpot, A.. (2022). Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi. in Book of Abstracts: 15th Photonics Workshop: Conference; 2022 Mar 13-16; Kopaonik, Serbia
Belgrade: Institute of Physics., 52.
https://hdl.handle.net/21.15107/rcub_ibiss_5735

Pajić T, Todorović N, Živić M, Rabasović M, Krmpot A. Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi. in Book of Abstracts: 15th Photonics Workshop: Conference; 2022 Mar 13-16; Kopaonik, Serbia. 2022;:52.
https://hdl.handle.net/21.15107/rcub_ibiss_5735 .

Pajić, Tanja, Todorović, Nataša, Živić, Miroslav, Rabasović, Mihailo, Krmpot, Aleksandar, "Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi" in Book of Abstracts: 15th Photonics Workshop: Conference; 2022 Mar 13-16; Kopaonik, Serbia (2022):52,
https://hdl.handle.net/21.15107/rcub_ibiss_5735 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Kozakijević, Suzana
AU  - Lukičić, Jovana
AU  - Živić, Miroslav
AU  - Krmpot, Aleksandar
AU  - Rabasović, Mihailo
AU  - Todorović, Nataša
PY  - 2022
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/5744
AB  - Biološka aktivnost selena zavisi od njegove doze i hemijskog oblika. U mikrotragovima povećava antioksidativni kapacitet, dok je u višim koncentracijama toksičan zbog stvaranja reaktivnih vrsta kiseonika i potencijalnog oštećenja mitohondrijalne membrane. Končaste gljive imaju sposobnost koncentrisanja selena u micelijumu i predstavljaju jedan od glavnih puteva za njegov ulazak u ekosisteme. U ovom radu je ispitivan uticaj hroničnog i akutnog dejstva natrijum selenita (uobičajen oblik selena u ishrani) na morfologiju i zastupljenost mitohondrija u živim ćelijama končaste gljive Phycomyces blakesleeanus tokom eksponencijalne faze rasta (EFR). Za in vivo oslikavanje morfologije mitohondrija i njihove dinamike u odgovoru na različite koncentracije selenita korišćena je multifotonska mikroskopija koja omogućava trodimenzionalno oslikavanje u visokoj rezoluciji i smanjeno fotooštećenje i fotoizbeljivanje uzorka zahvaljujući upotrebi infracrvenih ultrabrzih impulsnih lasera. Hronični tretmani selenitom su doveli do uočljivog trenda smanjenja zastupljenosti mitohondrija sa povećanjem koncentracije selenita. Izloženost hifa selenitu, tokom akutnih i hroničnih tretmana, rezultiralo je povećanjem zastupljenosti tubularne morfologije mitohondrija, koja je u akutnom tretmanu većim koncentracijama selenita (0,75 i 1 mM) bila povećana za 50% u odnosu na kontrolu. Kod mlađih hifa EFR izloženih hroničnom delovanju selenita (0,5 do 1 mM) tubularne mitohondrije su bile višestruko duže od onih u starijoj populaciji.
AB  - Биолошка активност селена зависи од његове дозе и хемијског облика. У
микротраговима повећава антиоксидативни капацитет, док је у вишим
концентрацијама токсичан због стварања реактивних врста кисеоника и
потенцијалног оштећења митохондријалне мембране. Кончасте гљиве имају
способност концентрисања селена у мицелијуму и представљају један од главних
путева за његов улазак у екосистеме. У овом раду је испитиван утицај хроничног и
акутног дејства натријум селенита (уобичајен облик селена у исхрани) на
морфологију и заступљеност митохондрија у живим ћелијама кончасте гљиве
Phycomyces blakesleeanus током експоненцијалне фазе раста (ЕФР). За in vivo
осликавање морфологије митохондрија и њихове динамике у одговору на
различите концентрације селенита коришћена је мултифотонска микроскопија која
омогућава тродимензионално осликавање у високој резолуцији и смањено
фотооштећење и фотоизбељивање узорка захваљујући употреби инфрацрвених
ултрабрзих импулсних ласера. Хронични третмани селенитом су довели до
уочљивог тренда смањења заступљености митохондрија са повећањем
концентрације селенита. Изложеност хифа селениту, током акутних и хроничних
третмана, резултирало је повећањем заступљености тубуларне морфологије
митохондрија, која је у акутном третману већим концентрацијама селенита (0,75 и
1 mМ) била повећана за 50% у односу на контролу. Код млађих хифа ЕФР
изложених хроничном деловању селенита (0,5 до 1 mМ) тубуларне митохондрије
су биле вишеструко дуже од оних у старијој популацији.
PB  - Belgrade: Serbian Biological Society
C3  - Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
T1  - In vivo oslikavanje pojedinačnih hifa Phycomyces blakesleeanus metodom multifotonske mikroskopije: uticaj selenita na morfologiju i funkciju mitohondrija
T1  - In vivo осликавање појединачних хифа Phycomyces blakesleeanus методом мултифотонске микроскопије: утицај селенита на морфологију и функцију митохондрија
SP  - 28
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_5744
ER  - 

@conference{
author = "Pajić, Tanja and Kozakijević, Suzana and Lukičić, Jovana and Živić, Miroslav and Krmpot, Aleksandar and Rabasović, Mihailo and Todorović, Nataša",
year = "2022",
abstract = "Biološka aktivnost selena zavisi od njegove doze i hemijskog oblika. U mikrotragovima povećava antioksidativni kapacitet, dok je u višim koncentracijama toksičan zbog stvaranja reaktivnih vrsta kiseonika i potencijalnog oštećenja mitohondrijalne membrane. Končaste gljive imaju sposobnost koncentrisanja selena u micelijumu i predstavljaju jedan od glavnih puteva za njegov ulazak u ekosisteme. U ovom radu je ispitivan uticaj hroničnog i akutnog dejstva natrijum selenita (uobičajen oblik selena u ishrani) na morfologiju i zastupljenost mitohondrija u živim ćelijama končaste gljive Phycomyces blakesleeanus tokom eksponencijalne faze rasta (EFR). Za in vivo oslikavanje morfologije mitohondrija i njihove dinamike u odgovoru na različite koncentracije selenita korišćena je multifotonska mikroskopija koja omogućava trodimenzionalno oslikavanje u visokoj rezoluciji i smanjeno fotooštećenje i fotoizbeljivanje uzorka zahvaljujući upotrebi infracrvenih ultrabrzih impulsnih lasera. Hronični tretmani selenitom su doveli do uočljivog trenda smanjenja zastupljenosti mitohondrija sa povećanjem koncentracije selenita. Izloženost hifa selenitu, tokom akutnih i hroničnih tretmana, rezultiralo je povećanjem zastupljenosti tubularne morfologije mitohondrija, koja je u akutnom tretmanu većim koncentracijama selenita (0,75 i 1 mM) bila povećana za 50% u odnosu na kontrolu. Kod mlađih hifa EFR izloženih hroničnom delovanju selenita (0,5 do 1 mM) tubularne mitohondrije su bile višestruko duže od onih u starijoj populaciji., Биолошка активност селена зависи од његове дозе и хемијског облика. У
микротраговима повећава антиоксидативни капацитет, док је у вишим
концентрацијама токсичан због стварања реактивних врста кисеоника и
потенцијалног оштећења митохондријалне мембране. Кончасте гљиве имају
способност концентрисања селена у мицелијуму и представљају један од главних
путева за његов улазак у екосистеме. У овом раду је испитиван утицај хроничног и
акутног дејства натријум селенита (уобичајен облик селена у исхрани) на
морфологију и заступљеност митохондрија у живим ћелијама кончасте гљиве
Phycomyces blakesleeanus током експоненцијалне фазе раста (ЕФР). За in vivo
осликавање морфологије митохондрија и њихове динамике у одговору на
различите концентрације селенита коришћена је мултифотонска микроскопија која
омогућава тродимензионално осликавање у високој резолуцији и смањено
фотооштећење и фотоизбељивање узорка захваљујући употреби инфрацрвених
ултрабрзих импулсних ласера. Хронични третмани селенитом су довели до
уочљивог тренда смањења заступљености митохондрија са повећањем
концентрације селенита. Изложеност хифа селениту, током акутних и хроничних
третмана, резултирало је повећањем заступљености тубуларне морфологије
митохондрија, која је у акутном третману већим концентрацијама селенита (0,75 и
1 mМ) била повећана за 50% у односу на контролу. Код млађих хифа ЕФР
изложених хроничном деловању селенита (0,5 до 1 mМ) тубуларне митохондрије
су биле вишеструко дуже од оних у старијој популацији.",
publisher = "Belgrade: Serbian Biological Society",
journal = "Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia",
title = "In vivo oslikavanje pojedinačnih hifa Phycomyces blakesleeanus metodom multifotonske mikroskopije: uticaj selenita na morfologiju i funkciju mitohondrija, In vivo осликавање појединачних хифа Phycomyces blakesleeanus методом мултифотонске микроскопије: утицај селенита на морфологију и функцију митохондрија",
pages = "28",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_5744"
}

Pajić, T., Kozakijević, S., Lukičić, J., Živić, M., Krmpot, A., Rabasović, M.,& Todorović, N.. (2022). In vivo oslikavanje pojedinačnih hifa Phycomyces blakesleeanus metodom multifotonske mikroskopije: uticaj selenita na morfologiju i funkciju mitohondrija. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia
Belgrade: Serbian Biological Society., 28.
https://hdl.handle.net/21.15107/rcub_ibiss_5744

Pajić T, Kozakijević S, Lukičić J, Živić M, Krmpot A, Rabasović M, Todorović N. In vivo oslikavanje pojedinačnih hifa Phycomyces blakesleeanus metodom multifotonske mikroskopije: uticaj selenita na morfologiju i funkciju mitohondrija. in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia. 2022;:28.
https://hdl.handle.net/21.15107/rcub_ibiss_5744 .

