Live-cell imaging

About this technique

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To study many functions of cells it is desirable to observe cells in their living state. There are many ways to do this with various levels of sophistication. For very quick imaging (seconds to a few minutes) you can study cell cultures on a slide with buffered media. For longer-term experiments it is adequate to use a heated stage and objective and buffered media. However, if possible, it is advisable to run your experiment in an enclosed environment where both temperature and gas are controlled, providing as normal an environment as possible for the cells. Therefore live-cell imaging systems have an incubation chamber built around the microscope.

Adherent cell lines are the easiest to work with but non-adherent ones can also be studied in these systems. The cells can be observed using fluorescence, differential interference contrast (DIC) or phase contrast, or a combination of the above, depending on the nature of the experiment. Cells can be fluorescently labeled through transfection of constructs carrying a marker such as green fluorescent protein (GFP) or by introducing vital dyes that are compatible with living cells and bind to specific cellular components such as calcium or DNA.

Some live-cell imaging systems have a multi-positioning stage allowing the observation of multiple cells over multiple wells. There is also considerable variation in the level of sophistication of the chambers in which the cells are mounted. These range from homemade chambers to commercial flow-through chambers. These set-ups allow compounds to be added to the cells and subsequent changes in cell behaviour to be studied, (e.g. calcium, pH, cellular movement etc.).

When capturing images of living cells, you need to be aware of the fact that all light is toxic to cells if they are exposed to it at high intensities. When using visible light, a green filter must be placed in the light path. Cells should only be exposed to the light for as long as it takes to gain a good image. The rule is always to expose your cells for as short a time as possible with as low light levels as possible to obtain a good image. This means that low-light-level cameras are essential.