Confocal and TIRF
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I have been using the TIRF adapter on our new Andor Spinning Disk confocal system and found that the focal plane of the confocal image and the image plane of the TIRF image are about 1-2um apart. This was determined by imaging small fluorescent beads attached to a coverslip and switching between the left port camera (confocal) and the right port camera (TIRF) with the same sample. I am having a hard time figuring out how that could happen with regard to the optics of the system and if I can make them parfocal. Dave
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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Well, as to making them parfocal I've no idea since I know
nothing of the optic path or the adjustments available. But it would seem
that your pinhole (ie the spinning disk) is not in the correct focal
plane. If so, this should have measurable effects on both XY and Z
resolution in confocal mode - that is, both will be worse than they
should be. Is this the case?
Guy Optical Imaging Techniques in Cell Biology From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht Sent: Thursday, 12 March 2009 11:20 PM To: [hidden email] Subject: Confocal and TIRF Dr. David Knecht
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax) No virus found in this incoming message. No virus found in this outgoing message. |
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Part of what I am trying to understand here is the optics of the spinning disk. I haven't found a good ray diagram of the excitation light path so I don't know how to think about the problem. I am presuming the excitation is essentially critical illumination, not koehler? If I move the while wheel slightly toward or away from the microscope, will that move the focal plane of the illumination spot, or make it out of focus? I am presuming that what is happening here is that the confocal excitation spot is coming into focus 1-2um higher in Z than the standard path. I had previously noticed that the confocal was different than the DIC and epi image position so I don't think it is the difference in the two cameras, but I will have to go back and convince myself that I am seeing the same thing in DIC, Epi and TIRF and all are different from the confocal. The confocal camera is "in focus" on the pinholes (imaging the stopped disk and moving the camera relative to it). I don't think this would matter as I would get a fuzzy out of focus image, not an in focus image in a different z position if it were the camera position or optics. So this issue is presumably about the optics between the disk and the objective. I guess the other key question is how much I should worry about this. If it is simply different, then it only matters if I am trying to compare images of the same sample taken in the different modes, and I am not sure I can think of a situation where it would matter if I were off by 1-2 um. However, if it matters to the resolution of the confocal system to be in correct "alignment" then I would worry more about it. Dave
On Mar 12, 2009, at 8:41 AM, Guy Cox wrote:
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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The excitation is basically Ploem, thus more like
Koehler. The spinning disk must be in an 'image' plane and therefore the
back focal plane should be evenly illuminated. The bit that puzzles me now
is that if the pinholes of the stopped disk are in focus (ie no refocussing
after collecting a widefield fluorescence image) then everything should be OK
and there should be no focal shift between the confocal and widefield
image. What is also probably fair to say is that if the defocus is only
1-2µm then optical aberrations are probably not too important, and you are not
likely to be losing any resolution. It may come down to the human factor
in accuracy of alignment.
Guy
Optical Imaging Techniques in Cell Biology From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht Sent: Friday, 13 March 2009 12:40 AM To: [hidden email] Subject: Re: Confocal and TIRF On Mar 12, 2009, at 8:41 AM, Guy Cox wrote:
Dr. David Knecht
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax) No virus found in this incoming message. No virus found in this outgoing message. |
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In reply to this post by Knecht, David
Perhaps the entire spinning disk assembly is not in the correct plane, so the
plane of focus in the specimen is shifted. If the distance between the objective (or rather the filed lens of the microscope) and the spinning disk is too short, you are focusing deeper into the specimen then you should. I suspect this introduces spherical aberration, just like it was on the old finite system when you did not put the eyepiece in the correct plane. Could you move the spinning disk head with the camera axially few mm further away rom the microscope? If there is an adjustable c-mount adaptor on your scope, it should be easy to do. Stan Vitha Microscopy and Imaging Center Texas A&M Univeristy On Thu, 12 Mar 2009 09:39:42 -0400, David Knecht <[hidden email]> wrote: >Part of what I am trying to understand here is the optics of the >spinning disk. I haven't found a good ray diagram of the excitation >light path so I don't know how to think about the problem. I am >presuming the excitation is essentially critical