Why no pinhole in transmission pathway?
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John Runions |
Why no pinhole in transmission pathway?
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Search the CONFOCAL archive at
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Hello folks, Here is one of those seemingly straightforward to answer questions that has me really stumped. You should hear all of the BS around here when I bring this up with the other confocal 'experts'. Why isn't there a pinhole in the transmission-image forming pathway? A confocal transmission image would be nice but I always tell people it's not possible. Is it not possible or just not done? Thanks for your help. John. --
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John Oreopoulos |
Re: Why no pinhole in transmission pathway?
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Search the CONFOCAL archive at
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This is a good question and I've wondered why the commercial systems don't have a transmission pinhole as well. I'm sure it must be possible because I think Minski's original confocal microscope had a transmission optical design. It's possible to form reflection confocal images with the backscattered laser light if you remove the emission filters in front of your detectors, and there isn't any tricky pinhole alignment associated with that. Perhaps that is the main reason - a transmission pathway pinhole would have to be aligned to the system and this might be difficult to maintain. In the epi configuration, the objective also acts as the condenser, whereas in the transmission pathway you have to align the objective, the condenser, and a pinhole.
John Oreopoulos, BSc, PhD Candidate University of Toronto Institute For Biomaterials and Biomedical Engineering Centre For Studies in Molecular Imaging On 6-Nov-07, at 10:03 AM, John Runions wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Tel: W:416-946-5022 |
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In reply to this post by John Runions
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, If it is a stage scan then there is no problem. :) Best, Peng Xi Dantus Research Group Department of Chemistry Michigan State University East Lansing, MI 48824 Tel: (517) 355-9715 x319 Email: [hidden email] http://www.msu.edu/~xipeng/ John Runions wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Hello folks, > > Here is one of those seemingly straightforward to answer questions > that has me really stumped. You should hear all of the BS around here > when I bring this up with the other confocal 'experts'. Why isn't > there a pinhole in the transmission-image forming pathway? A confocal > transmission image would be nice but I always tell people it's not > possible. Is it not possible or just not done? > > Thanks for your help. John. > -- > > ********************************* > C. John Runions, Ph.D. > School of Life Sciences > Oxford Brookes University > Oxford, UK > OX3 0BP > > email: [hidden email] <mailto:[hidden email]> > phone: +44 (0) 1865 483 964 > > web: http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html! > <http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html%21> > > > > > New - Oxford Brookes Master's in Bioimaging with Molecular Technology > <http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt> > |
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In reply to this post by John Runions
Search the CONFOCAL archive at
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Maybe the main usefulness of confocal imaging
comes when observing point luminous sources and not in situations where
contrast results from light passing through the entire specimen. But I am
pretty certain I’ve seen papers describing transmission confocal. Mike From:
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Andrew Resnick |
Re: Why no pinhole in transmission pathway?
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In reply to this post by John Runions
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John,
If you like, you could stop down the field diaphram to a pinhole, but you would then need to either scan the pinhole (or sample), or have some alternative like a spinning disk setup to obtain the image. There's no reason why it cannot be done. The main difference is that in fluorescence imaging, the excited fluorophore molecules are considered a source, and that source is confocal to the aperture pinhole. In transmission, there is no comparable source. But in principle, any imaging method can be performed in a 'confocal' style. That said, what's the advantage? Andy At 10:03 AM 11/6/2007, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Instructor Department of Physiology and Biophysics Case Western Reserve University 216-368-6899 (V) 216-368-4223 (F) |
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Jean-Yves Tinevez |
Re: Why no pinhole in transmission pathway?
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In reply to this post by John Oreopoulos
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I am not an expert, but you can think of re-using the pinhole in the reflected light path. For example just by using a plain 50%/50% mirror in the filter cube rather than a dichroic and filters. Thus you would have a pinhole-d view of your sample, with a transmitted-like light. Actually i have never tried, but i wonder if it could work. jy -- Jean-Yves Tinevez Max Planck Institute of Molecular Cell Biology and Genetics - Dresden |
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Michael Weber-4 |
Re: Why no pinhole in transmission pathway?
