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.
Highlights
Because of the transparent nature of a cell, microscopy techniques either use fluorescent markers or transform optical properties of the sample into an observable contrast
Our holo-tomographic microscopy (HTM) device is equipped with simultaneous holotomography and epifluorescence imaging (Fig 1A); we could compare fluorescence imaging of subcellular markers with refractive index (RI) maps to determine whether specific organelles have a recognizable RI signature that would allow label-free analysis of organelle biology in control conditions or upon perturbations
HTM performances were compared to other label-free methods such as fixed HeLa cells imaged with brightfield microscopy, in which contrast is created by the attenuation of the transmitted light; phase contrast [25], in which contrast is created by the capacity of the sample to scatter light; differential interference contrast (DIC) [26], which relies on two rays of polarized light, a reference and a sampling ray, that interfere to create contrast; and HTM (S1 Fig)
Summary
Because of the transparent nature of a cell, microscopy techniques either use fluorescent markers or transform optical properties of the sample into an observable contrast (for example, phase contrast, differential interference contrast [DIC]). Each of these techniques comes with limitations. Classical label-free imaging techniques, while less perturbing, provide images with low information content because of poor contrast and resolution. In this context, holo-tomographic microscopy (HTM) [1] is of great interest because it can provide label-free, high-content images using a very low-power light source that.
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