Phase Contrast problems
Locked
|
Hi all, I have a problem when viewing 6-well plates using a Leica DMIRB invert microscope and imaging using phase contrast. When I go to the live camera feed in our acquisition software I can see what looks like a reflection or the image of the condenser annulus in the image. It looks as though the light is passing through the annulus and then projects its image onto the CCD. This doesn't happen when imaging slides and only happens sometimes with 6-well plates. The plates are plastic, is phase contrast possible through plastic or does it have to be glass? I have aligned the microscope for phase contrast imaging using the Bertrand lens, but this does not appear to solve the problem. I am using a 5x Ph0 objective and the corresponding Ph0 condenser annulus. I would appreciate any thoughts on this. thanks, Matt Pearson. |
|
|
Hi Matt,
We have the same microscope and do not have any problems imaging phase through culture plates. Phase works fine on plastic, DIC does not usually work well (or at all). You say you have aligned the microscope for phase, does this include Kohler Illumination? What camera are you using? Can you see the same effect down the eye peices of the scope? Cheers Cam Cameron J Nowell Microscopy Research and Imaging Core Facility Peter MacCallum Cancer Centre 12 St Andrews Place East Mebourne, 3002 Victoria, Australia Phone: +61396561243 Fax: +61396561411 Mobil: +614122882700 ________________________________ From: Confocal Microscopy List on behalf of Matthew Pearson Sent: Wed 24/09/2008 9:16 PM To: [hidden email] Subject: Phase Contrast problems Hi all, I have a problem when viewing 6-well plates using a Leica DMIRB invert microscope and imaging using phase contrast. When I go to the live camera feed in our acquisition software I can see what looks like a reflection or the image of the condenser annulus in the image. It looks as though the light is passing through the annulus and then projects its image onto the CCD. This doesn't happen when imaging slides and only happens sometimes with 6-well plates. The plates are plastic, is phase contrast possible through plastic or does it have to be glass? I have aligned the microscope for phase contrast imaging using the Bertrand lens, but this does not appear to solve the problem. I am using a 5x Ph0 objective and the corresponding Ph0 condenser annulus. I would appreciate any thoughts on this. thanks, Matt Pearson. This email (including any attachments or links) may contain confidential and/or legally privileged information and is intended only to be read or used by the addressee. If you are not the intended addressee, any use, distribution, disclosure or copying of this email is strictly prohibited. Confidentiality and legal privilege attached to this email (including any attachments) are not waived or lost by reason of its mistaken delivery to you. If you have received this email in error, please delete it and notify us immediately by telephone or email. Peter MacCallum Cancer Centre provides no guarantee that this transmission is free of virus or that it has not been intercepted or altered and will not be liable for any delay in its receipt. |
 
|
Asson-Batres, Mary Ann |
Re: Phase Contrast problems
|
|
We have had a similar problem when viewing sections. Try removing the plate and you will likely see the abberant image even without a specimen in view. It turned out for us that this was due to condensation on the lens of the CCD camera. We had to have
a technician clean the lens and reseal it. No problems since then. Department of Biological Sciences Tennessee State University 3500 John A Merritt Blvd Nashville, TN 37209
Office 615-963-5779 FAX 615-963-2142
|
|
|
Aryeh Weiss |
imaging problem with LWD objective on IX81
|
|
In reply to this post by Nowell, Cameron
I am a problem imaging with the Olympus LWD (long working distance) objectives
on an IX81 frame. I have image well with the high NA objectives (oil and air), which are used when imaging through thin coverslips. However, with the LWD objectives, I cannot raise the turret enough to get an image. This is puzzling, because I thought that the objectives were designed to be parfocal, so that if I have focus for one objective, I will have focus for the other at the same turret position. Yet, if I put a regular slide with a coverlip on the scope, I can image it with the 20x close focus air objective, but then when I switch to the LWD objective ( and set the cover thickness ring appropriately for a coverslip, I cannot even get close to an in-focus image. The stage is a LUDL motorized stage, and it may be higher than the regular mechanical stage, but this does not explain why the close focus high NA objectives can focus, and the LWD objective dont. Thanks in advance for putting me straight on what is probably something obvious. --aryeh -- Aryeh Weiss School of Engineering Bar Ilan University Ramat Gan 52900 Israel Ph: 972-3-5317638 FAX: 972-3-7384050 |