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In reply to this post by John Oreopoulos
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal But how do you de-scan in the transmission beam path? You would need a second scanner behind the condenser which has to be synchronized with the main one, to bring the light to the pinhole. cheers, Michael John Oreopoulos wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal This is a good > question and I've wondered why the commercial systems don't have > a transmission pinhole as well. I'm sure it must be possible because I > think Minski's original confocal microscope had a transmission optical > design. It's possible to form reflection confocal images with the > backscattered laser light if you remove the emission filters in front of > your detectors, and there isn't any tricky pinhole alignment associated > with that. Perhaps that is the main reason - a transmission pathway > pinhole would have to be aligned to the system and this might be > difficult to maintain. In the epi configuration, the objective also acts > as the condenser, whereas in the transmission pathway you have to align > the objective, the condenser, and a pinhole. > > > John Oreopoulos, BSc, > PhD Candidate > University of Toronto > Institute For Biomaterials and Biomedical Engineering > Centre For Studies in Molecular Imaging > > > On 6-Nov-07, at 10:03 AM, John Runions wrote: > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> Hello folks, >> >> Here is one of those seemingly straightforward to answer questions >> that has me really stumped. You should hear all of the BS around here >> when I bring this up with the other confocal 'experts'. Why isn't >> there a pinhole in the transmission-image forming pathway? A confocal >> transmission image would be nice but I always tell people it's not >> possible. Is it not possible or just not done? >> >> Thanks for your help. John. >> -- >> >> ********************************* >> C. John Runions, Ph.D. >> School of Life Sciences >> Oxford Brookes University >> Oxford, UK >> OX3 0BP >> >> email: [hidden email] <mailto:[hidden email]> >> phone: +44 (0) 1865 483 964 >> >> web: http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html! >> <http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html%21> >> >> >> >> >> New - Oxford Brookes Master's in Bioimaging with Molecular Technology >> <http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt> >> >> > > > > > Tel: W:416-946-5022 |
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John Oreopoulos |
Re: Why no pinhole in transmission pathway?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Good point. You'd also have to align two sets of galvometric mirrors to each other (more $$$) and make sure that they scan the same way. Someone else just pointed out that this can be solved by using a stage-scanning system, however. But I think Andrew Resnick pointed out a better reason for there not being a transmission pathway pinhole. John On 6-Nov-07, at 10:43 AM, Michael Weber wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > But how do you de-scan in the transmission beam path? You would > need a second scanner behind the condenser which has to be > synchronized with the main one, to bring the light to the pinhole. > > cheers, > Michael > > > John Oreopoulos wrote: >> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/ >> cgi-bin/wa?S1=confocal This is a good question and I've wondered >> why the commercial systems don't have a transmission pinhole as >> well. I'm sure it must be possible because I think Minski's >> original confocal microscope had a transmission optical design. >> It's possible to form reflection confocal images with the >> backscattered laser light if you remove the emission filters in >> front of your detectors, and there isn't any tricky pinhole >> alignment associated with that. Perhaps that is the main reason - >> a transmission pathway pinhole would have to be aligned to the >> system and this might be difficult to maintain. In the epi >> configuration, the objective also acts as the condenser, whereas >> in the transmission pathway you have to align the objective, the >> condenser, and a pinhole. >> John Oreopoulos, BSc, >> PhD Candidate >> University of Toronto >> Institute For Biomaterials and Biomedical Engineering >> Centre For Studies in Molecular Imaging >> On 6-Nov-07, at 10:03 AM, John Runions wrote: >>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/ >>> cgi-bin/wa?S1=confocal Hello folks, >>> >>> Here is one of those seemingly straightforward to answer >>> questions that has me really stumped. You should hear all of the >>> BS around here when I bring this up with the other confocal >>> 'experts'. Why isn't there a pinhole in the transmission-image >>> forming pathway? A confocal transmission image would be nice but >>> I always tell people it's not possible. Is it not possible or >>> just not done? >>> >>> Thanks for your help. John. >>> -- >>> >>> ********************************* >>> C. John Runions, Ph.D. >>> School of Life Sciences >>> Oxford Brookes University >>> Oxford, UK >>> OX3 0BP >>> >>> email: [hidden email] <mailto:[hidden email]> >>> phone: +44 (0) 1865 483 964 >>> >>> web: http://www.brookes.ac.uk/lifesci/runions/HTMLpages/ >>> index.html! <http://www.brookes.ac.uk/lifesci/runions/HTMLpages/ >>> index.html%21> >>> >>> >>> >>> New - Oxford Brookes Master's in Bioimaging with Molecular >>> Technology <http://www.brookes.ac.uk/studying/courses/ >>> postgraduate/2007/bmt> >>> >>> >> Tel: W:416-946-5022 |
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Barbara Foster |
Re: Why no pinhole in transmission pathway?
