reducing size of illumination in epi-fluorescence
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Badri Ananth |
reducing size of illumination in epi-fluorescence
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi all, I have a question regarding epi-fluorescence microscopy and I'm hoping to get an answer here - I apologize for posting a question that is not related to confocal microscopy. I am a graduate student at UCSB and my technical knowledge of microscopy is quite basic so I'd appreciate any guidance you can give me. I want to do FRAP (fluorescence photobleaching) experiments on lipid bilayers. Currently we use our microscope - an old one, a Nikon Eclipse TE300 inverted microscope with a TE-FM epi-fluorescence attachment, to image lipid bilayers (and also for cellular immunofluorescence work). This is hooked up to a Coolsnap ES2 cooled-CCD imaging system. I use a 100x objective to bleach a small spot in the lipid bilayer using light from a 100w Mercury lamp. I use a 10x objective to monitor the recovery in a wider field. My problem is that even with the field diaphragm fully stopped down, the smallest field of illumination I can achieve is ~60 microns in diameter. Since the diffusion coefficient of the lipids is of the order of 1 sq. micron/sec., the recovery time is extremely long - approx. 1 hr. I would like to reduce this by bleaching smaller spots. Is there a way to reduce the size of the illuminated spot, e.g. by using a pinhole in the light path? Where and how would I place this pinhole? Thanks in advance - I will also try to contact Nikon technical support for help with this, but I suspect they'll try to sell me a new microscope instead. regards, badri |
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Julio Vazquez |
Re: reducing size of illumination in epi-fluorescence
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Badri, You can try to remove the field diaphragm insert, place a disc of aluminum foil over the diaphragm, and drill a pinhole in the foil. The diaphragm fully closed has a diameter of about 1-2 mm. With a pinhole, you could probably get a bleached field maybe ten times smaller. I'd be very careful not to damage the diaphragm! A more elegant option would be to have a piece of aluminum or steel that would fit in the slot occupied by the field diaphragm, and drill pinholes at the right location (or a series of pinholes of different sizes, so you could change the bleached area by sliding the metal piece (or using different ones). This may be more costly to manufacture, but might be doable if you have a good machine shop. -- Julio Vazquez, Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 = On Jul 21, 2008, at 2:48 PM, Badri Ananth wrote:
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Craig Brideau |
Re: reducing size of illumination in epi-fluorescence
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Depending on how tight the focus is at the field diaphragm you might need to use an additional lens to focus the lamp light onto your pinhole. A tighter focus on the pinhole will help it pass more light; otherwise you may lose too much power going through the pinhole.
Craig On Mon, Jul 21, 2008 at 5:24 PM, Julio Vazquez <[hidden email]> wrote:
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Badri Ananth |
Re: reducing size of illumination in epi-fluorescence
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In reply to this post by Badri Ananth
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, Thanks a lot for your suggestions! I need this pinhole to be positioned such that it can be removed (e.g. for other users doing normal epi-fluorescence). The best way to do this without re-engineering the whole field diaphragm unit, seems to be to replace one of the ND filter sliders with a pinhole. However, the plane of this filter is about 5 mm distant from the field diaphragm. I guess that means I'll have to re-focus the lamp light onto the pinhole, instead of the field diaphragm? The only focusable element in the light path is a 'collector' lens on the lamphouse itself - I don't know if that will suffice to refocus the light. There are two lenses in the main epi-fl unit; one rear and one front. It looks like the front one (nearest the objective), while not 'focusable' per se, can be translated by unscrewing a nut that holds it in place. Any ideas on how best to refocus? The lamp-house we use is mounted on the rear end of the microscope and that leaves no room for inserting new optics in the light path, so the only option is to replace one of the existing lenses. Is this feasible, e.g. replacing the lamphouse collector lens? thanks again for your help! best badri |
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Raghu Parthasarathy |
Re: reducing size of illumination in epi-fluorescence
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In reply to this post by Badri Ananth
