Due to reactive species generated by illumination at high intensities, fluorescent imaging can be fatal to cells. Cellular death or damage resulting from the fluorescent illumination is referred to as photodamage or phototoxicity. This kind of damage can be detected by the characteristic circular shape of the affected area.
In this example, CHO cells were illuminated for 30 min at maximum intensity through the 20x objective. As the image shows, phototoxicity is dramatic and all the illuminated cells are dead (observation through 5x objective).
The amount of light intensity absorbed by the sample depends on many aspects of the experiment but in general, the following settings should be taken into account:
- The intensity leaving the light source can be directly controlled by the mechanical shutter (black knob on the mercury lamp) or the speedDIAL of the X-Cite unit.
- This outgoing intensity passes through an attenuator controlled by the microscope controller. The settings go in 6 steps from 0 % to 100 % transmittance.
- Different objectives result in different energy densities with larger magnifications meaning higher likelihood of photodamage.
- Different filters also influence the absorbed energy, with lower excitation wavelength resulting in more damage.
- The time of illumination is directly proportional to the absorbed intensity.
- The level of phototoxicity also depends on the sensitivity of the cell line used.
Therefore, to avoid phototoxicity, one should adjust these settings towards less damaging values. The easiest approach is to decrease the transmittance of the attenuator while increasing the exposure time in the View settings. This change should result in a similar fluorescence brightness while reducing the intensity of the illumination. Also, it is important to execute every live-cell imaging experiment with minimizing the illumination-ovearhead. In practice this means that the illumination should be switched on as briefly as possible, preferably only for the time of image acquisition.
It is important to note that in case the parameters are not correctly set, even half a minute of illumination can cause drastic levels of cell death.
On the example below, CHO cells were illuminated with a 10x objective at maximal illumination level using the GFP filter. The shutter was open for 20, 10, 2 and 0.5 min with attenuator settings of T=12.5%, 25% and 100 .