What is photo-toxicity?

Under appropriate conditions of temperature (37°C), high humidity

, and a controlled atmosphere (5%

CO2), it is possible to grow cells in vitro.

However, observing these cells under

a microscope can have unintended effects. This situation is

analogous to

exposing yourself to

sunlight: if the sunlight is too intense or the exposure lasts too long, you may suffer a sunburn. Similarly, cells can be affected by prolonged or intense light exposure during microscopy. While transmitted light (bright-field, phase-contrast, DIC) generally does not

interfere with cell biology, fluorescence excitation light can

cause significant phototoxic effects.

What does phototoxicity

look like?

Prolonged exposure to intense illumination can cause significant cellular damage, resulting in retraction, detachment, and eventual cell death. The example below demonstrates this process (click on the image to watch the movie).

How can I identify

if phototoxicity has occurred?

The

most effective way to

detect phototoxicity is to

capture a larger field-of-view image after your acquisition.

Since phototoxicity is confined to the illuminated area,

comparing adjacent non-illuminated cells with those in the exposed region offers a good—though not perfect—approach.

By stepping back, you can observe that the damage is limited to the illuminated area. However, it’s important to note that the recorded region may be smaller than the actual exposed area.

It’s also crucial to understand that this method is not a flawless

control. A more ideal control would

involve using a separate dish with cells maintained

under identical conditions but without illumination.

This is because, in the image above, we

cannot definitively conclude whether

the affected circular

region has no impact on

nearby cells.

It’s possible that cell death

in the illuminated

region may release molecules that

influence the surrounding

cells. Therefore, the

most reliable control would be a

completely separate dish with unexposed cells.

What are the important factors

to consider when discussing phototoxicity?

Several key factors influence phototoxicity:

• Amount of light:

• Strong illumination causes more damage compared to dimmer light.
• Wavelength: The energy carried by

• light depends on its wavelength. Shorter

• wavelengths carry higher energy and

• tend to be more harmful.
• Illuminated area: Concentrating

• the same amount of light

• on a

• smaller area

• results in

• more localized damage.

• Duration of illumination:

• Prolonged exposure (e.g., 1 second vs. 10 ms) increases the risk of damage.
• Repetition of illumination: Frequent exposure (e.g., 10 ms pulses applied 20 times per minute) is

• more

• damaging than less frequent exposure (e.g., once per minute).

How can you detect phototoxicity?

Empirically, phototoxicity can be identified by observing cell behavior. If

cells are not dividing

, retracting, or detaching, it may indicate phototoxicity.

To assess phototoxicity:

• Acquire

• a larger field-of-view image of the recorded area to

• ensure no phototoxicity has occurred and to evaluate photobleaching.

• Use a power meter

• to measure the energy your cells are

• exposed to.

• 1. Measure the power at the objective using your

• 1. usual imaging settings.

• 1. Record the value in mW (

• 1. milliwatts = Joules/second).

• 1. Divide this value by the field of view

• 1. area (in cm²) to calculate the irradiance

• 1. in mW/

• 1. cm².

How to determine the maximum acceptable irradiance:
Finding an irradiance level that is stress-free

for your cells is

critical:

1. Expose your cells continuously to a defined irradiance.
2. Observe them over several hours. If they show no signs of phototoxicity, gradually increase the irradiance and repeat the observation.
3. Identify the maximum continuous irradiance that does not cause damage.

Keep in mind that this value provides a baseline. Since most experiments do not involve continuous exposure, it is possible to exceed this threshold briefly. However, doing so may induce temporary stress in the cells. It is up to you to decide whether this level of stress is acceptable for your specific experiment and whether it might interfere with the biological processes you are studying.