Pajić, Tanja, Kozakijević, Suzana, Lukičić, Jovana, Živić, Miroslav, Krmpot, Aleksandar, Rabasović, Mihailo, Todorović, Nataša, "In vivo oslikavanje pojedinačnih hifa Phycomyces blakesleeanus metodom multifotonske mikroskopije: uticaj selenita na morfologiju i funkciju mitohondrija" in Knjiga sažetaka: Treći Kongres biologa Srbije: Osnovna i primenjena istraživanja: Metodika nastave; 2022 Sep 21-25; Zlatibor, Serbia (2022):28,
https://hdl.handle.net/21.15107/rcub_ibiss_5744 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Krmpot, Aleksandar J.
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Stevanović, Katarina
AU  - Rabasović, Mihailo D.
PY  - 2021
UR  - https://iupab2020.sbbq.org.br/
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4719
AB  - Nonlinear laser scanning microscopy (NLSM), is an advanced optical technique that utilizes ultrashort laser pulses 
for structural and functional imaging, as well as laser manipulation of live organisms and cells. Two modalities of 
NLSM, two photon excitation fluorescence (TPEF) and third harmonic generation (THG) were applied for in vivo and 
label-free study of oxidative and lipid metabolism of individual cells of filamentous fungus Phycomyces 
blakesleeanus. Cell membranes and lipid droplets (LDs) are major sources of THG signal. TPEF allows us to 
determine the redox ratio (reflecting metabolic activity of cells) of the metabolic cofactors FAD and NAD(P)H 
autofluorescence. In addition, slight modifications of the experimental setup, mostly on software, enabled utilization 
of femtosecond laser pulses for precise cell microsurgery of hyphal cell wall. The optimized microsurgery procedure 
we than utilized to obtain protoplasts suitable for patch-clamp electrophysiological recording. Cell surgery of 
filamentous fungus Phycomyces blakesleeanus, were performed by ultrafast Ti:Sa laser (160 fs pulses). The same 
laser was used for in vivo autoTPEF imaging of NAD(P)H and FAD at different wavelengths. For in vivo THG imaging 
of label-free hyphae, we used 1040 nm, 200 fs pulses from Yb KGW laser. In vivo and label-free application of THG 
imaging enabled, accurately and reliably, detection of changes in distribution, total number, and size of LDs in control 
and treatment group of cells. Two-photon microscopy made it possible to obtain a redox ratio using autofluorescences 
of NAD(P)H and FAD in the same regions of live hyphae. The cell microsurgery procedure has been optimized and 
developed, which enabled the subsequent registration of currents on otherwise unaccessible membrane.
PB  - São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular
C3  - 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq
T1  - In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities
SP  - 358
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4719
ER  - 

@conference{
author = "Pajić, Tanja and Krmpot, Aleksandar J. and Todorović, Nataša and Živić, Miroslav and Stevanović, Katarina and Rabasović, Mihailo D.",
year = "2021",
abstract = "Nonlinear laser scanning microscopy (NLSM), is an advanced optical technique that utilizes ultrashort laser pulses 
for structural and functional imaging, as well as laser manipulation of live organisms and cells. Two modalities of 
NLSM, two photon excitation fluorescence (TPEF) and third harmonic generation (THG) were applied for in vivo and 
label-free study of oxidative and lipid metabolism of individual cells of filamentous fungus Phycomyces 
blakesleeanus. Cell membranes and lipid droplets (LDs) are major sources of THG signal. TPEF allows us to 
determine the redox ratio (reflecting metabolic activity of cells) of the metabolic cofactors FAD and NAD(P)H 
autofluorescence. In addition, slight modifications of the experimental setup, mostly on software, enabled utilization 
of femtosecond laser pulses for precise cell microsurgery of hyphal cell wall. The optimized microsurgery procedure 
we than utilized to obtain protoplasts suitable for patch-clamp electrophysiological recording. Cell surgery of 
filamentous fungus Phycomyces blakesleeanus, were performed by ultrafast Ti:Sa laser (160 fs pulses). The same 
laser was used for in vivo autoTPEF imaging of NAD(P)H and FAD at different wavelengths. For in vivo THG imaging 
of label-free hyphae, we used 1040 nm, 200 fs pulses from Yb KGW laser. In vivo and label-free application of THG 
imaging enabled, accurately and reliably, detection of changes in distribution, total number, and size of LDs in control 
and treatment group of cells. Two-photon microscopy made it possible to obtain a redox ratio using autofluorescences 
of NAD(P)H and FAD in the same regions of live hyphae. The cell microsurgery procedure has been optimized and 
developed, which enabled the subsequent registration of currents on otherwise unaccessible membrane.",
publisher = "São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular",
journal = "20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq",
title = "In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities",
pages = "358",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4719"
}

Pajić, T., Krmpot, A. J., Todorović, N., Živić, M., Stevanović, K.,& Rabasović, M. D.. (2021). In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities. in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq
São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular., 358.
https://hdl.handle.net/21.15107/rcub_ibiss_4719

Pajić T, Krmpot AJ, Todorović N, Živić M, Stevanović K, Rabasović MD. In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities. in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq. 2021;:358.
https://hdl.handle.net/21.15107/rcub_ibiss_4719 .

Pajić, Tanja, Krmpot, Aleksandar J., Todorović, Nataša, Živić, Miroslav, Stevanović, Katarina, Rabasović, Mihailo D., "In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities" in 20th IUPAB Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq (2021):358,
https://hdl.handle.net/21.15107/rcub_ibiss_4719 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Živić, Miroslav
AU  - Rabasović, Mihailo D.
AU  - Clayton, Andrew H. A.
AU  - Krmpot, Aleksandar J.
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4712
AB  - Oleaginous fungi can accumulate significant amounts of lipids in their mycelium (up to 80% of their biomass), primarily in the form of lipid droplets (LDs). LDs have optical properties that differ from the surrounding aquatic environment, which causes sudden changes in the refractive index. Here, we present in vivo and label-free imaging of individual hyphae of the oleaginous filamentous fungus 
Phycomyces blakesleeanus by Third Harmonic Generation (THG) microscopy method, where LDs 
are the main source of contrast. The LDs quantification from THG images was performed by two image analysis techniques: Image Correlation Spectroscopy (ICS) and software particle counting – Particle Size Analysis (PSA). ICS measures the spatial variation of fluorescence intensity fluctuations in the images, which can then be related to particle density and aggregation state. In order to test and compare the two methods, we used hyphae that undergo nitrogen starvation, which is known to cause alterations in lipid metabolism and the increase in LDs number. For in vivo THG imaging of label-free, > 24-hour old hyphae, we used 1040 nm, 200 fs pulses from Yb KGW laser. Detection was performed in the transmission arm by PMT through Hoya glass UV filter with peak transmission at 340nm. The laser beam was focused with the Zeiss Plan Neofluar 40x1.3 objective lens. For the imaging, the fungi were placed between two cover glasses of 0.17 μm thickness to match the objective lens requirements and for better transmission of the THG signal. An increased number of LDs under nitrogen starvation was observed in THG images and their 
number and size were analyzed using two quantification methods. The comparison of LDs number and 
size obtained by ICS and PSA shows that the number of LDs is approximately the same on average, but 
that ICS consistently detects slightly larger LD number in older group. The mean ICS measured diameter was slightly lower. Using the THG method in vivo and label-free, we can accurately and reliably, over time, detect changes in the localization, total number, and size of LDs in hyphae of the oleaginous filamentous fungus Phycomyces blakesleeanus.
PB  - Belgrade : Institute of Physics
C3  - VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop
T1  - Label-free third harmonic generation imaging of lipid droplets in live filamentous fungi
SP  - 115
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4712
ER  - 

