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).

Click on the image to see the movie

Cells subjected to 1 second of 59 mW, 395 nm light and 1 s 750 mW 550nm every 5 minutes. The total duration of the movie is approximately 8 hours.

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 usual imaging settings.
  2. Record the value in mW (milliwatts = Joules/second).
  3. Divide this value by the field of view area (in cm²) to calculate the irradiance in mW/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.

How to proceed with my experiment to minimize phototoxicity?

To ensure minimal phototoxicity during your experiment, follow these steps:

1. Estimate the light output of your instrument:
   Use a power meter to measure the light intensity (in mW) provided by your instrument. Ideally, your microscopy platform manager has recently conducted a complete quality control and can supply data on the light output at the sample. (For more information on microscopy quality control, refer to the "Power Linearity" section.)

2. Run a pilot experiment:
   Determine the Maximum Acceptable Continuous Irradiance (mW/cm²/s) your sample can tolerate without showing signs of phototoxicity. This helps define safe imaging parameters for your experiment.

3. Include proper controls:
   Incorporate controls in your experimental design. Ideally, image independent regions at different frequencies to confirm that imaging frequency does not affect your results. While this may not always be feasible due to software or experimental constraints, the goal is to have a control condition with the lowest light intensity required to successfully image your sample.

4. Assess photobleaching:
   At the end of your experiment, acquire a 5x5 tile scan of the region to estimate photobleaching and ensure that your imaging conditions did not cause significant photo-damage.