|
|
Csúcs Gábor |
Re: imaging problem with LWD objective on IX81
|
|
Dear Aryeh,
We regularly use a 20x LWD on our IX81 without any problem. You should be aware that the working distance of a LWD objective is several mm, so you need to be considerably further away from your sample compared to high-NA imaging. Eventually you simply need to lower your objective (using the focus wheel). if you can't lower your objective enough (to have a proper image) then eventually a focus-limit has been set/activated on your microscope. Cheers Gabor > I am a problem imaging with the Olympus LWD (long working distance) > objectives on an IX81 frame. I have image well with the high NA > objectives (oil and air), > which are used when imaging through thin coverslips. However, with the > LWD objectives, I cannot raise the turret enough to get an image. > This is puzzling, because I thought that the objectives were designed > to be parfocal, so that if I have focus for one objective, I will have > focus for the other at the same turret position. Yet, if I put a > regular slide with a coverlip on the scope, I can image it with the > 20x close focus air objective, but then when I switch to the LWD > objective ( and set the cover thickness ring appropriately for a > coverslip, I cannot even get close to an in-focus image. > > The stage is a LUDL motorized stage, and it may be higher than the > regular mechanical stage, but this does not explain why the close > focus high NA objectives can focus, and the LWD objective dont. > > Thanks in advance for putting me straight on what is probably > something obvious. > > --aryeh -- Gabor Csucs Light Microscopy Centre, ETH Zurich Schafmattstrasse 18, HPM F16 CH-8093, Zurich, Switzerland Web: www.lmc.ethz.ch Phone: +41 44 633 6221 Fax: +41 44 632 1298 e-mail: [hidden email] |
|
|
Aryeh Weiss |
Re: imaging problem with LWD objective on IX81
|
|
Dear Gabor,
Thank you for the reply. The problem here is that I cannot even get within the much longer WD of the LWD objective. I also tried lowering the objective just in case I was closer than I thought, but as I expected, I was already too far away. The WD for the objective in question is 3-4.2mm, so I have some idea of where it should be. Regards, --aryeh Gabor Csucs wrote: > Dear Aryeh, > > We regularly use a 20x LWD on our IX81 without any problem. You should > be aware that the working distance of a LWD objective is several mm, so > you need to be considerably further away from your sample compared to > high-NA imaging. Eventually you simply need to lower your objective > (using the focus wheel). if you can't lower your objective enough (to > have a proper image) then eventually a focus-limit has been > set/activated on your microscope. > > Cheers Gabor > >> I am a problem imaging with the Olympus LWD (long working distance) >> objectives on an IX81 frame. I have image well with the high NA >> objectives (oil and air), >> which are used when imaging through thin coverslips. However, with the >> LWD objectives, I cannot raise the turret enough to get an image. >> This is puzzling, because I thought that the objectives were designed >> to be parfocal, so that if I have focus for one objective, I will have >> focus for the other at the same turret position. Yet, if I put a >> regular slide with a coverlip on the scope, I can image it with the >> 20x close focus air objective, but then when I switch to the LWD >> objective ( and set the cover thickness ring appropriately for a >> coverslip, I cannot even get close to an in-focus image. >> >> The stage is a LUDL motorized stage, and it may be higher than the >> regular mechanical stage, but this does not explain why the close >> focus high NA objectives can focus, and the LWD objective dont. >> >> Thanks in advance for putting me straight on what is probably >> something obvious. >> >> --aryeh > > |
|
|
In reply to this post by Nowell, Cameron
We are moving our multiphoton system into temporary quarters and need
a temporary light barrier for our space. Does anyone know of a source for black fireproof fabric that we can buy by the yard and just hang ourselves? I found lots of fancy curtains and free standing barriers on the web, but we would prefer to just buy fabric by the yard. thanks, Carol -- <><><><><><><><><><><><><><><><><><><><><> Carol Bayles, Manager Microscopy & Imaging Facility (MIF) Life Sciences Core Lab Center 160a Biotech Bldg 607-254-4860 607-254-6379 fax http://cores.lifesciences.cornell.edu Confocal and Multiphoton Microscopy Nanobiotechnology Center www.nbtc.cornell.edu Cornell University Ithaca NY 14853 |
|
|
I would recommend black plastic shower curtains- they are cheap and they don't shred. We ran a shower pole across the room and hung them as normal. I had a room set with fireproof fabric ones and they shed fibers all over the place, it was a disaster to clean the optics. save your money- get plastic ones.
