Overcoming phototoxicity

In vitro live cell imaging has become a promising tool to study dynamic cell behaviors, such as division and differentiation, cell death, cell-cell interaction (e.g. immune cells), or cell response to treatments, over extended periods of time. However, few technologies have been developed towards that specific application. Today, most live imaging techniques rely on either high illumination regimes or fluorescent labelling, both inducing phototoxicity and compromising the ability to keep cells unperturbed and alive over time. Since our knowledge of biology is driven by observation, it is key to minimize the perturbations induced by the imaging technique.

This page focuses on the Master Project of Hugo Marc Moreno which compares standard epifluorescence microscopy with Nanolive imaging (also known as tomographic phase microscopy) in live cell imaging of mouse pre-adipocytes, and discusses the perturbations induced by phototoxicity on living samples during experimentation. Surprisingly, very few studies have been highlighting or discussing the artifacts arising from phototoxicity during live cell imaging. With Nanolive imaging, a low-power laser beam gets phase-shifted when sent through samples, this phase shift is then used to generate 3D-images of refractive indices of the observed samples. The way this technology gets rid of invasive fluorescent markers, while using low energy exposure regimes is a clear added value that makes it well suited to observe living samples, when compared to epifluorescence imaging. Moreno’s et al results raised the question of how much of our knowledge, particularly about mitochondria, may have been biased by artifacts induced by fluorescence imaging.

Moreno et al compared the possible phototoxic effects induced by either Nanolive imaging or epifluorescence imaging on mouse pre-adipocytes. Various parameters may impact the final phototoxic effect observed on living sample. (i) Excitation light wavelength, (ii) intensity, (iii) exposure time and (iv) labelling will all affect the energy quantity sent to the imaged sample.

To appreciate the impact of each actor (excitation light wavelength, intensity and exposure time), parameters were varied one by one.

Nanolive microscopy alone did not induce any detectable effect on mouse pre-adipocytes after one-hour acquisition at a frequency of 1 image every 6 seconds. The overall spreading and motility of the cell remained apparently uncompromised: cell shape did not drastically change nor shrink over the imaging period, the ability of filopodia to spread and adhere looked unaffected and main adhesion sites were not impaired. Shape and density of nucleoli (bright granules in the nucleus) did not provide any sign of perturbation neither. Moreover, mitochondrial network did not show any change in phenotype: no extended fusion nor fission was observed, the overall dynamic of the mitochondria remained constant during the full hour of acquisition, and the shape of mitochondria was conserved, no swelling of mitochondria was seen.

Imaging regimen: 1-hour acquisition at a frequency of 1 image every 6 seconds.

Cy5 & TritC: excitation light 10% intensity; 100ms exposure time

Cy5 & TritC exposure did not induce any detectable effect on mouse pre-adipocytes after one-hour acquisition at a frequency of 1 image every 6 seconds when excitation light at 10% intensity and 100ms exposure time were used in combination with tomographic phase microscopy. Increasing Cy5 and TritC intensities and exposure times did not affect the observed outcome.

Imaging regimen: 1-hour acquisition at a frequency of 1 image every 6 seconds.

FitC: excitation light 10% intensity; 100ms exposure time

FitC excitation light (474nm) exposure induced observable effects on cell homeostasis already at 10% intensity and 100ms exposure time. Loss of mitochondrial dynamics followed by loss of adherence and blebbing was observed at 20% and 40% intensities.

Imaging regimen: 1-hour acquisition at a frequency of 1 image every 6 seconds.

FitC: excitation light 10% intensity; 400ms exposure time

Increasing exposure time from 100ms to 400ms led to more serious cell damages. At 10% intensity and 400ms exposure time, the first sign of phototoxicity was the progressive slowdown of mitochondrial dynamics until an almost complete stop of mitochondrial movement reached after 30 min of acquisition, mitochondrial swelling was also observed in one replicate. This dynamic effect is obvious when looking at the full movie.

Imaging regimen: 1-hour acquisition at a frequency of 1 image every 6 seconds.

Dapi: excitation light 10% intensity; 100ms exposure time

Dapi exposition led to complete cell shrinkage and rounding, followed by extensive blebbing at all intensities with 100ms exposure time. At 10% intensity and 100ms exposure time, loss of cell adhesion and cell shrinkage took place in 8 minutes. The cell reached complete shrinkage and blebbing before 30min of acquisition, the acquisition was therefore stopped at 30min.

Imaging regimen: 30 minutes acquisition at a frequency of 1 image every 6 seconds.

In this second step, a fluorescence marker was added to excitation light to perform epifluorescence microscopy. MitoNIR was chosen and used as fluorescent probe for various reasons: 1) it is designed to work with Cy5 excitation light regimes in which Moreno’s et al did not observe phototoxic effect in the absence of probe. 2) Cy5 has the longest wavelength of the excitation lights Moreno’s et al tested, and longer wavelengths are known to be less toxic for living samples.

PreA labelled with MitoNIR and imaged with Nanolive imaging – without any fluorescence excitation

To begin, MitoNIR was imaged with Nanolive microscopy without any excitation light, to control for the possible effect of the addition of the molecule itself. Few mitochondria transiently swelled, before recovering their original shape 3-6 minutes later. Overall mitochondrial dynamics did not change during the acquisition time. Shift in the mitochondrial fusion-fission balance was not observed. Adherence and overall shape of the cell was maintained, this can be illustrated by the capacity of the cell to extend its membrane towards the left direction in between minutes 40:00 and 60:00.

PreA labelled with MitoNIR and imaged with Nanolive imaging + Cy5 excitation light with 10% intensity, 100ms exposure time and 1 acquisition every 6 seconds

Cells looked affected very fast:

1. Appearance of round-shaped or swollen mitochondria was observed with Nanolive technology.
2. Loss of mitochondrial specific fluorescent signal and high background or cytosolic fluorescent signal could be observed on the fluorescent image.
3. Mitochondrial dynamics slowed down and reached an almost complete stop after 38 minutes of exposure.
4. Very slow cell shrinkage was observed until timepoint 00:58:00, at which a consequent proportion of the cell lost adherence.

MitoNIR signal was concentrated in the nuclear periphery at later timepoints.

If you are interested in learning more, please download Moreno’s et al. Master thesis here: