M. In vivo metabolic imaging and micromanipulation of individual filamentous fungus cells using different nonlinear laser scanning microscopy modalities

Abstract

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.