Best parameters for optical slicing
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Pedro Camello |
Best parameters for optical slicing
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I´m trying to image a 3D map of mitotracker-stained cells. I´m using a Biorad 1024, 60x, NA 1.4. My first attempts generated acceptable images with iris 1.6 -2 (close to the theoretical optimal iris indicated by Biorad with this objective). My 2 questions are: 1) Using zoom 2.6 (the theoretical optimum zoom for this objective) I get pixel size 166 µm. Since published images with my cell type (pancreatic acini) indicate that most of the mitochondria are around 500 nm diameter (similar to the measured resolution of mi micro with iris 2) my intend is to sample at 150 nm to meet Nyquist. However, this creates a non-cubic voxel (166x166x150). Will be this a problem to make posterior 3D visualization and volume measurements? 2) If I need deconvolution to posterior processing and measurements, is it essential PSF measurements or will blinded methods be enough? Thanks in advance -- Dr Pedro J Camello Dpt Physiology Faculty of Veterinary Sciences University of Extremadura 10071 Caceres Spain Ph: 927257100 Extension 1321/1290 Fax:927257110 |
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Julio Vazquez |
Re: Best parameters for optical slicing
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Search the CONFOCAL archive at
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Hi Pedro, I am not sure how you or Biorad are calculating the optimal pixel sizes, or what the specific constraints for the Biorad 1024 are. For a 60x/1.4NA objective, the theoretical resolution in x/y at 580 nm (if you are using a red mito tracker) is around 200 microns (I am using the formula d = 0.5 x lambda/ NA). Therefore, your optimal pixel size, if you use a Nyquist factor of 2.5, should be around 80 microns. Obviously, you may get slightly different values depending on how your choice of wavelength. Regarding the sampling along the z axis, it is not so much the dimensions of the structures (mitochondria) that matter, but the optical properties of your scope. The axial resolution is about half the x/y resolution, and therefore 160 micron spacing between sections would be about optimal. If you are trying to look at very fine structures, oversampling a little (both in x, y, and z) may not be a bad idea, as long as your sample can take it and doesn't bleach... I don't think it as an issue that your x/y pixel dimensions are different from your z section spacing. The PSF itself is not spherically symmetrical (but elongated along z) and your sampling along x/y and z should be set accordingly. Secondly, when you load your images into whatever program you use for doing the 3-D measurements, the image should be calibrated to reflect the actual x,y and z dimensions. As long as you used Nyquist sampling, and you have good signal-to-noise, you should be able to get accurate measurements. For deconvolution, we've had good results with both measured PSF and calculated PSFs; blind should work fine too, but I haven't tried. If you have some reference beads, you could image them under conditions similar to those used to image your sample, deconvolve them with the same method, and do some measurements. That should give you an idea of how well your deconvolution alrgorithm is performing. If it doesn't look good, you can always re-deconvolve your data with a different method. Julio. -- Julio Vazquez, PhD Director of Scientific Imaging Fred Hutchinson Cancer Research Center 1100 Fairview Ave. N., mailstop DE-512 Seattle, WA 98109-1024 Tel: Office: 206-667-1215/ Lab: 206-667-4205 FAX: 206-667-6845 -------------------------------------------------- This message is confidential, intended only for the named recipient(s) and may contain information that is privileged or exempt from disclosure under applicable law. If you are not the intended recipient(s), you are notified that the dissemination, distribution or copying of this information is strictly prohibited. If you received this message in error, please notify the sender then delete this message. On Feb 9, 2008, at 9:30 AM, Pedro J Camello wrote:
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Search the CONFOCAL archive at
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Hello All,
To avoid more confusion, you both meant nanometers, not micrometers, when talking about xy-, z-resolution and z-stepsize, correct?
