Automated Holotomographic Microscopy
agree, fluorescence imag- ing works best on fixed cells.
Let’s evaluate these new
image-analysis capabilities in a concrete scenario. We have seen that the CX-A allows screening of multi- well plates. It acquisition of
also allows large fields
of view for studying single cells in their population context over very long time- lapse experiments. Again, efficient autofocus is essen- tial to ensure that the same z position, with a precision of less than a micrometer, is kept over distances of up to a millimeter apart in the case of a 10 × 10 gridscan. Figure 5 shows that it is possible to image mamma- lian cells over multiple days with perfect focus. Coupled with EA we demonstrate in Figure 5 that single cells in such an advanced time- lapse imaging scenario are well-segmented from the beginning to the end of the time-lapse experiment with no loss of objects due to image stitching. Besides
HTM, Figure 5: (c) Insets 1 and 2 show details of cell segmentations using the CX-A and EVE Analytics after 12 and 84 hours of acquisition.
death before the completion of 2 hours of acquisition. Exchang- ing either Draq5 for Hoechst or similar blue nuclear dyes, or CellTrace VioletTM
for another CFSE dye, led to premature
cell death. Tis is explained both by the higher cellular tox- icity of the alternative compounds and the greater generation of phototoxicity using blue dyes, which require UV excitation. We voluntarily did not use any chemical mitigation strategy for dealing with phototoxicity. Te use of antioxidants such as vitamin C, vitamin E phosphate, Trolox, or glutathione are far from neutral and perturb cells [29–32]. We developed the best CellProfiler 4 (CP4) [24] pipeline we
could in order to segment the same cells as those segmented with EA, using the low D+C fluorescent signal (Figure 4b). Tanks to the alliance of Draq5 and CellTrace VioletTM
, well-
defined nuclei could be detected and further analyzed within a less intense cytosolic signal (Figure 4b, red outlines). Overlaying the low D+C outlines on the RI signal shows how much detail of the cell’s morphology was missed using live fluo-imaging com- pared to RI-based segmentation. Someone not familiar with live cell imaging could be forgiven for thinking that the fluores- cent signal is of poor quality compared to the sharp immuno- or chemical staining one can obtain with fixed samples. We
30 the
CX-A is equipped with an epi-fluorescence system.
Tis allows investigation of the effect of fluorescence imaging on live cell growth dynamics in a quantitative manner using HTM as a reference, since this technology does not generate phototoxic stress. We prepared 3T3-derived pre-adipocytes as follows: 1) grown without any treatment as a control; 2) grown with MitoTracker but without fluorescence excitation; 3) grown with MitoTracker and 200 ms of 1% fluorescence exposure; and 4) grown with MitoTracker and 5% of the maximal excitation provided by the CoolLED fluorescence unit. Te investigation revealed a swiſt phototoxic effect even
at low excitation power (Figure 6a, blue and brown frames). It is worth noting that such CoolLED powers are the standard in the field of mitochondria imaging. Te presence of Mito- Tracker alone within cells is able to arrest cell growth and trigger apoptosis aſter 10.5 hours of imaging (Figure 6a, green frames) without killing all cells. Altogether the quantified data depict interesting phenomena. While the control cells show an increase in size with spreading and accumulation of dry mass, the addition of MitoTracker stops cell growth and spreading, but dry mass accumulates (Figure 6b). When the MitoTracker fluorophore is excited at 1% power, cells shrink, but the dry mass of cells is stable, indicating that the cell death we observe
www.microscopy-today.com • 2021 September
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