Plan Apochromat 20x Zeiss
Locked
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear list;
Recently, to check for the system calibration,I used a homogeneous sample with Apochromat 20x/.8 zeiss dry lens for our confocal microscope(diode source).The illumination pattern by 20x lens is very strange and it seems that it is not illuminating evenly and it only illuminates the center (I checked with 2 different homogeneous samples).I also checked with other lenses and this effect was not observed with them.I am just wondering if any one may know what could be the reason?is it because that the beam is not filling the lens back aperture or is it because of some design error in the lens? I am also including the acquired images. Appreciate your help Sarah |
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Was there oil on the lens? Today a facility user couldn't get decent images with the Nikon 20X air on our BioRad Radiance 2000 and, of course, the reason was that some bozo had put oil on the objective. _________________________________________ Michael Cammer http://www.aecom.yu.edu/aif/ |
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
no, no oil or water on the lens!
On Thu, Jul 17, 2008 at 11:20 PM, Michael Cammer <[hidden email]> wrote: Search the CONFOCAL archive at |
|
|
In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Does this only happen at the lowest zoom
setting (which means it is vignetting) or is it the same at all
zoom settings?
Guy Optical Imaging Techniques in Cell Biology From:
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear list; |
 
|
Michael Weber-4 |
Re: Plan Apochromat 20x Zeiss
|
|
In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Sarah, the illumination doesn't look good at all, also not with the other lenses. How did you acquire these images, what kind of sample did you use? I recommend either a mirror/glas slide in reflection, or a Chroma fluorescent slide. Both should bring you very bright signal, and you can acquire less noisy images. Are you sure you got the focus right? Did you use a LSM 510 for that? Open or closed pinhole? Seeing the image from the 20x, I would also guess that there is something on the front lens of your objective. Take it out and look through it from the back, towards a normal room light source. In addition, take an ocular as magnification glass and check the surface of the front lens. Michael Sarah Kefayati wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Dear list; > > Recently, to check for the system calibration,I used a homogeneous > sample with Apochromat 20x/.8 zeiss dry lens for our confocal > microscope(diode source).The illumination pattern by 20x lens is very > strange and it seems that it is not illuminating evenly and it only > illuminates the center (I checked with 2 different homogeneous > samples).I also checked with other lenses and this effect was not > observed with them.I am just wondering if any one may know what could be > the reason?is it because that the beam is not filling the lens back > aperture or is it because of some design error in the lens? > I am also including the acquired images. > > Appreciate your help > Sarah > http://sara61.googlepages.com/diode10x-0000.jpg > http://sara61.googlepages.com/diode20x-0001.jpg > http://sara61.googlepages.com/diode40x-0002.jpg > http://sara61.googlepages.com/diode60x-0003.jpg |
|
|
In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
One simple way to check the lens and
illumination is to start a time scan at a single point and put a piece of paper
a cm or so away from the lens. It should be a uniform circular spot. Mike Model From: Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear list; |
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
The homogeneous sample was a thick red glass block.the effect is there all the way in and out through the sample.I also checked with other homogeneous samples( a piece of paper and prepared mouse kidney slide), I could see the effect for them too.we use IX701 Olympus and Fluoview 300 scanner unit.Also pinhole was closed( on the largest one though).I took out the lens,looking through it, a surface of the lens is a bit damaged.But could it be the reason for this illumination pattern?
On Fri, Jul 18, 2008 at 9:27 AM, MODEL, MICHAEL <[hidden email]> wrote:
|
|
|
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal The homogeneous sample was a thick red glass block.the effect is there all the way in and out through the sample.I also checked with other homogeneous samples( a piece of paper and prepared mouse kidney slide), I could see the effect for them too.we use IX701 Olympus and Fluoview 300 scanner unit.Also pinhole was closed( on the largest one though).I took out the lens,looking through it, a surface of the lens is a bit damaged.But could it be the reason for this illumination pattern? All sorts of problems here. All your specimens have different
indices of refraction hence different spherical aberration and the
results will them very with focus depth.
But the main problem is that you can't expect to get good results
using a Zeiss ICS lens on an Olympus stand.
Lots of info on this in Chapter 7 of the Handbook.
Cheers,
Jim P.
On Fri, Jul 18, 2008 at 9:27 AM, MODEL, MICHAEL <[hidden email]> wrote: -- Jim Pawley (Summer address) c/o Postmaster, Egmont, BC, Canada,
V0N-1N0 604-883-2095, [hidden email]
|
|
|
In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
-
Sarah, Sure, the 20x image looks quite different from the others, and if you can see a scratch on the lens, that probably won't help getting a good image. All suggestions that have been made are valid, but I'd like to add this: You don't say how you acquired your images, and there may have been some image degradation due to the fact that you probably converted a monochrome, single channel image into RGB, and used a JPEG compression. But even so, if you separate the channels and look just at the red, your image is basically a salt and pepper pattern of pixels (much like amplified noise), where you can have pixels with very low values (below ten) right next to pixels with very high values (200 or more). Are you using reflected light mode? fluorescence mode? transmitted light mode? If you are using reflected light mode, then a piece of paper is NOT a homogeneous sample, since you are basically imaging the topology of the paper surface... not quite that smooth at the microscopic level. If you are using transmitted light mode, you need a very clean, homogeneous sample, such as a coverslip or similar... A pice of paper, again, is NOT a suitable homogeneous sample for transmitted light. If you are using fluorescence mode, then you need a very homogenous fluorescent sample, such as a Molecular Probes fluorescent plexiglas slide, and you need to focus inside, as has been pointed out, to avoid surface effects (scratches, etc...). Again, I don't see how a piece of paper would be a suitable sample in this case either... I don't see how a mouse kidney can be considered a homogenous sample either... If you are collecting good images of a properly illuminated, homogeneous fluorescent sample, you should get very even pixel intensities across your image, with smooth variations (a smooth line profile, not a saw tooth pattern as in yours) and a very narrow Histogram (with a small standard deviation). This would be true also for a transmitted light image of a suitable homogeneous sample, or for a reflected light image or a perfectly flat reflective sample (such as a mirror, or the surface of a coverslip or slide). If you used a thick homogenous piece of glass as a sample, your images don't look like any images I would expect for either imaging mode. They look more like background (noise) images that have been extremely amplified. They are neither homogeneous images with smooth intensity changes from pixel to pixel, nor do they show any structure (scratches or similar things), just random pixel intensities. My conclusion is that there may be some problem with your objective (or it is not suitable for your microscope, although we have used Zeiss objectives on Olympus stands with pretty good results... I don't think that in itself is the cause of your problem), but there might also be some other problem with the way you are collecting your images. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 On Jul 17, 2008, at 8:29 PM, Sarah Kefayati wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
| Free forum by Nabble | Edit this page |