Posted by
Julio Vazquez on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Fluorophore-excitable-with-405nm-and-488nm-lines-tp6432486p6432846.html
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Quantum dots come to mind.
Otherwise, I would suggest a few other options:
1. Fluorescent plexiglas slides (such as offered by Ted Pella and others:
http://www.tedpella.com/histo_html/fluor.htm ). These tend to come in sets of four for blue/green/orange and red fluorescence, but they do show some level of cross-excitation. Best of all, they will last a long time.
2. Get a set of two appropriate fluorescent dyes, mix them and mount the mix under a sealed coverslip. I suppose this may not be as stable over time as the plastic slide. Reproducibility may be an issue due to differential bleaching and other issues.
3. Just bounce the lasers off a mirror slide (or surface of a glass slide) and measure the reflected light. In this mode, you need to use a long pass or bandpass emission filter that includes the laser lines (or two separate filters). I use this method on point scanning confocal scopes.... it is easy and quite sensitive. Ideal for measuring laser power stability over time. Did not use it to measure illumination intensity ratios, but don't see why it wouldn't work.
4. Use a power meter and just measure the power at the objective (not as practical as (3).
5. Use a mix of fluorescence intensity calibration beads, such as the Inspek calibration kit from Invitrogen.
We use beads primarily for spatial calibration, i.e to measure image registration, focal shifts, etc, rather than for intensity calibrsations, but according to the product description, these should work (I would just measure enough beads).
If using an AOTF or other system to attenuate the laser intensity, you'll probably need to measure the intensity ratios at the actual settings, since it is not certain that the AOTF attenuation will be linear, and/or that it will be similar for both wavelengths. This should be easy to test though.
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, USA.
http://www.fhcrc.org/On Jun 2, 2011, at 11:59 AM, Daniel Gitler wrote:
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> Dear Confocalists/microscopists,
> I am in need of either beads or a fluorophore in solution which is exited consistently by both the 405nm and 488nm lines of a confocal microscope. What I really need is that the ratio of excitation should be constant, in which case two separate dyes are probably not a good choice, unless their molar ratio can be quite consistent (perhaps in beads?). I also need that the efficiency of excitation for both lines be quite decent (doesn't have to be maximal, just decent). Finally (it appears I have a lot of requisites) the dye/beads should be as insensitive as possible to environmental changes (i.e., pH etc). The idea is to have a good standard in order to calibrate the ratio of the power of these two lines when imaging a sample (I do not need to know the actual number, just a relative measure will do fine).
> I would appreciate any advice,
> Thanks,
> Daniel
>
>
> Daniel Gitler, Ph.D.
> Department of Physiology and Neurobiology
> Faculty of Health Sciences
> Ben Gurion University of the Negev
> Beer-Sheva 84105
> Israel
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