PSF with DIC
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While I'm here...has anyone properly investigated the effect of the DIC objective prism in confocal fluorescence imaging? I had always assumed (rightly or wrongly) that it's presence didn't influence the PSF, but last week I was imaging some subresolution
beads and found that, particularly on our IX81-based FV1000 confocals, the DIC objective prism had quite a pronounced effect on the psf. Specifically the psf was distorted along a diagonal axis and at the point of focus, the bead appeared significantly larger
with the prism in place. The implication of this is that for confocal fluorescence imaging, the resolution of the microscope is reduced when the DIC objective prism is in place. I've also looked on our Zeiss Axiovert 200 and Nikon TE-2000 based systems which
employ a slightly different method of DIC and there the effect is much less pronounced although noticeable.
Simon
Simon - thanks for your input on the noise issue - and as for the DIC prism, I haven't looked at the PSF but have noticed slight deterioration in resolution of fluorescent details, and try to remember to take it out when not needed.
(Besides, the handle gets on the way of our Prior stage and has been broken several times).
Mike
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Mike
We have seen this effect on Leica, Nikon, and Zeiss confocal microscopes. There is an incompatibly between the optics of a confocal microscope and the optics to generate DIC images (described below). A short description of the observation has been published in a review article on Evaluation of Confocal Performance(1). For optimum resolution it is essential to remove the interference DIC filters from the light path. I have included the description below from the review article. There is also an image of PSF beads and 0.5 beads in the article. Please contact me for additional information. Best wishes Bob 1. Zucker, R.M. Evaluation of Confocal Microscopy System Performance. Cell Imaging Techniques. Douglas Taajets editor Humana Press Chapter 5 77-135 2005 4.23. Interference Contrast and Confocal Interference contrast is a very useful parameter in microscopy and it can be combined with fluorescence. However, because the microscope system was designed for light to traverse through two interference filters, when this optical system is applied to a confocal microscope there is distortion in the fluorescence signals. The fluorescent light traverses the interference contrast filter and excites the sample, and then the emitted fluorescence travels back down through the same interference contrast filter and back through the scan head. The resulting image shows a duplication of very small particles (0.17 μ m, PSF beads) and a distortion of larger particles. PSF beads show two spots and 0.5 μm beads show an egg shaped image instead of a round image. The same distortion that is observed on beads will occur on biological structures in cells ( see Fig. 15). For optimum resolution of data that will be deconvoluted later, it is recommended to remove the interference filters when acquiring an image. Robert M. Zucker, PhD U.S. Environmental Protection Agency Office of Research and Development National Health and Environmental Effects Research Laboratory. Toxicology Assessment Division Telephone: 919-541-1585 Fax: 919-541-4017 e-mail: [hidden email] Mail address: USEPA,ORD,NHEERL,TAD Developmental Biology Branch ( MD 67) Research Triangle Park, North Carolina, 27711 Shipping address: 2525 E.NC Highway 54 Durham, NC, 27713 From: "MODEL, MICHAEL" <[hidden email]> To: [hidden email] Date: 10/07/2009 09:43 AM Subject: PSF with DIC Sent by: Confocal Microscopy List <[hidden email]> While I'm here...has anyone properly investigated the effect of the DIC objective prism in confocal fluorescence imaging? I had always assumed (rightly or wrongly) that it's presence didn't influence the PSF, but last week I was imaging some subresolution beads and found that, particularly on our IX81-based FV1000 confocals, the DIC objective prism had quite a pronounced effect on the psf. Specifically the psf was distorted along a diagonal axis and at the point of focus, the bead appeared significantly larger with the prism in place. The implication of this is that for confocal fluorescence imaging, the resolution of the microscope is reduced when the DIC objective prism is in place. I've also looked on our Zeiss Axiovert 200 and Nikon TE-2000 based systems which employ a slightly different method of DIC and there the effect is much less pronounced although noticeable. Simon Simon - thanks for your input on the noise issue - and as for the DIC prism, I haven't looked at the PSF but have noticed slight deterioration in resolution of fluorescent details, and try to remember to take it out when not needed. (Besides, the handle gets on the way of our Prior stage and has been broken several times). Mike |
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In reply to this post by mmodel
hey,
We had to remove our prism in our 100x DIC objective (in a nikon te2000) when doing sub-difraction fluorescence microscopy. We were getting the same you saw with the beads. So, imaging beads will affect also widefield microscopy when using the prism.