E.g., "optimal" xy resolution is 200 nm, not microns, with pixel size at 80 nm with Nyquist, 160 nm z-stepsize, etc. Zoltan
On 2/9/08, Julio Vazquez <[hidden email]> wrote:
Search the CONFOCAL archive at <a onclick="return top.js.OpenExtLink(window,event,this)" href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank">http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal -- -- Zoltan Cseresnyes Facility manager, Imaging Suite Dept. of Zoology University of Cambridge Downing Street, Cambridge CB2 3EJ UK Tel.: (++44) (0)1223 769282 Fax.: (++44) (0)1223 336676 |
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Julio Vazquez |
Re: Best parameters for optical slicing
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
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Yes, nanometers, of course.... my apologies. Julio. |
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Pedro Camello |
Re: Best parameters for optical slicing
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In reply to this post by Zoltan
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Yes, nm. Julio, you are right about lateral resolution. My reasoning is that although the optical performance does not depend on the structure, 150 nm is supposed to be good enough to sample structures of 500 nm. I would like to avoid too much oversampling. I don´t know how Biorad calculated that 2.6 zoom is the maximum advisable for this objective (it is a shame, but I never worried before about sampling rate, resolution, etc) Many thanks for your help. > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello All, > > To avoid more confusion, you both meant nanometers, not micrometers, when > talking about xy-, z-resolution and z-stepsize, correct? > E.g., "optimal" xy resolution is 200 nm, not microns, with pixel size at > 80 > nm with Nyquist, 160 nm z-stepsize, etc. > > Zoltan > > > On 2/9/08, Julio Vazquez <[hidden email]> wrote: >> >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal = Hi Pedro, >> >> >> I am not sure how you or Biorad are calculating the optimal pixel sizes, >> or what the specific constraints for the Biorad 1024 are. For a >> 60x/1.4NA >> objective, the theoretical resolution in x/y at 580 nm (if you are using >> a >> red mito tracker) is around 200 microns (I am using the formula d = 0.5 >> x >> lambda/ NA). Therefore, your optimal pixel size, if you use a Nyquist >> factor >> of 2.5, should be around 80 microns. Obviously, you may get slightly >> different values depending on how your choice of wavelength. >> >> >> Regarding the sampling along the z axis, it is not so much the >> dimensions >> of the structures (mitochondria) that matter, but the optical properties >> of >> your scope. The axial resolution is about half the x/y resolution, and >> therefore 160 micron spacing between sections would be about optimal. >> If >> you are trying to look at very fine structures, oversampling a little >> (both >> in x, y, and z) may not be a bad idea, as long as your sample can take >> it >> and doesn't bleach... >> >> >> I don't think it as an issue that your x/y pixel dimensions are >> different >> from your z section spacing. The PSF itself is not spherically >> symmetrical >> (but elongated along z) and your sampling along x/y and z should be set >> accordingly. Secondly, when you load your images into whatever program >> you >> use for doing the 3-D measurements, the image should be calibrated to >> reflect the actual x,y and z dimensions. As long as you used Nyquist >> sampling, and you have good signal-to-noise, you should be able to get >> accurate measurements. >> >> >> For deconvolution, we've had good results with both measured PSF and >> calculated PSFs; blind should work fine too, but I haven't tried. If you >> have some reference beads, you could image them under conditions similar >> to >> those used to image your sample, deconvolve them with the same method, >> and >> do some measurements. That should give you an idea of how well your >> deconvolution alrgorithm is performing. If it doesn't look good, you can >> always re-deconvolve your data with a different method. >> >> >> >> >> >> >> Julio. >> >> >> >> -- >> Julio Vazquez, PhD >> Director of Scientific Imaging >> Fred Hutchinson Cancer Research Center >> 1100 Fairview Ave. N., mailstop DE-512 >> Seattle, WA 98109-1024 >> >> >> Tel: Office: 206-667-1215/ Lab: 206-667-4205 >> FAX: 206-667-6845 >> >> >> [hidden email] >> http://www.fhcrc.org/science/shared_resources/imaging/ >> >> >> >> -------------------------------------------------- >> *DISCLAIMER:* >> >> This message is confidential, intended only for the named recipient(s) >> and >> may contain information that is privileged or exempt from disclosure >> under >> applicable law. If you are not the intended recipient(s), you are >> notified >> that the dissemination, distribution or copying of this information is >> strictly prohibited. If you received this message in error, please >> notify >> the sender then delete this message. >> >> >> >> >> >> >> >> On Feb 9, 2008, at 9:30 AM, Pedro J Camello wrote: >> >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> >> I´m trying to image a 3D map of mitotracker-stained cells. I´m using a >> Biorad 1024, 60x, NA 1.4. My first attempts generated acceptable images >> with iris 1.6 -2 (close to the theoretical optimal iris indicated by >> Biorad with this objective). >> >> >> My 2 questions are: >> >> >> 1) Using zoom 2.6 (the theoretical optimum zoom for this objective) I >> get >> pixel size 166 µm. Since published images with my cell type (pancreatic >> acini) indicate that most of the mitochondria are around 500 nm diameter >> (similar to the measured resolution of mi micro with iris 2) my intend >> is >> to sample at 150 nm to meet Nyquist. However, this creates a non-cubic >> voxel (166x166x150). Will be this a problem to make posterior 3D >> visualization and volume measurements? >> >> >> 2) If I need deconvolution to posterior processing and measurements, is >> it >> essential PSF measurements or will blinded methods be enough? >> >> >> Thanks in advance >> >> >> -- >> Dr Pedro J Camello >> Dpt Physiology >> Faculty of Veterinary Sciences >> University of Extremadura >> 10071 Caceres >> Spain >> Ph: 927257100 Extension 1321/1290 >> Fax:927257110 >> >> >> > > > -- > -- > Zoltan Cseresnyes > Facility manager, Imaging Suite > Dept. of Zoology University of Cambridge > Downing Street, Cambridge > CB2 3EJ UK > > Tel.: (++44) (0)1223 769282 > Fax.: (++44) (0)1223 336676 > -- Dr Pedro J Camello Dpt Physiology Faculty of Veterinary Sciences University of Extremadura 10071 Caceres Spain Ph: 927257100 Extension 1321 Fax:927257110 |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
The 2.6 x max zoom for a 63x immersion lens sounds about right, beyond this you would only get "empty" resolution increase, but real increase in photodamage. If you want to severely oversample, e.g. while studying very fine structures combined with post-processing algorithms (reconstructing, e.g.), then up to 5 x might be considered in my opinion.
Zoltan
On 2/9/08, Pedro J Camello <[hidden email]> wrote:
Search the CONFOCAL archive at -- -- Zoltan Cseresnyes Facility manager, Imaging Suite Dept. of Zoology University of Cambridge Downing Street, Cambridge CB2 3EJ UK Tel.: (++44) (0)1223 769282 Fax.: (++44) (0)1223 336676 |
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M. van de corput |
Re: Best parameters for optical slicing
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In reply to this post by Julio Vazquez
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Julio Vazquez wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal = > > Yes, nanometers, of course.... my apologies. > > Julio. I would not use blind deconvolution. When your system has an asymmetrical PSF (mostly due to RI mismatches) blind deconvolution can create artifacts that might look like mitochondria (fusion of signals into linear structures). So image a 100nm or 200nm bead and use that for deconvolution. Mariette |
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Paul Rigby |
Re: Best parameters for optical slicing
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In reply to this post by Pedro Camello
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Pedro, On our Biorad 1024UV system, using the Nikon 60x NA1.4 PlanApo objective, the final pixel size is 310nm x 310nm. Assuming optimal pixel size of 80nm for Nyquist sampling to resolve 200nm in x,y then the maximum "useful" zoom is 310/80 = 3.8. On different confocal systems the final pixel size could be quite different. I have always understood this is how Biorad calculated their optimal zoom value for that objective. Cheers Paul Dr Paul Rigby Senior Lecturer Centre for Microscopy, Characterisation and Analysis (M510) The University of Western Australia 35 Stirling Highway Crawley WA 6009 ________________________________ From: Confocal Microscopy List on behalf of Pedro J Camello Sent: Sun 10/02/2008 8:30 AM To: [hidden email] Subject: Re: Best parameters for optical slicing Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Yes, nm. Julio, you are right about lateral resolution. My reasoning is that although the optical performance does not depend on the structure, 150 nm is supposed to be good enough to sample structures of 500 nm. I would like to avoid too much oversampling. I don´t know how Biorad calculated that 2.6 zoom is the maximum advisable for this objective (it is a shame, but I never worried before about sampling rate, resolution, etc) Many thanks for your help. > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello All, > > To avoid more confusion, you both meant nanometers, not micrometers, when > talking about xy-, z-resolution and z-stepsize, correct? > E.g., "optimal" xy resolution is 200 nm, not microns, with pixel size at > 80 > nm with Nyquist, 160 nm z-stepsize, etc. > > Zoltan |
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Mayandi Sivaguru |
Re: Best parameters for optical slicing
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Pedro, I hope you have got the attachment, if not let me know as the
server rejected the post with word document attchment.
