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  - 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 .