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ibidi Stage Top Incubator Slide/Dish, CO2 – Silver Line
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System Components
The ibidi Stage Top Incubators – Silver Line are ideal for live cell imaging experiments that require physiologic conditions on the microscope.
Combining the ibidi Gas Incubation System and the ibidi Heating System, they precisely control essential parameters such as temperature, humidity, and CO2 levels on the microscope to get reproducible results of, for example:
The ibidi Stage Top Incubators – Silver Line are optimized for high-resolution imaging applications that require extra focus stability, for example:
Time lapse TIRF imaging showing focal adhesion dynamics of a mutant NIH-3T3 mouse embryonic fibroblast cell, which was transfected with EGFP-Paxillin, in a µ-Dish 35 mm, high Glass Bottom. TIRF microscopy images were acquired over a period of 12 hours in the ibidi Stage Top Incubator Slide/Dish, CO2 – Silver Line using a TIRF 100x immersion objective on a Nikon Eclipse microscope.
Data by Jonas Scholz and Prof. Jan Faix, Hannover Medical School, Germany.
Cells react sensitively to changes in their environment. Factors such as temperature, humidity/evaporation, and CO2/O2 levels significantly influence the outcome of cell culture assays. Therefore, it is crucial to maintain optimal conditions on the microscope stage during live cell imaging experiments to achieve biologically relevant and reproducible results.
The ibidi Stage Top Incubators – Silver Line combine the ibidi Heating System – Silver Line and the ibidi Gas Incubation System – Silver Line.
The ibidi Heating System creates stable and homogenous temperatures for high-resolution live cell microscopy. It fits into inverted microscope stages with a universal mounting frame for multiwell plates. When placed in the ibidi Heated Chamber, the cell culture vessels are kept at a stable, defined temperature during the live cell imaging experiment on the microscope.
The ibidi Gas Incubation System provides humid and CO2-rich air for stage top incubators like the ibidi Heating System. The gas mixture is continuously flushed through the stage top incubator, ensuring maximum humidity and an optimal pH for bicarbonate-buffered liquids.
For hypoxia assays, the ibidi Stage Top Incubators, CO2/O2 – Silver Line allow for the precise control of the oxygen levels in your cell culture vessel. The ambient O2 level can be controlled within a range of 1%–21%, and easily adapted to the experimental requirements.
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.
Cell behavior is strongly influenced by changes in medium components, such as salts and nutrients. Any evaporation from the cell culture vessels increases the substance concentration in the medium in an undefined way. Therefore, creating ideal humidity conditions—especially during live cell imaging—helps avoid evaporation and leads to reproducible experimental conditions.
The ibidi Stage Top Incubators – Silver Line use the patent-protected ibidi Humidity Control. The unique technology actively humidifies the gas mixture in a fast and reliable way before it enters the stage top incubator. The ibidi Humidity Control allows for very high humidity values of around “real” 90–95%, which provides the most reproducible environmental conditions for cells. Further, in contrast to other systems, the humidity sensor is located close to the sample, which makes the measurement far more transferable to the direct sample/cell environment.
By using the absolute humidity rather than the relative humidity for the feedback control, the ibidi Stage Top Incubators – Silver Line enable an even more precise regulation of the relative humidity (RH) inside the Incubation Chamber. Since the relative humidity is strongly temperature-dependent, determining the absolute humidity allows to compensate for any temperature differences between the humidity sensor and the sample.
Using the ibidi Humidity Control ensures a constant and very high relative humidity (RH) level inside the Incubation Chamber—identical to the conditions in standard cell culture incubators. This reliably prevents evaporation, which is one of the keys to artifact-free results in live cell imaging.
What Makes the ibidi Humidity Control Special?
The temperature of the Heated Lid of the ibidi Stage Top Incubators – Silver Line is independently controlled and optimally distributed, thereby solving the frequently occurring problem of condensation during live cell imaging.
A vertical temperature gradient is created by heating the lid to a temperature higher than the plate (e.g., 40°C). This gradient and the active humidity control prevent condensation on the lid of the cell culture vessel, which is crucial for non-disturbed high-quality microscopy images. The Heated Plate maintains the sample temperature at a constant 37 °C.
The ibidi Stage Top Incubators – Silver Line ensure a precisely controlled, stable temperature in the Incubation Chamber with homogeneous distribution across the wells of the cell culture vessel. The temperature is controlled via continuous current, which reliably prevents position fluctuations and makes the system optimal for high-resolution live cell microscopy with a stable focus.
