Live Cell Imgaing - cells dying due to phototoxicity

[adi]

Hi,

I am trying to chase the trafficking of a receptor-GFP protein in HEK 293 cells. Its darn slow receptor so chasing is over the period of hours. I find that cells start dying in the live cell imaging set up by 3 hrs....I have BSA, ascorbate in the medium .... what should I do to increase the lifespan of my cells?

Aditi

[Jon Moulton]

Your subject suggests phototoxicity might be the problem. If so, why not take an occasional image but then block the excitation light so the cells remain in the dark between images?

- Jon

[adi]

Hi,

I take a 2sec exposure once every 20mins. I need to chase the cells for 6 hrs...by 4 hrs the membranes of the cells start to bleb and everything goes awry. I image in DMEM without phenol red with BSA and Ascorbate in the solution. Is there something else I could do?

Thanks

Aditi

[Jon Moulton]

If you are blocking the light path between those 2 sec 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 (with a lot of short wavelengths) to cause so much damage.

It would be interesting to set up the cells in the same way have been doing, but then take no images until the last time point. That way you can see whether the blebbing is due to the light or another factor.

- Jon

[adi]

Hello again,

Yes I do block the light path at the time between two exposures.
I shall try just incubating the cells in the set up for 6 hrs and take an image right at the end....great thought!
But what I do find is that blebbing almost totally occurs in the cells that are in the field of view. If I shift to another set of the cells in another field I can continue to images these for sometime before blebbing sets in again.

Cheers

Aditi

[creepster]

im just thinking of environmental problems when tracking live cells during microscopic observation:
- do you think you would need a special media which keeps its buffer capacity since the cells are not in an CO2 atmosphere (incubator) ?
- do you think you need to keep the plate of cells warm during microscopy (37 celsius) ?

6 hours are pretty long, and i have no clue how hek293 stand changes from their regular incubation
although these conditions would not explain the change of ells within the visual field, but not the surrounding cells:

But what I do find is that blebbing almost totally occurs in the cells that are in the field of view. If I shift to another set of the cells in another field I can continue to images these for sometime before blebbing sets in again.

[Jon Moulton]

But what I do find is that blebbing almost totally occurs in the cells that are in the field of view. If I shift to another set of the cells in another field I can continue to images these for sometime before blebbing sets in again.

That's pretty convincing. What about the illumination of the cells -- have you filtered the excitation light with a bandpass filter so that only the band needed for your imaging illuminates the cells? Look at the transmittance spectrum of your excitation filter -- often there are higher-energy wavelengths 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.

[ChongOrr]

I expect that the illumination source would have to be extremely intense (with a lot of short wavelengths) to cause so much damage.

[brianrasnow]

Might try a Lumascope (http://www.bulldog-bio.com/lumascope.html#) for imaging. Can put it in the incubator and toggle on the LED for a fraction of a second. Turn up the sensor gain as high as possible and the light intensity way down. I suspect that will work.