Julia Julia E. Sable Lab Manager Sheetz Lab Columbia University Dept. of Biological Sciences (646) 283 4421 On Sep 24, 2008, at 11:41 AM, Carol Bayles wrote:
|
|
|
Aryeh Weiss |
Re: imaging problem with LWD objective on IX81
|
|
In reply to this post by Csúcs Gábor
I am reposting this because I was notified that it was rejected as having been
already posted. If it went through the first time, I apologize for the redundancy. Apparently, even through it adds a reply to someone who replied ot me, it looks too similar to the previous post. That can be a problem, because sometimes it is appropriate to leave the thread in place to make the post more understandable. Perhaps the list manager might comment on this issue, and proper net etiquette when this happens. --aryeh Dear Gabor, Thank you for the reply. The problem here is that I cannot even get within the much longer WD of the LWD objective. I also tried lowering the objective just in case I was closer than I thought, but as I expected, I was already too far away. The WD for the objective in question is 3-4.2mm, so I have some idea of where it should be. Regards, --aryeh Gabor Csucs wrote: > Dear Aryeh, > > We regularly use a 20x LWD on our IX81 without any problem. You should > be aware that the working distance of a LWD objective is several mm, so > you need to be considerably further away from your sample compared to > high-NA imaging. Eventually you simply need to lower your objective > (using the focus wheel). if you can't lower your objective enough (to > have a proper image) then eventually a focus-limit has been > set/activated on your microscope. > > Cheers Gabor > |
|
|
In reply to this post by Carol Bayles
Thorlabs have black curtain material, I don't know for sure if its by the yard though.
Neil > Date: Wed, 24 Sep 2008 11:41:19 -0400 > From: [hidden email] > Subject: laser curtains > To: [hidden email] > > We are moving our multiphoton system into temporary quarters and need > a temporary light barrier for our space. Does anyone know of a > source for black fireproof fabric that we can buy by the yard and > just hang ourselves? I found lots of fancy curtains and free > standing barriers on the web, but we would prefer to just buy fabric > by the yard. > > thanks, > Carol > -- > <><><><><><><><><><><><><><><><><><><><><> > Carol Bayles, Manager > Microscopy & Imaging Facility (MIF) > Life Sciences Core Lab Center > 160a Biotech Bldg > 607-254-4860 > 607-254-6379 fax > http://cores.lifesciences.cornell.edu > > Confocal and Multiphoton Microscopy > Nanobiotechnology Center > www.nbtc.cornell.edu > > Cornell University > Ithaca NY 14853 Try Facebook in Windows Live Messenger! Try it Now! |
|
|
In reply to this post by js1719
Uh... have you ever had the laser directly strike one of these plastic curtains? In our old lab we had such a setup, and the curtain had some interesting holes in it; not very safe as the point of these things is to keep the laser out of other areas. Our new lab has triple-layer fire-retardant curtains. They were installed by the builders and custom tailored locally so I doubt our supplier would be much help to anyone else. I can ask around though if anyone is interested. They can handle the beam from the laser (~1W) for several seconds with no significant damage and most importantly, no penetration of the curtain. You could probably get away with a heavy canvas or denim for the short term (sprayed black?), although they would only be suitable as a temporary measure because as Julia mentioned they will eventually shed fibers.
Craig On Wed, Sep 24, 2008 at 9:48 AM, js1719 <[hidden email]> wrote:
|
|
|
Oh, bit of a possible brainstorm: use the thin black ones stitched to a sturdier backing. The plastic will help contain the fibers and give you a black surface on the laser side, while the backing will handle the energy from the laser when it punches through the plastic layer.
Craig On Wed, Sep 24, 2008 at 10:28 AM, Craig Brideau <[hidden email]> wrote:
|
|
|
In reply to this post by Neil Kad
If you mean the BK5 stuff, they actually say on their website that it's not intended for laser curtain applications. Again it's not very sturdy. It's mainly meant as a light block for sensitive equipment, like keeping stray light out of your PMTs. We have some of our detector boxes wrapped in similar stuff so we don't have to worry so much about ambient light sneaking into our system.
