Far-red filter set
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Hi, we're considering buying a Y5 filter cube from Leica to see the far-red Alexas, DRAQ5 etc. but then someone mentioned to me that many (most?) people are unable to see far-red light. Does anyone know if this is true, or have experience with this (or a similar) filter cube? It has a BP 700/75 suppression filter. Kenneth |
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Generally the emission of these dyes is
considered to be beyond the range of the human eye, but there are papers which
describe direct observation of the shorter emission wavelengths, e.g. J
Histochem Cytochem. 2000 Mar 48(3):437-44 Simon From:
Hi,
we're
considering buying a Y5 filter cube from Leica to see the far-red Alexas, DRAQ5
etc. but then someone mentioned to me that many (most?) people are unable to
see far-red light. Does anyone know if this is true, or have experience with
this (or a similar) filter cube? It has a BP 700/75 suppression filter. Kenneth
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In reply to this post by Kenneth Bowitz Larsen
Search the CONFOCAL archive at
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We have a y5, can see 594, cy5 etc. Dark red, strong signal more apparent when
viewing, haven’t met anyone yet who can’t see it. Peter Imaging Facility Manager Wellcome Trust Centre for
Stem Cell Research Tennis Court Road CB2 1QR From: Hi,
we're
considering buying a Y5 filter cube from Leica to see the far-red Alexas, DRAQ5
etc. but then someone mentioned to me that many (most?) people are unable to
see far-red light. Does anyone know if this is true, or have experience with
this (or a similar) filter cube? It has a BP 700/75 suppression filter. Kenneth
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Search the CONFOCAL archive at
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We were recently imaging bacteria labelled with Atto
647. Nobody was
able to see them by eye, though the signal was excellent in
confocal,
particularly with APD detection. This was a Leica
filter set - I suppose
it was Y5 but don't know for sure. But the light
source may not have been
ideal for excitation either.
Guy
Optical Imaging Techniques in Cell Biology From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Peter Humphreys Sent: Friday, 7 March 2008 11:03 PM To: [hidden email] Subject: Re: Far-red filter set We have a y5, can see
594, cy5 etc. Dark red, strong signal
more apparent when viewing, haven’t met anyone yet who can’t see
it. Peter Imaging Facility
Manager Wellcome Trust Centre
for Stem Cell Research Tennis Court
Road CB2
1QR From:
Hi, we're considering buying a Y5 filter
cube from Leica to see the far-red Alexas, DRAQ5 etc. but then someone mentioned
to me that many (most?) people are unable to see far-red light. Does anyone know
if this is true, or have experience with this (or a similar) filter cube? It has
a BP 700/75 suppression filter. Kenneth
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Schwartz, Owen (NIH/NIAID) [E] |
Re: Far-red filter set
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In reply to this post by Kenneth Bowitz Larsen
Search the CONFOCAL archive at
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I have observed as well that people in
general cannot see out into the far red wavelengths. Sometimes if the sample
is very bright, you can see it, but in most cases not. The far-red cubes do
work very well however for most ccd cameras. Be aware that many cameras have a
blue filter installed to reduce IR reaching the ccd. This might have to be
removed (consult manufacturer) in order to image in the far-red. Cheers, Owen Owen M. Schwartz PhD From: Kenneth Bowitz
Larsen [mailto:[hidden email]] Hi,
we're
considering buying a Y5 filter cube from Leica to see the far-red Alexas, DRAQ5
etc. but then someone mentioned to me that many (most?) people are unable to
see far-red light. Does anyone know if this is true, or have experience with
this (or a similar) filter cube? It has a BP 700/75 suppression filter. Kenneth
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In reply to this post by Kenneth Bowitz Larsen
Hi, Ken,
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.
Good luck,
George
George Ring, Ph.D.