@conference{
author = "Pajić, Tanja and Todorović, Nataša and Živić, Miroslav and Rabasović, Mihailo D. and Clayton, Andrew H. A. and Krmpot, Aleksandar J.",
year = "2021",
abstract = "Oleaginous fungi can accumulate significant amounts of lipids in their mycelium (up to 80% of their biomass), primarily in the form of lipid droplets (LDs). LDs have optical properties that differ from the surrounding aquatic environment, which causes sudden changes in the refractive index. Here, we present in vivo and label-free imaging of individual hyphae of the oleaginous filamentous fungus 
Phycomyces blakesleeanus by Third Harmonic Generation (THG) microscopy method, where LDs 
are the main source of contrast. The LDs quantification from THG images was performed by two image analysis techniques: Image Correlation Spectroscopy (ICS) and software particle counting – Particle Size Analysis (PSA). ICS measures the spatial variation of fluorescence intensity fluctuations in the images, which can then be related to particle density and aggregation state. In order to test and compare the two methods, we used hyphae that undergo nitrogen starvation, which is known to cause alterations in lipid metabolism and the increase in LDs number. For in vivo THG imaging of label-free, > 24-hour old hyphae, we used 1040 nm, 200 fs pulses from Yb KGW laser. Detection was performed in the transmission arm by PMT through Hoya glass UV filter with peak transmission at 340nm. The laser beam was focused with the Zeiss Plan Neofluar 40x1.3 objective lens. For the imaging, the fungi were placed between two cover glasses of 0.17 μm thickness to match the objective lens requirements and for better transmission of the THG signal. An increased number of LDs under nitrogen starvation was observed in THG images and their 
number and size were analyzed using two quantification methods. The comparison of LDs number and 
size obtained by ICS and PSA shows that the number of LDs is approximately the same on average, but 
that ICS consistently detects slightly larger LD number in older group. The mean ICS measured diameter was slightly lower. Using the THG method in vivo and label-free, we can accurately and reliably, over time, detect changes in the localization, total number, and size of LDs in hyphae of the oleaginous filamentous fungus Phycomyces blakesleeanus.",
publisher = "Belgrade : Institute of Physics",
journal = "VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop",
title = "Label-free third harmonic generation imaging of lipid droplets in live filamentous fungi",
pages = "115",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4712"
}

Pajić, T., Todorović, N., Živić, M., Rabasović, M. D., Clayton, A. H. A.,& Krmpot, A. J.. (2021). Label-free third harmonic generation imaging of lipid droplets in live filamentous fungi. in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop
Belgrade : Institute of Physics., 115.
https://hdl.handle.net/21.15107/rcub_ibiss_4712

Pajić T, Todorović N, Živić M, Rabasović MD, Clayton AHA, Krmpot AJ. Label-free third harmonic generation imaging of lipid droplets in live filamentous fungi. in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop. 2021;:115.
https://hdl.handle.net/21.15107/rcub_ibiss_4712 .

Pajić, Tanja, Todorović, Nataša, Živić, Miroslav, Rabasović, Mihailo D., Clayton, Andrew H. A., Krmpot, Aleksandar J., "Label-free third harmonic generation imaging of lipid droplets in live filamentous fungi" in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop (2021):115,
https://hdl.handle.net/21.15107/rcub_ibiss_4712 .

TY  - CONF
AU  - Živić, Miroslav
AU  - Stevanović, Katarina
AU  - Pajić, Tanja
AU  - Rabasović, Mihailo D.
AU  - Krmpot, Aleksandar J.
AU  - Todorović, Nataša
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4713
AB  - Electrophysiology of cell membrane ion channels in filamentous fungi, unlike that in yeast, plant and 
animal cells is still in its infancy. The only two reports on single channel patch clamp recordings from 
native cell membrane of filamentous fungi were made on protoplasts obtained after cell wall 
microsurgery with UV pulsed laser more than 20 years ago. Both pioneering papers reported 
fairly high success rates of obtaining gigaohm contacts, but neither was followed by more studies. 
Advanced imaging techniques that enable more controllable surgery process and utilizing the 
femtosecond pulses, after optimizing the protocol, could result in minimally damaging cell wall 
microsurgery. The end result would be reproducibly high quality membrane of “de-walled” protoplasts. 
The membrane quality, property that is of utmost importance for application of patch clamp method, is 
a term describing not only mechanical integrity and cleanliness of the membrane, but the physiological 
fitness of the cell as well, as cells about to enter apoptosis or necrosis, or that were subjected to oxidative 
stress do not have it. We are presenting here, to the best of our knowledge, the first electrophysiological snapshot obtained on filamentous fungi protoplast after cell wall removal by a femtosecond laser microsurgery. Utilization 
of the Ti:Sa femtosecond laser with optimizations of the cell wall microsurgery protocol explained in this conference resulted in protoplasts that were prone to form contacts of high electrical resistance (GΩ) with a patch pipette. Ti:Sa laser operating at 730 nm (76 MHz, 160 fs pulse duration) combined with homemade nonlinear laser scanning microscope, physiological 40x 1.0 NA objective was employed for microsurgery and imaging. Standard patch-clamp set up was used for electrophysiology. In single channel recordings from more than 30 patches, 11 different channel types were distinguished, based on the reversal potential in asymmetric ionic conditions and on the conductance. By far, the most frequent types of conductance were anionic. We have found four groups of ion channel currents, based 
on ion selectivity: 1. Unselective anion currents (not discriminating between chloride and glutamate) 44% of all recorded currents. 2. Anionic currents selective for chloride (carried exclusively or mostly by chloride) 35%. 3. Organic acid permeable anionic currents (discriminating for glutamate over chloride) 17%; 4. Calcium cationic current was recorded once. The range of conductance size (g) was variable, with unselective anionic currents encompassing the
smallest (5 pS) and largest (160 pS) recorded conductances. Calcium conductance was small (6 pS), while organic acid conductances and Cl-selective conductances had similar ranges (10-60 pS). Most of the conductances displayed linear current-voltage relationships.
PB  - Belgrade : Institute of Physics
C3  - VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop
T1  - First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery
SP  - 111
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4713
ER  - 

@conference{
author = "Živić, Miroslav and Stevanović, Katarina and Pajić, Tanja and Rabasović, Mihailo D. and Krmpot, Aleksandar J. and Todorović, Nataša",
year = "2021",
abstract = "Electrophysiology of cell membrane ion channels in filamentous fungi, unlike that in yeast, plant and 
animal cells is still in its infancy. The only two reports on single channel patch clamp recordings from 
native cell membrane of filamentous fungi were made on protoplasts obtained after cell wall 
microsurgery with UV pulsed laser more than 20 years ago. Both pioneering papers reported 
fairly high success rates of obtaining gigaohm contacts, but neither was followed by more studies. 
Advanced imaging techniques that enable more controllable surgery process and utilizing the 
femtosecond pulses, after optimizing the protocol, could result in minimally damaging cell wall 
microsurgery. The end result would be reproducibly high quality membrane of “de-walled” protoplasts. 
The membrane quality, property that is of utmost importance for application of patch clamp method, is 
a term describing not only mechanical integrity and cleanliness of the membrane, but the physiological 
fitness of the cell as well, as cells about to enter apoptosis or necrosis, or that were subjected to oxidative 
stress do not have it. We are presenting here, to the best of our knowledge, the first electrophysiological snapshot obtained on filamentous fungi protoplast after cell wall removal by a femtosecond laser microsurgery. Utilization 
of the Ti:Sa femtosecond laser with optimizations of the cell wall microsurgery protocol explained in this conference resulted in protoplasts that were prone to form contacts of high electrical resistance (GΩ) with a patch pipette. Ti:Sa laser operating at 730 nm (76 MHz, 160 fs pulse duration) combined with homemade nonlinear laser scanning microscope, physiological 40x 1.0 NA objective was employed for microsurgery and imaging. Standard patch-clamp set up was used for electrophysiology. In single channel recordings from more than 30 patches, 11 different channel types were distinguished, based on the reversal potential in asymmetric ionic conditions and on the conductance. By far, the most frequent types of conductance were anionic. We have found four groups of ion channel currents, based 
on ion selectivity: 1. Unselective anion currents (not discriminating between chloride and glutamate) 44% of all recorded currents. 2. Anionic currents selective for chloride (carried exclusively or mostly by chloride) 35%. 3. Organic acid permeable anionic currents (discriminating for glutamate over chloride) 17%; 4. Calcium cationic current was recorded once. The range of conductance size (g) was variable, with unselective anionic currents encompassing the
smallest (5 pS) and largest (160 pS) recorded conductances. Calcium conductance was small (6 pS), while organic acid conductances and Cl-selective conductances had similar ranges (10-60 pS). Most of the conductances displayed linear current-voltage relationships.",
publisher = "Belgrade : Institute of Physics",
journal = "VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop",
title = "First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery",
pages = "111",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4713"
}

Živić, M., Stevanović, K., Pajić, T., Rabasović, M. D., Krmpot, A. J.,& Todorović, N.. (2021). First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery. in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop
Belgrade : Institute of Physics., 111.
https://hdl.handle.net/21.15107/rcub_ibiss_4713

Živić M, Stevanović K, Pajić T, Rabasović MD, Krmpot AJ, Todorović N. First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery. in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop. 2021;:111.
https://hdl.handle.net/21.15107/rcub_ibiss_4713 .

