C. elegans YFP Imaging - Help Needed

[paul wright]

Hi, 

I am doing a project on the effect of certain compounds on the accumulation of a protein, aSynuclein, in C. elegans.  (aSynuclein being a common indicator of Parkinson's disease)

The C. elegans strains I am using are OW13 and NL5901.  In both strains, aSynuclein in the body walls of the worms is marked by Yellow Fluorescent Protein.  

I currently do not know how to image the worms.  (I assume that I need to shine UV light on the worms in a dark setting to see the glowing, but I am not sure) 

I do not have access to an epifluorescent microscope, but I do have access to a dissecting microscope and UV light. 

Any input on how to take pictures of the fluorescence of the YFP would be appreciated! 

[Avery louie]

Hey Paul,

You might want to figure out how much YFP your cells will produce.  If there is not enough, you will not be able to see it.  Some of the microscope experts in this group might hop on and explain about how much you would need to detect it.  My guess is that unless there is quite a bit of it (milligrams?) you will have a lot of trouble, and you might not want to use your eye as the imager- the UV will be going in as well as the Yellow.  You could try to build a scope with a yellow pass filter (yellow glass) and a uv stop filter, which might help you get a better signal to noise ratio (although it would make the overall signals smaller).

One option you might want to look at is going to a place that does have an 'scope that would work, and getting access to it.  Generally you want to deal with one really hard thing (studying a protein in this case) at a time, instead of trying to study a protein and build a fluorescent microscope.

--A

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[Nathan McCorkle]

Check out the input and output wavelengths for a few fluorescent proteins (including YFP):

http://zeiss-campus.magnet.fsu.edu/articles/probes/images/aequoreafpintrofigure4.jpg

This photo/video industry filter sample pack is $2 and is probably a good place to start (an iGEM project recommended trying these before settling on more expensive filters)

http://shop.leefiltersusa.com/Swatch-Book-Designers-Edition-SWB.htm

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-Nathan

[paul wright]

I found a link for a previous researcher who made a DIY set-up for observing GFP: http://wormlab.rice.edu/LED/

Do you think that if I replaced the filter for GFP observation with one for YFP observation, it would work? 

[Nathan McCorkle]

On Mon, Feb 16, 2015 at 12:05 AM, paul wright <paulwrightp...@gmail.com> wrote:

I found a link for a previous researcher who made a DIY set-up for observing GFP: http://wormlab.rice.edu/LED/

Do you think that if I replaced the filter for GFP observation with one for YFP observation, it would work? 

You'd have to swap the filter /and/ LED... looks like 505nm may be the closest to 514, but maybe you or others know more about who has the best LED prices these days. Based on that link, I found luxeon's website and they have a color selection panel with nanometers on the left side:

http://www.luxeonstar.com/505nm