Dept. of Cell and Developmental Biology SUNY Upstate Medical University 750 E. Adams St. Syracuse NY 13210 Tel. (315) 464-8595 FAX (315) 464-8535 email: [hidden email] >>> Kenneth Bowitz Larsen <[hidden email]> 3/7/08 6:42:44 AM >>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi, we're considering buying a Y5 filter cube from Leica to see the far-red Alexas, DRAQ5 etc. but then someone mentioned to me that many (most?) people are unable to see far-red light. Does anyone know if this is true, or have experience with this (or a similar) filter cube? It has a BP 700/75 suppression filter. Kenneth |
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Hege Avsnes Dale |
Fixing GFP positive tissue after freeze sectioning
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In reply to this post by Schwartz, Owen (NIH/NIAID) [E]
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi. Do anyone have a protocol for good preservation of GFP in tissue after freeze sectioning? The tissue is a tumor with EGFP-expressing cells and need only a fixation that will preserve GFP (no permabilization needed). Would it also be a better idea to fix before sectioning? Hege |
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In reply to this post by George Ring
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 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 -- Martin Wessendorf, Ph.D. office: (612) 626-0145 Assoc Prof, Dept Neuroscience lab: (612) 624-2991 University of Minnesota Preferred FAX: (612) 624-8118 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 Minneapolis, MN 55455 **MY E-MAIL ADDRESS HAS CHANGED. PLEASE USE [hidden email] ** |
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A nice paper on it here for wide field: Â http://www.jhc.org/cgi/reprint/48/3/437
The Jackson website discusses Cy5 a bit here:Â http://www.jacksonimmuno.com/technical/f-cy3-5.asp On Mar 7, 2008, at 9:27 AM, Martin Wessendorf wrote:
Michael J. Herron,  U of MN, Dept. of Entomology   [hidden email]     612-624-3688 (office) 612-625-5299 (FAX) |
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Search the CONFOCAL archive at
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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:
-- Jennifer Waters, Ph.D. Director, Nikon Imaging Center at Harvard Medical School |
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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 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 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:
-- Jennifer Waters, Ph.D. Director, Nikon Imaging Center at Harvard Medical School No virus found in this incoming message. No virus found in this outgoing message. |
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In reply to this post by Kenneth Bowitz Larsen
Search the CONFOCAL archive at
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Kenneth, in my experience, using a M200, D640/20 (Ex), D680/30 (Em), 660 DCLP (BS), a spot size of around 10 micron diameter having an 8 bit intensity of over 200 is clearly visible to my eyes as well as to our graduate student. But when you project this image to MRm camera (1388x1040) we typically set a exposure of 30-50 ms (which yield spot intensity of over 200 in 8 bits). Shiv At 05:42 AM 3/7/2008, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Microscopy Facility Manager 8, Institute for Genomic Biology University of Illinois at Urbana-Champaign 1206 West Gregory Dr. Urbana, IL 61801 USA Office: 217.333.1214 Fax: 217.244.2496 [hidden email] http://core.igb.uiuc.edu |
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Kirill Ukhanov |
Re: Fixing GFP positive tissue after freeze sectioning
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In reply to this post by Hege Avsnes Dale
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Hege, GFP could be quenched by aldehyde fixation, on the other hand you need to fix the tissue to avoid wash out of GFP as it is mostly located in cytoplasm (well, depending how its expression is controlled). Poor fixation may result in a loss of the GFP from the tissue even if you think of not using permeabilization etc. I would recommend fixing your tissue before cryosectioning and then use anti-GFP antibody to enhance native GFP (if you find the signal too weak). I remember I found an extensive discussion on this issue here in the list or elsewhere in microscopy forums. good luck Kirill Ukhanov University of Florida On Fri, 7 Mar 2008 16:22:06 +0100, Hege Avsnes Dale <[hidden email]> wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hi. >Do anyone have a protocol for good preservation of GFP in tissue after >freeze sectioning? >The tissue is a tumor with EGFP-expressing cells and need only a >fixation that will preserve GFP (no permabilization needed). Would it >also be a better idea to fix before sectioning? > >Hege >========================================================================= |
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Kirill Ukhanov |
Re: Fixing GFP positive tissue after freeze sectioning
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In reply to this post by Hege Avsnes Dale
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Hege, GFP could be quenched by aldehyde fixation, on the other hand you need to fix the tissue to avoid wash out of GFP as it is mostly located in cytoplasm (well, depending how its expression is controlled). Poor fixation may result in a loss of the GFP from the tissue even if you think of not using permeabilization etc. I would recommend fixing your tissue before cryosectioning and then use anti-GFP antibody to enhance native GFP (if you find the signal too weak). I remember I found an extensive discussion on this issue here in the list or elsewhere in microscopy forums. good luck Kirill Ukhanov University of Florida On Fri, 7 Mar 2008 16:22:06 +0100, Hege Avsnes Dale <[hidden email]> wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hi. >Do anyone have a protocol for good preservation of GFP in tissue after >freeze sectioning? >The tissue is a tumor with EGFP-expressing cells and need only a >fixation that will preserve GFP (no permabilization needed). Would it >also be a better idea to fix before sectioning? > >Hege >========================================================================= |
