Visikol offers a wide variety of cell-based assays, ranging from 2D to 3D and many of these assays draw on Visikol’s expertise in imaging and image analysis. Most live cell imaging assays are performed in 2D such as wound healing and calcium flux assays, however live cell imaging may be appropriate for some 3D assays as well. In particular, 3D invasion assays (angiogenesis assays) and immune cell infiltration assays can be suitable candidates for live cell imaging, but something like a cell viability assay, while able to be evaluated using live cell imaging may ultimately be more suited for a different approach. So, when is an appropriate time to use live cell imaging verses a more traditional fixed-cell approach?

Living Cell Imaging vs. Fixing Cells

The choice of live cell imaging verses fixing cells at set time points is entirely dependent on the type of study being done and the questions being asked. In general, live cell imaging is advantageous when the experiment requires multiple time points, and the desired outputs can’t be adequately determined with one final endpoint or other non-destructive endpoints. For instance, in a wound healing assay (scratch wound assay) to determine how the cells are moving, images need to be taken of the same scratch at the beginning and the end of the study at a minimum, so that differences in how “healed” the wound is can be determined. However, doing the assay this way only gives a snapshot into how the wound healing took place. To get a better idea of what is happening to the cells, and to be able to better quantify things like cellular velocity and cell proliferation, it can become necessary to increase the number of timepoints, where instead of imaging only at 0 hours and 24 hours, a researcher may instead opt to image at 0, 6, 12, 18 and 24 hours. While it is possible to have someone take a sample in and out of an imager for each time point, it becomes burdensome when time points occur outside of standard work hours or when the time points are close together, this is where a live-cell imaging system can come in handy. Live cell imaging systems incorporate incubation, which allows for temperature, CO2, and humidity levels to be maintained and thus cell cultures can be imaged for days or even weeks.

Live Cell Imaging Considerations

While live cell imaging is useful in some instances, it can become cumbersome, as live cell imaging can take up a lot more microscope time and become cost prohibitive, both in terms of imager time and in the costs associated with the generation of large amounts of data (i.e. storage and analysis). Thus, for longer term experiments it can become necessary to weigh the pros and cons of live cell imaging. For instance, a four-week long study that wants to look at viability over time could be done with a viability dye and live cell imaging, but it would likely be more cost effective to collect supernatant samples over the four weeks and evaluate them for the release of cytotoxicity markers.

Another thing to consider is the target of interest. While there are many live cell dyes on the market that can allow for the visualization of the nucleus, organelles, cytoskeleton etc. without causing harm to the cells themselves this list is not all inclusive and some of the dyes do not hold-up well for long term imaging. An alternative way to visualize targets of interest in cells is through transfection of the cells so that the cells produce a fluorescent version of the target of interest. This can be a useful method, but is often time consuming to achieve stable cell lines for use in a study. Before moving forward with these methods, it should be considered whether fixing cells and using immunofluorescent labeling could accomplish the same outcomes.

Ultimately it is useful to take a step back and think about whether a study truly requires live cell imaging because while it can be key to understanding certain processes it can add significant costs to a study when relevant answers could be obtained with more traditional and less expensive fixed end points. If you would like to learn more about whether live cell imaging is right for you or if you are interested in any of the assays Visikol offers, please reach out.

2022-02-22T07:24:27-05:00

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