How do you trust your live cell imaging correlates with experimental conditions?

DO YOU CONTROL FOR PHOTOTOXICITY?

A major problem with label-based imaging techniques is phototoxicity. Phototoxicity leads to cell death, or (more dangerously as its harder to control), influences cell morphology and dynamics. 

While acknowledged, this issue still receives too little attention by researchers as highlighted in this month’s Nature Methods Editorial (1). Even if cells don’t die, it is impossible, without adequate controls, to tell if cell dynamics are in response to experimental conditions, or to phototoxic effects.

The mitigation of phototoxicity leads to poor time resolution of time lapse approaches. This is particularly true for small organelles like mitochondria or lipid droplets that are extremely sensitive to photo-induced oxidation. Last but not least, the use of chemical or genetically-encoded fluorescent markers perturb the targeted biological processes.

The 3D Cell Explorer overcomes these problems as it injects in the sample ~100 times less energy (~0.2 nW/µm2) than light sheet microscopes (~1nW/µm2) that are the reference in the matter. With a resolution below 200 nm, it enables high resolution and high-frequency imaging even with sensitive material, giving access to organelle dynamics that were previously out of reach. What is more important, label-free means that no sample preparation is required, saving time and money.

Compare the results on the videos below:

Caption video 1: mouse Pre-adipocytes unlabeled and labeled with MitoTracker (dilution 1:5000) have been imaged for 3 hrs at 1 image every 6s with holotomography (left panel) and holotomography + Cy5 fluorescence channel (right panel). We chose to use this type of cells because pre-adipocytes are known to be resistant and quite proliferative. Moreover, we use a mild fluorescence acquisition protocol, Cy5 is one of the least phototoxic light in fluorescence microscopy (long wavelenght). After less than an hour, cells on the right, exposed to fluorescence start dying through apoptosis. While, the ones on the left, imaged just through holotomography, show no sign of stress. This lack of phototoxicity can be even better appreciated in longer movies showing very sensitive cells like stem cells imaged for more than 50 hours as shown at this link: https://nanolive.ch/stem-cell-research/ .

Caption video 2: we see a loss of mitochondrial potential (fluorescence intensity is progressively reducing) over the time of the movie. On shorter time scales, there is some staining blinking that we attribute to bursts of mitochondrial function attempting to compensate while the whole mitochondrial network is globally failing. We can observe all cellular mitochondria becoming apoptotic in the right panel (classical donut shape) while they remain unchanged.

(1) https://doi.org/10.1038/s41592-018-0170-4

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