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 .

TY  - CONF
AU  - Stevanović, Katarina
AU  - Križak, Strahinja
AU  - Todorović, Nataša
AU  - Živić, Miroslav
PY  - 2018
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6375
AB  - Jonski kanali filamentoznih gljiva su još uvek u velikoj meri neistraženi, uglavnom usled prisustva brzo regenerišućeg ćelijskog zida. Naš model sistem citoplazmatskih kapi dobijenih iz sporangiofora gljive Phycomyces blakesleeanus, odnosno ćelijske membrane koja još uvek nije pokrivena ćelijskim zidom, nam omogućava da registrujemo struje metodom nametnute voltaže na deliću membrane. U hipoosmotskim uslovima, pored izlazno ispravljene anjonske struje (ORIC), u familiji struja sa cele membrane su uočljive i ulazno ispravljene anjonske struje koje su propustljive za glukonat. Ovde će biti opisane po prvi put. U hipoosmotskoj sredini (kada je prisutan i ORIC), sa hlorom simetrično zamenjenim glukonatom, glukonat propustljiva struja se javlja u 50% eksperimenata (n=10). U izoosmotskim uslovima, kada ORIC nije aktivan, ulazna struja je 2.5 puta veća kada je simetrični glukonat provodni jon nego kada je prisutan hlor (n=11, prisutna u 72% zapisa). Fit voltažne zavisnosti struje Bolcmanovom funkcijom pokazuje jasno ulazno ispravljanje, sa vrednošću nalektrisanja vratnica -0.55±0.10 (p<0.001). Upadljiva osobina glukonat provodljive struje je spora deaktivacija i odložena aktivacija (na potencijalu -150 mV struja se aktivira posle 137±36 ms (n=8)). Snimanje aktivnosti pojedinačnih kanala u asimetričnim anjonskim uslovima (glukonat/hlor) pokazalo je da u membrani citoplazmatske kapi postoje najmanje tri različita jonska kanala (u 3 od 30 eksperimenata) koji su propustljiviji za glukonat nego za hlor sa sledećim karakteristikama: provodljivost (u pS) 83, 60 i 75; potencijal reverzije (mV) -13,13, 9,42, -5,28; propustljivost za glukonat/propustljivost za hlor 1.80, 1.53, 1.27.
PB  - Belgrade: Serbian Biological Society
C3  - Drugi kongres biologa Srbije; 2018 Sep 25-30; Kladovo, Srbija
T1  - Ulazno ispravljena anjonska struja u membrani citoplazmatskih kapi iz gljive Phycomyces blakesleeanus
SP  - 34
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_6375
ER  - 

@conference{
author = "Stevanović, Katarina and Križak, Strahinja and Todorović, Nataša and Živić, Miroslav",
year = "2018",
abstract = "Jonski kanali filamentoznih gljiva su još uvek u velikoj meri neistraženi, uglavnom usled prisustva brzo regenerišućeg ćelijskog zida. Naš model sistem citoplazmatskih kapi dobijenih iz sporangiofora gljive Phycomyces blakesleeanus, odnosno ćelijske membrane koja još uvek nije pokrivena ćelijskim zidom, nam omogućava da registrujemo struje metodom nametnute voltaže na deliću membrane. U hipoosmotskim uslovima, pored izlazno ispravljene anjonske struje (ORIC), u familiji struja sa cele membrane su uočljive i ulazno ispravljene anjonske struje koje su propustljive za glukonat. Ovde će biti opisane po prvi put. U hipoosmotskoj sredini (kada je prisutan i ORIC), sa hlorom simetrično zamenjenim glukonatom, glukonat propustljiva struja se javlja u 50% eksperimenata (n=10). U izoosmotskim uslovima, kada ORIC nije aktivan, ulazna struja je 2.5 puta veća kada je simetrični glukonat provodni jon nego kada je prisutan hlor (n=11, prisutna u 72% zapisa). Fit voltažne zavisnosti struje Bolcmanovom funkcijom pokazuje jasno ulazno ispravljanje, sa vrednošću nalektrisanja vratnica -0.55±0.10 (p<0.001). Upadljiva osobina glukonat provodljive struje je spora deaktivacija i odložena aktivacija (na potencijalu -150 mV struja se aktivira posle 137±36 ms (n=8)). Snimanje aktivnosti pojedinačnih kanala u asimetričnim anjonskim uslovima (glukonat/hlor) pokazalo je da u membrani citoplazmatske kapi postoje najmanje tri različita jonska kanala (u 3 od 30 eksperimenata) koji su propustljiviji za glukonat nego za hlor sa sledećim karakteristikama: provodljivost (u pS) 83, 60 i 75; potencijal reverzije (mV) -13,13, 9,42, -5,28; propustljivost za glukonat/propustljivost za hlor 1.80, 1.53, 1.27.",
publisher = "Belgrade: Serbian Biological Society",
journal = "Drugi kongres biologa Srbije; 2018 Sep 25-30; Kladovo, Srbija",
title = "Ulazno ispravljena anjonska struja u membrani citoplazmatskih kapi iz gljive Phycomyces blakesleeanus",
pages = "34",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6375"
}

Stevanović, K., Križak, S., Todorović, N.,& Živić, M.. (2018). Ulazno ispravljena anjonska struja u membrani citoplazmatskih kapi iz gljive Phycomyces blakesleeanus. in Drugi kongres biologa Srbije; 2018 Sep 25-30; Kladovo, Srbija
Belgrade: Serbian Biological Society., 34.
https://hdl.handle.net/21.15107/rcub_ibiss_6375

Stevanović K, Križak S, Todorović N, Živić M. Ulazno ispravljena anjonska struja u membrani citoplazmatskih kapi iz gljive Phycomyces blakesleeanus. in Drugi kongres biologa Srbije; 2018 Sep 25-30; Kladovo, Srbija. 2018;:34.
https://hdl.handle.net/21.15107/rcub_ibiss_6375 .

Stevanović, Katarina, Križak, Strahinja, Todorović, Nataša, Živić, Miroslav, "Ulazno ispravljena anjonska struja u membrani citoplazmatskih kapi iz gljive Phycomyces blakesleeanus" in Drugi kongres biologa Srbije; 2018 Sep 25-30; Kladovo, Srbija (2018):34,
https://hdl.handle.net/21.15107/rcub_ibiss_6375 .

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

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

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

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

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

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

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

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

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

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