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  - Stevanović, Katarina
AU  - Todorović, Nataša
AU  - Krmpot, Aleksandar
AU  - Rabasović, Mihailo
AU  - Jelenković, Branislav
AU  - Živić, Miroslav
PY  - 2019
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6290
AB  - Label-free two photon nonlinear microscopy is well established as a powerful tool for monitoring
metabolic state of the various cell types due to its non-perturbative nature and fairly low phototoxicity,
while application of third harmonic generation (THG) for three-dimensional (3D) cell and tissue
microscopy was enabled more recently. THG occurs at structural interfaces, such as local transitions
of the refractive index, most generally speaking at interfaces that are formed between aqueous
interstitial fluids and lipid-rich structures. Here we present preliminary data obtained by capturing
both THG and optical redox ratio signal from the same regions of the hyphae of Phycomyces
blakesleeanus.
Label- free metabolic intravital microscopy through application of both THG and NAD(P)H+/FAD+
autofluorescence ratio was used in alternating sequence on the same field of view on a fungal cells of
a model filamentous fungus Phycomyces blakesleeanus. The glass coverslip with collagen coating
bearing unstained hyphae was mounted on custom built microscope. Laser beams for both imaging
modalities were focused with the same objective lens, Zeiss Plan Neofluar 40x1.3. The
autofluorescence of NAD(P)H was excited by Ti:Sa laser pulses at 730 nm, 160 fs duration and signal
was collected through 479/40 filter, while for autofluorescence of FAD we used excitation by the
same laser pulses at 860 nm, 160 fs duration and 530/43 filter.
For THG, we used 1040 nm, 200 fs pulses from Yb KGW laser, and detection was performed by
PMT through Hoya glass UV filter with peak transmission at 340nm. As a control for perturbation of
optical redox ratio, rothenone (complex I inhibitor) was applied in some experiments. Nile red
staining was used to confirm that the brightest structures of round shape in THG images consist of
lipids and probably represent lipid droplets that serve as energy deposits in hyphea.
PB  - Belgrade: Institute for Biological Research "Siniša Stanković"– National Institute of Republic of Serbia, University of Belgrade
C3  - Proceedings: 14th Multinational Congress on Microscopy; 2019 Sep 15-20; Belgrade, Serbia
T1  - Combined two photon excitation fluorescence and third harmonic generation imaging of redox ratio for monitoring metabolic state of live cells of fungus Phycomyces blakesleeanus
SP  - 122
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6290
ER  - 

@conference{
author = "Pajić, Tanja and Stevanović, Katarina and Todorović, Nataša and Krmpot, Aleksandar and Rabasović, Mihailo and Jelenković, Branislav and Živić, Miroslav",
year = "2019",
abstract = "Label-free two photon nonlinear microscopy is well established as a powerful tool for monitoring
metabolic state of the various cell types due to its non-perturbative nature and fairly low phototoxicity,
while application of third harmonic generation (THG) for three-dimensional (3D) cell and tissue
microscopy was enabled more recently. THG occurs at structural interfaces, such as local transitions
of the refractive index, most generally speaking at interfaces that are formed between aqueous
interstitial fluids and lipid-rich structures. Here we present preliminary data obtained by capturing
both THG and optical redox ratio signal from the same regions of the hyphae of Phycomyces
blakesleeanus.
Label- free metabolic intravital microscopy through application of both THG and NAD(P)H+/FAD+
autofluorescence ratio was used in alternating sequence on the same field of view on a fungal cells of
a model filamentous fungus Phycomyces blakesleeanus. The glass coverslip with collagen coating
bearing unstained hyphae was mounted on custom built microscope. Laser beams for both imaging
modalities were focused with the same objective lens, Zeiss Plan Neofluar 40x1.3. The
autofluorescence of NAD(P)H was excited by Ti:Sa laser pulses at 730 nm, 160 fs duration and signal
was collected through 479/40 filter, while for autofluorescence of FAD we used excitation by the
same laser pulses at 860 nm, 160 fs duration and 530/43 filter.
For THG, we used 1040 nm, 200 fs pulses from Yb KGW laser, and detection was performed by
PMT through Hoya glass UV filter with peak transmission at 340nm. As a control for perturbation of
optical redox ratio, rothenone (complex I inhibitor) was applied in some experiments. Nile red
staining was used to confirm that the brightest structures of round shape in THG images consist of
lipids and probably represent lipid droplets that serve as energy deposits in hyphea.",
publisher = "Belgrade: Institute for Biological Research "Siniša Stanković"– National Institute of Republic of Serbia, University of Belgrade",
journal = "Proceedings: 14th Multinational Congress on Microscopy; 2019 Sep 15-20; Belgrade, Serbia",
title = "Combined two photon excitation fluorescence and third harmonic generation imaging of redox ratio for monitoring metabolic state of live cells of fungus Phycomyces blakesleeanus",
pages = "122",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6290"
}

