Automated Holotomographic Microscopy


improved carboxyfluorescein succinimidyl ester (CFSE)-based dye that diffuses into cells to be processed by esterases. Tis modification allows the fluorescent compound to covalently bind to intracellular amines, providing a stable, non-specific fluorescent staining of whole cells. CellTrace VioletTM


is less


toxic for staining live cells thanks to a low impact on cell proliferation [28] and its relative low phototoxicity. Draq5 and CellTrace VioletTM


were used at respective con- centrations of 1:4000 and 1:1000 for 15 minutes. Cells were then


washed 30 minutes prior to image acquisition. Tis alliance of cell dyes, used at a low concentration and emitting in the same fluo- rescent channel, proved to be the only solution for the cells to be able to sustain the epifluorescence acquisition regime described above (2 hours; 1 image per 4 minutes), with a laser excitation set at 10% and 200 ms of exposure. Altogether, this fluorescent stain- ing protocol and acquisition regime is called “low D+C.” An increase in dye concentration, staining time, laser power, exposure time, or acquisition frequency resulted in cell


Figure 5: Long time-lapse imaging with the CX-A and robust cell segmentation. (a) Image acquisition stays in focus over days. (b) EVE Analytics precisely segments single cells over a range of cell densities (continued on the next page).


2021 September • www.microscopy-today.com 29


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