TY  - JOUR
AU  - Pajić, Tanja
AU  - Stevanović, Katarina
AU  - Todorović, Nataša
AU  - Krmpot, Aleksandar J
AU  - Živić, Miroslav
AU  - Savić-Šević, Svetlana
AU  - Lević, Steva M
AU  - Stanić, Marina
AU  - Pantelić, Dejan
AU  - Jelenković, Brana
AU  - Rabasović, Mihailo D
PY  - 2024
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6666
AB  - Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens. However, their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall. Here, we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi, which enabled patch-clamp measurements on protoplasts released from hyphae. A reproducible and highly precise (diffraction-limited, submicron resolution) method for obtaining viable released protoplasts was developed. Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae. The plasma membrane of the obtained protoplasts formed tight and high-resistance (GΩ) contacts with the recording pipette. The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour. Compared to previous studies using heterologously expressed channels, this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.
PB  - Springer Nature
T2  - Microsystems & Nanoengineering
T1  - In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi
VL  - 10
DO  - 10.1038/s41378-024-00664-x
SP  - 47
ER  - 

@article{
author = "Pajić, Tanja and Stevanović, Katarina and Todorović, Nataša and Krmpot, Aleksandar J and Živić, Miroslav and Savić-Šević, Svetlana and Lević, Steva M and Stanić, Marina and Pantelić, Dejan and Jelenković, Brana and Rabasović, Mihailo D",
year = "2024",
abstract = "Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens. However, their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall. Here, we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi, which enabled patch-clamp measurements on protoplasts released from hyphae. A reproducible and highly precise (diffraction-limited, submicron resolution) method for obtaining viable released protoplasts was developed. Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae. The plasma membrane of the obtained protoplasts formed tight and high-resistance (GΩ) contacts with the recording pipette. The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour. Compared to previous studies using heterologously expressed channels, this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.",
publisher = "Springer Nature",
journal = "Microsystems & Nanoengineering",
title = "In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi",
volume = "10",
doi = "10.1038/s41378-024-00664-x",
pages = "47"
}

Pajić, T., Stevanović, K., Todorović, N., Krmpot, A. J., Živić, M., Savić-Šević, S., Lević, S. M., Stanić, M., Pantelić, D., Jelenković, B.,& Rabasović, M. D.. (2024). In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi. in Microsystems & Nanoengineering
Springer Nature., 10, 47.
https://doi.org/10.1038/s41378-024-00664-x

Pajić T, Stevanović K, Todorović N, Krmpot AJ, Živić M, Savić-Šević S, Lević SM, Stanić M, Pantelić D, Jelenković B, Rabasović MD. In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi. in Microsystems & Nanoengineering. 2024;10:47.
doi:10.1038/s41378-024-00664-x .

Pajić, Tanja, Stevanović, Katarina, Todorović, Nataša, Krmpot, Aleksandar J, Živić, Miroslav, Savić-Šević, Svetlana, Lević, Steva M, Stanić, Marina, Pantelić, Dejan, Jelenković, Brana, Rabasović, Mihailo D, "In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi" in Microsystems & Nanoengineering, 10 (2024):47,
https://doi.org/10.1038/s41378-024-00664-x . .

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  - 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  - 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  - 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 .