Pajić, T., Stevanović, K., Todorović, N., Krmpot, A., Rabasović, M., Jelenković, B.,& Živić, M.. (2019). Combined two photon excitation fluorescence and third harmonic generation imaging of redox ratio for monitoring metabolic state of live cells of fungus Phycomyces blakesleeanus. in Proceedings: 14th Multinational Congress on Microscopy; 2019 Sep 15-20; Belgrade, Serbia
Belgrade: Institute for Biological Research "Siniša Stanković"– National Institute of Republic of Serbia, University of Belgrade., 122.
https://hdl.handle.net/21.15107/rcub_ibiss_6290

Pajić T, Stevanović K, Todorović N, Krmpot A, Rabasović M, Jelenković B, Živić M. Combined two photon excitation fluorescence and third harmonic generation imaging of redox ratio for monitoring metabolic state of live cells of fungus Phycomyces blakesleeanus. in Proceedings: 14th Multinational Congress on Microscopy; 2019 Sep 15-20; Belgrade, Serbia. 2019;:122.
https://hdl.handle.net/21.15107/rcub_ibiss_6290 .

Pajić, Tanja, Stevanović, Katarina, Todorović, Nataša, Krmpot, Aleksandar, Rabasović, Mihailo, Jelenković, Branislav, Živić, Miroslav, "Combined two photon excitation fluorescence and third harmonic generation imaging of redox ratio for monitoring metabolic state of live cells of fungus Phycomyces blakesleeanus" in Proceedings: 14th Multinational Congress on Microscopy; 2019 Sep 15-20; Belgrade, Serbia (2019):122,
https://hdl.handle.net/21.15107/rcub_ibiss_6290 .

TY  - CONF
AU  - Pajić, Tanja
AU  - Stevanović, Katarina
AU  - Todorović, Nataša
AU  - Krmpot, Aleksandar
AU  - Rabasović, Mihailo
AU  - Jelenković, Branislav
AU  - Živić, Miroslav
PY  - 2019
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6291
AB  - Third Harmonic Generation is a nonlinear optical effect in which the incident laser beam
interacts with a medium producing the light of exactly three times shorter wavelength than the
incidental one. THG is generated in medium that have third order nonlinearity, but it is
particularly pronounced at the interfaces where the steep change of refractive index takes a place
[1]. THG phenomenon is employed in laser scanning microscopy that utilizes ultrashort laser
pulses for imaging. The THG microscopy is a label free techniques that provides important
information on the sample such as membrane imaging and lipid droplets distribution [2]. It is
mostly used for in vivo imaging of small model organisms like zebrafish [3] and C. Elegance [4].
We present THG imaging of filamentous fungus organism Phycomyces blakesleeanus, combined with
Two Photon Excitation fluorescence (TPEF). The hyphae were grown in various conditions on
glass coverslips coated with collagen and concanavalin A. For THG imaging of label-free, 16-24
hour old hyphae, we used 1040 nm, 200 fs pulses from Yb KGW laser, while for TPEF, Ti:Sa
pulses at 730 nm, 160 fs duration, were used. Both laser beams were focused with the same
objective lens, Zeiss Plan Neofluar 40x1.3. Detection of THG was performed by PMT through
Hoya glass UV filter with peak transmission at 340nm, while for TPEF 400-700 nm band pass
filter was used. THG images revealed the chitinous cell wall and the membrane that are clearly
separated. The appearance of the cell wall was confirmed by colocalization with TPEF images.
Most prominent observation on the THG images is presence of numerous, seemingly randomly
dispersed, round shiny features throughout the cytoplasm, for which we suspect that could be
lipid droplets as indicated in Débarre et al., 2006.
PB  - Belgrade: Vinča Institute of Nuclear Sciences – National Institute of the Republic of Serbia, University of Belgrade
C3  - Book of Abstracts: The Seventh International School and Conference on Photonics: PHOTONICA 2019; 2019 Aug 26-30; Belgrade, Serbia
T1  - In vivo third harmonic generation imaging of Phycomyces blakesleeanus
SP  - 130
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6291
ER  - 