b -- Bruno Afonso http://brunoafonso.com (personal, mostly portuguese) http://openwetware.org/wiki/User:BrunoAfonso (Professional, english) On Wed, Oct 7, 2009 at 09:42, MODEL, MICHAEL <[hidden email]> wrote:
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Dear all, I have a –maybe stupid- and not confocal but “basic optic” question: As you can read in all textbooks concerning microscopy and geometric optics the objective produces a real, inverted and magnified image since the distance from the object to the object front lens is bigger than one but less than two focal lengths of that given objective. This intermediate image is then magnified by the eyepiece. Since the intermediate image lies exactly in the front focal plane of the eyepiece the result is a virtual, true sided and magnified image which occur in the infinite space. Our eye with its optical components is then producing a real image on the retina. So far, so good. By definition, a virtual image can not be captured on a screen. BUT: When I hold a piece of paper in front of the eyepiece in a distance bigger or smaller than the back focal plane of the eyepiece (the distance I use when I look through it with my eyes) I am able to capture a pretty sharp image of my object on the paper. Why is this? I should not since it is a virtual image? Thanks for your input! Joachim Joachim Hehl LMC-Light Microscopy Centre, ETH Zurich Hönggerberg Schafmattstrasse 18, HPM F16.1 CH-8093, Zurich, Switzerland Web: www.lmc.ethz.ch Phone: +41 44 633 6202 Natel: +41 44 658 1679 Fax: +41 44 632 1298 e-mail: Joachim.Hehl@... |
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You can always get a real image from an eyepiece by
refocussing so that the first image is in front of the focal plane of the
eyepiece - either adjust the microscope focus a little or lift the eyepiece
slightly in its tube. If you see a sharp image without refocussing from
your normal viewing position, it probably means that you - like me - are
long-sighted!
Guy
Optical Imaging Techniques in Cell Biology From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Joachim Hehl Sent: Thursday, 8 October 2009 7:36 PM To: [hidden email] Subject: Eyepiece and virtual image Dear all, I have a maybe stupid- and not confocal but basic optic question: As you can read in all textbooks concerning microscopy and geometric optics the objective produces a real, inverted and magnified image since the distance from the object to the object front lens is bigger than one but less than two focal lengths of that given objective. This intermediate image is then magnified by the eyepiece. Since the intermediate image lies exactly in the front focal plane of the eyepiece the result is a virtual, true sided and magnified image which occur in the infinite space. Our eye with its optical components is then producing a real image on the retina. So far, so good. By definition, a virtual image can not be captured on a screen. BUT: When I hold a piece of paper in front of the eyepiece in a distance bigger or smaller than the back focal plane of the eyepiece (the distance I use when I look through it with my eyes) I am able to capture a pretty sharp image of my object on the paper. Why is this? I should not since it is a virtual image? Thanks for your input! Joachim Joachim Hehl LMC-Light Microscopy Centre, ETH Zurich Hönggerberg Schafmattstrasse 18, HPM F16.1 CH-8093, Zurich, Switzerland Web: www.lmc.ethz.ch Phone: +41 44 633 6202 Natel: +41 44 658 1679 Fax: +41 44 632 1298 e-mail: Joachim.Hehl@... |
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The point is that I neither adjust the focus nor lift the eyepiece. I look through the eyepiece (without my glasses, I am short-sighted), focusing and then I only hold a piece of paper in front of the eyepiece and can see the picture on it. And this also happens to my normal-sighted colleagues. Joachim On [DATE], "Guy Cox" <[ADDRESS]> wrote: You can always get a real image from an eyepiece by refocussing so that the first image is in front of the focal plane of the eyepiece - either adjust the microscope focus a little or lift the eyepiece slightly in its tube. If you see a sharp image without refocussing from your normal viewing position, it probably means that you - like me - are long-sighted! |
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But is this image truly in focus? If you adjust the
microscope focus, does it get sharper?