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In reply to this post by John Runions
Search the CONFOCAL archive at
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Hi, John
Possible... and has been done, just not commercially. Barbara Foster, President We've moved! Microscopy/Microscopy Education 7101 Royal Glen Trail, Suite A McKinney TX 75070 P: (972)924-5310 Skype: fostermme W: www.MicroscopyEducation.com MME is now scheduling customized, on-site courses through December. Call us today for details. P. S. Need a good general reference or light microscopy text for next semester? Call us today to learn more about "Optimizing LIght Microscopy". Copies still available through MME... even for class-room lots ... and we give quantity discounts. Just call us here in the MME office for details. At 03:13 AM 11/6/2007, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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lechristophe |
Re: Why no pinhole in transmission pathway?
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In reply to this post by Jean-Yves Tinevez
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal What you get from doing that (using the confocal with reflected light) is very close to what is called RICM (Reflexion Interference Contrast Microscopy). You get nice dark areas where the cells are closely attached to the substrate, and it's even simpler to do than regular RCIM on a widefield microscope. Christophe Jean-Yves Tinevez a écrit : > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > > >>> >>> Here is one of those seemingly straightforward to answer questions >>> that has me really stumped. You should hear all of the BS around >>> here when I bring this up with the other confocal 'experts'. Why >>> isn't there a pinhole in the transmission-image forming pathway? A >>> confocal transmission image would be nice but I always tell people >>> it's not possible. Is it not possible or just not done? >>> > > > I am not an expert, but you can think of re-using the pinhole in the > reflected light path. For example just by using a plain 50%/50% mirror > in the filter cube rather than a dichroic and filters. > Thus you would have a pinhole-d view of your sample, with a > transmitted-like light. > Actually i have never tried, but i wonder if it could work. > jy > > > -- > Jean-Yves Tinevez > Max Planck Institute of Molecular Cell Biology and Genetics - Dresden > |
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John Oreopoulos |
Re: Why no pinhole in transmission pathway?
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Search the CONFOCAL archive at
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There is an article in Biotechniques 2002 describing this type of imaging. I think the proper name for it is "confocal reflection" or "confocal backscatter" microscopy: But now that you mention it, I've often wondered if the interpretation of the spatial contrast in a reflection/backscatter confocal image is the same as that seen in a RICM image. Can you really compare them like that? John Oreopoulos On 6-Nov-07, at 11:33 AM, Christophe Leterrier wrote:
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James Pawley |
Re: Why no pinhole in transmission pathway?
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In reply to this post by John Oreopoulos
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi all, Yes, scanning the specimen does make transmission confocal almost possible: as long as the refractive index of the specimen is entirely constant. (i.e., the specimen is dead and stained) focal plane and the coverslip on the "in" direction than they are on the "out" direction, then even if you initially line up the illumination optics to coincide with the detection path, you will find that this overlap will change as the specimen is moved past the spot. Consequently, the amount of signal actually passing through the detection pinhole depends not only on how much absorbing material is located at the focus of the laser but also (negatively) on the degree of misalignment. i.e. the output signal is no longer dependent on events occurring at the focal spot and therefore the resulting data no longer can be thought of as coming from an optical section. There have been suggestions that this "dynamic misalignment" problem could be avoided if the detector were not a fixed pinhole but a mini-image recording device (say a 5x5 CCD). As this spot was displaced from the axis by the inhomogeneities of the specimen, it would be tracked, the centroid of the spot located and its intensity measured. Sort of a movable pinhole to track the displaced spot to keep the whole system "confocal". This would be neat (In fact is was one proposed uses for the "EM-CCiode" detector proposed by me and others) but it would be really complicated to sort out the data. Probably for this reason, it has not been done. The other reason is that it really isn't clear what useful information would be obtained from the transmitted light signal that could not be obtained far more easily by detecting Backscattered light. Using any laser line, BSL will give you a lovely, fully-confocal darkfield image that shows all inhomogeneities as white. For more information, you might look at the chapter on Transmission Confocal by Carol Cogswell in the Second edition (but not the Third) of the Handbook. Cheers, Jim P. -- ********************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 [hidden email] 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2008 "If it ain't diffraction, it must be statistics." Anon. |
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John Oreopoulos |
