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  - 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 .