Guy
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Joachim Hehl Sent: Thursday, 8 October 2009 8:03 PM To: [hidden email] Subject: Re: Eyepiece and virtual image The point is that I neither adjust the focus nor lift the eyepiece. I look through the eyepiece (without my glasses, I am short-sighted), focusing and then I only hold a piece of paper in front of the eyepiece and can see the picture on it. And this also happens to my normal-sighted colleagues. Joachim On [DATE], "Guy Cox" <[ADDRESS]> wrote: You can always get a real image from an eyepiece by refocussing so that the first image is in front of the focal plane of the eyepiece - either adjust the microscope focus a little or lift the eyepiece slightly in its tube. If you see a sharp image without refocussing from your normal viewing position, it probably means that you - like me - are long-sighted! |
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In reply to this post by Joachim Hehl
Might you be looking at the Ramsden disc? If so, that is sharp image of the field diaphragm and condenser diaphragm planes, not the specimen plane. The point is that I neither adjust the focus nor lift the eyepiece. I look through the eyepiece (without my glasses, I am short-sighted), focusing and then I only hold a piece of paper in front of the eyepiece and can see the picture on it. And this also happens to my normal-sighted colleagues. Gary Radice |
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In reply to this post by Guy Cox
Joachim On [DATE], "Guy Cox" <[ADDRESS]> wrote: But is this image truly in focus? If you adjust the microscope focus, does it get sharper? |
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In reply to this post by gradice
On [DATE], "gradice" <[ADDRESS]> wrote: Might you be looking at the Ramsden disc? If so, that is sharp image of the field diaphragm and condenser diaphragm planes, not the specimen plane. |
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In reply to this post by Joachim Hehl
Oops, typed too fast. The Ramsden disc is conjugate with the condenser
diaphragm and back focal plane of the objective, not the field diaphragm. Gary Radice |
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In reply to this post by Zucker.Robert
[hidden email] writes:
> 4.23. Interference Contrast and Confocal > Interference contrast is a very useful parameter in microscopy and > it can be combined with fluorescence. However, because the > microscope system was designed for light to traverse through two > interference filters, when this optical system is applied to a > confocal microscope there is distortion in the fluorescence > signals. The fluorescent light traverses the interference contrast > filter and excites the sample, and then the emitted fluorescence > travels back down through the same interference contrast filter and > back through the scan head. The resulting image shows a duplication > of very small particles (0.17 μ m, PSF beads) and a distortion of > larger particles. PSF beads show two spots and 0.5 μm beads show an > egg shaped image instead of a round image. The same distortion that > is observed on beads will occur on biological structures in cells ( > see Fig. 15). For optimum resolution of data that will be > deconvoluted later, it is recommended to remove the interference > filters when acquiring an image. On my first reading of this I thought by interference contrast filter Robert was referring to the polariser. On a second reading I realise that it refers to the DIC prism. I wrote this extended reply before realising that we are saying the same thing but I am posting this anyway as a second description might help people understand what is going on and why this happens. DIC works by sheering the two polarizations relative to each other with the condenser prism. The beams then pass through slightly different sections of the sample, and are recombined with the second (objective) prism. This produces an image of relative phase shift between the two beams. In epi-fluorescence the excitation beam passes through the DIC (objective) prism and is split into two beams, offset relative to one another. The fluorescence from these two regions is them shifted back as the emission passes back through the (objective) DIC prism. This produces a double image shifted by the sheer in the DIC prism. The sheer tends to be a fraction of the resolution, say 1/3rd but varies with lens, manufacture etc... In conventional wide field this is generally not noticeable. On a properly set up confocal this leads to a pronounced broadening of the PSF in the sheer direction, at 45 degrees to the x and y sample axis. As Robert says, the take home message is it is best to remove any DIC optics before taking confocal images. Ian |
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In reply to this post by Joachim Hehl
Try removing the ocular and repeating the test. I think that you will
