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Excitation of fluorphores using UV for microscope vs lasers for cytometry


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#1 seanspotatobusiness

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Posted 12 May 2014 - 07:27 AM

For assessing fluorophore protein expression in cells I can use a flow cytometer equipped with four lasers of wavelengths 405, 488, 561 and 640. To view the cells under a microscope I can use a UV light source (recently upgraded from a mercury vapour lamp to an LED (the Lumencor Sola)). Since my current protein of interest is mCherry which has an optimum excitation at about 587 nm (I'd used 561 nm laser because that's what we have), does that mean the microscope is hopeless for this purpose? The cells are very dimly lit and I can only view them with an extended exposure setting on the camera. The same set up has worked for mApple but excitation spectrum is shifted about 25 nm lower.


Edited by seanspotatobusiness, 12 May 2014 - 07:33 AM.


#2 bob1

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Posted 12 May 2014 - 01:03 PM

It should still work - you need to use the appropriate filter for mCherry.  If you can see white light coming out of the light source on your microscope - then it should be fine.



#3 seanspotatobusiness

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Posted 15 May 2014 - 01:55 PM

I've come to realise that referring to the mercury vapour lamp as a "UV light" is misleading! (it produces light in multiple wavelengths)

 

My cells are simply very low-expressing of mCherry; it's more readily apparent using a flow cytometer.



#4 bob1

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Posted 15 May 2014 - 03:34 PM

This may not be the case - the laser is probably higher power and more specific in its excitation - hence it seems brighter.






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