Coverslip
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Hello All:
I need to know how I can measure the depth from the cover slip,is there any marked or fluorescent coverslip that I can use for this purpose?
Thanks for any suggestion
Sarah
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello all, I would like some input into an idea I have for obtaining a PSF. I study peroxisomes using yeast as a model system for understanding how they are created. In yeast, peroxisomes have a well characterized morphology, being spherical organelles with a diameter of 100 to 200nm. Other than this size variability I think they are excellent candidates for obtaining a PSF as they can be easily, fluorescently labelled (by targeting fluorescent protein chimeras to their matrix), are similar in size to what is typically used to obtain PSF's, and are "embedded" in the sample. I do a lot of live cell imaging, using a LSM510 Meta and am always looking for ways to improve my system. As a result most of my images are fairly noisy and I rely on deconvolution to remove the noise, and improve contrast and resolution. My initial attempts at using peroxisomes for this purpose have provided me with a PSF that is slightly different from what I obtain with fluorescent beads (the peroxisome derived PSF is less symmetrical) and provides, in my estimation, a more realistic result. Your thoughts and concerns on this idea would be most welcome. Fred Fred D. Mast Department of Cell Biology Medical Sciences Building Room 5-14 University of Alberta Edmonton, Alberta, T6G 2H7 Canada Tel: 1-780-492-7407 [hidden email] |
 
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John Oreopoulos |
Re: Use of a fluorescent organelle in determining a PSF
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal This sounds like a great idea to me, Fred, but how do you account for the movement of the peroxisome and the eventual photobleaching of some of the fluorescent labels during the PSF acquisition? John Oreopoulos On 22-Feb-08, at 4:54 PM, Fred Mast wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello all, > > I would like some input into an idea I have for obtaining a PSF. I > study peroxisomes using yeast as a model system for understanding > how they are created. In yeast, peroxisomes have a well > characterized morphology, being spherical organelles with a > diameter of 100 to 200nm. Other than this size variability I think > they are excellent candidates for obtaining a PSF as they can be > easily, fluorescently labelled (by targeting fluorescent protein > chimeras to their matrix), are similar in size to what is typically > used to obtain PSF's, and are "embedded" in the sample. I do a lot > of live cell imaging, using a LSM510 Meta and am always looking for > ways to improve my system. As a result most of my images are fairly > noisy and I rely on deconvolution to remove the noise, and improve > contrast and resolution. My initial attempts at using peroxisomes > for this purpose have provided me with a PSF that is slightly > different from what I obtain with fluorescent beads (the peroxisome > derived PSF is less symmetrical) and provides, in my estimation, a > more realistic result. Your thoughts and concerns on this idea > would be most welcome. > > Fred > > Fred D. Mast > Department of Cell Biology > Medical Sciences Building Room 5-14 > University of Alberta > Edmonton, Alberta, T6G 2H7 > Canada > > Tel: 1-780-492-7407 > [hidden email] |
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Sarah Kefayati |
Re: Use of a fluorescent organelle in determining a PSF
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In reply to this post by Fred Mast
Search the CONFOCAL archive at
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Dear Fred:
You may find this article useful,they also extract the PSF from the spherical shape objects from the sample.
