What is photo-toxicity?

Under appropriate temperature (37°C), humidity (hight) and atmosphere (5% CO₂) it is possible to grow cells in vitro.

Healthy cells

But looking at these cells under the microscope is not without impact. This is similar to when you are exposing yourself to the sun light. If the sun is too strong and/or if you are exposed for a too long period you can have a bad sun burn... The same applies to cells under a microscope. While transmitted light (bright-field, phase-contrast, DIC) does not affect cell biology, fluorescence excitation light can be deleter.

How does photo-toxicity look like?

Strong illumination light will eventually burn your cells. Adhering cells will retract, detach and eventually die. See the example below (Click on the image to see the movie). These are cells exposed to 1 second of 70mW of 550nm light every 5 minutes. The total movie is about 3h. The cells retract, detach and eventually die.

Click on the image to see the movie

Below is another example of cells exposed to 1 second of 59mW of 395nm and 1s of 70mW of 550nm light every 5 minutes. The total duration is about 8h.

Click on the image to see the movie

How can I identify photo-toxicity has occured?

The best way to do this is to take a larger field of view image after your acquisition. Because photo-toxicity occurs only in the illuminated area, taking a picture of adjacent cells will provide with a very good (but not perfect) approach.

If you step back then you can see that the damaged area is actually limited to the exposed area. Note: The recorded area is smaller than the exposed area.
Importantly this is not a perfect control. A more ideal control would be to have another dish with cells maintained in the same condition but without illumination. In fact in the image above we can't conclude whether or not the affected circular area has no impact on the surrounding cells. It is possible that cell death occurring under the illuminated field of view can produce and release molecules that will affect the surrounding area. Therefore the ideal control would be a totally separated dish.

What are the important factors when talking about photo-toxicity?

The amount of light, the wavelength, the surface illuminated, the duration of illumination, the repetition of the illumination.

Amount of light: If you shine a strong light vs a dim light

The wavelength: The energy carried by laser light is dependent of the wavelength. Shorter wavelength have more energy and are more deleterious

The area illuminated: Shining the same amount of light into a small area result in stronger damage

The duration of illumination: Shining light for 1 second is doing more damage than 10 ms

The repetition: Shining 10 ms 20 times per minute is doing more damage than 1 time per minute

Empirically you can determine if photo-toxicity occur by looking at your cells. IF your cells are not dividing, if they detach then you may have photo-toxicity.

It is always good to acquire a larger field of view of the recorded area to make sure no photo-toxicity has occurred.

With a power meter you can also evaluate the amount of energy that your cells are comfortable with.

To do this measure the power at the objective using your regular imaging settings.

You will obtain a value in mW (which is Joule/second).

Then divide this value by the field of view of your objective. This will provide the irradiance expressed in mW/cm².

Finding the irradiance that is stress free or your cells is key. To do this you can shine continuously a defined irradiance and observe your cells for several hours. If your cells do not display any sign of photo-toxicity you can increase the irradiance and continue until finding the maximum acceptable continuous irradiance.

This will provide a good idea of how strong are your cells.

Obviously there is some flexibility since you will likely not continuously expose your cells. So you may pass above the maximum acceptable continuous irradiance value, which will stress your cells but eventually they will recover. Only you can determine if this amount of stress is experimentally acceptable and not modify the biology of what you want to measure.

Here is another discrete example.

Cells were imaged for 100ms with 70mW of 550nm light every 5 minutes. Cells are dividing faster than the effect of photo-toxicity that is occurring. Again here the best way to control is to take an overview image at the end of the acquisition and compare exposed cells to non-exposed cells.

Click on the image to see the movie