Combined two photon excitation fluorescence and third harmonic generation imaging of redox ratio for monitoring metabolic state of live cells of fungus Phycomyces blakesleeanus

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.