Live cell microscopy

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Live cell microscopy

Postby tyler1982 » Jun 16 2009 2:00 pm

Hi all!
has any of you ever worked on living cells imaging (no fixing with ethanol/formaline/whatever)?
Basically, I should microinject my cells with a fluorescent dye and see what happens. But, i need them to be alive and (but I don't know to what extent this is possible) healty after that.
If anybody has any kind of insight on the topic please let me know
Thanks in advance

Claudio Procaccianti
PhD student
University of Milano Bicocca
Milan, Italy
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Re: Live cell microscopy

Postby Astarte » Jul 08 2009 8:01 am

I think you should give some more info on what exactly you want to image in your cell.
Do you want to see the amount of cells, the shape of the cells, a fluorescent signal inside the cells?
There are different possibilities depending on what you want to see, so if you want your question answered, you should give more details.
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Re: Live cell microscopy

Postby Gnapponico » Aug 03 2009 5:35 pm

Confocal or realtime microscopy?
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Re: Live cell microscopy

Postby lln » Jul 15 2010 9:18 am

For live cell imaging, let say you are going to image them for more than 5 - 6 hours or even overnight, HEPES buffered medium should allow your cells to survive happily during and after imaging. Of course, you still need to supply CO2 and the right temperature during your imaging. Hope this is useful. :)
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Re: Live cell microscopy

Postby CrowSan » Jul 15 2010 3:20 pm

Hi I have never microinjected single cells (but I have mircoinjected Drosophila embryos which are much larger). One of the things to watch out for is "bursting" the cell when injecting. When we inject embryos we dehydrate them a little first so that the extra liquid (1% egg volume) does not cause this. I don't think you can do the same for your cell but if you kept the dye conc as high as possible and inject the minimum amount of liquid it may prevent this happening. Also, of course, imaging the same cell over a time course will cause the cell to heat up and "fry" unless you are using a low laser power....
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Re: Live cell microscopy

Postby WISBiomed » Aug 10 2010 10:33 pm

Sounds like you need to do a time resolved imaging to monitor the cell/fluorescent changes over defined time inside the incubation (so you will have the appropriate CO2,O2, moistures..). Digital Bio manufactures a small inverted digital microscope which can record images in time resolved mode. You can place the device inside your incubator and monitor the cell changes upto 60 hours.http://www.wisbiomed.com/ec/index.php?main_page=product_info&cPath=16&products_id=1308
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Re: Live cell microscopy

Postby BiochemRay » Oct 19 2010 8:25 pm

Cells are fine for an hour at room temp without C02. The acidosis will be quickly corrected upon re-incubation.

You should plate the cells on gridded slides, note the injected cell position, they go back and find the cells.

If there is a short time course involved, just image the cells, they are fine for hours.

Be careful with HEPES, cells do not have HEPES pumps, and this buffer could lead to artifacts.
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Re: Live cell microscopy

Postby JMinCA » Oct 19 2010 10:21 pm

You do not mention how long you want to image your cells. If you do not have access to a stage incubator for CO2 control, or the microscopes that fit into tissue culture incubator, you can try keeping your cells in a CO2-independent media. Depending on your cell type, you can try L-15. Gibco also makes Hybernate A, which is good for neurons. If you are able to make your own media from powder, you can also play with the ratio of Sodium Bicarbonate to Hepes. I make a Hank's balanced salt solution with 5mM Hepes, but only 1mM Sodium Bicarbonate. Many basal media formulations are based on Hank's BSS (others are based on Earl's BSS), so my mixture may be a good place to start.
For better signal-to-noise ratio when visualizing a fluorescent protein, you may also want to use a media without phenol red.
Good luck with your microinjections!
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