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Hi, First, you should certainly be able to get a bleached FRAP spot *far* smaller than 60 microns using the setup you've described. (I've done this routinely, on lipid bilayers, on the same sort of microscope.) So I suspect something else is wrong. Think about this: your stated bleaching objective lens magnification is 100X, and your camera has approx. 10 micron pixels, so a 60 micron spot should be 600 pixels wide on your image -- i.e. about half the total field of view (for a 1200x1200 px camera)! If your spots really are this wide, either your field diaphragm isn't closing, or it's not much of a diaphragm! Second, even if your spot is 60 microns wide (which I stress it should not be) it's incorrect to conclude that you necessarily need 1hour to see diffusion. The edges of the spot should blur well before this, and you can extract the diffusion coefficient from how the shape of the whole spot changes with time. If your sample really needs 1 hour to recover, then it's not fluid. best wishes, Raghu Raghuveer Parthasarathy [hidden email] Assistant Professor Department of Physics 1274 University of Oregon Eugene, OR 97403-1274 http://physics.uoregon.edu/~raghu/ ----- Original Message ---- From: Badri Ananth <[hidden email]> To: [hidden email] Sent: Monday, July 21, 2008 2:48:28 PM Subject: reducing size of illumination in epi-fluorescence Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi all, I have a question regarding epi-fluorescence microscopy and I'm hoping to get an answer here - I apologize for posting a question that is not related to confocal microscopy. I am a graduate student at UCSB and my technical knowledge of microscopy is quite basic so I'd appreciate any guidance you can give me. I want to do FRAP (fluorescence photobleaching) experiments on lipid bilayers. Currently we use our microscope - an old one, a Nikon Eclipse TE300 inverted microscope with a TE-FM epi-fluorescence attachment, to image lipid bilayers (and also for cellular immunofluorescence work). This is hooked up to a Coolsnap ES2 cooled-CCD imaging system. I use a 100x objective to bleach a small spot in the lipid bilayer using light from a 100w Mercury lamp. I use a 10x objective to monitor the recovery in a wider field. My problem is that even with the field diaphragm fully stopped down, the smallest field of illumination I can achieve is ~60 microns in diameter. Since the diffusion coefficient of the lipids is of the order of 1 sq. micron/sec., the recovery time is extremely long - approx. 1 hr. I would like to reduce this by bleaching smaller spots. Is there a way to reduce the size of the illuminated spot, e.g. by using a pinhole in the light path? Where and how would I place this pinhole? Thanks in advance - I will also try to contact Nikon technical support for help with this, but I suspect they'll try to sell me a new microscope instead. regards, badri |
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Julio Vazquez |
Re: reducing size of illumination in epi-fluorescence
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On our Nikon E800, with the 100x objective, the fully closed field diaphragm (~1 mm diameter) gives a spot of about 40 microns, so not too far off from the 60 microns reported by Badri. This is actually a quite small spot when compared to the full field of view (but significant compared to the area covered by the camera, which is much smaller). I agree with Raghu's second point though... you can indeed measure the diffusion properties of your sample by looking at a small region near the edge (or anywhere for that matter) of the bleached area, not necessarily the entire bleached spot, so it doesn't really matter which size or shape your bleached area is... the calculations may be a bit more difficult though, because an arbitrary region will be located asymmetrically inside your bleached area. Maybe then the simplest would be to tape a square piece of foil to your field diaphragm holder, on the outside, so that half of your field is bleached. You can then look at recovery in the center (near the bleached edge), where you can approximate your situation to a two vessel system separated by a wall. This would be even more true if using a lower power objective. -- Julio Vazquez, Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 On Jul 22, 2008, at 7:24 AM, Raghu Parthasarathy wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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Raghu Parthasarathy |
Re: reducing size of illumination in epi-fluorescence
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In reply to this post by Badri Ananth
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About the size of a FRAP spot for microscopy: The comments from Julio Vazquez may certainly be more correct than my memory of the Nikon aperture size, since for a while now we've been simply using our own set of field apertures -- simply pieces of metal with holes drilled in that slide into the appropriate slot on the microscope. This is simple to do, and gives considerable freedom. (I had forgotten about this, but it is still certainly the case that I was still able to happily do FRAP with Nikon's field diaphragm for years before these home-made apertures.) About extracting diffusion coefficients from the recovery of a spot that's large enough so that only the edges are effectively blurred in a reasonable time: The diffusion of the entire irregular spot doesn't follow any nice function, but does obey Fick's laws (by assumption!) and can be modeled as such. So, you can take a picture, take another picture later, and ask what diffusion coefficient would cause the first picture to be transformed into the second over the time between the two acquisitions. This