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PAULINE YU - I have not worked with fluorescent samples and confocal imaging while using a safe light. But from my many years of film work, I can suggest 2 things to try:

    1) A "safe light" is not as good as a "sodium light" for insuring that the light that you are working in will not affect your material. So try what you are doing using something called a sodium light and see if it helps.

    2) Even with a sodium light, the amount of light that impinges upon your sample has a negative effect. To minimize the amount of light that hits your sample, turn on your sodium light source for 20-30 minutes before you want to work in that space. The light takes a while to get to maximum brightness. Once that has happened, set the 'windows' that let the light out on the sodium light to the lowest level (and therefore the least light) that you can manage to work in. And remember that amount of light and distance from light are related, so the farther your sample is from the light, the less the amount of light impinging on this.

The above suggestions will only help your situation if it is, in fact, the light that is your problem. If none of the above helps, you should look in another direction for improvement of your situation.


ANDREA ELBERGER




pauline yu wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear List-peoples,
> I'm quasi-self taught when it comes to using confocal, so please bear with me for asking an elementary question. When it comes to working with samples after they've been stained(in my case, Syto 24 for DNA and Alexa Fluor568 antibody), is it unsafe(i.e. damaging to the fluorochromes) to use a "darkroom safelight" or a red light bulb to illuminate samples when trying to mount them? I figured that if the wavelength of the red light was substantially longer than the excitation wavelength, I wouldn't have to worry too much about bleaching. Is this totally untrue? Should I just be working under the *absolute minimum illumination* with my samples prior to imaging?
>
> My main problem is that I'm seeing very rapid bleaching of my Syto dye, from just scanning a z-series through my sample(sea urchin eggs and embryos) even though I'm using only 1-3% transmission(ancient Biorad MRC600). The only other possibility I could think of is that my dye lot is "old"--purchased about a year ago, but stored concentrated at -20C this whole time until before use. My samples are mounted in 70% glycerol. Should I invest in some fluorochrome stabilizing media or try a different mounting medium? My mounted slides aren't nearly as "archival" for imaging purposes as I'd like them to be.
>
> Many thanks for any pointers. Literature recommendations are greatly appreciated.
>
> Pauline Yu
> [log in to unmask]
> Graduate Student
> Manahan Research Laboratory

--
Dr. Andrea J. Elberger
Professor, Anatomy and Neurobiology
Director, Confocal Laser Scanning Microscope Facility
The University of Tennessee, Memphis
855 Monroe Avenue
Memphis, TN  38163  U.S.A.
tel: 901-448-4101
FAX: 901-448-7193
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