Re: Why no pinhole in transmission pathway?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Jim, you've just referred to backscatter confocal imaging as something akin to darkfield confocal, and someone else earlier today compared it to reflection interference microscopy. Which mode of microscopy is backscatter imaging more like then? Is possible to interpret the contrast in a backscatter image? I've tried backscatter imaging a few times with adherent fibroblasts, and the images do show interesting features, but how am I to tell what causes one region of the image to look bright and another to look dark? It's a very complicated light pattern often. John Oreopoulos On 6-Nov-07, at 5:16 PM, James Pawley wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi all, > > Yes, scanning the specimen does make transmission confocal almost > possible: as long as the refractive index of the specimen is > entirely constant. (i.e., the specimen is dead and stained) > focal plane and the coverslip on the "in" direction than they are > on the "out" direction, then even if you initially line up the > illumination optics to coincide with the detection path, you will > find that this overlap will change as the specimen is moved past > the spot. > > Consequently, the amount of signal actually passing through the > detection pinhole depends not only on how much absorbing material > is located at the focus of the laser but also (negatively) on the > degree of misalignment. i.e. the output signal is no longer > dependent on events occurring at the focal spot and therefore the > resulting data no longer can be thought of as coming from an > optical section. > > There have been suggestions that this "dynamic misalignment" > problem could be avoided if the detector were not a fixed pinhole > but a mini-image recording device (say a 5x5 CCD). As this spot was > displaced from the axis by the inhomogeneities of the specimen, it > would be tracked, the centroid of the spot located and its > intensity measured. Sort of a movable pinhole to track the > displaced spot to keep the whole system "confocal". > > This would be neat (In fact is was one proposed uses for the "EM- > CCiode" detector proposed by me and others) but it would be really > complicated to sort out the data. Probably for this reason, it has > not been done. > > The other reason is that it really isn't clear what useful > information would be obtained from the transmitted light signal > that could not be obtained far more easily by detecting > Backscattered light. Using any laser line, BSL will give you a > lovely, fully-confocal darkfield image that shows all > inhomogeneities as white. > > For more information, you might look at the chapter on > Transmission Confocal by Carol Cogswell in the Second edition (but > not the Third) of the Handbook. > > Cheers, > > Jim P. > > -- > ********************************************** > Prof. James B. Pawley, Ph. 608-263-3147 > Room 223, Zoology Research Building, FAX 608-265-5315 > 1117 Johnson Ave., Madison, WI, 53706 [hidden email] > 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, > Vancouver Canada > Info: http://www.3dcourse.ubc.ca/ Applications due by March > 15, 2008 > "If it ain't diffraction, it must be statistics." Anon. |
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James Pawley |
Re: Why no pinhole in transmission pathway?
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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 > >Jim, you've just referred to backscatter confocal imaging as >something akin to darkfield confocal, and someone else earlier today >compared it to reflection interference microscopy. Which mode of >microscopy is backscatter imaging more like then? Is possible to >interpret the contrast in a backscatter image? I've tried >backscatter imaging a few times with adherent fibroblasts, and the >images do show interesting features, but how am I to tell what >causes one region of the image to look bright and another to look >dark? It's a very complicated light pattern often. > >John Oreopoulos All good questions John. To start with, the bad news: the BSL (actually it really is reflected light!) from the interface of the coverslip to the medium gives a HUGE signal. So huge that it actually sets up interference patterns with the incoming light. So I don't try to do BSL close to this interface. And you DO have to use the Anti-flex pol techniques to eliminate a background signal from light reflected back at all the out-of-focus optical surfaces in the system. (This light is always there, but is usually removed by the barrier filter on the basis of its wavelength.) I use the term darkfield because it fits: the undiffracted ray doesn't enter the detector, only scattered light does. More to the point it produces an image that is almost identical to widefield darkfield. The thing that is different is that, as lasers are far more coherent than WF light sources, you get a lot of interference effects in confocal BSL than in widefield darkfield when imaging features that are large and smooth with respect to the wavelength of light. So it works best with objects that are small and at least a few microns away from the nearest glass-medium interface. The contrast is simple: Brightness depends on particle size (until the particle is big enough to be "resolved") and varies with the square of the difference of its RI from that of the medium. It is slightly annoying that features that have a lower RI look just like those having a higher RI but if what you want is "structure," the BSL image can't be beat. And its free. No additional light need strike the specimen. Cheers, Jim P. >On 6-Nov-07, at 5:16 PM, James Pawley wrote: > >>Search the CONFOCAL archive at >>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >>Hi all, >> >>Yes, scanning the specimen does make transmission confocal almost >>possible: as long as the refractive index of the specimen is >>entirely constant. (i.e., the specimen is dead and stained) >>focal plane and the coverslip on the "in" direction than they are >>on the "out" direction, then even if you initially line up the >>illumination optics to coincide with the detection path, you will >>find that this overlap will change as the specimen is moved past >>the spot. >> >>Consequently, the amount of signal actually passing through the >>detection pinhole depends not only on how much absorbing material >>is located at the focus of the laser but also (negatively) on the >>degree of misalignment. i.e. the output signal is no longer >>dependent on events occurring at the focal