Živić, Miroslav, Stevanović, Katarina, Pajić, Tanja, Rabasović, Mihailo D., Krmpot, Aleksandar J., Todorović, Nataša, "First glance at a multitude of ion currents on filamentous fungus P. blakesleeanus protoplasts obtained by femtosecond laser microsurgery" in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop (2021):111,
https://hdl.handle.net/21.15107/rcub_ibiss_4713 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Živić, Miroslav
AU  - Rabasović, Mihailo D.
AU  - Krmpot, Aleksandar J.
AU  - Todorović, Nataša
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4722
AB  - Lipid droplets (LDs) are small mobile organelles conserved in all eukaryotic cells. We wanted to 
test if the LD movement can be muffled by an incomplete inhibition of mitochondrial respiration, 
induced by treating hyphae of filamentous fungus Phycomyces blakesleeanus with 0.5 mM sodium 
azide. Nitrogen starved hyphae were used, in order to obtain LDs in larger sizes and numbers. The data 
obtained unequivocally showed: 1. Sodium azide treatment dramatically reduces the LD velocity and 
the distances LDs travel; 2. LDs in both controls and in azide-treated hyphae oscillate in a small 
confined space instead of travelling through the cell; 3. Azide-treated LDs oscillate less frequently and 
in smaller confinement than controls.
PB  - Kragujevac, Serbia: Institute for Information Technologies, University of Kragujevac
C3  - 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021
T1  - The dampening of lipid droplet oscillatory movement in nitrogen starved filamentous fungi by a low dose of mitochondrial respiration inhibitor
DO  - 10.46793/ICCBI21.226P
SP  - 226
EP  - 229
ER  - 

@conference{
author = "Pajić, Tanja and Živić, Miroslav and Rabasović, Mihailo D. and Krmpot, Aleksandar J. and Todorović, Nataša",
year = "2021",
abstract = "Lipid droplets (LDs) are small mobile organelles conserved in all eukaryotic cells. We wanted to 
test if the LD movement can be muffled by an incomplete inhibition of mitochondrial respiration, 
induced by treating hyphae of filamentous fungus Phycomyces blakesleeanus with 0.5 mM sodium 
azide. Nitrogen starved hyphae were used, in order to obtain LDs in larger sizes and numbers. The data 
obtained unequivocally showed: 1. Sodium azide treatment dramatically reduces the LD velocity and 
the distances LDs travel; 2. LDs in both controls and in azide-treated hyphae oscillate in a small 
confined space instead of travelling through the cell; 3. Azide-treated LDs oscillate less frequently and 
in smaller confinement than controls.",
publisher = "Kragujevac, Serbia: Institute for Information Technologies, University of Kragujevac",
journal = "1st International Conference on Chemo and BioInformatics, ICCBIKG 2021",
title = "The dampening of lipid droplet oscillatory movement in nitrogen starved filamentous fungi by a low dose of mitochondrial respiration inhibitor",
doi = "10.46793/ICCBI21.226P",
pages = "226-229"
}

Pajić, T., Živić, M., Rabasović, M. D., Krmpot, A. J.,& Todorović, N.. (2021). The dampening of lipid droplet oscillatory movement in nitrogen starved filamentous fungi by a low dose of mitochondrial respiration inhibitor. in 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021
Kragujevac, Serbia: Institute for Information Technologies, University of Kragujevac., 226-229.
https://doi.org/10.46793/ICCBI21.226P

Pajić T, Živić M, Rabasović MD, Krmpot AJ, Todorović N. The dampening of lipid droplet oscillatory movement in nitrogen starved filamentous fungi by a low dose of mitochondrial respiration inhibitor. in 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021. 2021;:226-229.
doi:10.46793/ICCBI21.226P .

Pajić, Tanja, Živić, Miroslav, Rabasović, Mihailo D., Krmpot, Aleksandar J., Todorović, Nataša, "The dampening of lipid droplet oscillatory movement in nitrogen starved filamentous fungi by a low dose of mitochondrial respiration inhibitor" in 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021 (2021):226-229,
https://doi.org/10.46793/ICCBI21.226P . .

TY  - CONF
AU  - Stevanović, Katarina
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Krmpot, Aleksandar J.
AU  - Živić, Miroslav
AU  - Rabasović, Mihailo D.
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4714
AB  - We have developed an advanced protocol for laser cell surgery to obtain protoplasts of filamentous 
fungi, suitable for investigation of ion channels, relying on few attempts already made in the past. Among obtained protoplasts, 32% were shown to be “patchable”, meaning that formation of the 
gigaseal by the micropipette was possible. Protoplasts were produced by first making an incision on the 
cell wall of plasmolysed hyphae by a tightly focused femtosecond laser beam. Cell surgery 
is followed by a reduction of solution osmolarity to promote extrusion of protoplast (or more often, a 
part of it) through the cut. The two key points- cell surgery parameters and the proper sequence of the 
solutions used – were subjected to variations to gain insight into parameters that contribute to protoplast 
production and stability. The proper selection of the pipette size and shape with respect to the protoplast 
size was also of great importance. Cell surgery and hyphae imaging was performed by a nonlinear laser scanning microscope. Ti:Sa laser was operating at 730nm, with 76MHz repetition rate and 160fs pulse duration. Water dipping objective lens (40 x 1.0, Zeiss W Plan-Apochromat) on the upright non-linear microscope system made possible to perform laser surgery and patch clamp on two separate systems, while working in the same microscopic chamber. Prior to microsurgery, two-photon excitation fluorescence was used to scan the hyphae cell wall stained with Calcofluor white dye, using the same wavelength as for the surgery. Two-step plasmolysis, with increased concentration of calcium in the more hyperosmotic solution was both efficient for microsurgery to be performed and had a stabilizing effect on protoplasts. Subtle deplasmolysis prior to the patch clamping effective enough to stimulate protoplasts to exit, without 
making the membrane overstretched to interact with the pipette was employed. Optimized concentration 
and type of chemical agens for inhibition of the cell wall production was continuously present in all 
solutions, as an indispensable factor for success.
PB  - Belgrade : Institute of Physics
C3  - VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop
T1  - Laser microsurgery of filamentous fungi: The latest protocol enabling patch-clamp amenable protoplasts
SP  - 104
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4714
ER  - 

@conference{
author = "Stevanović, Katarina and Pajić, Tanja and Todorović, Nataša and Krmpot, Aleksandar J. and Živić, Miroslav and Rabasović, Mihailo D.",
year = "2021",
abstract = "We have developed an advanced protocol for laser cell surgery to obtain protoplasts of filamentous 
fungi, suitable for investigation of ion channels, relying on few attempts already made in the past. Among obtained protoplasts, 32% were shown to be “patchable”, meaning that formation of the 
gigaseal by the micropipette was possible. Protoplasts were produced by first making an incision on the 
cell wall of plasmolysed hyphae by a tightly focused femtosecond laser beam. Cell surgery 
is followed by a reduction of solution osmolarity to promote extrusion of protoplast (or more often, a 
part of it) through the cut. The two key points- cell surgery parameters and the proper sequence of the 
solutions used – were subjected to variations to gain insight into parameters that contribute to protoplast 
production and stability. The proper selection of the pipette size and shape with respect to the protoplast 
size was also of great importance. Cell surgery and hyphae imaging was performed by a nonlinear laser scanning microscope. Ti:Sa laser was operating at 730nm, with 76MHz repetition rate and 160fs pulse duration. Water dipping objective lens (40 x 1.0, Zeiss W Plan-Apochromat) on the upright non-linear microscope system made possible to perform laser surgery and patch clamp on two separate systems, while working in the same microscopic chamber. Prior to microsurgery, two-photon excitation fluorescence was used to scan the hyphae cell wall stained with Calcofluor white dye, using the same wavelength as for the surgery. Two-step plasmolysis, with increased concentration of calcium in the more hyperosmotic solution was both efficient for microsurgery to be performed and had a stabilizing effect on protoplasts. Subtle deplasmolysis prior to the patch clamping effective enough to stimulate protoplasts to exit, without 
making the membrane overstretched to interact with the pipette was employed. Optimized concentration 
and type of chemical agens for inhibition of the cell wall production was continuously present in all 
solutions, as an indispensable factor for success.",
publisher = "Belgrade : Institute of Physics",
journal = "VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop",
title = "Laser microsurgery of filamentous fungi: The latest protocol enabling patch-clamp amenable protoplasts",
pages = "104",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4714"
}

Stevanović, K., Pajić, T., Todorović, N., Krmpot, A. J., Živić, M.,& Rabasović, M. D.. (2021). Laser microsurgery of filamentous fungi: The latest protocol enabling patch-clamp amenable protoplasts. in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop
Belgrade : Institute of Physics., 104.
https://hdl.handle.net/21.15107/rcub_ibiss_4714

Stevanović K, Pajić T, Todorović N, Krmpot AJ, Živić M, Rabasović MD. Laser microsurgery of filamentous fungi: The latest protocol enabling patch-clamp amenable protoplasts. in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop. 2021;:104.
https://hdl.handle.net/21.15107/rcub_ibiss_4714 .

