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

PLoS Biol. 2019 Dec 19;17(12):e3000553. doi: 10.1371/journal.pbio.3000553. eCollection 2019 Dec.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • HeLa Cells
  • Humans
  • Image Processing, Computer-Assisted / methods
  • Imaging, Three-Dimensional / methods
  • Lipid Metabolism
  • Microscopy, Fluorescence / methods*
  • Mitochondria / metabolism
  • Organelles / physiology*
  • Refractometry / methods*

Grants and funding

This work was supported by a grant from the Swiss National Science Foundation (SNSF) and the Swiss SystemsX.ch initiative evaluated by the Swiss National Science Foundation (LipidX). MF was funded by Nanolive SA. PS, CT, and FGvdG were funded by LipidX and SNSF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.