spot and therefore the >>resulting data no longer can be thought of as coming from an >>optical section. >> >>There have been suggestions that this "dynamic misalignment" >>problem could be avoided if the detector were not a fixed pinhole >>but a mini-image recording device (say a 5x5 CCD). As this spot was >>displaced from the axis by the inhomogeneities of the specimen, it >>would be tracked, the centroid of the spot located and its >>intensity measured. Sort of a movable pinhole to track the >>displaced spot to keep the whole system "confocal". >> >>This would be neat (In fact is was one proposed uses for the >>"EM-CCiode" detector proposed by me and others) but it would be >>really complicated to sort out the data. Probably for this reason, >>it has not been done. >> >>The other reason is that it really isn't clear what useful >>information would be obtained from the transmitted light signal >>that could not be obtained far more easily by detecting >>Backscattered light. Using any laser line, BSL will give you a >>lovely, fully-confocal darkfield image that shows all >>inhomogeneities as white. >> >>For more information, you might look at the chapter on >>Transmission Confocal by Carol Cogswell in the Second edition (but >>not the Third) of the Handbook. >> >>Cheers, >> >>Jim P. >> >>-- >> ********************************************** >>Prof. James B. Pawley, Ph. 608-263-3147 >>Room 223, Zoology Research Building, FAX 608-265-5315 >>1117 Johnson Ave., Madison, WI, 53706 [hidden email] >>3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada >>Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2008 >> "If it ain't diffraction, it must be statistics." Anon. -- **************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-262-9083 250 N. Mills St., Madison, WI, 53706 [hidden email] "A scientist is not one who can answer questions but one who can question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39 |
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Stephen Cody |
Re: Why no pinhole in transmission pathway?
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In reply to this post by James Pawley
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal One of the reasons why confocal microscopy became so successful all of a sudden was that the light path of epillumination wass so simple and reliable compared with the transmitted path: No need for two perfectly matched objectives, one acting as a condenser, perfectly focused in XY and Z (Confocal) on the same point. Changing objectives would have been quite time consuming. Cheers Stephen H. Cody Microscopy Manager Central Resource for Advanced Microscopy Ludwig Institute for Cancer Research PO Box 2008 Royal Melbourne Hospital Victoria, 3050 Australia Tel: 61 3 9341 3155 Fax: 61 3 9341 3104 email: [hidden email] www.ludwig.edu.au/labs/confocal.html www.ludwig.edu.au/confocal Tip: Learn how to receive reminders about you microscope booking: www.ludwig.edu.au/confocal/Local/Booking_Hint.htm -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley Sent: Wednesday, 7 November 2007 9:16 AM To: [hidden email] Subject: Re: Why no pinhole in transmission pathway? Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi all, Yes, scanning the specimen does make transmission confocal almost possible: as long as the refractive index of the specimen is entirely constant. (i.e., the specimen is dead and stained) focal plane and the coverslip on the "in" direction than they are on the "out" direction, then even if you initially line up the illumination optics to coincide with the detection path, you will find that this overlap will change as the specimen is moved past the spot. Consequently, the amount of signal actually passing through the detection pinhole depends not only on how much absorbing material is located at the focus of the laser but also (negatively) on the degree of misalignment. i.e. the output signal is no longer dependent on events occurring at the focal spot and therefore the resulting data no longer can be thought of as coming from an optical section. There have been suggestions that this "dynamic misalignment" problem could be avoided if the detector were not a fixed pinhole but a mini-image recording device (say a 5x5 CCD). As this spot was displaced from the axis by the inhomogeneities of the specimen, it would be tracked, the centroid of the spot located and its intensity measured. Sort of a movable pinhole to track the displaced spot to keep the whole system "confocal". This would be neat (In fact is was one proposed uses for the "EM-CCiode" detector proposed by me and others) but it would be really complicated to sort out the data. Probably for this reason, it has not been done. The other reason is that it really isn't clear what useful information would be obtained from the transmitted light signal that could not be obtained far more easily by detecting Backscattered light. Using any laser line, BSL will give you a lovely, fully-confocal darkfield image that shows all inhomogeneities as white. For more information, you might look at the chapter on Transmission Confocal by Carol Cogswell in the Second edition (but not the Third) of the Handbook. Cheers, Jim P. -- ********************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 [hidden email] 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2008 "If it ain't diffraction, it must be statistics." Anon. This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error. The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research. |
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In reply to this post by James Pawley
Search the CONFOCAL archive at
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Comments on Jim's two posts:
1st post - why do transmitted confocal? One reason is that you can
in principle do phase contrast that way, and optical sectioning with
phase contrast would be rather neat. But the idea of using a small
array of detectors has the possibly fatal flaw that if the point in focus
is dark and there's something bright out of focus it will probably 'find'
the wrong point.