see the same image on your paper, except it may be brighter. The very small depth of focus of your objective translates into a very large image depth. There ia table that shows this at: http://www.microscopyu.com/articles/formulas/formulasfielddepth.html It may be that on your paper, you are seeing an undeviated component of the real image from the objective/tube lens combination, which passed though the center of your ocular. If I am correct, then you will not see a very sharp image if you use a low mag/low NA objective, and you will see the best images with high mag/high NA objectives. --aryeh Joachim Hehl wrote: > I would say it is in focus. But yes it gets a bit sharper when adjusting > the focus. And the wider I go back with the paper the bigger the image > is, like a projector. Maybe you try it out with one of your scopes? > > Joachim > > On [DATE], "Guy Cox" <[ADDRESS]> wrote: > > But is this image truly in focus? If you adjust the microscope > focus, does it get sharper? > > Guy > > > ------------------------------------------------------------------------ > *From:* Confocal Microscopy List > [mailto:[hidden email]] *On Behalf Of *Joachim Hehl > *Sent:* Thursday, 8 October 2009 8:03 PM > *To:* [hidden email] > *Subject:* Re: Eyepiece and virtual image > > Dear Guy, > The point is that I neither adjust the focus nor lift the eyepiece. > I look through the eyepiece (without my glasses, I am > short-sighted), focusing and then I only hold a piece of paper in > front of the eyepiece and can see the picture on it. And this also > happens to my normal-sighted colleagues. > Joachim > > On [DATE], "Guy Cox" <[ADDRESS]> wrote: > > You can always get a real image from an eyepiece by refocussing > so that the first image is in front of the focal plane of the > eyepiece - either adjust the microscope focus a little or lift > the eyepiece slightly in its tube. If you see a sharp image > without refocussing from your normal viewing position, it > probably means that you - like me - are long-sighted! > > 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 <http://www.guycox.net/> > > > > > ------------------------------------------------------------------------ > *From:* Confocal Microscopy List > [mailto:[hidden email]] *On Behalf Of > *Joachim Hehl > *Sent:* Thursday, 8 October 2009 7:36 PM > *To:* [hidden email] > *Subject:* Eyepiece and virtual image > > > > > Dear all, > > I have a –maybe stupid- and not confocal but “basic optic” > question: > > As you can read in all textbooks concerning microscopy and > geometric optics the objective produces a real, inverted and > magnified image since the distance from the object to the > object front lens is bigger than one but less than two focal > lengths of that given objective. > This intermediate image is then magnified by the eyepiece. > Since the intermediate image lies exactly in the front focal > plane of the eyepiece the result is a virtual, true sided and > magnified image which occur in the infinite space. Our eye > with its optical components is then producing a real image on > the retina. > So far, so good. > By definition, a virtual image can not be captured on a screen. > BUT: When I hold a piece of paper in front of the eyepiece in a > distance bigger or smaller than the back focal plane of the > eyepiece (the distance I use when I look through it with my > eyes) I am able to capture a pretty sharp image of my object on > the paper. Why is this? I should not since it is a virtual image? > Thanks for your input! > > Joachim > > > Joachim Hehl > LMC-Light Microscopy Centre, ETH Zurich Hönggerberg > Schafmattstrasse 18, HPM F16.1 > CH-8093, Zurich, Switzerland > > Web: www.lmc.ethz.ch > Phone: +41 44 633 6202 > Natel: +41 44 658 1679 > Fax: +41 44 632 1298 > e-mail: [hidden email] > > > -- Aryeh Weiss School of Engineering Bar Ilan University Ramat Gan 52900 Israel Ph: 972-3-5317638 FAX: 972-3-7384050 |
 
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Julian Smith III |
Re: Eyepiece and virtual image
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In reply to this post by Joachim Hehl
Yes--what you describe is quite true--but the point is that you do have
to refocus the microscope to make the projected image sharp. In fact, there are old methods for using that to take photos with plate-film cameras, where the image was refocused on the camera's ground-glass screen. But I think the answer to your original question is that the intermediate image is not quite at the front focal point of the eyepiece, so the eyepiece isn't really projecting an image to infinity. In fact, when you look in the eyepiece, the apparent (virtual) image distance is roughly 25cm away. My understanding is that this is (historically speaking) so that you could look in your upright monocular microscope with your left eye and at your drawing paper on the table next to the microscope with your right eye. That said, I think the physical distance between the actual front focal plane (image at infinity) and the intermediate image (image at 25 cm) is probably pretty small. I'm not able to walk through the math to get there.... Julian Joachim Hehl wrote: > I would say it is in focus. But yes it gets a bit sharper when > adjusting the focus. And the wider I go back with the paper the bigger > the image is, like a projector. Maybe you try it out with one of your > scopes? > > Joachim > > On [DATE], "Guy