Image-based analysis of living mammalian cells using label-free 3D refractive index maps reveals new organelle dynamics and dry mass flux

Abstract

Holo-tomographic microscopy (HTM) is a label-free microscopy method reporting the fine changes of a cell’s refractive indices (RIs) in three dimensions at high spatial and temporal resolution. By combining HTM with epifluorescence, we demonstrate that mammalian cellular organelles such as lipid droplets (LDs) and mitochondria show specific RI 3D patterns. To go further, we developed a computer-vision strategy using FIJI, CellProfiler3 (CP3), and custom code that allows us to use the fine images obtained by HTM in quantitative approaches. We could observe the shape and dry mass dynamics of LDs, endocytic structures, and entire cells’ division that have so far, to the best of our knowledge, been out of reach. We finally took advantage of the capacity of HTM to capture the motion of many organelles at the same time to report a multiorganelle spinning phenomenon and study its dynamic properties using pattern matching and homography analysis. This work demonstrates that HTM gives access to an uncharted field of biological dynamics and describes a unique set of simple computer-vision strategies that can be broadly used to quantify HTM images.

Quantitative Phase Imaging of Spreading Fibroblasts Identifies the Role of Focal Adhesion Kinase in the Stabilization of the Cell Rear

“…The increase in dry mass density at the leading edge was associated with membrane protrusion or with structures resembling peripheral ruffles [ 63 ]. Hence, ours and other recent reports [ 62 , 68 , 69 ] stress the potential of label-free non-invasive microscopy in the identification of novel aspects of cell behavior and physiology.…”

Seipin-Mediated Contacts as Gatekeepers of Lipid Flux at the Endoplasmic Reticulum–Lipid Droplet Nexus

“…While direct observation of NL fluxes within the ER is technically challenging, rapid advances in imaging and probe technologies (Valm et al, 2017;Adhikari et al, 2019) are likely to facilitate this in the near future. Findings from a recent study, employing elegant imaging techniques (holo-tomographic microscopy), support the notion of a fluid connection of LDs through the ER (Sandoz et al, 2019). It was found that LD growth in a given cell during fatty acid administration was highly synchronous, with both shrinking and growing of LDs happening in concert, as if sharing a common pool of lipid precursors.Furthermore, upon initial LD assembly, preexisting LDs actually initially slightly shrunk in size at the same time as numerous new LDs emerged.…”

Super-resolution fluorescence-assisted diffraction computational tomography reveals the three-dimensional landscape of the cellular organelle interactome

“…Increased spatial resolution or prolonged exposure would lead to the distribution of the LE/LY signal across an even larger field of view and the ultimate disappearance of the structure into the background noise. Similarly, because ER tubules and junctions also undergo rapid motions in live cells 41 , they have never been observed by any ODT microscopes in live cells previously, and such an observation has even been deemed to be impossible 10 . Therefore, the spatial resolution must be matched with the corresponding temporal resolution to enable the maximal resolution achievable in live-cell ODT imaging, which has been overlooked in previous designs 6,9,13 .…”

“…S8), we used Wiener filtering (Eq. (10) in Supplementary Note 3) to reconstruct the 3D distribution of the RIs within the cell.…”

“…S1). Therefore, despite the claimed~100nm spatial resolution, which should be sufficient to resolve most organelles in cells, lipid droplets (LDs), chromosomes, and mitochondria are the organelles seen by the current ODT microscopes 5,6,9,10 . In addition, a lack of molecular specificity in label-free ODT microscopy also hinders the interpretation of imaging results.…”