illumination, not >koehler? If I move the while wheel slightly toward or away from the >microscope, will that move the focal plane of the illumination spot, >or make it out of focus? I am presuming that what is happening here >is that the confocal excitation spot is coming into focus 1-2um higher >in Z than the standard path. I had previously noticed that the >confocal was different than the DIC and epi image position so I don't >think it is the difference in the two cameras, but I will have to go >back and convince myself that I am seeing the same thing in DIC, Epi >and TIRF and all are different from the confocal. The confocal camera >is "in focus" on the pinholes (imaging the stopped disk and moving the >camera relative to it). I don't think this would matter as I would >get a fuzzy out of focus image, not an in focus image in a different z >position if it were the camera position or optics. So this issue is >presumably about the optics between the disk and the objective. I >guess the other key question is how much I should worry about this. >If it is simply different, then it only matters if I am trying to >compare images of the same sample taken in the different modes, and I >am not sure I can think of a situation where it would matter if I were >off by 1-2 um. However, if it matters to the resolution of the >confocal system to be in correct "alignment" then I would worry more >about it. Dave > >On Mar 12, 2009, at 8:41 AM, Guy Cox wrote: > >> Well, as to making them parfocal I've no idea since I know nothing >> of the optic path or the adjustments available. But it would seem >> that your pinhole (ie the spinning disk) is not in the correct focal >> plane. If so, this should have measurable effects on both XY and Z >> resolution in confocal mode - that is, both will be worse than they >> should be. Is this the case? >> >> Guy >> >> Optical Imaging Techniques in Cell Biology >> by Guy Cox CRC Press / Taylor & Francis >> http://www.guycox.com/optical.htm >> ______________________________________________ >> Associate Professor Guy Cox, MA, DPhil(Oxon) >> Electron Microscope Unit, Madsen Building F09, >> University of Sydney, NSW 2006 >> ______________________________________________ >> Phone +61 2 9351 3176 Fax +61 2 9351 7682 >> Mobile 0413 281 861 >> ______________________________________________ >> http://www.guycox.net >> >> >> >> From: Confocal Microscopy List >> ] On Behalf Of David Knecht >> Sent: Thursday, 12 March 2009 11:20 PM >> To: [hidden email] >> Subject: Confocal and TIRF >> >> I have been using the TIRF adapter on our new Andor Spinning Disk >> confocal system and found that the focal plane of the confocal image >> and the image plane of the TIRF image are about 1-2um apart. This >> was determined by imaging small fluorescent beads attached to a >> coverslip and switching between the left port camera (confocal) and >> the right port camera (TIRF) with the same sample. I am having a >> hard time figuring out how that could happen with regard to the >> optics of the system and if I can make them parfocal. Dave >> >> Dr. David Knecht >> Department of Molecular and Cell Biology >> Co-head Flow Cytometry and Confocal Microscopy Facility >> U-3125 >> 91 N. Eagleville Rd. >> University of Connecticut >> Storrs, CT 06269 >> 860-486-2200 >> 860-486-4331 (fax) >> >> >> >> No virus found in this incoming message. >> Checked by AVG. >> Version: 7.5.557 / Virus Database: 270.11.10/1995 - Release Date: >> 11/03/2009 8:28 AM >> >> >> No virus found in this outgoing message. >> Checked by AVG. >> Version: 7.5.557 / Virus Database: 270.11.10/1995 - Release Date: >> 11/03/2009 8:28 AM >> > >Dr. David Knecht >Department of Molecular and Cell Biology >Co-head Flow Cytometry and Confocal Microscopy Facility >U-3125 >91 N. Eagleville Rd. >University of Connecticut >Storrs, CT 06269 >860-486-2200 >860-486-4331 (fax) > > > |
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This is my conclusion as well but I haven't had time to try to move the disk yet and see how far it needs to move to correct the 1-2 microns the focal plane is off. I will report back when I have time to do the realignment. My plan is to dry fluorescent 0.5um beads to a coverslip, get them in focus on the TIRF port, then adjust the eyepieces to that spot in widefield, and then adjust the Yokogawa position while imaging through the disk. Dave
On Mar 13, 2009, at 11:41 AM, Stanislav Vitha wrote:
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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In reply to this post by Knecht, David
Here in Amsterdam we have the same problem. We fiddled around with the
optics and it turned out that the camera is for the TIRF image is not at the right position. Andor is now working hard on it to get it right together with Nikon. One of the problems is that we have a doubly raised stage: One layer (bottom) with long pass dichroics for the TIRF and EPI excitation and visual emission and a second layer (top) with short-pass dichroics for the TIRF emission (camera). The optics for the second camera is still new and, I think, still under development. So, my advice, pull out the camera a little and see if it improves. And ask Andor to solve the problem for you. If you have a Nikon Ti with the two layers, you can copy our set-up as soon as it is working properly. Success! Kind regards, Erik Manders |
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