The problem

Live cells get sick and unhealthy during time-lapse imaging

Cells can become damaged from both the low- and high-wavelength light that is used to excite fluorophores. Most cells used in research are not adapted to handle the number of photons that are directed toward them during a typical imaging experiment.

While you are imaging, if you see cells detaching from the culturing vessel, showing plasma membrane blebbing or large vacuoles, enlarged mitochondria, or fluorescent protein aggregation, these are all clues that you have stressed, unhealthy cells.

Stained cell image showing two cells: one unhealthy cell showing blebbing next to a healthy cell.   Figure 1. The cell in the top of the figure shows catastrophic blebbing of the cell membrane, while its neighbor remains relatively healthy.

Additionally, photoexcited fluorophores can release reactive oxygen species, which can damage nearby cellular structures.

Cell image montage showing how extended illumination renders a sample GFP-expressing cells unable to fluoresce and a sample of cells unable to proliferate.

Figure 2. Top panel: brightfield and green channel overlay of a field of live HeLa cells transduced with CellLight® H2B-GFP reagent. Cells in the illuminated area have undergone repeated illumination for 10 hours prior to the capture of this image; cells in the non-illuminated area were illuminated only for the capture of this image. For the sample subjected to repeated illumination, we see dimming and loss of the GFP signal (green channel image), and we can also observe significant cell damage, including cell shrinking, cell rounding, and mitochondrial enlargement (brightfield channel image). Bottom panel: scratch wound in a culture of HDFn cells loaded with CellTracker® Deep Red reagent. The illuminated area was subjected to repeated illumination for 10 hr. Cells in this area shows a loss of viability were not able to grow into the wound, while cells in the non-illuminated area show viable cell growth into the wound.

Both of these processes are referred to as phototoxicity, sometimes without distinguishing between the two. The main recourse for phototoxicity is to optimize both your microscope setup and your experiment to use the lowest amount of illumination.

What you can do 

Optimize the light path in your fluorescence microscope

One approach is to design your imaging system to be very sensitive and able to capture most of the emitted light, since you will be using lower amounts of illumination to excite your fluorophores to minimize cell damage. This is usually accomplished by optimizing the light path in your fluorescence microscope setup to be as efficient as possible, using the most sensitive detector possible (usually a CCD camera), and using as little light as possible for excitation, and at the most optimal wavelength for the fluorophore you are using.

Try lower intensity and shorter exposure times

Sometimes you have to do your best with the equipment you have. You will still want to use the illumination that gives you the best signal over background with the lowest level of fluorophore excitation. This means optimizing your experiment for the lowest intensity and shortest exposure times possible on your system. In some cases, particularly when you wish to image over a long period of time, it is advisable to sacrifice resolution in exchange for healthier cells. This may mean shorter exposure times, binning, or a lower magnification. You can also try using fluorophores that are red-shifted to see if that helps preserve the health of your cells. See the live-cell imaging section.

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