Cox" <[ADDRESS]> wrote: > > But is this image truly in focus? If you adjust the microscope > focus, does it get sharper? > > Guy > > > ------------------------------------------------------------------------ > *From:* Confocal Microscopy List > [mailto:[hidden email]] *On Behalf Of *Joachim Hehl > *Sent:* Thursday, 8 October 2009 8:03 PM > *To:* [hidden email] > *Subject:* Re: Eyepiece and virtual image > > Dear Guy, > The point is that I neither adjust the focus nor lift the > eyepiece. I look through the eyepiece (without my glasses, I am > short-sighted), focusing and then I only hold a piece of paper in > front of the eyepiece and can see the picture on it. And this also > happens to my normal-sighted colleagues. > Joachim > > On [DATE], "Guy Cox" <[ADDRESS]> wrote: > > You can always get a real image from an eyepiece by > refocussing so that the first image is in front of the focal > plane of the eyepiece - either adjust the microscope focus a > little or lift the eyepiece slightly in its tube. If you see a > sharp image without refocussing from your normal viewing > position, it probably means that you - like me - are long-sighted! > > 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 <http://www.guycox.net/> > > > > > ------------------------------------------------------------------------ > *From:* Confocal Microscopy List > [mailto:[hidden email]] *On Behalf Of > *Joachim Hehl > *Sent:* Thursday, 8 October 2009 7:36 PM > *To:* [hidden email] > *Subject:* Eyepiece and virtual image > > > > > Dear all, > > I have a –maybe stupid- and not confocal but “basic optic” > question: > > As you can read in all textbooks concerning microscopy and > geometric optics the objective produces a real, inverted and > magnified image since the distance from the object to the > object front lens is bigger than one but less than two focal > lengths of that given objective. > This intermediate image is then magnified by the eyepiece. > Since the intermediate image lies exactly in the front focal > plane of the eyepiece the result is a virtual, true sided and > magnified image which occur in the infinite space. Our eye > with its optical components is then producing a real image on > the retina. > So far, so good. > By definition, a virtual image can not be captured on a > screen. BUT: When I hold a piece of paper in front of the > eyepiece in a distance bigger or smaller than the back focal > plane of the eyepiece (the distance I use when I look through > it with my eyes) I am able to capture a pretty sharp image of > my object on the paper. Why is this? I should not since it is > a virtual image? > Thanks for your input! > > Joachim > > > Joachim Hehl > LMC-Light Microscopy Centre, ETH Zurich Hönggerberg > Schafmattstrasse 18, HPM F16.1 > CH-8093, Zurich, Switzerland > > Web: www.lmc.ethz.ch > Phone: +41 44 633 6202 > Natel: +41 44 658 1679 > Fax: +41 44 632 1298 > e-mail: [hidden email] > > > -- Julian P.S. Smith III Director, Winthrop Microscopy Facility Dept. of Biology Winthrop University 520 Cherry Rd. Rock Hill, SC 29733 803-323-2111 x6427 (vox) 803-323-3448 (fax) 803-524-2347 (cell) |
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In reply to this post by Joachim Hehl
Joachim-
According to my understanding, some oculars produce the virtual image and some do not. If not, then you can see the image projected on a sheet of paper. Carol Center for Microscopy & Microanalysis Bowling Green State University ________________________________________ From: Confocal Microscopy List [[hidden email]] On Behalf Of Joachim Hehl [[hidden email]] Sent: Thursday, October 08, 2009 5:35 AM To: [hidden email] Subject: Eyepiece and virtual image Dear all, I have a –maybe stupid- and not confocal but “basic optic” question: As you can read in all textbooks concerning microscopy and geometric optics the objective produces a real, inverted and magnified image since the distance from the object to the object front lens is bigger than one but less than two focal lengths of that given objective. This intermediate image is then magnified by the eyepiece. Since the intermediate image lies exactly in the front focal plane of the eyepiece the result is a virtual, true sided and magnified image which occur in the infinite space. Our eye with its optical components is then producing a real image on the retina. So far, so good. By definition, a virtual image can not be captured on a screen. BUT: When I hold a piece of paper in front of the eyepiece in a distance bigger or smaller than the back focal plane of the eyepiece (the distance I use when I look through it with my eyes) I am able to capture a pretty sharp image of my object on the paper. Why is this? I should not since it is a virtual image? Thanks for your input! Joachim Joachim Hehl LMC-Light Microscopy Centre, ETH Zurich Hönggerberg Schafmattstrasse 18, HPM F16.1 CH-8093, Zurich, Switzerland Web: www.lmc.ethz.ch Phone: +41 44 633 6202 Natel: +41 44 658 1679 Fax: +41 44 632 1298 e-mail: [hidden email] |
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In reply to this post by Julian Smith III
Hi all,
This topic causes lots of confusion at the UBC Live Cell
Course.