is a very brief sketch of the method I've employed for FRAP analysis. A slightly longer one can be found on the last page of this paper: http://pubs.acs.org/cgi-bin/abstract.cgi/jacsat/2007/129/i17/abs/ja067819i.html . A description of the 1d version of this sort of treatment can be found in Howard Berg's excellent book, "Random Walks in Biology," in one of the appendices. And, I'm happy to write more later. Good luck! Raghu Raghuveer Parthasarathy [hidden email] Assistant Professor Department of Physics 1274 University of Oregon Eugene, OR 97403-1274 http://physics.uoregon.edu/~raghu/ ----- Original Message ---- From: Julio Vazquez <[hidden email]> To: [hidden email] Sent: Tuesday, July 22, 2008 9:42:29 AM Subject: Re: reducing size of illumination in epi-fluorescence Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal = On our Nikon E800, with the 100x objective, the fully closed field diaphragm (~1 mm diameter) gives a spot of about 40 microns, so not too far off from the 60 microns reported by Badri. This is actually a quite small spot when compared to the full field of view (but significant compared to the area covered by the camera, which is much smaller). I agree with Raghu's second point though... you can indeed measure the diffusion properties of your sample by looking at a small region near the edge (or anywhere for that matter) of the bleached area, not necessarily the entire bleached spot, so it doesn't really matter which size or shape your bleached area is... the calculations may be a bit more difficult though, because an arbitrary region will be located asymmetrically inside your bleached area. Maybe then the simplest would be to tape a square piece of foil to your field diaphragm holder, on the outside,
so that half of your field is bleached. You can then look at recovery in the center (near the bleached edge), where you can approximate your situation to a two vessel system separated by a wall. This would be even more true if using a lower power objective. -- Julio Vazquez, Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 On Jul 22, 2008, at 7:24 AM, Raghu Parthasarathy wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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Richard Cole |
Re: reducing size of illumination in epi-fluorescence
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In reply to this post by Badri Ananth
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Badri, I concur with Raghu, we have a TE200 in the
core and with a 60X (1.4NA) we bleach 5-10µM spots in lipid bilayers .
You scope should have an aperture in the fluorescence path, if that does
not close down small enough I have some other suggestions, which I would be
more than happy to share off the list.
Cheers Rich Cole
Website www.wadsworth.org/cores/alm/index.htm From: Raghu
Parthasarathy [mailto:[hidden email]]
-- Assistant Professor ----- Original Message ---- IMPORTANT NOTICE: This e-mail and any attachments may contain confidential or sensitive information which is, or may be, legally privileged or otherwise protected by law from further disclosure. It is intended only for the addressee. If you received this in error or from someone who was not authorized to send it to you, please do not distribute, copy or use it or any attachments. Please notify the sender immediately by reply e-mail and delete this from your system. Thank you for your cooperation. |
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Badri Ananth |
Re: reducing size of illumination in epi-fluorescence
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi all, Thanks again, Raghu, Julio, Richard. I went back to lab to determine again what my microscope can do. Raghu is right, the minimum bleach spot I can get is actually about 23 microns, which is indeed the size of the field diaphragm when fully stopped down and focused through a 100x lens. The mistake I had made earlier was to measure a spot on a bilayer that I had bleached previously with the diaphragm fully open. So it appears that modifying the Nikon scope with a custom aperture is not necessary. Also, thanks for the suggestions about how exactly to go about measuring diffusion. My original intention was to simply follow the average intensity over the whole spot with time and use that to calculate D. I have done this previously (on a different microscope and setup). One way was to simply use the recovery half-time to calculate an order of magnitude estimate for D. The other was to solve the 1-D diffusion equation analytically and fit the solution to the recovery curve to extract D (following the famous Axelrod paper, Biophys J 1976 if I'm not mistaken). I have not tried modeling/fitting the solution only to the time-varying edge profile - it may have some advantages I suppose. Apart from an estimate of D, I want to see if the fluorescence recovers completely - this is because I am studying diffusion of lipopeptides that are anchored in the bilayer - and it may be interesting to see if they have a 100% 'mobile fraction'. Any suggestions/insight on this? Once again, thanks for your help. best badri |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear All, Yesterday I have tested our new 585 nm laser in conjuction with 514 nm argon and 437 nm diode under TIR conditions. Even though I have not noticed the need for the critical angle re-adjustment between 437 nm and 514 nm lines (77 nm difference), however, when the critical angle was set for the yellow laser (585 nm), switching from 585 nm to 514 nm line (71 nm difference) required the (manual) resetting of the critical angle. I used the 89006 triple beamsplitter (A/R coated), which seemed to reflect the 514 argon line (up to 70%). I am looking for help with the device that would allow quick, low msec range, critical angle re-adjustment or any other suggestions will be appreciated. Thanks a lot in advance, Vitaly Vitaly P. Boyko, NCI-Frederick, 301-846-6575 |
