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Molecular Biology Techniques Q&A Live cell imaging


This month’s questions from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) come from the “Microscopy and Imaging Techniques” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but do not represent endorsements by, or the opinions of, BioTechniques.


How can I keep my cells healthy for prolonged imaging? (Thread 17621) Q


I am trying to follow the trafficking of a GFP-tagged receptor in HEK 293 cells. The process takes hours, so it requires long imaging sessions. For my live cell imaging experiments, I use DMEM without phenol red, but with


BSA and ascorbate. The cells begin to die near the three hour time point. Is there anything I can do to increase the lifespan of my cells?


incubator, you may need a special media that maintains its buffering capacity. How do you maintain the temperature of the cells? You may need a platform to keep the cells warm during microscopy.


A There are several environmental problems that can affect tracking live cells by microscopy. Since the cells are not in a CO2


Six hours is a long time for cells to be away from their routine incubation conditions. I am not sure how HEK293 cells stand up to the environmental changes.


A It sounds like phototoxicity might be the problem. I suggest that you take an occasional image and block the excitation light between imaging events so the cells remain in the dark most of the time.


A I currently use 2 second exposures once every 20 minutes and block the light path between each exposure. By hour four, cell membranes bleb and everything goes awry.


A If you are blocking the light path between those 2 second exposures so that the cells are in the dark when not being imaged, I expect that the illumination source would have to be extremely intense at short wavelengths to cause so much damage.


It would be interesting to set up the cells using the same protocol, but not take any images until the final time point. In this way, you can see whether the blebbing is due to light or another factor.


Q I will try the experiment you recommend. I should note that I find blebbing occurs only in the cells in the field of view. When I shift to another set of cells in another field, I can image those for some time before blebbing begins.


A That’s pretty convincing evidence that phototoxicity is the problem here. You might be able to modify the illumination of the cells. Did you use a bandpass filter for excitation so only the band you need for imaging illuminates the cells? You can look at the transmittance spectrum of your excitation filter because often higher-energy wavelengths are allowed through the filter some distance from the bandpass region you want. Perhaps a UV-blocking longpass filter should be added to the excitation light path to get rid of unneeded high-energy photons.


Vol. 54 | No. 6 | 2013 307 www.BioTechniques.com


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