I've been looking at the emission spectrum of the p300 and see that it is not great for excitation of Alexa488 and similiar dyes, but fits Cy2 (and FITC!) much better. Has anyone done a comparison of different fluors at this wavelength for immunofluorescence?
We have pE-300 mounted on olympus CKX53 in our cell culture room. No problem for EGFP. From the spectrum, pE300 fits better with EGFP. The excitation spectrum of FITC is actually very closed to Alexa488. There should be no problem for either three unless the florescence intensity is low. That is to say we only use this setup for routine checking and documenting of cell cultures but not for quantification purpose.
Alexa488 works, but the p300 only hits the shoulder of the excitation peak, so the match is not ideal. GFP is fine, as you say, and FITC excitation peak is also a good match. I'm just wondering if we can improve our existing imaging at this wavelength.
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