Parameters for Live Cell Imaging

Carbon Dioxide (CO2) Levels and pH of the Medium

A pH of 7.2–7.4 provides optimal growth conditions for most mammalian cells. The ambient carbon dioxide (CO2) levels are an important parameter for live cell imaging, since they influence the pH of the cell culture medium.

For a stable pH, many cell culture media use the bicarbonate buffer system, which is the main physiological buffer system in our blood. Typically, the medium contains sodium bicarbonate (NaHCO3), which balances with the carbonic acid (H2CO3) and the CO2 in the air. In a standard 5% CO2 incubation atmosphere, bicarbonate-buffered cell culture media exhibit a pH of 7.4. However, the required CO2 level in the ambient air can be different when using a special medium. Please consider the manufacturer’s manual for the pH requirements of your applied cell culture medium.


Equation of the bicarbonate-buffered system.

Changes in the atmospheric CO2 lead to the alteration of pH in the medium. Constant CO2 levels are therefore crucial for reproducible cell culture assays. Most commercial cell culture media contain phenol red as a color indication for the pH. The medium should be replaced if the color turns yellow (acidic) or purple (alkaline).

Apart from the bicarbonate buffer system, organic chemical buffering using HEPES is possible. In this system, a controlled amount of CO2 in the ambient air is not required. However, HEPES can be toxic for some cell types.

ibidi Solution

ibidi Stage Top Incubation System

The ibidi Stage Top Incubation Systems provide stable CO2 levels in the cell culture chamber directly on the microscope. The ambient CO2 level is changeable within a range of 0%–15% and can be easily adapted to the requirements of the cell type and the medium being used.

Read on and learn more about Humidity and Evaporation in live cell imaging.