Parameters for Live Cell Imaging
Humidity and Evaporation
ibidi’s Patented Humidity Control
The ibidi Humidity Control ensures a constant and very high relative humidity (RH) level inside the ibidi Stage Top Incubator—identical to the conditions in standard cell culture incubators. This unique and patent-protected technology actively humidifies the gas mixture in a fast and reliable way before it enters the Stage Top Incubator.
How Does it Work?
First, almost water-free gas from bottles or the laboratory line is mixed in the ibidi Gas Mixer. Then, the gas mixture is pumped through a heated water column for humidification, before it finally enters the ibidi Stage Top Incubator. There, the humidity is constantly measured by a humidity sensor. This enables the comparison of the preset to the actual humidity values, allowing for feedback control of the system.
What Makes the ibidi Humidity Control Special?
Standard humidity control systems use only heated water columns to control the gas humidity in the stage top incubator. By changing the water temperature, they control the water concentration in the gas that enters the stage top incubator. Because the water temperature in the heated column changes slowly, it takes a lot more time for the humidity level to adapt. Therefore, these systems respond very slowly to disturbances like opening the stage top incubator or condensation.
In contrast, ibidi uses the following patented technology for the fast and accurate regulation of humidity: Dry and humid gas streams are mixed in a specific and actively-controlled ratio that exactly matches the preset humidity parameters.
To achieve this, the dry gas mixture from the Gas Mixer is divided: one part bubbles through the water column for humidification, the other part remains dry by flowing through a bypass. This not only makes the control much faster, but also produces much smaller fluctuations in the air humidity. The ibidi Humidity Control's most important advantage is that it allows for very high humidity values of around “real” 90–95%, which provides the best and most reproducible environmental conditions for cells.
The ratio of dry gas and humidified gas is determined by the actual and preset humidity, and controlled by a feedback mechanism. In a steady-state system (e.g., when both the set and the actual RH are 90%), a mixture of humidified and dry gas enters the ibidi Stage Top Incubator.
When the humidity is too low, which causes evaporation, only humidified gas from the humidification column enters the Stage Top Incubator. This increases the humidity and prevents from evaporation.
When the humidity is too high, which causes condensation, only dry gas enters the Stage Top Incubator. This decreases the humidity and prevents from condensation.
Why Is Humidification Important? Why Must Evaporation Be Avoided?
Constant levels of salts, nutrients, and other cell culture medium components are essential for reproducible cell behavior, and are crucial for consistent and reliable in vitro results. Any evaporation from the cell culture vessels increases the substance concentration in the medium in an undefined way.
This dramatically influences the vital functions of living cells, which leads to impaired cell behavior of all kinds, such as increased or decreased proliferation, up- and downregulation of gene expression, and induction of apoptosis/cell death. This happens particularly fast in low-volume cultures.
The consequences of evaporation caused by humidity that is too low are frequently underestimated. Cells even react more sensitively to concentration changes than to temperature changes, which can markedly distort the results. Therefore, evaporation from cell culture vessels during live cell imaging experiments should be absolutely avoided.
A constant and high level of humidity (>90% RH) in the stage top incubator is one of the keys to artifact-free results in live cell imaging.
Rat fibroblast cells cultured in full growth medium at different levels of relative humidity (RH) 48 hours after seeding. Phase contrast microscopy, 20x objective lens.
Humidity and Evaporation Control in Different Systems
In the CO2 Incubator
Cell culture vessels in the CO2 incubator are normally cultivated in an open system (e.g., gases can diffuse through the cell culture plastics), and are therefore in an equilibrium with the surrounding air. In standard CO2 incubators, the humidity is created by evaporation from a water tray. Optimally, the relative humidity (RH) within an incubation system should be within the range of 90%–95%.
In the Stage Top Incubator
During a live cell imaging experiment, the evaporation in the stage top incubator should be kept as low as possible, underlining the need for a reliable humidity control. A constant and high level of humidity (>90% RH) in the stage top incubator is one of the keys to artifact-free results in live cell imaging.
The ibidi Stage Top Incubation Systems provide a patented and best-in-class, feedback-controlled humidity regulation. The humidity in this system is constant and can be smoothly adapted to specific experimental needs. With a possible humidity of up to nearly 100%, the evaporation within the ibidi Stage Top Incubation System is securely prevented.
The design of the ibidi μ-Dishes is optimized for low evaporation. Their lids can be set to a lock position, ensuring minimal evaporation.
For long-term studies, evaporation can be additionally reduced by using ibidi Anti-Evaporation Oil. This silicone oil is overlaid onto the culture medium, thus decreasing evaporation. Silicone oil is highly gas permeable for O2 and CO2, but effectively blocks humidity loss.
Find out more about how to avoid evaporation in the Application Note AN 12: Avoiding Evaporation (PDF).
Read on and learn more about Oxygen (O2) Levels and Hypoxia in live cell imaging.