Stevanović, Katarina, Pajić, Tanja, Todorović, Nataša, Krmpot, Aleksandar J., Živić, Miroslav, Rabasović, Mihailo D., "Laser microsurgery of filamentous fungi: The latest protocol enabling patch-clamp amenable protoplasts" in VIII International School and Conference on Photonics PHOTONICA2021 & HEMMAGINERO workshop (2021):104,
https://hdl.handle.net/21.15107/rcub_ibiss_4714 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Stefanović, Dunja
AU  - Rabasović, Mihailo D.
AU  - Krmpot, Aleksandar J.
AU  - Živić, Miroslav
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4721
AB  - Selenium salts have been known for long time to have a potential for both beneficial and harmful 
effects on living organisms. It is present in the environment, where it can be readily assimilated by 
plants and fungi, thus entering the food chain. We investigated the cell growth dynamics in the presence 
of selenite which is considered to have more toxic potential than selenate. The effects of selenite (1 
mM) on the growth of fungi from the activated spores to the end of the exponential growth were 
measured on several hypha morphological parameters by microscopy in vivo. Phycomyces 
blaekesleneeanus was used as model filamentous fungus. The most striking effect of Se+4 treatment was 
inhibition of hypha growth, resulting in more than four times shorter hypha in Se+4 –treatment group 
than in the control (200 ± 50 µm, n = 50 vs 900 ± 100 µm, n = 40 respectively) at the end of exponential 
growth period under controlled conditions. The Se+4 effect was an inhibition and not a simple delay in 
growth, as hypha length did not change significantly from 27th to 30th hour of culture in Se+4
-treatment group. Since the microscopy was performed on live cultured cells, undisturbed cytoplasmic streaming was observed, confirming that hyphae were alive at all time points measured. 30h old spore diameters were also significantly reduced by Se+4 treatment (p = 0.0365), while hypha diameters were not 
significantly altered.
PB  - Kragujevac, Serbia: Institute for Information Technologies, University of Kragujevac
C3  - 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021
T1  - The influence of selenite on fillamentous fungi hypha morphometry parameters
DO  - 10.46793/ICCBI21.308P
SP  - 308
EP  - 311
ER  - 

@conference{
author = "Pajić, Tanja and Todorović, Nataša and Stefanović, Dunja and Rabasović, Mihailo D. and Krmpot, Aleksandar J. and Živić, Miroslav",
year = "2021",
abstract = "Selenium salts have been known for long time to have a potential for both beneficial and harmful 
effects on living organisms. It is present in the environment, where it can be readily assimilated by 
plants and fungi, thus entering the food chain. We investigated the cell growth dynamics in the presence 
of selenite which is considered to have more toxic potential than selenate. The effects of selenite (1 
mM) on the growth of fungi from the activated spores to the end of the exponential growth were 
measured on several hypha morphological parameters by microscopy in vivo. Phycomyces 
blaekesleneeanus was used as model filamentous fungus. The most striking effect of Se+4 treatment was 
inhibition of hypha growth, resulting in more than four times shorter hypha in Se+4 –treatment group 
than in the control (200 ± 50 µm, n = 50 vs 900 ± 100 µm, n = 40 respectively) at the end of exponential 
growth period under controlled conditions. The Se+4 effect was an inhibition and not a simple delay in 
growth, as hypha length did not change significantly from 27th to 30th hour of culture in Se+4
-treatment group. Since the microscopy was performed on live cultured cells, undisturbed cytoplasmic streaming was observed, confirming that hyphae were alive at all time points measured. 30h old spore diameters were also significantly reduced by Se+4 treatment (p = 0.0365), while hypha diameters were not 
significantly altered.",
publisher = "Kragujevac, Serbia: Institute for Information Technologies, University of Kragujevac",
journal = "1st International Conference on Chemo and BioInformatics, ICCBIKG 2021",
title = "The influence of selenite on fillamentous fungi hypha morphometry parameters",
doi = "10.46793/ICCBI21.308P",
pages = "308-311"
}

Pajić, T., Todorović, N., Stefanović, D., Rabasović, M. D., Krmpot, A. J.,& Živić, M.. (2021). The influence of selenite on fillamentous fungi hypha morphometry parameters. in 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021
Kragujevac, Serbia: Institute for Information Technologies, University of Kragujevac., 308-311.
https://doi.org/10.46793/ICCBI21.308P

Pajić T, Todorović N, Stefanović D, Rabasović MD, Krmpot AJ, Živić M. The influence of selenite on fillamentous fungi hypha morphometry parameters. in 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021. 2021;:308-311.
doi:10.46793/ICCBI21.308P .

Pajić, Tanja, Todorović, Nataša, Stefanović, Dunja, Rabasović, Mihailo D., Krmpot, Aleksandar J., Živić, Miroslav, "The influence of selenite on fillamentous fungi hypha morphometry parameters" in 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021 (2021):308-311,
https://doi.org/10.46793/ICCBI21.308P . .

TY  - CONF
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Stefanović, Dunja
AU  - Rabasović, Mihailo D.
AU  - Krmpot, Aleksandar J.
AU  - Živić, Miroslav
PY  - 2021
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/4680
AB  - Third Harmonic Generation (THG) microscopy was employed as a method of choice for lipid droplet (LD) measurements and quantification of the effect of selenite on LDs. Nonlinear laser scanning microscopy (NLSM) employs ultra-short laser pulses for imaging. THG microscopy is the modality of NLSM. Strong THG signals can only be observed from regions with non-uniformities with respect to their refractive index. Such regions in biological samples are lipid-water interfaces, and by far the brightest features in cells are LDs. For that reason, THG microscopy is the appropriate method for imaging of LDs from live unfixed cells, without the need for additional labeling. The biological effects of spore- to- end- of- exponential- phase duration (27 - 30 h) of exposure to 1 mM selenite were monitored in vivo on the cells of filamentous fungi in liquid culture. We measured the lipid droplet density and size distribution in a model fungi Phycomyces blakesleeanus. The in-house built microscope frame complemented with Yb KGW laser (1040 nm, 200 fs pulses) was used, while detection was enabled in the transmission arm by PMT through the Hoya glass UV filter (peak at 340 nm). From THG images of control and Se+4–treated hyphae, LD size and number were measured, showing that LD density was increased by more than 60% in Se+4–treated hyphae, compared to control. The average LD size distribution seemed slightly changed by Se+4-treatment. The obtained results suggest that 1 mM selenite treatment probably induces cellular stress response in filamentous fungi.
PB  - Kragujevac, Serbia: Institute for Information Technologies, University of Kragujevac
C3  - 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021; 2021 Oct 26-27; Kragujevac, Serbia
T1  - The effects of selenite on filamentous fungi lipid droplets monitored in vivo label free using advanced nonlinear microscopy technique
DO  - 10.46793/ICCBI21.300P
SP  - 300
EP  - 303
ER  - 

@conference{
author = "Pajić, Tanja and Todorović, Nataša and Stefanović, Dunja and Rabasović, Mihailo D. and Krmpot, Aleksandar J. and Živić, Miroslav",
year = "2021",
abstract = "Third Harmonic Generation (THG) microscopy was employed as a method of choice for lipid droplet (LD) measurements and quantification of the effect of selenite on LDs. Nonlinear laser scanning microscopy (NLSM) employs ultra-short laser pulses for imaging. THG microscopy is the modality of NLSM. Strong THG signals can only be observed from regions with non-uniformities with respect to their refractive index. Such regions in biological samples are lipid-water interfaces, and by far the brightest features in cells are LDs. For that reason, THG microscopy is the appropriate method for imaging of LDs from live unfixed cells, without the need for additional labeling. The biological effects of spore- to- end- of- exponential- phase duration (27 - 30 h) of exposure to 1 mM selenite were monitored in vivo on the cells of filamentous fungi in liquid culture. We measured the lipid droplet density and size distribution in a model fungi Phycomyces blakesleeanus. The in-house built microscope frame complemented with Yb KGW laser (1040 nm, 200 fs pulses) was used, while detection was enabled in the transmission arm by PMT through the Hoya glass UV filter (peak at 340 nm). From THG images of control and Se+4–treated hyphae, LD size and number were measured, showing that LD density was increased by more than 60% in Se+4–treated hyphae, compared to control. The average LD size distribution seemed slightly changed by Se+4-treatment. The obtained results suggest that 1 mM selenite treatment probably induces cellular stress response in filamentous fungi.",
publisher = "Kragujevac, Serbia: Institute for Information Technologies, University of Kragujevac",
journal = "1st International Conference on Chemo and BioInformatics, ICCBIKG 2021; 2021 Oct 26-27; Kragujevac, Serbia",
title = "The effects of selenite on filamentous fungi lipid droplets monitored in vivo label free using advanced nonlinear microscopy technique",
doi = "10.46793/ICCBI21.300P",
pages = "300-303"
}

Pajić, T., Todorović, N., Stefanović, D., Rabasović, M. D., Krmpot, A. J.,& Živić, M.. (2021). The effects of selenite on filamentous fungi lipid droplets monitored in vivo label free using advanced nonlinear microscopy technique. in 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021; 2021 Oct 26-27; Kragujevac, Serbia
Kragujevac, Serbia: Institute for Information Technologies, University of Kragujevac., 300-303.
https://doi.org/10.46793/ICCBI21.300P

Pajić T, Todorović N, Stefanović D, Rabasović MD, Krmpot AJ, Živić M. The effects of selenite on filamentous fungi lipid droplets monitored in vivo label free using advanced nonlinear microscopy technique. in 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021; 2021 Oct 26-27; Kragujevac, Serbia. 2021;:300-303.
doi:10.46793/ICCBI21.300P .