We try to emphasize three things:
1. That any lens will create a real image of any object on its
"front" side at some distance on its "back" side
as long as the object is more than one focal length away from the
optical center of the lens.
2. The magnification of this image will be directly proportional
to the ratio of the distance between the central plane of the lens and
the image divided by the distance between the object and the central
plane. (i.e, With any converging lens, you can get any
magnification as long as you are flexible about where your object and
your images planes are.)
3. Therefore, the three reasons for using different lenses for
different magnifications are
So yes, an infinity objective (plus its tube lens but either in
the presence or the absence of an occular) will form real images of
the structures found at any plane in the object that is more than one
focal length beyond its optical center. The location of this image
along the optical axis will determine what plane in the object will be
in focus there.
BUT such an image will only be free from aberrations as
long as it is focused on one plane in the object. For
infinity-conjugate objectives, the ideal object plane is that found at
the front focal plane of the objective and it will focus light from
this plane into an (almost) perfect "intermediate" image on
a plane 1 cm below the lip of the tube holding the ocular.
The ocular itself is located so that its optical center is one
focal length above this intermediate image plane meaning that this
image emerges from the ocular as though it were coming from an
object an infinite distance away. This is important because, on a
binocular viewing unit, the two ocular tubes are parallel to each
other and so you would like the optical axes of your eyes to also be
parallel, a condition that will only occur when your eyes think that
they are focused on distant object. (The idea that this set up was
designed to allow one superimpose the image from the microscope with
that of an image being drawn by hand about one meter away is a useful
approximation, but isn't quite true) .
Yes, you can find real images of any plane in the object
projected onto some plane above the "eye point" or
"exit pupil" of the ocular, (or if you remove the ocular, at
some plane down the tube) but any such image will only be free from
aberrations if the image plane is at infinity (Which is why one uses a
special projection lens rather than an ocular to project the image
onto, for instance, a CCD camera).
I hope that this helps,
Cheers,
Jim Pawley
Yes--what you describe is quite true--but the point is that you do have to refocus the microscope to make the projected image sharp. In fact, there are old methods for using that to take photos with plate-film cameras, where the image was refocused on the camera's ground-glass screen. But I think the answer to your original question is that the intermediate image is not quite at the front focal point of the eyepiece, so the eyepiece isn't really projecting an image to infinity. In fact, when you look in the eyepiece, the apparent (virtual) image distance is roughly 25cm away. My understanding is that this is (historically speaking) so that you could look in your upright monocular microscope with your left eye and at your drawing paper on the table next to the microscope with your right eye.
