Label-Free Analysis of Organelle Interactions Using Organelle-Specific Phase Contrast Microscopy (OS-PCM)

Abstract

Organelles are highly dynamic and fulfill their function by constant motion and cooperation with each other. Current methods rely on fluorescence, leading to short observation time (via photobleaching) and experimental complexity (via multiple labeling). While label-free microscopes promise a paradigm change in this regard, the spatiotemporal resolutions and specificity are still insufficient to study organelle interactions. Using mitochondria and lysosome as examples, we demonstrate that our organelle-specific phase contrast microscopy (OS-PCM) can achieve automatic analysis of dynamic metrics of multiple organelles as well as their interactions from unlabeled cells for the first time. Compared to fluorescence-based methods, our method is gentle and holds great promise for label-free visualization and analysis of pan-organelle dynamics and interactions, with minimum perturbation to the cell.

Rapid Intracellular Detection and Analysis of Lipid Droplets’ Morpho-Chemical Composition by Phase-Guided Raman Sampling

“…Such a microscope, as reported previously, allows high-resolution, high-contrast visualization of all of the membrane-bound organelles (such as LDs and mitochondria) within the cell (example shown in Figure S10). As shown in Figure B, once the phase image of the cell has been collected, an LD mask is obtained through a deep-learning-based segmentation method that we have dubbed OS-PCM (organelle-specific phase contrast microscopy) . Then, we can calculate the morphological parameters (size, number, positions, etc.)…”

“…Building upon our recent work in organelle-specific phase microscopy (OS-PCM), a U-net model was constructed to identify LDs. Compared to the other conventional methods such as intensity thresholding, the U-net can provide more accurate LD recognition results, especially for the recognition of lipid droplet clusters and small lipid droplets (example results shown in Figure S6).…”

“…Briefly, an annular light source composed of blue LEDs (central wavelength, 432 nm) and a custom-made 3D printed bracket provided high-angle light incident to the sample. The ultraoblique illumination is critical to the microscope achieving the high spatial resolution (276 nm) . Unscattered light was attenuated to improve the overall image contrast by using an annular attenuator fabricated from an optical density filter.…”

“…In this “phase-guided Raman sampling” approach (abbreviated as PRS), we utilize our recently developed, high-resolution phase imaging platform , to capture high-resolution images of the interior organelle structure of a cell. Through a deep-learning framework, we identify the position and morphology of each individual lipid droplet in the cell. Feeding that information to a galvanometric mirror pair, we steer a Raman sampling beam one by one to each LD within the cell.…”

“…Here, we will use phase imaging to guide the Raman analysis of subcellular organelles. In this “phase-guided Raman sampling” approach (abbreviated as PRS), we utilize our recently developed, high-resolution phase imaging platform , to capture high-resolution images of the interior organelle structure of a cell. Through a deep-learning framework, we identify the position and morphology of each individual lipid droplet in the cell.…”