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In reply to this post by Kenneth Bowitz Larsen
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Kenneth, does the Leica Y5 set consist of hard-coated filters and dichromatic mirrors? I recommend to go for this type of filters (higher transmission, less backreflection). Chroma and Semrock do offer them. Michael Kenneth Bowitz Larsen wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi, > > we're considering buying a Y5 filter cube from Leica to see the far-red > Alexas, DRAQ5 etc. but then someone mentioned to me that many (most?) > people are unable to see far-red light. Does anyone know if this is > true, or have experience with this (or a similar) filter cube? It has a > BP 700/75 suppression filter. > > > Kenneth |
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Caroline Bass |
Re: Fixing GFP positive tissue after freeze sectioning
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In reply to this post by Kirill Ukhanov
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I regularly fix brain tissue that is expressing GFP (via injection of a viral vector). Standard NBF works fine and I have no problems. I looked into this issue extensively a few years ago, and I can say that two things killed my signal: not fixing the tissue, and allowing fresh tissue to dehydrate. I have fixed my brains in two ways, perfusion of the animal and immersion in NBF. Since the brain (mouse) is a large organ I cut it in three parts and just dropped it in for a quick and easy fix. This worked fine, but I only used it when I needed to get a quick look at the expression zone. On 3/10/08 9:34 AM, "Kirill Ukhanov" <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear Hege, > > GFP could be quenched by aldehyde fixation, on the other hand you need to > fix the tissue to avoid wash out of GFP as it is mostly located in cytoplasm > (well, depending how its expression is controlled). Poor fixation may result > in a loss of the GFP from the tissue even if you think of not using > permeabilization etc. I would recommend fixing your tissue before > cryosectioning and then use anti-GFP antibody to enhance native GFP (if you > find the signal too weak). I remember I found an extensive discussion on > this issue here in the list or elsewhere in microscopy forums. > > good luck > > Kirill Ukhanov > University of Florida > > > On Fri, 7 Mar 2008 16:22:06 +0100, Hege Avsnes Dale > <[hidden email]> wrote: > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hi. >> Do anyone have a protocol for good preservation of GFP in tissue after >> freeze sectioning? >> The tissue is a tumor with EGFP-expressing cells and need only a >> fixation that will preserve GFP (no permabilization needed). Would it >> also be a better idea to fix before sectioning? >> >> Hege >> ========================================================================= |
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Melissa Gonzalez |
Re: Fixing GFP positive tissue after freeze sectioning
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Can you directly observe GFP in fresh frozen tissues? I always fix in 4% PFA, cryoprotect, and then freeze. Those sections always look great. But any time I have received fresh frozens they turn up negative. Post fixing in PFA doesn't work either. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Caroline Bass Sent: Monday, March 10, 2008 6:55 AM To: [hidden email] Subject: Re: Fixing GFP positive tissue after freeze sectioning Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I regularly fix brain tissue that is expressing GFP (via injection of a viral vector). Standard NBF works fine and I have no problems. I looked into this issue extensively a few years ago, and I can say that two things killed my signal: not fixing the tissue, and allowing fresh tissue to dehydrate. I have fixed my brains in two ways, perfusion of the animal and immersion in NBF. Since the brain (mouse) is a large organ I cut it in three parts and just dropped it in for a quick and easy fix. This worked fine, but I only used it when I needed to get a quick look at the expression zone. On 3/10/08 9:34 AM, "Kirill Ukhanov" <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear Hege, > > GFP could be quenched by aldehyde fixation, on the other hand you need to > fix the tissue to avoid wash out of GFP as it is mostly located in cytoplasm > (well, depending how its expression is controlled). Poor fixation may result > in a loss of the GFP from the tissue even if you think of not using > permeabilization etc. I would recommend fixing your tissue before > cryosectioning and then use anti-GFP antibody to enhance native GFP (if you > find the signal too weak). I remember I found an extensive discussion on > this issue here in the list or elsewhere in microscopy forums. > > good luck > > Kirill Ukhanov > University of Florida > > > On Fri, 7 Mar 2008 16:22:06 +0100, Hege Avsnes Dale > <[hidden email]> wrote: > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hi. >> Do anyone have a protocol for good preservation of GFP in tissue >> freeze sectioning? >> The tissue is a tumor with EGFP-expressing cells and need only a >> fixation that will preserve GFP (no permabilization needed). Would it >> also be a better idea to fix before sectioning? >> >> Hege >> ======================================================================== = |
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