-- James and Christine Pawley, 21 N. Prospect Ave. Madison, WI,
53726 Phone: 608-238-3953
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In reply to this post by Ian Dobbie
After reading Ian and Robert's comments, I appreciate that there might be degradation of the PSF if DIC optics are in the confocal image forming pathway. I am just a bit confused about which optical parts should be removed. Different manufacturers have different names for equivalent bits. I usually think of there being four components in the image forming pathway for DIC - two polarisers, and two DIC prisms. These have various names depending on who you talk to, e.g. analyser, Wollaston prism etc. My question is Zeiss specific. In their microscopes, there is a piece of glass that I call the objective prism in the back focal plane of the objective. Will it affect the PSF of confocal images. It is a fiddly and expensive bit to remove and I worry about doing so if there is not going to be image degradation. Thanks for your help, John. Ian Dobbie wrote: [hidden email] writes: --
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In reply to this post by mmodel
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In reply to this post by John Runions
When using DIC with a confocal setup, there is only one part which
interferes with the laser/emission: the objective Nomarski prism. The two other parts (condenser Nomarski prism, analyzer) sit in the condenser which is not part of the confocal beam path. A polarizer is not necessary, since the laser is already polarized. In Zeiss setups, you put the objective prism manually in a slit below the objective. And yes, in my experience it does degrade image quality. This is especially true if you are working at the resolution limit. For best image quality, you have to remove the prism. That's actually one thing I like at the Leica stands (i.e. DMI6000): the prism is motorized and can be moved out automatically between two images, which means you can set up a time-lapse with DIC and fluorescence images w/o the latter being worse than necessary. Michael > Hi All, > > After reading Ian and Robert's comments, I appreciate that there might be > degradation of the PSF if DIC optics are in the confocal image forming > pathway. I am just a bit confused about which optical parts should be > removed. Different manufacturers have different names for equivalent > bits. I usually think of there being four components in the image > forming pathway for DIC - two polarisers, and two DIC prisms. These > have various names depending on who you talk to, e.g. analyser, Wollaston > prism etc. > > My question is Zeiss specific. In their microscopes, there is a piece > of glass that I call the objective prism in the back focal plane of the > objective. Will it affect the PSF of confocal images. It is a fiddly > and expensive bit to remove and I worry about doing so if there is not > going to be image degradation. > > Thanks for your help, John. > > > Ian Dobbie wrote: [hidden email] writes: > 4.23. Interference Contrast and Confocal > Interference contrast is a very useful parameter in microscopy and it can > be combined with fluorescence. However, because the microscope system was > designed for light to traverse through two interference filters, when > this optical system is applied to a confocal microscope there is > distortion in the fluorescence signals. The fluorescent light traverses > the interference contrast filter and excites the sample, and then the > emitted fluorescence travels back down through the same interference > contrast filter and back through the scan head. The resulting image > shows a duplication of very small particles (0.17 μ m, PSF beads) and a > distortion of larger particles. PSF beads show two spots and 0.5 μm > beads show an egg shaped image instead of a round image. The same > distortion that is observed on beads will occur on biological structures > in cells ( see Fig. 15). For optimum resolution of data that will be > deconvoluted later, it is recommended to remove the interference filters > when acquiring an image. On my first reading of this I thought > by interference contrast filter Robert was referring to the polariser. On > a second reading I realise that it refers to the DIC prism. I wrote this > extended reply before realising that we are saying the same thing but I > am posting this anyway as a second description might help people > understand what is going on and why this happens. DIC works by sheering > the two polarizations relative to each other with the condenser prism. > The beams then pass through slightly different sections of the sample, > and are recombined with the second (objective) prism. This produces an > image of relative phase shift between the two beams. In epi-fluorescence > the excitation beam passes through the DIC (objective) prism and is split > into two beams, offset relative to one another. The fluorescence from > these two regions is them shifted back as the emission passes back > through the (objective) DIC prism. This produces a double image shifted > by the sheer in the DIC prism. The sheer tends to be a fraction of the > resolution, say 1/3rd but varies with lens, manufacture etc... In > conventional wide field this is generally not noticeable. On a properly > set up confocal this leads to a pronounced broadening of the PSF in the > sheer direction, at 45 degrees to the x and y sample axis. As Robert > says, the take home message is it is best to remove any DIC optics before > taking confocal images. Ian > -- > Runions signature (Sent from my cra%#y non-Blackberry > electronic device that still has wires)  > ********************************* > John Runions, Ph.D. > School of Life Sciences > Oxford Brookes University > Oxford, UK > OX3 0BP > > email: [hidden email] > phone: +44 (0) 1865 483 964 Runionsâ lab web site  Visit The > Illuminated Plant Cell dot com > Oxford Brookes Master's in Bioimaging with Molecular Technology |