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John Oreopoulos |
Re: TIR critical angle re-adjustent
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Hi Vitaly,
I'm not sure if you have a commercial instrument or a home-built TIRF microscope on an optical bench, but generally this is an issue that always comes up anytime you have multi-color TIRF. The problem stems from the fact that the critical angle for each laser line is different because of the characteristics and wavelength dependence of the substrate-sample refractive interface (ie: the critical angle is slightly different for each laser line) and also the wavelength dependence of the glass inside the objective. Note also that the equation for the evanescent penetration depth is wavelength dependent as well such that even if you had all laser lines impinging on the interface at the same angle, each penetration depth would be different. (There's an article on this topic here: http://www.photonics.com/content/bio/2008/May/features/91651.aspx) I know that this effect was nearly impossible to deal with in the early commercial TIRF systems, but I think now that all or some of the major microscope vendors have developed a system where each laser line is directed towards the microscope down a different path and then each line merges after passing through or reflecting off a dichroic mirror before entering the microscope. Each laser sits on an adjustable platform so that you can line them all up differently and independently. For an example of this, see page 15 of this .pdf document: I've seen home-built systems at other labs that are essentially the same thing. If you're in the situation where all laser lines are funneled down the same optical path (ie: through a single optical fiber) and there is only one way to adjust the angle of incidence for all three laser lines at the same time, then the best you can do is replace the manual micrometer screw with some kind of fast motorized screw and synchronize this to your image acquisition software somehow (maybe using something like Micro-Manager - http://www.micro-manager.org/ ?). Thorlabs sells motorized actuators that could do the job: http://www.thorlabs.com/Navigation.cfm?Guide_ID=83 I tried this once on our system but the actuator I was using was too slow. I think it's also important to emphasize that even after setting up all laser lines the way you want them to go into the scope or after you've found a way to adjust the beam incident angle rapidly, you should measure the actual penetration depth somehow. In objective-based TIRF systems, the penetration depth can sometimes deviate significantly far away from the predicted depth given by the equation. I think about a year or two ago there was a listing on the server about this topic which I posted a reply to. Here it is: Confocal listserver item 027568 posted on 2007-02-22 John Oreopoulos, BSc, PhD Candidate University of Toronto Institute For Biomaterials and Biomedical Engineering Centre For Studies in Molecular Imaging Tel: W:416-946-5022 On 24-Jul-08, at 10:48 AM, Vitaly Boyko wrote:
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In reply to this post by vb-2
Speaking of TIRF issues, perhaps list members would like to comment on some differing opinions I've been hearing about the usefulness of a TIRF lens for standard fluorescence. I've heard two extremes on the appropriateness/usefulness of a 1.49 NA TIRF lens for high mag/high res/high sensitivity imaging away from the substratum (for example, intranuclear structures). Although I'm a bit physics-challenged, it seems to make sense to me that the NA would not be realized in a glycerol mounting medium due to RI mismatch, and could be counter productive, in fact. Seems the vendors that sell TIRF lens-systems say they are great for every use, pumping up the high NA as a cure for everything, and vendors that don't sell them say they're only good for a true TIRF application. |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have had very good luck using a 100x / 1.49 NA TIRF lens for imaging in aqueous samples - typically yeast or mammalian cells grown on coverslips - on our spinning disk confocal. I'm not sure how much the larger NA helps, but having the correction collar is really nice for correcting the spherical aberration that comes from using an oil immersion lens to image into an aqueous sample. If you put the time into setting the correction collar, the Z-profiles obtained with the TIRF lens are much more symmetric and show less intensity fall off into the sample than do images taken with a 100x/1.4 lens. We've not, however, compared this to using a water immersion lens. The lens we're using are Nikon ones. So from my limited experience, there can be some