Re: Far-red filter set

Posted by Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Far-red-filter-set-tp592652p592671.html

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Well, that depends where you are exciting.  I guess if you excite in the yellow

that could work but exciting at 640 rather rules it out.  I'm sure it is just a

sensitivity issue - I can see 750nm laser light easily enough.  And if you

have a cell layer or similar there will be enough glow around to find things.

But little bacteria are difficult.  We had 3 young men, I old man (me) and

1 young woman try and none of us could see them.   We had to find them

by phase - even that wasn't so easy since we'd worked hard to match

the refractive index!

 

                                                                                           Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
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Electron Microscope Unit, Madsen Building F09,
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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jennifer Waters
Sent: Saturday, 8 March 2008 5:07 AM
To: [hidden email]
Subject: Re: Far-red filter set

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Here's my guess:
While the peak intensity of Cy5 is not visible, it starts emitting (if you look at the spectra) around 620nm which most of us can see.  So if there is a high enough concentration of probe, the small percentage of photons that emit from Cy5 in the lower wavelengths will be detectable.
Jennifer

On Fri, Mar 7, 2008 at 11:11 AM, Michael Herron <[hidden email]> wrote:

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A nice paper on it here for wide field:  http://www.jhc.org/cgi/reprint/48/3/437

The Jackson website discusses Cy5 a bit here: http://www.jacksonimmuno.com/technical/f-cy3-5.asp

On Mar 7, 2008, at 9:27 AM, Martin Wessendorf wrote:

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George Ring wrote:

SUPPOSEDLY the human eye can see up to 700nm, and women can see longer wavelengths than men.  That said, I agree with the others who responded - I haven't met anyone who can see it.  A camera, however, or confocal can detect far red.

I think that the human eye has been shown to be capable of detecting photons out beyond 1000 nm--it's just the probability of detection gets pretty low.

--Based on my own lab's experience, I agree with Peter's comment--I haven't met many people who can't see Cy5, for instance.  However, intensity is all-important.  Filled cells that've been stained with Cy5-streptavidin are easy to see with a 20x 0.7 NA lens by eye and occasionally visible even at 4x.  However, smaller structures with weaker labeling (e.g. terminals) are much harder to see and even with brightly labeled cells, the details can get lost.

Re: IR filters on CCD cameras--they indeed can be deadly when trying to image far-red fluorophores!

Martin

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