Shiv At 10:35 AM 2/11/2008, Mayandi Sivaguru wrote: Pedro, if I remember right the Biorad system calculates the optimum resolutions based on the Raleigh criterion based on the standard formulas as mentioned by Julio. But it does not accomodates the Nyquist sampling criteria, at least as far as the Radiance 2000 system, but I imagine the same in the case of 1024. Here I have a attached a table compiled with optimum, xy resolution, Z resolution, pixel sizes and zoom levels for Olympus Objectives for two different resolution settings 512x512 and three different excitation lambdas. The most commonly used objectives are highlighted. As you know the you cannot zoom the system electronically indefinitely beyond the resolving power (resolution in XY) of the objective, as this will yield empty magnification. I would suggest you try to use far field optical techniques such as STORM, STED (Stimulated emission depletion) or structured illumination coupled with linear deconcolution to resolve structures less than the diffraction limit i.e., lower than 200 nm in xy. Microscopy Facility Manager 8, Institute for Genomic Biology University of Illinois at Urbana-Champaign 1206 West Gregory Dr. Urbana, IL 61801 USA Office: 217.333.1214 Fax: 217.244.2496 [hidden email] http://core.igb.uiuc.edu |
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Glen MacDonald-2 |
Re: Best parameters for optical slicing
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In reply to this post by Pedro Camello
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal The pinhole diameter is based upon the the diameter of the Airy disk at the detector. The usual definition of optimal pinhole diameter is that the confocal aperture is set to the outside edge of the first dark ring of the Airy disk. This diameter depends upon the Magnification, emission wavelength and distance between the back focal plane of the objective and the detector. The table in your 1024 manual has converted measurements in optical units to mm of pinhole diameter. The 1.3 meter folded optical path in the scanhead allowed these systems to avoid the issues of pinhole alignment that plagued other instruments of that era. The table assumes you are using a Nikon 160 mm tube length microscope, such as a Diaphot. Notice that the equation gives two examples, a standard brightfield scope or with addition of fluorescence and DIC. If you only have epi- fluorescence on your scope, and no DIC, then use assume an total mag of 63 times lens mag and use the average of the 2 values given in the table for your lens. Nyquist sampling at 2.3 times the object frequency is adequate for detection, but to resolve the point to point Rayleigh resolution, you need to double it again to 4.6X the object frequency, especially if deconvolving. Regards, glen Glen MacDonald Core for Communication Research Virginia Merrill Bloedel Hearing Research Center Box 357923 University of Washington Seattle, WA 98195-7923 USA (206) 616-4156 [hidden email] ************************************************************************ ****** The box said "Requires WindowsXP or better", so I bought a Macintosh. ************************************************************************ ****** On Feb 9, 2008, at 3:30 PM, Pedro J Camello wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Yes, nm. > > Julio, you are right about lateral resolution. My reasoning is that > although the optical performance does not depend on the structure, > 150 nm > is supposed to be good enough to sample structures of 500 nm. I > would like > to avoid too much oversampling. > > I don´t know how Biorad calculated that 2.6 zoom is the maximum > advisable > for this objective (it is a shame, but I never worried before about > sampling rate, resolution, etc) > > Many thanks for your help. > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hello All, >> >> To avoid more confusion, you both meant nanometers, not >> micrometers, when >> talking about xy-, z-resolution and z-stepsize, correct? >> E.g., "optimal" xy resolution is 200 nm, not microns, with pixel >> size at >> 80 >> nm with Nyquist, 160 nm z-stepsize, etc. >> >> Zoltan >> >> >> On 2/9/08, Julio Vazquez <[hidden email]> wrote: >>> >>> Search the CONFOCAL archive at >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal = Hi Pedro, >>> >>> >>> I am not sure how you or Biorad are calculating the optimal pixel >>> sizes, >>> or what the specific constraints for the Biorad 1024 are. For a >>> 60x/1.4NA >>> objective, the theoretical resolution in x/y at 580 nm (if you >>> are using >>> a >>> red mito tracker) is around 200 microns (I am using the formula d >>> = 0.5 >>> x >>> lambda/ NA). Therefore, your optimal pixel size, if you use a >>> Nyquist >>> factor >>> of 2.5, should be around 80 microns. Obviously, you may get >>> slightly >>> different values depending on how your choice of wavelength. >>> >>> >>> Regarding the sampling along the z axis, it is not so much the >>> dimensions >>> of the structures (mitochondria) that matter, but the optical >>> properties >>> of >>> your scope. The axial resolution is about half the x/y >>> resolution, and >>> therefore 160 micron spacing between sections would be about >>> optimal. >>> If >>> you are trying to look at very fine structures, oversampling a >>> little >>> (both >>> in x, y, and z) may not be a bad idea, as long as your sample can >>> take >>> it >>> and doesn't bleach... >>> >>> >>> I don't think it as an issue that your x/y pixel dimensions are >>> different >>> from your z section spacing. The PSF itself is not spherically >>> symmetrical >>> (but elongated along z) and your sampling along x/y and z should >>> be set >>> accordingly. Secondly, when you load your images into whatever >>> program >>> you >>> use for doing the 3-D measurements, the image should be >>> calibrated to >>> reflect the actual x,y and z dimensions. As long as you used Nyquist >>> sampling, and you have good signal-to-noise, you should be able >>> to get >>> accurate measurements. >>> >>> >>> For deconvolution, we've had good results with both measured PSF and >>> calculated PSFs; blind should work fine too, but I haven't tried. >>> If you >>> have some reference beads, you could image them under conditions >>> similar >>> to >>> those used to image your sample, deconvolve them with the same >>> method, >>> and >>> do some measurements. That should give you an idea of how well your >>> deconvolution alrgorithm is performing. If it doesn't look good, >>> you can >>> always re-deconvolve your data with a different method. >>> >>> >>> >>> >>> >>> >>> Julio. >>> >>> >>> >>> -- >>> Julio Vazquez, PhD >>> Director of Scientific Imaging >>> Fred Hutchinson Cancer Research Center >>> 1100 Fairview Ave. N., mailstop DE-512 >>> Seattle, WA 98109-1024 >>> >>> >>> Tel: Office: 206-667-1215/ Lab: 206-667-4205 >>> FAX: 206-667-6845 >>> >>> >>> [hidden email] >>> http://www.fhcrc.org/science/shared_resources/imaging/ >>> >>> >>> >>> -------------------------------------------------- >>> *DISCLAIMER:* >>> >>> This message is confidential, intended only for the named >>> recipient(s) >>> and >>> may contain information that is privileged or exempt from disclosure >>> under >>> applicable law. If you are not the intended recipient(s), you are >>> notified >>> that the dissemination, distribution or copying of this >>> information is >>> strictly prohibited. If you received this message in error, please >>> notify >>> the sender then delete this message. >>> >>> >>> >>> >>> >>> >>> >>> On Feb 9, 2008, at 9:30 AM, Pedro J Camello wrote: >>> >>> Search the CONFOCAL archive at >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>> >>> I´m trying to image a 3D map of mitotracker-stained cells. I´m >>> using a >>> Biorad 1024, 60x, NA 1.4. My first attempts generated acceptable >>> images >>> with iris 