benefit to using a TIRF lens as compared to an equivalent non-TIRF lens. Kurt Mancini, Michael A wrote: > > Speaking of TIRF issues, perhaps list members would like to comment on > some differing opinions I've been hearing about the usefulness of a > TIRF lens for standard fluorescence. I've heard two extremes on the > appropriateness/usefulness of a 1.49 NA TIRF lens for high mag/high > res/high sensitivity imaging away from the substratum (for example, > intranuclear structures). Although I'm a bit physics-challenged, it > seems to make sense to me that the NA would not be realized in a > glycerol mounting medium due to RI mismatch, and could be counter > productive, in fact. Seems the vendors that sell TIRF lens-systems say > they are great for every use, pumping up the high NA as a cure for > everything, and vendors that don't sell them say they're only good for > a true TIRF application. > > Comments are welcome both on/off the list. > > Cheers, > > Mike > _______________________ > Michael A. Mancini, Ph.D > Department of Molecular and Cellular Biology > Baylor College of Medicine > Houston, TX 77030 > [hidden email] > 713.408.0179 cell > -- Kurt Thorn, PhD Director, Nikon Imaging Center University of California San Francisco UCSF MC 2140 Genentech Hall Room S252 600 16th St. San Francisco, CA 94158-2517 http://nic.ucsf.edu phone 415.514.9709 fax 415.514.4300 |
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In reply to this post by Mancini, Michael A
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I'm going to say that in general, the TIRF objectives can be used for intranuclear structures both with confocal and regular epifluorescence, but I think most people on the listserver are going to say their preference would always be for a water objective with a live-cell specimen. Reducing aberrations caused by refractive index mismatch is probably more important to maintain BOTH resolution and high signal count. Jim Pawley has discussed a lot of these issues in his book, and at his live-cell microscopy course the detrimental effects of refractive mis-match were emphasized several times. High NA oil objectives perform the best when you're concerned with what's happening at or very close to the substrate. That's why TIRF is great for single-molecule in vitro experiments as well that require the sample to be diluted and immobilized on the surface.
John On 24-Jul-08, at 2:32 PM, Mancini, Michael A wrote:
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In reply to this post by Kurt Thorn
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I second Kurt's assessment. At the coverslip surface, the higher NA = higher res and more light gather. As you go into the sample, the RI of the sample begins to dominate. This can be corrected with a collar, or if there is no collar, using oils of a different RI can also correct (you have to test empirically for your system and the room temp etc.). Paul Paul S. Maddox, PhD Assistant Professor Institute for Research in Immunology and Cancer Dept of Pathology and Cell Biol, U. de Montreal P.O. Box 6128, Station Centre-Ville Montréal QC H3C 3J7 CANADA Courier: 2900, boulevard Édouard-Montpetit Pavillon Marcelle-Coutu, Quai 20 Montreal QC H3T 1J4 CANADA [hidden email] Ph: 514-343-7894 Fax: 514-343-6843 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kurt Thorn Sent: Thursday, July 24, 2008 2:46 PM To: [hidden email] Subject: Re: TIRF Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have had very good luck using a 100x / 1.49 NA TIRF lens for imaging in aqueous samples - typically yeast or mammalian cells grown on coverslips - on our spinning disk confocal. I'm not sure how much the larger NA helps, but having the correction collar is really nice for correcting the spherical aberration that comes from using an oil immersion lens to image into an aqueous sample. If you put the time into setting the correction collar, the Z-profiles obtained with the TIRF lens are much more symmetric and show less intensity fall off into the sample than do images taken with a 100x/1.4 lens. We've not, however, compared this to using a water immersion lens. The lens we're using are Nikon ones. So from my limited experience, there can be some benefit to using a TIRF lens as compared to an equivalent non-TIRF lens. Kurt Mancini, Michael A wrote: > > Speaking of TIRF issues, perhaps list members would like to comment on > some differing opinions I've been hearing about the usefulness of a > TIRF lens for standard fluorescence. I've heard two extremes on the > appropriateness/usefulness of a 1.49 NA TIRF lens for high mag/high > res/high sensitivity imaging away from the substratum (for example, > intranuclear structures). Although I'm a bit physics-challenged, it > seems to make sense to me that the NA would not be realized in a > glycerol mounting medium due to RI mismatch, and could be counter > productive, in fact. Seems the vendors that sell TIRF lens-systems say > they are great for every use, pumping up the high NA as a cure for > everything, and vendors that don't sell them say they're only good for > a true TIRF application. > > Comments are welcome both on/off the list. > > Cheers, > > Mike > _______________________ > Michael A. Mancini, Ph.D > Department of Molecular and Cellular Biology > Baylor College of Medicine > Houston, TX 77030 > [hidden email] > 713.408.0179 cell > -- Kurt Thorn, PhD Director, Nikon Imaging Center University of California San Francisco UCSF MC 2140 Genentech Hall Room S252 600 16th St. San Francisco, CA 94158-2517 http://nic.ucsf.edu phone 415.514.9709 fax 415.514.4300 |
