Phototoxicity in Live Cell Imaging - do you know the pyramid of frustration?

This video shows 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 in the left.

DO YOU CONTROL FOR PHOTOTOXICITY?

Label-based imaging is essential for many areas of cell discovery research and assay. But an undeniable reality with label-based imaging techniques is phototoxicity. Phototoxicity leads to cell death but prior to death influences cell morphology, behaviour and dynamics (hard to measure and control for). How can we know to what extent the art of observing influences the observation? 

While acknowledged, this issue still receives too little attention by researchers as highlighted in a recent Nature Methods Editorial (ref 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 authors write ...

… microscopy methods can allow fast processes to be tracked in nearly real time and in super-resolution … achieving faster, higher-resolution, higher signal-to-noise, and deeper imaging often requires greater light doses relative to those a biological specimen might experience naturally. It is now well understood that such light doses, and in some cases any light at all, can negatively affect sample fitness and viability….

And notably for all who publish live cell imaging

…For some time at Nature Methods we have been asking authors of manuscripts describing live-imaging methods to assay phototoxicity. We will continue to encourage such assessment, with a strong preference that it be included prior to peer review…

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

The figure below highlights the trade offs in live cell imaging (ref 2). The authors write

...To obtain relevant data in bioimaging, one must acquire images of sufficient contrast, spatial resolution, and temporal resolution, under conditions that do not affect the biology of interest (Fig. 2). Contrast, spatial resolution, and temporal resolution are interdependent—no single factor can be changed without affecting the other two—and optimization of these factors often is coupled to increased exposure to light, which usually comes at a cost to sample health. In our opinion, sample health is the highest priority in live imaging..

(2) https://www.bme.unc.edu/files/2019/01/Laissue-PP_Assessing-Phototoxicity-in-Live-Fluorescence-Imaging_Nat-Meth-2017.pdf

One solution to the pyramid of frustration is to use label free imaging, such as the 3D Cell Explorer system. It negates 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 good resolution and high-frequency imaging even with sensitive material such as stem cells https://nanolive.ch/stem-cell-research/, 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. In addition, the bias free, data rich images produced lend themselves exceptionally well to analytic analysis, with multiple cell and population metrics, including dry mass (impossible to calculate from a fluorescence signal) available enabling both simple and complex assay and phenotypic screening https://www.nanolive.ch/products/live-cell-analytics/eve-analytics/ .

This video shows mouse Pre-adipocytes unlabeled and labeled with MitoTracker (dilution 1:5000) and 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/.

From Organelles (e.g. mitochondria https://nanolive.ch/cellular-metabolism-research/ )

To Single Cells (e.g. macrophage https://nanolive.ch/macrophages/ )

To Cell Populations (e.g. immuno-oncology https://nanolive.ch/immuno-oncology/ )

Label Free & 3D (e.g. https://nanolive.ch/the-ultimate-live-cell-imaging-tool/)

With superior, un biased analytics that are not impacted by phototoxicity ( https://www.nanolive.ch/products/live-cell-analytics/eve-analytics/ )

Unperturbed imaging

No phototoxicity

No photobleaching

See what you have been missing

#nanolive #cellbiology #microscopy #livecellimaging