Sarah Kefayati
Biophysics MSc student
Brock university
St. Catharines
CANADA
On Fri, Feb 22, 2008 at 4:54 PM, Fred Mast <[hidden email]> wrote: Search the CONFOCAL archive at |
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Glen MacDonald-2 |
Re: Use of a fluorescent organelle in determining a PSF
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In reply to this post by Fred Mast
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Fred, Do you know for certain that the chosen peroxisome has a homogenous distribution of fluoropore? Looking at isolated peroxisomes might give a hint and provide a nice control as to whether the distortion of the PSF in comparison to plain beads is due to the optical properties of the sample. And, as you estimate, presenting a more realistic PSF. I'm assuming you are looking at fixed yeasties, or John's concern regarding motion would be very valid. 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 22, 2008, at 1:54 PM, Fred Mast wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello all, > > I would like some input into an idea I have for obtaining a PSF. I > study peroxisomes using yeast as a model system for understanding > how they are created. In yeast, peroxisomes have a well > characterized morphology, being spherical organelles with a > diameter of 100 to 200nm. Other than this size variability I think > they are excellent candidates for obtaining a PSF as they can be > easily, fluorescently labelled (by targeting fluorescent protein > chimeras to their matrix), are similar in size to what is typically > used to obtain PSF's, and are "embedded" in the sample. I do a lot > of live cell imaging, using a LSM510 Meta and am always looking for > ways to improve my system. As a result most of my images are fairly > noisy and I rely on deconvolution to remove the noise, and improve > contrast and resolution. My initial attempts at using peroxisomes > for this purpose have provided me with a PSF that is slightly > different from what I obtain with fluorescent beads (the peroxisome > derived PSF is less symmetrical) and provides, in my estimation, a > more realistic result. Your thoughts and concerns on this idea > would be most welcome. > > Fred > > Fred D. Mast > Department of Cell Biology > Medical Sciences Building Room 5-14 > University of Alberta > Edmonton, Alberta, T6G 2H7 > Canada > > Tel: 1-780-492-7407 > [hidden email] |
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Michael Schell |
Re: Use of a fluorescent organelle in determining a PSF
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In reply to this post by Fred Mast
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Since some parts of the peroxisomal lumen are comprised of a "crystalline matrix" of protein, would this affect the refractive index and thus create spherical aberration in the PSF? Michael J. Schell, Ph.D., CIV, USUHS Assist. Professor Dept. of Pharmacology Uniformed Services University 4301 Jones Bridge Rd. Bethesda, MD 20814-3220 tel: (301) 295-3249 [hidden email] >>> Glen MacDonald <[hidden email]> 02/22/08 5:47 PM >>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Fred, Do you know for certain that the chosen peroxisome has a homogenous distribution of fluoropore? Looking at isolated peroxisomes might give a hint and provide a nice control as to whether the distortion of the PSF in comparison to plain beads is due to the optical properties of the sample. And, as you estimate, presenting a more realistic PSF. I'm assuming you are looking at fixed yeasties, or John's concern regarding motion would be very valid. 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 22, 2008, at 1:54 PM, Fred Mast wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello all, > > I would like some input into an idea I have for obtaining a PSF. I > study peroxisomes using yeast as a model system for understanding > how they are created. In yeast, peroxisomes have a well > characterized morphology, being spherical organelles with a > diameter of 100 to 200nm. Other than this size variability I think > they are excellent candidates for obtaining a PSF as they can be > easily, fluorescently labelled (by targeting fluorescent protein > chimeras to their matrix), are similar in size to what is typically > used to obtain PSF's, and are "embedded" in the sample. I do a lot > of live cell imaging, using a LSM510 Meta and am always looking for > ways to improve my system. As a result most of my images are fairly > noisy and I rely on deconvolution to remove the noise, and improve > contrast and resolution. My initial attempts at using peroxisomes > for this purpose have provided me with a PSF that is slightly > different from what I obtain with fluorescent beads (the peroxisome > derived PSF is less symmetrical) and provides, in my estimation, a > more realistic result. Your thoughts and concerns on this idea > would be most welcome. > > Fred > > Fred D. Mast > Department of Cell Biology > Medical Sciences Building Room 5-14 > University of Alberta > Edmonton, Alberta, T6G 2H7 > Canada > > Tel: 1-780-492-7407 > [hidden email] |
