Optimized Optical Conditions

Apart from temperature stability, buffer, and humidity control, some important technical conditions for live cell imaging also need to be considered. For optimal results in long-term assays (of 12 hours or more), condensation and focus stability are crucial parameters. The magnification of the optical system defines the resolution and the amount of cellular details that can be visualized.

Focus Stability

The requirements for focus stability strongly depend on the temperature changes of the optical system and the objective’s numerical aperture (NA). The ibidi Heating & Incubation System provides crucial focus stability in live cell imaging by maintaining a continuous, smooth temperature regulation.

In general, when using an extremely high magnification and TIRF, an optical stabili­ty of better than 1 μm can more easily be achieved by using glass coverslips (e.g., the µ-Dish ^{35 mm, high} Glass Bottom). When using high-resolution time-lapse microscopy, ibidi recommends the application of an auto-focus system. This may be an optical focus system like the Nikon Perfect Focus System (PFS), or a software-based auto-focus.

Condensation

Humidity in ambient air can lead to condensation on all surfaces. If these surfaces are in the opti­cal pathway, small water droplets will cause light scattering. This di­minishes the optical quality of transmitted light microscopy (i.e., phase contrast and DIC).

The independently controlled, heated glass lid of the ibidi Heating & Incubation System solves the problem of condensation in live cell imaging.

By heating the lid to a temperature higher than the plate, a vertical temperature gradient is created. This gradient and an active humidity control prevent the formation of condensation on the lid of the Petri dish. The temperature at the cells’ site is maintained at a constant 37 °C.

In the ibidi channel slides, condensation inside the optical pathway is intrinsically impossible. The example on the right shows this result after the sample is removed from the incubator.

Magnification and Resolution

Choosing the right magnification is a compromise between either having a higher resolution or more cells and statistics.

Using low-resolution microscopy with a 4x or 10x objective can be advantageous in wound healing, chemotaxis, and tube formation assays, because focusing is less delicate. On the other hand, a high magnification is useful for imaging subcellular details in immunofluorescence.

Illumination and Photobleaching

When using fluorescence-based microscopy techniques, it is especially crucial to minimize cell stress that is caused by the excitation light. Phototoxic effects and photobleaching happen quickly in living cells and might alter the outcome of the experiment. The selection of the right parameters, listed below, is a mutual compromise between an optimal signal-to-noise ratio and good cell viability.

Here are some recommendations for achieving the best cell viability in live cell imaging:

• Minimize the time the cells are exposed to excitation light by using shutters.
• Minimize the exposure time per image.
• Minimize the area in which the cells are exposed to excitation light by closing the field diaphragm as far as possible.
• Minimize the intensity of the excitation light.
• Maximize the time between the images for cell recovery.
• Optimize the signal-to-noise ratio by using a highly sensitive CCD camera.
• Match fluorescence filters and fluorophores as closely as possible.
• Use longer wavelengths (e.g., green or red) instead of UV or blue light excitation for fewer phototoxic effects.
• Use objective lenses with the highest numerical apertures available for optimal signal detection.

Calculating the Time Lapse Interval (Δt) for Cell Tracking

For efficient time lapse assays the optimal time lapse interval is necessary. The optimal time between the frames is mainly dependent on the cells` velocity and morphology changes but also on the magnification and the type of analysis. E.g. for automated cell tracking we recommend using a higher overlap. For cell tracking the time interval (Δt) defines the overlap, thus allowing you to follow single cells during migration and chemotaxis experiments. The time interval between two images should be chosen so as to allow a cell movement between sequent images that will not exceed 40% of its average diameter. Additionally to this 40% overlap, a security factor should also be used to follow cells that are twice as fast as the average, or cells that undergo big morphology changes. We estimate, typically, that the maximal speed of a cell is twice the average speed, leading to a security factor of 0.5.

For calculation, it is recommended to use the velocity values and cell diameter values from literature or preliminary experiments.

In our example calculation, HT-1080 cells are migrating in a 3D collagen gel matrix with a velocity of approximately 30 µm/h (0.5 µm/min) with a cell size of ca. 25 µm. With a 40% average overlap and a safety factor of 0.5, the time lapse interval is 10 min.

The following example demonstrates the effect of imaging cells with different time lapse intervals (Δt).