Heat-up Curve
Heat-up curve of the ibidi Heating System Slide/Dish – Silver Line with Dish Holder and µ-Dish 35 mm, high, filled with cell culture medium (Sample). The heat-up curve of the ibidi Heating System was recorded at an environmental temperature of 24°C (± 0.3°C). Plate and Lid reached their target temperature within 10 minutes. After complete equilibration of the sample to 37°C within 40 min, the sample temperature stayed at 37.0°C (± 0.2°C).
Sample Temperature Stability
Sample temperature stability inside the ibidi Heating System Slide/Dish – Silver Line with Dish Holder and µ-Dish 35 mm, high, filled with cell culture medium (Sample). After equilibration of the sample inside the Heating System, the environment temperatures were increased stepwise (17°C → 20°C → 23°C → 26°C; max 27.7 °C). The temperature of the sample inside the Heating System stably stayed at 36.9°C (± 0.15°C).
Temperature Distribution
Stable and consistent temperature distribution in the µ-Slide 8 Well high (left) and the µ-Dish 35 mm, high (right) during the incubation in the ibidi Heating System Slide/Dish – Silver Line. Images were acquired with a FLIR thermal camera.
The ibidi Stage Top Incubators Slide/Dish – Silver Line can be extended with the ibidi Objective Heater Universal – Silver Line. Microscope objectives that require close approximation to the sample (e.g., water or oil immersion objectives) might cause cooling of the sample by thermal transfer from the sample to the objective, when the objective is not heated. Adequate heating of the objective by the Objective Heater prevents cooling of the sample, enabling long-term high-resolution imaging of living cells.
The oxygen (O2) concentration at normal atmospheric pressure is around 20–21%. However, the O2 levels in healthy body tissues and our blood are much lower. Variations in the oxygen concentration are part of the normal body system, ranging from 14% in the alveoli to 5% in peripheral tissues.
The oxygen levels can be even lower in pathological situations (hypoxia). Hypoxia especially occurs in tumor tissue, which is characterized by markedly low O2 levels that range from approximately 4% to a minimum of below 0.5%.
The ibidi Stage Top Incubator Slide/Dish, CO2/O2 – Silver Line allows for the precise control of oxygen levels in the incubation chamber between the range of 1% to 21%, creating an ideal solution for live cell imaging experiments under hypoxic conditions.
McKeown SR (2014) Defining normoxia, physoxia and hypoxia in tumours-implications for treatment response. Br J Radiol 87(1035):20130676. 10.1259/bjr.20130676.
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O2 partial pressure in several tissue types (1 kPa ≙ 1 % in gas mix ≙ 7.5 mm Hg).
Time lapse of a B16-F1 mouse melanoma cell migrating on the laminin-coated surface of the µ-Dish 35 mm, high Glass Bottom. Phase contrast microscopy images were acquired over a a period of 10 minutes in the ibidi Stage Top Incubator Slide/Dish, CO2 – Silver Line using a 100x immersion objective on an Olympus X-81 microscope.
Data by Jonas Scholz and Prof. Jan Faix, Hannover Medical School, Germany.
Time lapse TIRF imaging showing focal adhesion dynamics of a mutant NIH-3T3 mouse embryonic fibroblast cell, which was transfected with EGFP-Paxillin, in a µ-Dish 35 mm, high Glass Bottom. TIRF microscopy images were acquired over a period of 12 hours in the ibidi Stage Top Incubator Slide/Dish, CO2 – Silver Line using a TIRF 100x immersion objective on a Nikon Eclipse microscope.
Data by Jonas Scholz and Prof. Jan Faix, Hannover Medical School, Germany.
Brightfield ROCS (Rotating Coherent Scattering) microscopy of living A549 lung cancer cells using a self-made ROCS microscope, recorded with a 445 nm laser within 10 minutes (2 images per second). The cells were cultured in the µ-Dish 35 mm, high Glass Bottom. Live cell imaging was conducted using the ibidi Stage Top Incubator Slide/Dish – Silver Line.
Data by Dominik Huber and Alexander Rohrbach, Department of Microsystems Engineering (IMTEK), Albert-Ludwigs-Universität Freiburg, Germany.
Jünger, F., Ruh, D., Strobel, D., Michiels, R., Huber, D., Brandel, A., Madl, J., Gavrilov, A., Mihlan, M., Daller, C. C., Rog-Zielinska, E. A., Römer, W., Lämmermann, T., Rohrbach, A. 100 Hz ROCS microscopy correlated with fluorescence reveals cellular dynamics on different spatiotemporal scales. Nature Communications, 2022, 10.1038/s41467-022-29091-0.
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