Pajić, Tanja, Todorović, Nataša, Stefanović, Dunja, Rabasović, Mihailo D., Krmpot, Aleksandar J., Živić, Miroslav, "The effects of selenite on filamentous fungi lipid droplets monitored in vivo label free using advanced nonlinear microscopy technique" in 1st International Conference on Chemo and BioInformatics, ICCBIKG 2021; 2021 Oct 26-27; Kragujevac, Serbia (2021):300-303,
https://doi.org/10.46793/ICCBI21.300P . .

TY  - CONF
AU  - Pajić, Tanja
AU  - Todorović, Nataša
AU  - Krmpot, Aleksandar J.
AU  - Rabasović, Mihailo D.
AU  - Jelenković, Branislav
AU  - Živić, Miroslav
PY  - 2020
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/4643
AB  - Nonlinear laser scanning microscopy (NLSM) enables non-invasive, label-free visualization of living cell, their internal structures and metabolic state (which is a general marker of cell health). The metabolic activity of the cell can be monitored through the optical redox ratio of endogenous fluorophores in cytoplasm and mitochondria. Optical redox ratio provides relative changes in NAD(P)H and FAD concentrations, i.e. oxidation-reduction state in the cell. Lipid droplets (LDs) also play an important role in cellular energy metabolism. LDs are dynamic organelles that interact with molecules involved in metabolism and change their numbers and size depending on the metabolic state of the cell. In stressed cells, LDs maintain energy and redox homeostasis. Here we combined two powerful modalities of NLSM, Two Photon Excitation fluorescence (TPEF) and Third Harmonic Generation (THG), to optically detect how the molecules and organelles, involved in energy metabolism, are distributed. THG is a nonlinear coherent optical effect in which the incident laser beam interacts with a medium producing the light of exactly three times shorter wavelenght than the incidental one. THG mostly ocuurs at interfaces where  change of refractive index is steep, like water-lipid structures. Therefore, cell membranes and lipid droplets are major sources of THG signal. For observation of a map of the oxidation-reduction ratio in a living cell, how the NADH and FAD ratio changes, we used two photon nonlinear microscopy. For in vivo TPEF imaging of optical redox ratio from the same region on hyphae of filamentous fungus Phycomyces blakesleeanus, we used ultrafast Ti:Sa laser, 160 fs pulses. The autofluorescence of NAD(P)H was excited by laser pulses at 730 nm and signal was collected through 479/40 filter, while for autofluorescence of FAD we used excitation by the same laser pulses at 860 nm, 530/43 filter. For THG imaging, we used 1040 nm, 200 fs pulses from Yb KGW laser, and detection was performed by PMT through Hoya glass UV filter with peak transmission at 340nm. Both laser beams were focused with the same objective lens, Zeiss Plan Neofluar 40x1.3. With our nonlinear microscope and its different modalities, we were able to localize the two metabolic molecules, NAD(P)H and FAD, clearly see the lipid droplets, their distribution, number and size, and to characterize cell metabolism. In stressed cells, we obtained a decrease in the redox ratio and a higher number of lipid droplets under nitrogen starvation.
PB  - Belgrade : Institute of Physics
C3  - Book of Abstracts: 13th Photonics Workshop: Conferenece; 2020 Mar 8-12; Kopaonik, Serbia.
T1  - In vivo metabolic imaging of fungus Phycomyces blakesleeanus using label-free nonlinear laser scanning microscopy
SP  - 23
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4643
ER  - 

@conference{
author = "Pajić, Tanja and Todorović, Nataša and Krmpot, Aleksandar J. and Rabasović, Mihailo D. and Jelenković, Branislav and Živić, Miroslav",
year = "2020",
abstract = "Nonlinear laser scanning microscopy (NLSM) enables non-invasive, label-free visualization of living cell, their internal structures and metabolic state (which is a general marker of cell health). The metabolic activity of the cell can be monitored through the optical redox ratio of endogenous fluorophores in cytoplasm and mitochondria. Optical redox ratio provides relative changes in NAD(P)H and FAD concentrations, i.e. oxidation-reduction state in the cell. Lipid droplets (LDs) also play an important role in cellular energy metabolism. LDs are dynamic organelles that interact with molecules involved in metabolism and change their numbers and size depending on the metabolic state of the cell. In stressed cells, LDs maintain energy and redox homeostasis. Here we combined two powerful modalities of NLSM, Two Photon Excitation fluorescence (TPEF) and Third Harmonic Generation (THG), to optically detect how the molecules and organelles, involved in energy metabolism, are distributed. THG is a nonlinear coherent optical effect in which the incident laser beam interacts with a medium producing the light of exactly three times shorter wavelenght than the incidental one. THG mostly ocuurs at interfaces where  change of refractive index is steep, like water-lipid structures. Therefore, cell membranes and lipid droplets are major sources of THG signal. For observation of a map of the oxidation-reduction ratio in a living cell, how the NADH and FAD ratio changes, we used two photon nonlinear microscopy. For in vivo TPEF imaging of optical redox ratio from the same region on hyphae of filamentous fungus Phycomyces blakesleeanus, we used ultrafast Ti:Sa laser, 160 fs pulses. The autofluorescence of NAD(P)H was excited by laser pulses at 730 nm and signal was collected through 479/40 filter, while for autofluorescence of FAD we used excitation by the same laser pulses at 860 nm, 530/43 filter. For THG imaging, we used 1040 nm, 200 fs pulses from Yb KGW laser, and detection was performed by PMT through Hoya glass UV filter with peak transmission at 340nm. Both laser beams were focused with the same objective lens, Zeiss Plan Neofluar 40x1.3. With our nonlinear microscope and its different modalities, we were able to localize the two metabolic molecules, NAD(P)H and FAD, clearly see the lipid droplets, their distribution, number and size, and to characterize cell metabolism. In stressed cells, we obtained a decrease in the redox ratio and a higher number of lipid droplets under nitrogen starvation.",
publisher = "Belgrade : Institute of Physics",
journal = "Book of Abstracts: 13th Photonics Workshop: Conferenece; 2020 Mar 8-12; Kopaonik, Serbia.",
title = "In vivo metabolic imaging of fungus Phycomyces blakesleeanus using label-free nonlinear laser scanning microscopy",
pages = "23",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4643"
}

Pajić, T., Todorović, N., Krmpot, A. J., Rabasović, M. D., Jelenković, B.,& Živić, M.. (2020). In vivo metabolic imaging of fungus Phycomyces blakesleeanus using label-free nonlinear laser scanning microscopy. in Book of Abstracts: 13th Photonics Workshop: Conferenece; 2020 Mar 8-12; Kopaonik, Serbia.
Belgrade : Institute of Physics., 23.
https://hdl.handle.net/21.15107/rcub_ibiss_4643

Pajić T, Todorović N, Krmpot AJ, Rabasović MD, Jelenković B, Živić M. In vivo metabolic imaging of fungus Phycomyces blakesleeanus using label-free nonlinear laser scanning microscopy. in Book of Abstracts: 13th Photonics Workshop: Conferenece; 2020 Mar 8-12; Kopaonik, Serbia.. 2020;:23.
https://hdl.handle.net/21.15107/rcub_ibiss_4643 .