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In reply to this post by Fred Mast
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Image-Adaptive Deconvolution for Three-Dimensional Deep Biological Imaging by Monvel et al
They used naturally occuring fluorescent spots to find real PSF From: Confocal Microscopy List on behalf of Fred Mast Sent: Fri 2/22/2008 4:54 PM To: [hidden email] Subject: Use of a fluorescent organelle in determining a PSF Search the CONFOCAL archive at |
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In reply to this post by Michael Schell
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Thank-you for all those who replied... Yeast don't have a paracrystalline matrix core like other organisms, however, it is packed with protein so it would have a higher refractive index than it's surroundings. That being said, would it be any different than using beads, which have a refractive index closer to that of glass? Glen your point about homogenous distribution is a good one. While I don't believe it to be an issue here (as the peroxisome matrix of yeast is a homogenous compartment), I'm not really sure how I would go about evaluating this... Thanks again for the input, Fred On 22-Feb-08, at 5:03 PM, Michael Schell wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Since some parts of the peroxisomal lumen are comprised of a > "crystalline matrix" of protein, would this affect the refractive > index and thus create spherical aberration in the PSF? > > Michael J. Schell, Ph.D., CIV, USUHS > Assist. Professor > Dept. of Pharmacology > Uniformed Services University > 4301 Jones Bridge Rd. > Bethesda, MD 20814-3220 > tel: (301) 295-3249 > [hidden email] >>>> Glen MacDonald <[hidden email]> 02/22/08 5:47 PM >>> > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi Fred, > Do you know for certain that the chosen peroxisome has a homogenous > distribution of fluoropore? Looking at isolated peroxisomes might > give a hint and provide a nice control as to whether the distortion > of the PSF in comparison to plain beads is due to the optical > properties of the sample. And, as you estimate, presenting a more > realistic PSF. I'm assuming you are looking at fixed yeasties, or > John's concern regarding motion would be very valid. > > 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 22, 2008, at 1:54 PM, Fred Mast wrote: > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hello all, >> >> I would like some input into an idea I have for obtaining a PSF. I >> study peroxisomes using yeast as a model system for understanding >> how they are created. In yeast, peroxisomes have a well >> characterized morphology, being spherical organelles with a >> diameter of 100 to 200nm. Other than this size variability I think >> they are excellent candidates for obtaining a PSF as they can be >> easily, fluorescently labelled (by targeting fluorescent protein >> chimeras to their matrix), are similar in size to what is typically >> used to obtain PSF's, and are "embedded" in the sample. I do a lot >> of live cell imaging, using a LSM510 Meta and am always looking for >> ways to improve my system. As a result most of my images are fairly >> noisy and I rely on deconvolution to remove the noise, and improve >> contrast and resolution. My initial attempts at using peroxisomes >> for this purpose have provided me with a PSF that is slightly >> different from what I obtain with fluorescent beads (the peroxisome >> derived PSF is less symmetrical) and provides, in my estimation, a >> more realistic result. Your thoughts and concerns on this idea >> would be most welcome. >> >> Fred >> >> Fred D. Mast >> Department of Cell Biology >> Medical Sciences Building Room 5-14 >> University of Alberta >> Edmonton, Alberta, T6G 2H7 >> Canada >> >> Tel: 1-780-492-7407 >> [hidden email] > Fred D. Mast Department of Cell Biology Medical Sciences Building Room 5-14 University of Alberta Edmonton, Alberta, T6G 2H7 Canada Tel: 1-780-492-7407 [hidden email] |
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In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
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Dear Sarah,
you could use reflections from the coverglass which are
characteristic for upper and lower edge of the cover glass and easy to
spot.
regards, jens
--- Dr.
Jens Rietdorf[hidden email] From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sarah Kefayati Sent: Freitag, 22. Februar 2008 21:42 To: [hidden email] Subject: Coverslip Hello All:
I need to know how I can measure the depth from the cover slip,is there any
marked or fluorescent coverslip that I can use for this purpose?
