Live cell Imaging with fluoview 1000
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Jean-Pierre CLAMME-2 |
Live cell Imaging with fluoview 1000
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Hi, I had my first experience today with this system for live cell imaging and all my cells under the laser didn't make it safely (the one around were just fine). So I'm curious to know if some of you used a fluoview 1000 for live cell imaging of hoechst or Fm4-64? If yes, what kind of range of power and aquisition repetition (z stack and repetition) did your cells take ? (I did a stack of 16 images in z every 2 minutes so around 1minute of imaging and 1 minute of resting) Thanks, JP - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Jean-Pierre CLAMME, PhD Senior Scientist Nitto Denko Technical 501 Via Del Monte Oceanside, CA 92058 E-mail: [hidden email] Phone: +760.435.7065 |
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Cameron Nowell |
Re: Live cell Imaging with fluoview 1000
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Hi JP, I have used an
FV1000 in the past to image live cells and larvae for 12-24 hours with no
bleaching/phototoxicity.This was done with laser power setting in the <10%
range using a multi ion argon laser and a 405nm solid state laser. Can you supply some more information? -
Which lasers do you have on your machine (Argpn Ion, Solid state
etc.)? -
How long are you imaging for -
What is the sample? -
What media are you using Cheers Cam Cameron J. Nowell Office: +61 3 9341 3155 From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Jean-Pierre
CLAMME
No virus
found in this incoming message. |
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Naomi Book |
Re: Live cell Imaging with fluoview 1000
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In reply to this post by Jean-Pierre CLAMME-2
Hi,
I have used the FV-1000 many times, for long
(up to 72 hours) live cell imaging, using HeLa cells, cos and other cell lines.
I use 2 excitations 488 and 543. the cells survive with no problem. the LIS
incubator is doing a great job.
Naomi Book
--------------------------------------------
Naomi Melamed-Book, PhD
Bio-Imaging Unit, Life Science
Institute
Hebrew University, Jerusalem, Israel.
E-mail: [hidden email]
Phone: 972-2 6585453
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----- Original Message -----
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Steffen Dietzel |
percentile calculation, ImageJ or otherwise?
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Dear all,
I have a stack of images (16bit) for which I need some measurement of the average signal intensity, to describe signal loss with increasing depth. Since the particular signal is sort of repetitive (Collagen matrix), I thought of just going for the 5-percentile (5% of all pixels in an image plane have the grayvalue X or higher). ImageJ readily calculates the median (50-percentile) but I didn't find an option for other percentiles in the program or on the plug-ins web page. It is probably easy to program it if one knows how and I am sure others have done it before but programming isn't one of my capabilities. Other suggestions to solve this problems also would be welcome Best regards Steffen -- --------------------------------------------------------------------------------------------------- Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy Mail room (for letters etc.): Marchioninistr. 15, D-81377 München Building location and address for courier, parcel services etc: Marchioninistr. 27, D-81377 München (Großhadern) Phone: +49/89/2180-76509 Fax-to-email: +49/89/2180-9976509 skype: steffendietzel e-mail: [hidden email] |
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You can set the threshold at your chosen value and check the "Limit to threshold" box in Set Measurements. Then Analyze - Measure.
-----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel Sent: Friday, January 30, 2009 5:25 AM To: [hidden email] Subject: percentile calculation, ImageJ or otherwise? Dear all, I have a stack of images (16bit) for which I need some measurement of the average signal intensity, to describe signal loss with increasing depth. Since the particular signal is sort of repetitive (Collagen matrix), I thought of just going for the 5-percentile (5% of all pixels in an image plane have the grayvalue X or higher). ImageJ readily calculates the median (50-percentile) but I didn't find an option for other percentiles in the program or on the plug-ins web page. It is probably easy to program it if one knows how and I am sure others have done it before but programming isn't one of my capabilities. Other suggestions to solve this problems also would be welcome Best regards Steffen -- --------------------------------------------------------------------------------------------------- Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy Mail room (for letters etc.): Marchioninistr. 15, D-81377 München Building location and address for courier, parcel services etc: Marchioninistr. 27, D-81377 München (Großhadern) Phone: +49/89/2180-76509 Fax-to-email: +49/89/2180-9976509 skype: steffendietzel e-mail: [hidden email] |
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Steffen Dietzel |
Re: percentile calculation, ImageJ or otherwise?