@conference{
author = "Pajić, Tanja and Stevanović, Katarina and Todorović, Nataša and Krmpot, Aleksandar and Rabasović, Mihailo and Jelenković, Branislav and Živić, Miroslav",
year = "2019",
abstract = "Third Harmonic Generation is a nonlinear optical effect in which the incident laser beam
interacts with a medium producing the light of exactly three times shorter wavelength than the
incidental one. THG is generated in medium that have third order nonlinearity, but it is
particularly pronounced at the interfaces where the steep change of refractive index takes a place
[1]. THG phenomenon is employed in laser scanning microscopy that utilizes ultrashort laser
pulses for imaging. The THG microscopy is a label free techniques that provides important
information on the sample such as membrane imaging and lipid droplets distribution [2]. It is
mostly used for in vivo imaging of small model organisms like zebrafish [3] and C. Elegance [4].
We present THG imaging of filamentous fungus organism Phycomyces blakesleeanus, combined with
Two Photon Excitation fluorescence (TPEF). The hyphae were grown in various conditions on
glass coverslips coated with collagen and concanavalin A. For THG imaging of label-free, 16-24
hour old hyphae, we used 1040 nm, 200 fs pulses from Yb KGW laser, while for TPEF, Ti:Sa
pulses at 730 nm, 160 fs duration, were used. Both laser beams were focused with the same
objective lens, Zeiss Plan Neofluar 40x1.3. Detection of THG was performed by PMT through
Hoya glass UV filter with peak transmission at 340nm, while for TPEF 400-700 nm band pass
filter was used. THG images revealed the chitinous cell wall and the membrane that are clearly
separated. The appearance of the cell wall was confirmed by colocalization with TPEF images.
Most prominent observation on the THG images is presence of numerous, seemingly randomly
dispersed, round shiny features throughout the cytoplasm, for which we suspect that could be
lipid droplets as indicated in Débarre et al., 2006.",
publisher = "Belgrade: Vinča Institute of Nuclear Sciences – National Institute of the Republic of Serbia, University of Belgrade",
journal = "Book of Abstracts: The Seventh International School and Conference on Photonics: PHOTONICA 2019; 2019 Aug 26-30; Belgrade, Serbia",
title = "In vivo third harmonic generation imaging of Phycomyces blakesleeanus",
pages = "130",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6291"
}

Pajić, T., Stevanović, K., Todorović, N., Krmpot, A., Rabasović, M., Jelenković, B.,& Živić, M.. (2019). In vivo third harmonic generation imaging of Phycomyces blakesleeanus. in Book of Abstracts: The Seventh International School and Conference on Photonics: PHOTONICA 2019; 2019 Aug 26-30; Belgrade, Serbia
Belgrade: Vinča Institute of Nuclear Sciences – National Institute of the Republic of Serbia, University of Belgrade., 130.
https://hdl.handle.net/21.15107/rcub_ibiss_6291

Pajić T, Stevanović K, Todorović N, Krmpot A, Rabasović M, Jelenković B, Živić M. In vivo third harmonic generation imaging of Phycomyces blakesleeanus. in Book of Abstracts: The Seventh International School and Conference on Photonics: PHOTONICA 2019; 2019 Aug 26-30; Belgrade, Serbia. 2019;:130.
https://hdl.handle.net/21.15107/rcub_ibiss_6291 .

Pajić, Tanja, Stevanović, Katarina, Todorović, Nataša, Krmpot, Aleksandar, Rabasović, Mihailo, Jelenković, Branislav, Živić, Miroslav, "In vivo third harmonic generation imaging of Phycomyces blakesleeanus" in Book of Abstracts: The Seventh International School and Conference on Photonics: PHOTONICA 2019; 2019 Aug 26-30; Belgrade, Serbia (2019):130,
https://hdl.handle.net/21.15107/rcub_ibiss_6291 .