Shea Miller wrote:
>
>    I have just started looking at a GFP-transformed fungus growing on plant > surfaces, using regular fluorescence microscopy.  I can clearly see the
> fungus using two filter sets: a UV set (ex/em 365/>420) and a blue filter
> set (ex/em 470/>515).  While the fluorescence in blue light is much
> brighter, stuff that is blue/blue-white with UV illumination is green in the
> blue light.  is the "extra" green in the blue light likely to be where the
> GFP is not as concentrated, and hence doesn't show up in the UV, or is it
> more likely to be some other compound that has similar excitation/emission
> characteristics?

Consider the possibility that the blue-white emission that you see with
UV excitation is autofluorescence.
It is also possible that the green emission that you see with the blue
excitation is also autofluorescence, which looks green because  your
emission filter set cuts off the blue emission when using blue
excitation.

GFP emission fades quickly in some preps, and if you look closely at
your sample you may find that in the first few seconds there is a green
component that fades out.

Unfortunately, it is often the case that when the emission looks too
good to be true, that is exactly what it is.

--aryeh
--
Aryeh Weiss                          | email: [log in to unmask]
Department of Electronics            | URL:
http://optics.jct.ac.il/~aryeh
Jerusalem College of Technology      | phone: 972-2-6751146
POB 16031                            | FAX:   972-2-6751275
Jerusalem, Israel                    | ham radio: 4X1PB/KA1PB