Pajić, Tanja, Todorović, Nataša, Krmpot, Aleksandar J., Rabasović, Mihailo D., Jelenković, Branislav, Živić, Miroslav, "In vivo metabolic imaging of fungus Phycomyces blakesleeanus using label-free nonlinear laser scanning microscopy" in Book of Abstracts: 13th Photonics Workshop: Conferenece; 2020 Mar 8-12; Kopaonik, Serbia. (2020):23,
https://hdl.handle.net/21.15107/rcub_ibiss_4643 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Stevanović, Katarina
AU  - Todorović, Nataša
AU  - Krmpot, Aleksandar
AU  - Rabasović, Mihailo
AU  - Lazović, Vladimir
AU  - Pantelić, Dejan
AU  - Jelenković, Branislav
AU  - Živić, Miroslav
PY  - 2018
UR  - https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&cad=rja&uact=8&ved=2ahUKEwj1h4zoze6BAxWUtqQKHec6BAsQFnoECBsQAQ&url=https%3A%2F%2Fskbs.sk%2Fwp-content%2Fuploads%2F2018%2F05%2Frbc_2018.pdf&usg=AOvVaw3s3FIWs-7Q4jE-gsL6G_U6&opi=89978449
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6292
AB  - Application of patch-clamp method for investigation of membrane ion channels of filamentous
fungi is nontrivial task due to presence of chitinous cell wall. Complete removal of the
wall patch is needed to make the membrane accessible to glass pipette. We use the model
filamentous fungus organism, Phycomyces blakesleeanus which is undertaken to the cell
surgery procedure by means of tightly focused femtosecond laser beam. The hyphae were
grown on glass coverslips coated with collagen plasmolysed and imaged by homemade nonlinear
laser scanning microscope by detecting two photon excitation fluorescence signal.
Although intrinsic autofluorescence of chitin enables imaging of the cell wall the hyphae
were stained by Calcofluor White dye prior to the imaging in order to improve signal to
noise ratio. Ti:Sa laser, used for both imaging and surgery, was operating at 730nm, with
76MHz repetition rate and 160fs pulse duration. Carl Zeiss, EC Plan-NEOFLUAR, 401.3
oil immersion objective was used for tight focusing of the laser beam and for the collection
of the fluorescence signal. A visible interference filter (415nm - 685nm) was placed in front
of detector in order to remove scattered laser light. The successful cutting of cell wall could
be achieved within the range of laser intensities and cutting speeds (dwell times). The
hyphae were kept in azide throughout the experiment in order to block the regeneration
of the cell wall. After the surgery, hyphae were slowly deplasmolysed to induce exit of a
portion of the protoplast through the laser made incision in the cell wall.
PB  - Ljubljana: Slovenian Biophysical Society
C3  - Book of abstracts: 8th Regional Biophysics Conference: RBC2018; 2018 May 16-20; Zreče, Slovenia
T1  - Successful Ti: Sapphire laser cell surgery of Phycomyces blakesleeanus cell wall
SP  - PS-43
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6292
ER  - 

@conference{
author = "Pajić, Tanja and Stevanović, Katarina and Todorović, Nataša and Krmpot, Aleksandar and Rabasović, Mihailo and Lazović, Vladimir and Pantelić, Dejan and Jelenković, Branislav and Živić, Miroslav",
year = "2018",
abstract = "Application of patch-clamp method for investigation of membrane ion channels of filamentous
fungi is nontrivial task due to presence of chitinous cell wall. Complete removal of the
wall patch is needed to make the membrane accessible to glass pipette. We use the model
filamentous fungus organism, Phycomyces blakesleeanus which is undertaken to the cell
surgery procedure by means of tightly focused femtosecond laser beam. The hyphae were
grown on glass coverslips coated with collagen plasmolysed and imaged by homemade nonlinear
laser scanning microscope by detecting two photon excitation fluorescence signal.
Although intrinsic autofluorescence of chitin enables imaging of the cell wall the hyphae
were stained by Calcofluor White dye prior to the imaging in order to improve signal to
noise ratio. Ti:Sa laser, used for both imaging and surgery, was operating at 730nm, with
76MHz repetition rate and 160fs pulse duration. Carl Zeiss, EC Plan-NEOFLUAR, 401.3
oil immersion objective was used for tight focusing of the laser beam and for the collection
of the fluorescence signal. A visible interference filter (415nm - 685nm) was placed in front
of detector in order to remove scattered laser light. The successful cutting of cell wall could
be achieved within the range of laser intensities and cutting speeds (dwell times). The
hyphae were kept in azide throughout the experiment in order to block the regeneration
of the cell wall. After the surgery, hyphae were slowly deplasmolysed to induce exit of a
portion of the protoplast through the laser made incision in the cell wall.",
publisher = "Ljubljana: Slovenian Biophysical Society",
journal = "Book of abstracts: 8th Regional Biophysics Conference: RBC2018; 2018 May 16-20; Zreče, Slovenia",
title = "Successful Ti: Sapphire laser cell surgery of Phycomyces blakesleeanus cell wall",
pages = "PS-43",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6292"
}

Pajić, T., Stevanović, K., Todorović, N., Krmpot, A., Rabasović, M., Lazović, V., Pantelić, D., Jelenković, B.,& Živić, M.. (2018). Successful Ti: Sapphire laser cell surgery of Phycomyces blakesleeanus cell wall. in Book of abstracts: 8th Regional Biophysics Conference: RBC2018; 2018 May 16-20; Zreče, Slovenia
Ljubljana: Slovenian Biophysical Society., PS-43.
https://hdl.handle.net/21.15107/rcub_ibiss_6292

Pajić T, Stevanović K, Todorović N, Krmpot A, Rabasović M, Lazović V, Pantelić D, Jelenković B, Živić M. Successful Ti: Sapphire laser cell surgery of Phycomyces blakesleeanus cell wall. in Book of abstracts: 8th Regional Biophysics Conference: RBC2018; 2018 May 16-20; Zreče, Slovenia. 2018;:PS-43.
https://hdl.handle.net/21.15107/rcub_ibiss_6292 .

Pajić, Tanja, Stevanović, Katarina, Todorović, Nataša, Krmpot, Aleksandar, Rabasović, Mihailo, Lazović, Vladimir, Pantelić, Dejan, Jelenković, Branislav, Živić, Miroslav, "Successful Ti: Sapphire laser cell surgery of Phycomyces blakesleeanus cell wall" in Book of abstracts: 8th Regional Biophysics Conference: RBC2018; 2018 May 16-20; Zreče, Slovenia (2018):PS-43,
https://hdl.handle.net/21.15107/rcub_ibiss_6292 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Stevanović, Katarina
AU  - Todorović, Nataša
AU  - Krmpot, Aleksandar
AU  - Rabasović, Mihailo
AU  - Lazović, Vladimir
AU  - Pantelić, Dejan
AU  - Jelenković, Branislav
AU  - Živić, Miroslav
PY  - 2017
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6293
AB  - The ion channels on the membrane of filamentous fungi remain uninvestigated to this day due to their inaccessibility to patch-clamp pipette, brought about by sturdy cell wall. Small number of described channels is from very specific types of cells (wall less mutants or sporangiophore aerial cell membrane). The enzymatic approaches of cell wall removal, albeit successful on plant cells, failed when applied to fungi. In order to obtain clean “patchable” membrane from any type of filamentous fungal cell we undertook a task of finding the conditions for cut of the small part of the hyphal wall by laser surgery on the model filamentous fungus organism, Phycomyces blakesleeanus. The successful wall surgery should result in exposure of large enough portion of cell membrane with a minimal damage to the protoplast. Therefore, we performed series of experiments with cell plasmolisys in hypoosmotic media and subsequent deplasmolisys, to determine the conditions for reliable retraction of cytoplasm that could be reversed. Next, hyphae, grown on glass coverslips coated with collagen, were plasmolysed and mounted on the stage of the homemade nonlinear laser scanning microscope for imaging and cell surgery [1]. The Ti:Sapphire laser (Coherent, Mira 900-F) has been used as a light source in the microscope. It has operated at 730nm. This wavelength enables two photon excitation of auto-fluorescence in cytoplasm, as well as dye (Calcofluor white), visualizing fungi wall. We have used Carl Zeiss, EC Plan-NEOFLUAR, 40×1.3 oil immersion objective for focusing of the laser beam and collection of fluorescence. A visible interference filter (415nm - 685 nm) in front of detector has been used to remove scattered laser light. The successful cutting of cell wall could be achieved within the range of laser intensities and cutting speeds (dwell times). Throughout the experiment, fungi were kept in azide or Brefeldin A in order to block the process of depositing the new wall material. Afterwards, hyphae were slowly deplasmolysed to induce exit of a portion of the protoplast through the laser-cut hole in the cell wall. However, in some instances, the part of the protoplast bulged through the hole immediately after cell surgery, while the cell was still in hypertonic solution. In other instances, the cytoplasm remained away from the cut hyphal apex even through series of slow incrementing hypotonic solutions. Finally, when laser cutting was applied on the side of the cell only, as to cut a small hole, the successful exit of a portion of protoplast through the hole during deplasmolisys could be reliably achieved.
PB  - Belgrade: Institute of Physics
C3  - Book of Abstracts: the Sixth International School and Conference on Photonics & COST actions: MP1406 and MP1402 & H2020-MSCA-RISE-2015 CARDIALLY workshop: PHOTONICA2017; 2017 Aug 28 - Sep 1; Belgrade, Serbia
T1  - Phycomyces blakesleeanus hypha cell wall surgery by Ti:Sapphire laser
SP  - 106
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6293
ER  - 

