Live-cell imaging reveals information of biological processes at a microscopic level. It requires that the specimen is kept under near physiological conditions. Some of them are mentioned in the paragraph below. Typical specimens are classical cell cultures, live tissues, organoids, or model organisms. Commonly the sample is investigated with fluorescence microscopy. To achieve this, cells are transfected and, consequently, transgenic organisms are produced which express fluorescently labelled proteins. In addition, live-cell dyes can be used.
Live-cell imaging conditions must mimic a physiological environment to achieve as realistic experimental results as possible. Optimal physiological conditions – meaning temperature, pH, oxygen level – differ between organisms. Whereas mammalian cells require ~37°C, insect cells grow best at ~27°C, fish prefer 28°C, while fission yeast is kept at ~30°C.
The pH within cell culture media is kept constantly at 7.0 – 7.4 with the help of a sodium bicarbonate buffer, which requires the presence of CO2 in the incubator atmosphere accordingly. In addition, the incubator atmosphere should be saturated with water, so that no culture medium can evaporate.
In vivo oxygen levels vary across tissues and cell types. Interestingly, currently oxygenation is not widely considered for cell culture incubators and live-cell imaging. Sub-optimal oxygen levels can result amongst others in decreased proliferation rates (hyperoxia) or reduced metabolic rates (hypoxia) (Hadanny, A.; Efrati, S.).
Light protection is another challenge for live-cell imaging, because light exposure can influence living cells per se, as well as the fluorophore. For example, high light doses can lead to DNA damage or photobleach the fluorophores.
Mica has been designed to cope with all these challenges:
The housing itself acts as an incubator. This means that the whole volume around the sample is heated up to the required temperature (5°C above room temperature up to 42°C) and equilibrated to the desired CO2 level and humidity.
The O2 levels are controlled within an additional stage-top chamber, if desired. All parameters can be controlled by the Mica