Craig On Wed, Sep 24, 2008 at 10:19 AM, Neil Kad <[hidden email]> wrote:
|
|
|
I am not aware of all the things people have brought up here, but I have had very good luck with Blackout curtains from http://www.blackoutcurtains.com/
They were very reasonable in price, helpful on the phone, and the quality was much better than I had expected. I have had the curtains for a couple years in a heavily trafficked area and they have held up nicely. I don't know if they sell by the yard or not, but they might be worth a call. Mike
Michael J. Herron, U of MN, Dept. of Entomology 612-624-3688 (office) 612-625-5299 (FAX) |
|
|
In reply to this post by Carol Bayles
Carol,
The answer depends on the purpose of the curtains. Blackout material from Thorlabs will block light, but IS REALLY NOT intended to handle a laser beam, expecially one from a Class 4 laser which has femtosecond pulses. Burning plastic is not healthy! Kentek, Rockwell Laser Industries, and other laser safety suppliers sell curtains and panels which can handle a Class 4 laser beams. Some suppliers offer different colours. If safety rules must be adhered to, call the safety companies. I heard of people using gyprock (sp?) to block laser beam, but it not the intended purpose. Good luck, Sophie P.S.: no financial gain with companies mentioned above. ____________________________________________________ Sophie M. K. Brunet, Ph. D. Research Officer Optical Spectroscopy, Laser Systems and Applications Chemistry 112 sessional lecturer [hidden email] 306-966-1719 (office) 306-966-1702 (fax) ____________________________________________________ Saskatchewan Structural Sciences Centre University of Saskatchewan Thorvaldson Bldg. 110 Science Place Saskatoon, Sk S7N 5C9 ____________________________________________________ Quoting Carol Bayles <[hidden email]>: > We are moving our multiphoton system into temporary quarters and need > a temporary light barrier for our space. Does anyone know of a > source for black fireproof fabric that we can buy by the yard and > just hang ourselves? I found lots of fancy curtains and free > standing barriers on the web, but we would prefer to just buy fabric > by the yard. > > thanks, > Carol > -- > <><><><><><><><><><><><><><><><><><><><><> > Carol Bayles, Manager > Microscopy & Imaging Facility (MIF) > Life Sciences Core Lab Center > 160a Biotech Bldg > 607-254-4860 > 607-254-6379 fax > http://cores.lifesciences.cornell.edu > > Confocal and Multiphoton Microscopy > Nanobiotechnology Center > www.nbtc.cornell.edu > > Cornell University > Ithaca NY 14853 > |
|
|
In reply to this post by Matthew Pearson-3
Hi Matt,
The
pictures you sent look like an image of the phase ring in the objective. There
are a couple of things to try to isolate where it is
occuring.
1. Does it happen on all objectives?
2. Does it come in and out of focus when you move the
condenser
3. Do you have another camera you could hook up and
try?
4. Does the microscope have any DIC/Normaski optics in it?
Are they in the light path?
5. What hppens when you adjust the focus on the camera (may
have to do this by sliding the camera tube in and out)
6. Does this happen if you put a glass slide (or no slide)
on the scope (ie is it a plastic related issue).
Cheers
Cam
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Matthew Pearson Sent: Wednesday, 24 September 2008 9:16 PM To: [hidden email] Subject: Phase Contrast problems Hi all, I have a problem when viewing 6-well plates using a Leica DMIRB invert microscope and imaging using phase contrast. When I go to the live camera feed in our acquisition software I can see what looks like a reflection or the image of the condenser annulus in the image. It looks as though the light is passing through the annulus and then projects its image onto the CCD. This doesn't happen when imaging slides and only happens sometimes with 6-well plates. The plates are plastic, is phase contrast possible through plastic or does it have to be glass? I have aligned the microscope for phase contrast imaging using the Bertrand lens, but this does not appear to solve the problem. I am using a 5x Ph0 objective and the corresponding Ph0 condenser annulus. I would appreciate any thoughts on this. thanks, Matt Pearson.
This email (including any attachments or links) may contain |
«
Return to Confocal Microscopy List
|
1 view|%1 views
| Free forum by Nabble | Edit this page |