2nd post - 'reflection' vs 'backscattered' The image will only approximate
a darkfield image if the objects are below the resolution limit. Larger objects
will just act as reflectors and undiffracted light will indeed reach the detector.
So there is a pure reflection imaging regime, a diffracted backscatter regime
and in between an intermediate regime. Lipid droplets, for example will have
some reflection component. And interference effects will always be important
when you get into the reflection regime - layers, in particular, will give tricky
contrast. On the other hand, you could get some neat interference images
of membranes next to the substrate. So it's not a question of 'who's right'
it's 'what are you looking at'.
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 |
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Rietdorf, Jens |
Re: Why no pinhole in transmission pathway?
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In reply to this post by Andrew Resnick
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Dear Andy,
good question, even more, because DIC optics would
provide some intrinsic sectioning. Some people in house have been using
oblique illumination (Dadt contrast) in combination with 2 photon
imaging.
regards, jens
--- Dr.
Jens Rietdorf[hidden email] From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andrew Resnick Sent: Dienstag, 6. November 2007 16:30 To: [hidden email] Subject: Re: Why no pinhole in transmission pathway? If you like, you could stop down the field diaphram to a pinhole, but you would then need to either scan the pinhole (or sample), or have some alternative like a spinning disk setup to obtain the image. There's no reason why it cannot be done. The main difference is that in fluorescence imaging, the excited fluorophore molecules are considered a source, and that source is confocal to the aperture pinhole. In transmission, there is no comparable source. But in principle, any imaging method can be performed in a 'confocal' style. That said, what's the advantage? Andy At 10:03 AM 11/6/2007, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Instructor Department of Physiology and Biophysics Case Western Reserve University 216-368-6899 (V) 216-368-4223 (F) |
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George McNamara |
Re: Why no pinhole in transmission pathway?
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In reply to this post by John Runions
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Hi John, As mentioned by others, you would need to a second, synchronized scanner to descan the transmission path. Fortunately, the transmission path is a great place to put non-descanned detectors for multiphoton microscopy. The "NDD-T" detectors can be for the laser wavelength (good idea to attenuate the signal when using a 3.3 W average power Ti:Sapphire laser line!), second harmonic generation, and/or fluorescence. Steve Vogel (NIH) put his TCSPC FLIM fluorescence detectors after a dual dry/oil high NA condenser for his Zeiss LSM510 multiphoton microscope. Steve said it was much easier to add the detectors to his system that way. With respect to SHG, Watt Webb's group has published comparisons of epi- vs transmission-SHG. With respect to transmission microscopy in general, what appears opaque to the eye may be almost transparent in the near infrared. For example, an ~6 mm thick mouse brain transmits enough light at >700 nm to see red polymer filled blood vessels using a standard tungsten-halogen lamp and good digital CCD camera. Same specimen is completely opaque in the visible or with white light source and no wavelength selection. This was exploited years ago by Dodt and Zieglgansberger in near infrared video enhanced differential interference contrast optical sectioning microscopy of brain slices. The principle is easily demonstrated with a red (but not green) laser pointer and finger. At 10:03 AM 11/6/2007, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal George McNamara, Ph.D. University of Miami, Miller School of Medicine Image Core Miami, FL 33010 [hidden email] [hidden email] 305-243-8436 office http://home.earthlink.net/~pubspectra/ http://home.earthlink.net/~geomcnamara/ http://www.sylvester.org/health_pro/shared_resources/index.asp (see Analytical Imaging Core Facility) |
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