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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.

you have the right instrumentation.

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

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.

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.

, O2 A I don’t think that is necessary. Your cells will be fine for several hours at room temperature without CO2

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

, and moisture. I . 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

you can trying placing your cells in CO2 A, which is good for neurons.

control or a microscope that fits into the tissue culture incubator, -independent media. Depending on your cell type, you might try L-15 or Hybernate

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.

Selected and edited by Kristie Nybo, Ph.D.

BioTechniques 54:307-309 (June 2013) doi 10.2144/000114038 To purchase reprints of this article, contact:

Vol. 54 | No. 6 | 2013 309

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