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This discussion prompted me to do a few simple
image quality tests on our Deltavision system, Olympus IX70 scope. One thing I found was if you collect fluorescence and DIC separately, so no DIC components are in the light path during fluorescence acquisition, the DIC image is noticeably shifted laterally and doesn't line up with the fluorescent image. Of course you can collect all the channels together with the DIC stuff in the light path and there is no shift (didn't notice any significant image degradation, will try some more involved tests) but fluorescent intensities are reduced by about 70%, mainly due to the analyzer on this system. Dave Dave McDonald Scientific Imaging Lab Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North, DE-512 Seattle, WA 98109 206-667-4205 http://www.fhcrc.org At 03:18 AM 10/9/2009, you wrote: >When using DIC with a confocal setup, there is only one part which >interferes with the laser/emission: the objective Nomarski prism. The two >other parts (condenser Nomarski prism, analyzer) sit in the condenser >which is not part of the confocal beam path. A polarizer is not necessary, >since the laser is already polarized. > >In Zeiss setups, you put the objective prism manually in a slit below the >objective. And yes, in my experience it does degrade image quality. This >is especially true if you are working at the resolution limit. For best >image quality, you have to remove the prism. That's actually one thing I >like at the Leica stands (i.e. DMI6000): the prism is motorized and can be >moved out automatically between two images, which means you can set up a >time-lapse with DIC and fluorescence images w/o the latter being worse >than necessary. > >Michael > > > > Hi All, > > > > After reading Ian and Robert's comments, I appreciate that there might be > > degradation of the PSF if DIC optics are in the confocal image forming > > pathway. I am just a bit confused about which optical parts should be > > removed. Different manufacturers have different names for equivalent > > bits. I usually think of there being four components in the image > > forming pathway for DIC - two polarisers, and two DIC prisms. These > > have various names depending on who you talk to, e.g. analyser, Wollaston > > prism etc. > > > > My question is Zeiss specific. In their microscopes, there is a piece > > of glass that I call the objective prism in the back focal plane of the > > objective. Will it affect the PSF of confocal images. It is a fiddly > > and expensive bit to remove and I worry about doing so if there is not > > going to be image degradation. > > > > Thanks for your help, John. > > > > > > Ian Dobbie wrote: [hidden email] writes: > > 4.23. Interference Contrast and Confocal > > Interference contrast is a very useful parameter in microscopy and it can > > be combined with fluorescence. However, because the microscope system was > > designed for light to traverse through two interference filters, when > > this optical system is applied to a confocal microscope there is > > distortion in the fluorescence signals. The fluorescent light traverses > > the interference contrast filter and excites the sample, and then the > > emitted fluorescence travels back down through the same interference > > contrast filter and back through the scan head. The resulting image > > shows a duplication of very small particles (0.17 μ m, PSF beads) and a > > distortion of larger particles. PSF beads show two spots and 0.5 μm > > beads show an egg shaped image instead of a round image. The same > > distortion that is observed on beads will occur on biological structures > > in cells ( see Fig. 15). For optimum resolution of data that will be > > deconvoluted later, it is recommended to remove the interference filters > > when acquiring an image. On my first reading of this I thought > > by interference contrast filter Robert was referring to the polariser. On > > a second reading I realise that it refers to the DIC prism. I wrote this > > extended reply before realising that we are saying the same thing but I > > am posting this anyway as a second description might help people > > understand what is going on and why this happens. DIC works by sheering > > the two polarizations relative to each other with the condenser prism. > > The beams then pass through slightly different sections of the sample, > > and are recombined with the second (objective) prism. This produces an > > image of relative phase shift between the two beams. In epi-fluorescence > > the excitation beam passes through the DIC (objective) prism and is split > > into two beams, offset relative to one another. The fluorescence from > > these two regions is them shifted back as the emission passes back > > through the (objective) DIC prism. This produces a double image shifted > > by the sheer in the DIC prism. The sheer tends to be a fraction of the > > resolution, say 1/3rd but varies with lens, manufacture etc... In > > conventional wide field this is generally not noticeable. On a properly > > set up confocal this leads to a pronounced broadening of the PSF in the > > sheer direction, at 45 degrees to the x and y sample axis. As Robert > > says, the take home message is it is best to remove any DIC optics before > > taking confocal images. Ian > > -- > > Runions signature (Sent from my cra%#y non-Blackberry > > electronic device that still has wires)  > > ********************************* > > John Runions, Ph.D. > > School of Life Sciences > > Oxford Brookes University > > Oxford, UK > > OX3 0BP > > > > email: [hidden email] > > phone: +44 (0) 1865 483 964 Runionsâ lab web site  Visit The > > Illuminated Plant Cell dot com > > Oxford Brookes Master's in Bioimaging with Molecular Technology |
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