In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities
2021
Аутори:
Pajić, TanjaKrmpot, Aleksandar J.
Todorović, Nataša
Živić, Miroslav
Stevanović, Katarina
Rabasović, Mihailo D.
Тип документа:
Конференцијски прилог (Објављена верзија)
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© 2021 Sociedade Brasileira de Bioquímica e Biologia Molecular (SBBq)
Метаподаци
Приказ свих података о документуАпстракт:
Nonlinear laser scanning microscopy (NLSM), is an advanced optical technique that utilizes ultrashort laser pulses
for structural and functional imaging, as well as laser manipulation of live organisms and cells. Two modalities of
NLSM, two photon excitation fluorescence (TPEF) and third harmonic generation (THG) were applied for in vivo and
label-free study of oxidative and lipid metabolism of individual cells of filamentous fungus Phycomyces
blakesleeanus. Cell membranes and lipid droplets (LDs) are major sources of THG signal. TPEF allows us to
determine the redox ratio (reflecting metabolic activity of cells) of the metabolic cofactors FAD and NAD(P)H
autofluorescence. In addition, slight modifications of the experimental setup, mostly on software, enabled utilization
of femtosecond laser pulses for precise cell microsurgery of hyphal cell wall. The optimized microsurgery procedure
we than utilized to obtain protoplasts suitable for patch-clamp electrophysiological recording. Cell surgery of
filamentous fungus Phycomyces blakesleeanus, were performed by ultrafast Ti:Sa laser (160 fs pulses). The same
laser was used for in vivo autoTPEF imaging of NAD(P)H and FAD at different wavelengths. For in vivo THG imaging
of label-free hyphae, we used 1040 nm, 200 fs pulses from Yb KGW laser. In vivo and label-free application of THG
imaging enabled, accurately and reliably, detection of changes in distribution, total number, and size of LDs in control
and treatment group of cells. Two-photon microscopy made it possible to obtain a redox ratio using autofluorescences
of NAD(P)H and FAD in the same regions of live hyphae. The cell microsurgery procedure has been optimized and
developed, which enabled the subsequent registration of currents on otherwise unaccessible membrane.
Кључне речи:
nonlinear imaging; cell surgeryФинансирање / пројекти:
- HEMMAGINERO - Hemoglobin-Based Spectroscopy and Nonlinear Imaging of Erythrocytes and Their Membranes As Emerging Diagnostic Tool (RS-ScienceFundRS-Promis-6066079)
У:
- 20th International Congress of the International Union for Pure Applied Biophysics (IUPAB); 50th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology (SBBq); 45th Congress of Brazilian Biophysics Society (SBBf); 13th Brazilian Society on Nuclear Biosciences Congress 2021; 2021 Oct 4-8; São Paulo, Brazil. São Paulo: Sociedade Brasileira de Bioquímica e Biologia Molecular; 2021. p. 358.