@conference{
author = "Pajić, Tanja and Todorović, Nataša and Krmpot, Aleksandar J. and Rabasović, Mihailo D. and Jelenković, Branislav and Živić, Miroslav",
year = "2020",
abstract = "Nonlinear laser scanning microscopy (NLSM) enables non-invasive, label-free visualization of living cell, their internal structures and metabolic state (which is a general marker of cell health). The metabolic activity of the cell can be monitored through the optical redox ratio of endogenous fluorophores in cytoplasm and mitochondria. Optical redox ratio provides relative changes in NAD(P)H and FAD concentrations, i.e. oxidation-reduction state in the cell. Lipid droplets (LDs) also play an important role in cellular energy metabolism. LDs are dynamic organelles that interact with molecules involved in metabolism and change their numbers and size depending on the metabolic state of the cell. In stressed cells, LDs maintain energy and redox homeostasis. Here we combined two powerful modalities of NLSM, Two Photon Excitation fluorescence (TPEF) and Third Harmonic Generation (THG), to optically detect how the molecules and organelles, involved in energy metabolism, are distributed. THG is a nonlinear coherent optical effect in which the incident laser beam interacts with a medium producing the light of exactly three times shorter wavelenght than the incidental one. THG mostly ocuurs at interfaces where change of refractive index is steep, like water-lipid structures. Therefore, cell membranes and lipid droplets are major sources of THG signal. For observation of a map of the oxidation-reduction ratio in a living cell, how the NADH and FAD ratio changes, we used two photon nonlinear microscopy. For in vivo TPEF imaging of optical redox ratio from the same region on hyphae of filamentous fungus Phycomyces blakesleeanus, we used ultrafast Ti:Sa laser, 160 fs pulses. The autofluorescence of NAD(P)H was excited by laser pulses at 730 nm and signal was collected through 479/40 filter, while for autofluorescence of FAD we used excitation by the same laser pulses at 860 nm, 530/43 filter. For THG imaging, 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. Both laser beams were focused with the same objective lens, Zeiss Plan Neofluar 40x1.3. With our nonlinear microscope and its different modalities, we were able to localize the two metabolic molecules, NAD(P)H and FAD, clearly see the lipid droplets, their distribution, number and size, and to characterize cell metabolism. In stressed cells, we obtained a decrease in the redox ratio and a higher number of lipid droplets under nitrogen starvation.",
publisher = "Belgrade : Institute of Physics",
journal = "Book of Abstracts: 13th Photonics Workshop: Conferenece; 2020 Mar 8-12; Kopaonik, Serbia.",
title = "In vivo metabolic imaging of fungus Phycomyces blakesleeanus using label-free nonlinear laser scanning microscopy",
pages = "23",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4643"
}
