Transformed Yeast colonies with a Plasmid having GFP, showed faded Fluorescence. It was light yellowish in colour rather than the usual Green Colour . What could be the reason ? The Microscope was found to be proper, as some other samples showed proper fluor.
Hello several reasons can cause problem. Please see points below.
1. Use a lens with as high a numerical aperture as possible, e.g. 100x 1.4 NA.
2. Bin your image 2x2 or even more depends what magnification you need.
3. Make sure you are using a filter set that is suitable for GFP.
4. Increase the exposure time, gain, etc.
5. Make sure your fluorescent light source is properly focused.
5. Be careful if you are using anti-fade mounting solution - they do prevent photobleaching but some also reduce the initial fluorescence.
Hello several reasons can cause problem. Please see points below.
1. Use a lens with as high a numerical aperture as possible, e.g. 100x 1.4 NA.
2. Bin your image 2x2 or even more depends what magnification you need.
3. Make sure you are using a filter set that is suitable for GFP.
4. Increase the exposure time, gain, etc.
5. Make sure your fluorescent light source is properly focused.
5. Be careful if you are using anti-fade mounting solution - they do prevent photobleaching but some also reduce the initial fluorescence.
Hi there,
Are you sure the GFP is actually efficiently expressed in these cells (ie. check protein level by WB)?
Hello Karthik
Could you please elaborate how you image these samples and whether the imaging is live of in fixed samples. In case you are imaging live and the GFP is the non-modified original sequence, then that GFP is highly photolabile and may be bleaching out during imaging. Additionally, why do you think the GFP emission you see is in the yellow range? Could you please indicate the detector ranges?
Best
Aparajita
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