During live cell imaging, environmental parameters, such as temperature, CO2 and humidity, should be controlled to optimize experimental results. A well- controlled environment not only provides cells the best condition they need to grow, but also minimize the focus drift caused by thermal fluctuation.
Types of microscope incubator (also known as environmental control chamber):
There are two main types of microscope incubators-
A. Cage incubator: An acrylic chamber which encloses the whole microscope. It provides stable temperature across the chamber. As it needs to warm up the air inside whole chamber, it usually takes 30 minutes for the temperature to stabilize at preset temperature.
B. Stage incubator: A compact metal chamber which can be mounted on the microscope stage. As the stage chamber can be easily removed from the stage, it’s convenient for shared microscope facility. On the other hand, inhomogeneous temperature distribution is usually a trade-off for this kind of incubator.
Typically, the temperature in the microscope incubator can be set from ambient to 50°C. The control accuracy depends on the design and type of incubators. For example, without advanced control, a typical stage incubator might have a 2 °C difference in temperature across the sample. On the other hand, a well-designed cage incubator yields a control precision of ± 0.2 °C.
During the time-lapse imaging, cell culture medium might evaporate and thus change cell conditions dramatically. To minimize medium evaporation, a small chamber connecting a water reservoirs is usually used to cover the sample. Passing through the water reservoir, the air gets humidified and then enter the small chamber. This technique should keep the relative humidity inside the small chamber between 90-100%
For live-cell imaging, 5% CO2 is usually supplied to a small chamber to keep the PH of culture medium constant. Most labs use 5% CO2 gas cylinder directly, while some others use 100% CO2 cylinder with a CO2 controller.
Temperature Sensor Setup:
The closer the distance between sensor and the sample, the better the temperature control. However, putting the sensor directly into the medium might alter the characteristics of the sensor and it will need calibration later on.
Microscope Incubator Customization:
As the microscope configuration varies widely, the microscope incubators usually need to be customized for individual setup.
Heat sources like mercury lamps should be isolated from the environmental control chamber to ensure optimal temperature control
Carson is a senior microscopist, who had built numerous high-speed two-photon, TIRF, SHG and OCT microscope systems. He is also an expert on environmental control for microscope systems, and is a consultant for the microscope incubator development at Haison Technology.