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.Molecular Biology Techniques Q&A
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?
A There are several environmental problems that can affect tracking live cells by microscopy. Since the cells are not in a CO2 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.
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.
What is the best approach for live cell imaging? (22869)
Q I would like to microinject cells with a fluorescent dye and then image the results. To do this, I will need to keep the cells alive and healthy during imaging. If anyone has done this before, I would appreciate some advice to get started.
A It would be easier to advise you if you provide more detail on what you want to image in your cell. Do you expect changes in the number of cells, their shape, or the fluorescent signal inside the cells? Will you be using confocal or real time microscopy? There are several approaches for live cell imaging and the best depends on what you would like to see.
A To begin live cell imaging, you should first select the time frame you need to image your process of interest. If you plan to image for more than five or six hours or up to overnight, HEPES buffered medium should allow your cells to survive happily during and after imaging. You will need to supply CO2 and the right temperature during imaging, so be sure that you have the right instrumentation.
A I have never microinjected single cells, but I have microinjected Drosophila embryos, which are much larger. During microinjection, one of the most important things to avoid is bursting the cell. For injecting embryos, we dehydrate them first so that the extra liquid (up to 1% of the volume of the egg) does not overburden the cell membranes. I'm not sure if you can do something similar to prepare your cells. You should certainly keep the dye concentration as high as possible so you can inject the minimal amount of liquid to avoid bursting the cells.
For imaging, a key problem is imaging the same cell over a long time course heats the cell and damages it. Be sure to use low laser power and filters.
A It sounds like you need to do time resolved imaging to monitor the fluorescent changes within the cell over a defined time. For this, it is best to image inside an incubator so you will have the appropriate levels of CO2, O2, and moisture. I once used a small inverted digital microscope that could record images in time resolved mode. The device fit right into the incubator and allowed me to monitor cell changes up to 60 hours.
A I don't think that is necessary. Your cells will be fine for several hours at room temperature without CO2. Any acidosis will be corrected quickly upon re-incubation. If you only need a short time course, just image the cells. They should remain healthy for a few hours outside the incubator.
If you need to image longer, one method that has worked well for me is to plate the cells on slides with grids. When you microinject the cell, just note its position. Then you can move the cells back and forth between the microscope and the incubator and easily find the cell you want to image.
Be careful with HEPES. Cells do not have HEPES pumps and the buffer often leads to artifacts.
A If you do not have access to a stage incubator for CO2 control or a microscope that fits into the tissue culture incubator, you can trying placing your cells in CO2-independent media. Depending on your cell type, you might try L-15 or Hybernate A, which is good for neurons.
If you are able to make your own media from powder, you can also optimize the ratio of sodium bicarbonate to HEPES. Hank's balanced salt solution with 5 mM HEPES and 1 mM sodium bicarbonate works well for me. Many basal media formulations are based on Hank's balanced salt solution or Earl's balanced salt solution, so my mixture may be a good place to start. For better signal-to-noise ratio when visualizing a fluorescent protein, you should also use a media without phenol red.