@conference{
author = "Pajić, Tanja and Stevanović, Katarina and Todorović, Nataša and Krmpot, Aleksandar and Rabasović, Mihailo and Lazović, Vladimir and Pantelić, Dejan and Jelenković, Branislav and Živić, Miroslav",
year = "2017",
abstract = "The ion channels on the membrane of filamentous fungi remain uninvestigated to this day due to their inaccessibility to patch-clamp pipette, brought about by sturdy cell wall. Small number of described channels is from very specific types of cells (wall less mutants or sporangiophore aerial cell membrane). The enzymatic approaches of cell wall removal, albeit successful on plant cells, failed when applied to fungi. In order to obtain clean “patchable” membrane from any type of filamentous fungal cell we undertook a task of finding the conditions for cut of the small part of the hyphal wall by laser surgery on the model filamentous fungus organism, Phycomyces blakesleeanus. The successful wall surgery should result in exposure of large enough portion of cell membrane with a minimal damage to the protoplast. Therefore, we performed series of experiments with cell plasmolisys in hypoosmotic media and subsequent deplasmolisys, to determine the conditions for reliable retraction of cytoplasm that could be reversed. Next, hyphae, grown on glass coverslips coated with collagen, were plasmolysed and mounted on the stage of the homemade nonlinear laser scanning microscope for imaging and cell surgery [1]. The Ti:Sapphire laser (Coherent, Mira 900-F) has been used as a light source in the microscope. It has operated at 730nm. This wavelength enables two photon excitation of auto-fluorescence in cytoplasm, as well as dye (Calcofluor white), visualizing fungi wall. We have used Carl Zeiss, EC Plan-NEOFLUAR, 40×1.3 oil immersion objective for focusing of the laser beam and collection of fluorescence. A visible interference filter (415nm - 685 nm) in front of detector has been used to remove scattered laser light. The successful cutting of cell wall could be achieved within the range of laser intensities and cutting speeds (dwell times). Throughout the experiment, fungi were kept in azide or Brefeldin A in order to block the process of depositing the new wall material. Afterwards, hyphae were slowly deplasmolysed to induce exit of a portion of the protoplast through the laser-cut hole in the cell wall. However, in some instances, the part of the protoplast bulged through the hole immediately after cell surgery, while the cell was still in hypertonic solution. In other instances, the cytoplasm remained away from the cut hyphal apex even through series of slow incrementing hypotonic solutions. Finally, when laser cutting was applied on the side of the cell only, as to cut a small hole, the successful exit of a portion of protoplast through the hole during deplasmolisys could be reliably achieved.",
publisher = "Belgrade: Institute of Physics",
journal = "Book of Abstracts: the Sixth International School and Conference on Photonics & COST actions: MP1406 and MP1402 & H2020-MSCA-RISE-2015 CARDIALLY workshop: PHOTONICA2017; 2017 Aug 28 - Sep 1; Belgrade, Serbia",
title = "Phycomyces blakesleeanus hypha cell wall surgery by Ti:Sapphire laser",
pages = "106",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6293"
}

Pajić, T., Stevanović, K., Todorović, N., Krmpot, A., Rabasović, M., Lazović, V., Pantelić, D., Jelenković, B.,& Živić, M.. (2017). Phycomyces blakesleeanus hypha cell wall surgery by Ti:Sapphire laser. in Book of Abstracts: the Sixth International School and Conference on Photonics & COST actions: MP1406 and MP1402 & H2020-MSCA-RISE-2015 CARDIALLY workshop: PHOTONICA2017; 2017 Aug 28 - Sep 1; Belgrade, Serbia
Belgrade: Institute of Physics., 106.
https://hdl.handle.net/21.15107/rcub_ibiss_6293

Pajić T, Stevanović K, Todorović N, Krmpot A, Rabasović M, Lazović V, Pantelić D, Jelenković B, Živić M. Phycomyces blakesleeanus hypha cell wall surgery by Ti:Sapphire laser. in Book of Abstracts: the Sixth International School and Conference on Photonics & COST actions: MP1406 and MP1402 & H2020-MSCA-RISE-2015 CARDIALLY workshop: PHOTONICA2017; 2017 Aug 28 - Sep 1; Belgrade, Serbia. 2017;:106.
https://hdl.handle.net/21.15107/rcub_ibiss_6293 .

Pajić, Tanja, Stevanović, Katarina, Todorović, Nataša, Krmpot, Aleksandar, Rabasović, Mihailo, Lazović, Vladimir, Pantelić, Dejan, Jelenković, Branislav, Živić, Miroslav, "Phycomyces blakesleeanus hypha cell wall surgery by Ti:Sapphire laser" in Book of Abstracts: the Sixth International School and Conference on Photonics & COST actions: MP1406 and MP1402 & H2020-MSCA-RISE-2015 CARDIALLY workshop: PHOTONICA2017; 2017 Aug 28 - Sep 1; Belgrade, Serbia (2017):106,
https://hdl.handle.net/21.15107/rcub_ibiss_6293 .

TY  - JOUR
AU  - Stanić, Marin
AU  - Križak, Strahinja
AU  - Jovanović, Mirna
AU  - Pajić, Tanja
AU  - Ćirić, Ana
AU  - Žižić, Milan
AU  - Zakrzewska, Joanna
AU  - Cvetić Antić, Tijana
AU  - Todorović, Nataša
AU  - Živić, Miroslav
PY  - 2017
UR  - http://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000429
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/2730
AB  - Increasing resistance of fungal strains to known fungicides has prompted identification of new candidates for fungicides among substances previously used for other purposes. We have tested the effects of known anion channel inhibitors anthracene-9-carboxylic acid (A9C) and niflumic acid (NFA) on growth, energy metabolism and anionic current of mycelium of fungus Phycomyces blakesleeanus. Both inhibitors significantly decreased growth and respiration of mycelium, but complete inhibition was only achieved by 100 and 500 μM NFA for growth and respiration, respectively. A9C had no effect on respiration of human NCI-H460 cell line and very little effect on cucumber root sprout clippings, which nominates this inhibitor for further investigation as a potential new fungicide. Effects of A9C and NFA on respiration of isolated mitochondria of P. blakesleeanus were significantly smaller, which indicates that their inhibitory effect on respiration of mycelium is indirect. NMR spectroscopy showed that both A9C and NFA decrease the levels of ATP and polyphosphates in the mycelium of P. blakesleeanus, but only A9C caused intracellular acidification. Outwardly rectifying, fast inactivating instantaneous anionic current (ORIC) was also reduced to 33±5 and 21±3% of its pre-treatment size by A9C and NFA, respectively, but only in the absence of ATP. It can be assumed from our results that the regulation of ORIC is tightly linked to cellular energy metabolism in P. blakesleeanus, and the decrease in ATP and polyphosphate levels could be a direct cause of growth inhibition.
T2  - Microbiology
T1  - Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites
IS  - 3
VL  - 163
DO  - 10.1099/mic.0.000429
SP  - 364
EP  - 372
ER  - 

@article{
author = "Stanić, Marin and Križak, Strahinja and Jovanović, Mirna and Pajić, Tanja and Ćirić, Ana and Žižić, Milan and Zakrzewska, Joanna and Cvetić Antić, Tijana and Todorović, Nataša and Živić, Miroslav",
year = "2017",
abstract = "Increasing resistance of fungal strains to known fungicides has prompted identification of new candidates for fungicides among substances previously used for other purposes. We have tested the effects of known anion channel inhibitors anthracene-9-carboxylic acid (A9C) and niflumic acid (NFA) on growth, energy metabolism and anionic current of mycelium of fungus Phycomyces blakesleeanus. Both inhibitors significantly decreased growth and respiration of mycelium, but complete inhibition was only achieved by 100 and 500 μM NFA for growth and respiration, respectively. A9C had no effect on respiration of human NCI-H460 cell line and very little effect on cucumber root sprout clippings, which nominates this inhibitor for further investigation as a potential new fungicide. Effects of A9C and NFA on respiration of isolated mitochondria of P. blakesleeanus were significantly smaller, which indicates that their inhibitory effect on respiration of mycelium is indirect. NMR spectroscopy showed that both A9C and NFA decrease the levels of ATP and polyphosphates in the mycelium of P. blakesleeanus, but only A9C caused intracellular acidification. Outwardly rectifying, fast inactivating instantaneous anionic current (ORIC) was also reduced to 33±5 and 21±3% of its pre-treatment size by A9C and NFA, respectively, but only in the absence of ATP. It can be assumed from our results that the regulation of ORIC is tightly linked to cellular energy metabolism in P. blakesleeanus, and the decrease in ATP and polyphosphate levels could be a direct cause of growth inhibition.",
journal = "Microbiology",
title = "Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites",
number = "3",
volume = "163",
doi = "10.1099/mic.0.000429",
pages = "364-372"
}

Stanić, M., Križak, S., Jovanović, M., Pajić, T., Ćirić, A., Žižić, M., Zakrzewska, J., Cvetić Antić, T., Todorović, N.,& Živić, M.. (2017). Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites. in Microbiology, 163(3), 364-372.
https://doi.org/10.1099/mic.0.000429

Stanić M, Križak S, Jovanović M, Pajić T, Ćirić A, Žižić M, Zakrzewska J, Cvetić Antić T, Todorović N, Živić M. Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites. in Microbiology. 2017;163(3):364-372.
doi:10.1099/mic.0.000429 .

Stanić, Marin, Križak, Strahinja, Jovanović, Mirna, Pajić, Tanja, Ćirić, Ana, Žižić, Milan, Zakrzewska, Joanna, Cvetić Antić, Tijana, Todorović, Nataša, Živić, Miroslav, "Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites" in Microbiology, 163, no. 3 (2017):364-372,
https://doi.org/10.1099/mic.0.000429 . .