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Rietdorf, Jens |
TIR critical angle re-adjustent
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In reply to this post by vb-2
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Vitaly, Both Leica and Zeiss (no commercial interest) provide solutions for automatic adjustment of the incident beam angle (provided you measure the tilt of the reflecting interface for each sample). We used simple stepper motors to automate the turning of the micrometer screw of an Olympus 3 line setup (again n.c.i) Be aware, the field is decaying exponentially and you can influence for example the half evanescence field depth by the angle, [so the distance at which the incident power has decreased to 50%], but not the depth of the field of view, which in turn also depends on the incident power and on the concentration and brightness of the fluorophores. Cheers, jens -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Vitaly Boyko Sent: Thursday, July 24, 2008 4:48 PM To: [hidden email] Subject: TIR critical angle re-adjustent Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear All, Yesterday I have tested our new 585 nm laser in conjuction with 514 nm argon and 437 nm diode under TIR conditions. Even though I have not noticed the need for the critical angle re-adjustment between 437 nm and 514 nm lines (77 nm difference), however, when the critical angle was set for the yellow laser (585 nm), switching from 585 nm to 514 nm line (71 nm difference) required the (manual) resetting of the critical angle. I used the 89006 triple beamsplitter (A/R coated), which seemed to reflect the 514 argon line (up to 70%). I am looking for help with the device that would allow quick, low msec range, critical angle re-adjustment or any other suggestions will be appreciated. Thanks a lot in advance, Vitaly Vitaly P. Boyko, NCI-Frederick, 301-846-6575 |
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John Oreopoulos |
Re: TIR critical angle re-adjustent
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Sorry, I forgot to add to my post earlier in the day "no commercial interest" as well. I just happened to pick two Olympus examples there as well. John Oreopoulos On 24-Jul-08, at 3:50 PM, Rietdorf, Jens wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear Vitaly, > > Both Leica and Zeiss (no commercial interest) provide solutions for > automatic adjustment of the incident beam angle (provided you measure > the tilt of the reflecting interface for each sample). We used simple > stepper motors to automate the turning of the micrometer screw of an > Olympus 3 line setup (again n.c.i) Be aware, the field is decaying > exponentially and you can influence for example the half evanescence > field depth by the angle, [so the distance at which the incident power > has decreased to 50%], but not the depth of the field of view, > which in > turn also depends on the incident power and on the concentration and > brightness of the fluorophores. > > Cheers, jens > > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]] On > Behalf Of Vitaly Boyko > Sent: Thursday, July 24, 2008 4:48 PM > To: [hidden email] > Subject: TIR critical angle re-adjustent > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear All, > > Yesterday I have tested our new 585 nm laser in conjuction with 514 nm > argon > and 437 nm diode under TIR conditions. > > Even though I have not noticed the need for the critical angle > re-adjustment > between 437 nm and 514 nm lines (77 nm difference), however, when the > critical angle was set for the yellow laser (585 nm), switching > from 585 > nm > to 514 nm line (71 nm difference) required the (manual) resetting of > the > critical angle. > I used the 89006 triple beamsplitter (A/R coated), which seemed to > reflect > the 514 argon line (up to 70%). > > I am looking for help with the device that would allow quick, low msec > range, critical angle re-adjustment or any other suggestions will be > appreciated. > > Thanks a lot in advance, > > Vitaly > > Vitaly P. Boyko, > NCI-Frederick, > 301-846-6575 > > > |
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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 Do plate readers (ie, machines that are supposed to detect fluorescence in 96 well plates) detect fluorescence in cells that are adherent to the bottom of the wells, or is the fluorescence detected mainly from the solution in the wells? I would have thought that the detect total fluorescence in a volume, and in principle it would not matter if the fluorescence is contained in adherent cells, or in the solution. However, recently we have the experience of seeing cells clearly with bright fluorescence in a microscope, and then getting no reading from the plate reader. I have very little experience with plate readers, so the collective wisdom of the group will be greatly appreciated. --aryeh -- Aryeh Weiss School of Engineering Bar Ilan University Ramat Gan 52900 Israel Ph: 972-3-5317638 FAX: 972-3-7384050 |
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