1.6 -2 (close to the theoretical optimal iris indicated by >>> Biorad with this objective). >>> >>> >>> My 2 questions are: >>> >>> >>> 1) Using zoom 2.6 (the theoretical optimum zoom for this >>> objective) I >>> get >>> pixel size 166 µm. Since published images with my cell type >>> (pancreatic >>> acini) indicate that most of the mitochondria are around 500 nm >>> diameter >>> (similar to the measured resolution of mi micro with iris 2) my >>> intend >>> is >>> to sample at 150 nm to meet Nyquist. However, this creates a non- >>> cubic >>> voxel (166x166x150). Will be this a problem to make posterior 3D >>> visualization and volume measurements? >>> >>> >>> 2) If I need deconvolution to posterior processing and >>> measurements, is >>> it >>> essential PSF measurements or will blinded methods be enough? >>> >>> >>> Thanks in advance >>> >>> >>> -- >>> Dr Pedro J Camello >>> Dpt Physiology >>> Faculty of Veterinary Sciences >>> University of Extremadura >>> 10071 Caceres >>> Spain >>> Ph: 927257100 Extension 1321/1290 >>> Fax:927257110 >>> >>> >>> >> >> >> -- >> -- >> Zoltan Cseresnyes >> Facility manager, Imaging Suite >> Dept. of Zoology University of Cambridge >> Downing Street, Cambridge >> CB2 3EJ UK >> >> Tel.: (++44) (0)1223 769282 >> Fax.: (++44) (0)1223 336676 >> > > > -- > Dr Pedro J Camello > Dpt Physiology > Faculty of Veterinary Sciences > University of Extremadura > 10071 Caceres > Spain > Ph: 927257100 Extension 1321 > Fax:927257110 |
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Martin Wessendorf-2 |
Re: Best parameters for optical slicing
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Glen MacDonald wrote: > Nyquist sampling at 2.3 times the object frequency is adequate for > detection, but to resolve the point to point Rayleigh resolution, you > need to double it again to 4.6X the object frequency, especially if > deconvolving. I may be wrong on this, but unless they fixed it sometime while my back was turned, the only "true" zooms possible on a BioRad are 1, 2, 4 and 8x. Intermediate values requires double-scanning some lines. Using such a value can result in artifacts when deconvolving. Take care! Martin Wessendorf -- Martin Wessendorf, Ph.D. office: (612) 626-0145 Assoc Prof, Dept Neuroscience lab: (612) 624-2991 University of Minnesota Preferred FAX: (612) 624-8118 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 Minneapolis, MN 55455 **MY E-MAIL ADDRESS HAS CHANGED. PLEASE USE [hidden email] ** |
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Glen MacDonald-2 |
Re: Best parameters for optical slicing
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Yes, I forgot about zooming by powers of 2 due to the galvos. There should be a thread in the archives on that. Our 1024 was mothballed for about 2 years and I just got it going again. I've got much recall, or mistakes to repeat, on that system. Regards Glen On Feb 11, 2008, at 9:50 AM, Martin Wessendorf wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Glen MacDonald wrote: > >> Nyquist sampling at 2.3 times the object frequency is adequate for >> detection, but to resolve the point to point Rayleigh resolution, >> you need to double it again to 4.6X the object frequency, >> especially if deconvolving. > > I may be wrong on this, but unless they fixed it sometime while my > back was turned, the only "true" zooms possible on a BioRad are 1, > 2, 4 and 8x. Intermediate values requires double-scanning some > lines. Using such a value can result in artifacts when deconvolving. > > Take care! > > Martin Wessendorf > -- > Martin Wessendorf, Ph.D. office: (612) 626-0145 > Assoc Prof, Dept Neuroscience lab: (612) 624-2991 > University of Minnesota Preferred FAX: (612) 624-8118 > 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 > Minneapolis, MN 55455 > **MY E-MAIL ADDRESS HAS CHANGED. PLEASE USE [hidden email] ** |
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