How to set up a fluorescence live-cell imaging experiment

Live-cell fluorescence imaging has many advantages and offers a variety of experimental design opportunities. However, as with any other method, there are some considerations that need to be taken into account when setting up the experiment to obtain meaningful data and to avoid damage to the cells and potential artifacts.

Keep cells in perfect conditions

First of all, cells need to be maintained in a physiological environment throughout the whole duration of the experiment. Some immortalized cell lines are robust enough to be imaged for the short term without any environmental control. However, changes in temperature, oxygen, and carbon dioxide (CO2) concentration can lead to changes in cell behavior and impact the observed processes. Therefore, in longer experiments, the imaging system with a humidified heated chamber and CO2 control can be used. However, fluorescence live-cell imaging devices that fit and work inside an incubator are preferred.

Choose the right fluorescent dye

The choice of the dye is equally important. Genetically encoded fluorescent proteins are more specific and stable over time in culture; however, they do imply genetic manipulation of the cells including all the possible negative effects that this process can have on cell physiology. In the case of the chemical fluorophores, their effects on cells in terms of cytotoxicity, permeability, and retention over time need to be considered. The selection will be obviously also influenced by the excitation and emission spectra of the probe depending on the fluorescence microscope filter setup.

Minimize the light intensity

Lastly, there are negative effects in cells caused by their exposure to high-intensity excitation light during the imaging process. The photon flux is damaging to cells by creating excessive amounts of reactive species, such as free radicals and ROS, in a process called phototoxicity. Therefore it is desirable to design experiments in such a way as to minimize the light (wavelength range and number of photons) that cells are exposed to, but at the same time to optimize the signal to noise ratio. This depends not only on the brightness of the fluorescent label itself but also on the light source of the fluorescent microscope (LED lights vs mercury lamps).

Mind non-invasiveness

All in all, awareness of the notion that fluorescence live microscopy is not entirely non-invasive can help us design experiments to minimize cell harm, non-physiological responses, and experimental artifacts and at the same time obtain detailed information about cellular processes over time.

기타 고려할 사항

There are several, standard components of cell culture media that can interfere with the live imaging of your cells. Phenol red, often found in DMEM, is used to visualize the pH in cell culture dishes. This compound, however, can quench the signal of some visible wavelength fluorescent dyes when used during live cell fluorescent microscopy. Another component of DMEM is riboflavin, which is autofluorescent and can influence the signal-to-noise ratio.

If your dye signal is bright, the background created by phenol red and other compounds should not be a problem. However, if your fluorescent signal is low, we recommend using a phenol red-free medium or a specialized medium developed for live cell imaging.

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