Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi

Abstract We report the utilization of the Third-Harmonic Generation (THG) microscopy, using home built nonlinear laser scanning microscope, for label-free live cell imaging of lipid droplets in the hypha of filamentous fungus Phycomyces blakesleeanus. THG microscopy images show bright spherical features dispersed throughout hypha cytoplasm in control conditions and a transient increase in the number of bright features after complete nitrogen starvation. We show that predominant source of THG signal in imaged hypha are lipid droplets and test side by side two methods for quantification of lipid droplet size and number from THG images: Particle Size Analysis (PSA) and Image Correlation Spectroscopy (ICS). The two analysis methods gave congruous results, showing expected increase of lipid droplet number after nitrogen starvation, while ICS showed somewhat lower mean diameter of lipid droplets than PSA. We conclude that THG imaging, followed by PSA and ICS, can be easily and reliably used on filamentous fungi for the in vivo quantification of lipid droplets since there is no need for sample labeling, fixation or any other specific preparation. In addition, we demonstrate that ICS is suitable for THG microscopy image analysis, although it is primarily developed and have been mostly used for fluorescence signals so far.