Thanks for any suggestion
Sarah
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Dear Sarah,
If you look at reflection passed through pin-hole you should be able to spot the air-glass and glass-air interface (i.e. top and bottom surfaces of the coverslip respectively) with sub-micron accuracy. As light would refract on entering through top surface, you will need to apply simple correction to calculate coverslip thickness from z-distance measured by motor. From simple calculation, the correction should be: t_coverslip/z_motor=cot(theta2)/cot(theat1), [just to avoid misreading cot is cotangent] where theta1= half-aperture angle as per NA of the objective in air (i.e. inversin(NA)) and theta2= half-aperture angle after refraction from air-glass interface (therefore, theta1=1.51theta2). But I am not sure whether the coverslip correction done by the objective will affect this correction factor!! May be those who understand internals of objectives more can comment on this. Hope this helps Shalin On Mon, Feb 25, 2008 at 4:29 AM, Rietdorf, Jens <[hidden email]> wrote:
-- ~~~~~~~~~~~~~~~~~~~~~~~~~ Shalin Mehta mobile: +65-90694182 blog: shalin.wordpress.com ~~~~~~~~~~~~~~~~~~~~~~~~~~ Bioimaging Lab, Block-E3A, #7-10 Div of Bioengineering, NUS Singapore 117574 website: http://www.bioeng.nus.edu.sg/optbioimaging/colin/index.html Liver Cancer Functional Genomics Lab, #6-05 National Cancer Centre, Singapore 169610 ~~~~~~~~~~~~~~~~~~~~~~~~~~~ What is light? ''Light behaves like Voltaire, the French genius, 250 years ago. He was born as a conservative Catholic, he lived as a liberal Protestant, and he returned to Catholicism when he faced the end of his life. Light is born as photons. It travels through space as a wave, and it dies finally as a photon.'' - Einstein as quoted in Lohmann's paper (JOSA-A, Vol. 17, No.10, pp.1755-1762) |
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In reply to this post by Rietdorf, Jens
Search the CONFOCAL archive at
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How to do you that? See it?
What is the microscope setup? Microscopie et imagerie Département de biochimie Université de Montréal C.P. 6128, succursale Centre-ville Montréal QC H3C
3J7 Canada tél. (514) 343-6111 poste 5148 De :
Confocal Microscopy List [mailto:[hidden email]] De la part de Rietdorf, Jens Dear Sarah, you could use reflections from the
coverglass which are characteristic for upper and lower edge of the cover glass
and easy to spot. regards,
jens --- Dr.
Jens Rietdorf[hidden email] From:
Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sarah Kefayati Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello All: I need to know how I can measure the depth from the cover slip,is there
any marked or fluorescent coverslip that I can use for this purpose? Thanks for any suggestion Sarah |
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Dear Monique,
We need to use confocal and collect reflected laser. One will need to setup a light path that allows passage of reflected laser to a detector which has pin-hole in front - which should be possible on most of the microscopes as the dichroic never completely gets rid of excitation laser. At air-glass and glass-air interfaces, laser will be reflected much more strongly than within coverslip. This residual laser from dichroic should be detectable if the emission filter is removed from the path. The confocal pin-hole will provide strong depth descrimination and allow measurement of coverslip to submicron accuracy. The correction factor that I mentioned assumes normal incidence of laser on coverslip, so the use 'beam-parking' or 'point-scanning' mode at the center of field of view is required. Regards Shalin On Mon, Feb 25, 2008 at 11:12 PM, Vasseur Monique <[hidden email]> wrote:
-- ~~~~~~~~~~~~~~~~~~~~~~~~~ Shalin Mehta mobile: +65-90694182 blog: shalin.wordpress.com ~~~~~~~~~~~~~~~~~~~~~~~~~~ Bioimaging Lab, Block-E3A, #7-10 Div of Bioengineering, NUS Singapore 117574 website: http://www.bioeng.nus.edu.sg/optbioimaging/colin/index.html Liver Cancer Functional Genomics Lab, #6-05 National Cancer Centre, Singapore 169610 ~~~~~~~~~~~~~~~~~~~~~~~~~~~ What is light? ''Light behaves like Voltaire, the French genius, 250 years ago. He was born as a conservative Catholic, he lived as a liberal Protestant, and he returned to Catholicism when he faced the end of his life. Light is born as photons. It travels through space as a wave, and it dies finally as a photon.'' - Einstein as quoted in Lohmann's paper (JOSA-A, Vol. 17, No.10, pp.1755-1762) |
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