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At 14:56 30.01.2009, you wrote:
>You can set the threshold at your chosen value >and check the "Limit to threshold" box in Set >Measurements. Then Analyze - Measure. That's the solution to a different problem. What I'd like to get an answer for is the question "At which level do I have to set the threshold so that 5% of all image pixels are above threshold?" Steffen >-----Original Message----- >From: Confocal Microscopy List >[mailto:[hidden email]] On Behalf Of Steffen Dietzel >Sent: Friday, January 30, 2009 5:25 AM >To: [hidden email] >Subject: percentile calculation, ImageJ or otherwise? > >Dear all, > >I have a stack of images (16bit) for which I need >some measurement of the average signal intensity, >to describe signal loss with increasing depth. >Since the particular signal is sort of repetitive >(Collagen matrix), I thought of just going for >the 5-percentile (5% of all pixels in an image >plane have the grayvalue X or higher). > >ImageJ readily calculates the median >(50-percentile) but I didn't find an option for >other percentiles in the program or on the >plug-ins web page. It is probably easy to program >it if one knows how and I am sure others have >done it before but programming isn't one of my capabilities. > >Other suggestions to solve this problems also would be welcome > >Best regards > >Steffen > >-- >--------------------------------------------------------------------------------------------------- >Steffen Dietzel, PD Dr. rer. nat >Ludwig-Maximilians-Universität München >Walter-Brendel-Zentrum für experimentelle Medizin (WBex) >Head of light microscopy > >Mail room (for letters etc.): >Marchioninistr. 15, D-81377 München > >Building location and address for courier, parcel services etc: >Marchioninistr. 27, D-81377 München (Großhadern) > >Phone: +49/89/2180-76509 >Fax-to-email: +49/89/2180-9976509 >skype: steffendietzel >e-mail: [hidden email] |
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Julio Vazquez |
Re: percentile calculation, ImageJ or otherwise?
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In reply to this post by Steffen Dietzel
Hi Steffen,
there are probably more elegant and/or faster ways, but you could: 1) with ImageJ, plot the Histogram (Analyze > Histogram ) and then get the values as a list. You can paste the list in Excel and use the Excel "Sum" function to determine where the top 5% intensities are. Not elegant, bit will give you an answer. 2) with Photoshop, open your image (in greyscale mode) and use the curves tool. In the Curves window, use the "Options" button for the Auto adjustment to set the minimum at zero and the maximum clipping at 5%. Apply to your image and then look in the curves window where Photoshop set the thresholds. -- 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 Jan 30, 2009, at 2:25 AM, Steffen Dietzel wrote:
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Jeff Reece |
Re: percentile calculation, ImageJ or otherwise?
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Dear Steffen and List,
ImageJ does something very much like Julio's option 2: choose
Image->Adjust->Brightness/Contrast, and every click on the "Auto"
button clips a certain % of pixels from the high end of the display
contrast (and showing the pixel value where that occurs). I don't know
what the % is but wouldn't be hard to figure out.
Cheers,
Jeff
--------------------
Jeff M. Reece
Reecent Technologies, LLC
Honing the edge of quantitative microscopy
919-672-4681 ----- Original Message -----
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Shalin Mehta |
Re: percentile calculation, ImageJ or otherwise?
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Dear Steffen and list,
Unless I am missing a point here, I think the simple and best strategy is just to compute the average intensity of each plane to examine how signal decays with depth. Averaging p = mxn pixels will take time to the order of 'p' operations (in algorithm complexity parlance averaging is an O(n) operation). Obtaining percentile involves sorting of all pixels which is an O(nlogn) operation in general. i.e., sorting p=mxn pixels will take approximately p*log(p) operations. Computing histogram is therefore slower than computing average, but that's the intuitive way if one really needs to compute percentile. best regards shalin On Sat, Jan 31, 2009 at 3:26 AM, Jeff Reece <[hidden email]> wrote:
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Claire Brown |
Re: Live cell Imaging with fluoview 1000
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In reply to this post by Jean-Pierre CLAMME-2
I just finished writing a paper on live cell imaging. It will be published
in J. Cell Science in early March. I sent Jean-Pierre a copy if anyone is interested please contact me offline. I would be happy to share a copy of the final copy of the manuscript. We went through all the imaging platforms and made suggestions on how to improve light throughput and give suggested laser powers among other things. I hope it will be a useful contribution to the community. Sincerely, Claire |
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Elberger, Andrea June |
Re: Live cell Imaging with fluoview 1000
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CLAIRE - I would appreciate a copy of your article. Your posting to the
confocal listserv did not list your email contact. Thanks. Dr. Andrea J. Elberger Professor, Anatomy and Neurobiology Director, Confocal Laser Scanning Microscope Facility The University of Tennessee Health Science Center 855 Monroe Avenue Memphis, TN 38163 U.S.A. tel: 901-448-4101 FAX: 901-448-7193 <mailto: [hidden email]> -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Claire Brown Sent: Saturday, January 31, 2009 7:34 AM To: [hidden email] Subject: Re: Live cell Imaging with fluoview 1000 I just finished writing a paper on live cell imaging. It will be published in J. Cell Science in early March. I sent Jean-Pierre a copy if anyone is interested please contact me offline. I would be happy to share a copy of the final copy of the manuscript. We went through all the imaging platforms and made suggestions on how to improve light throughput and give suggested laser powers among other things. I hope it will be a useful contribution to the community. Sincerely, Claire |
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Elberger, Andrea June |
Re: Live cell Imaging with fluoview 1000 - My Apologies
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In reply to this post by Claire Brown
My apologies to the Confocal Listserv members for my recent reply
posted. The email format set up on my work computer did not show the complete title line - there was an email address listed. Dr. Andrea J. Elberger Professor, Anatomy and Neurobiology Director, Confocal Laser Scanning Microscope Facility The University of Tennessee Health Science Center 855 Monroe Avenue Memphis, TN 38163 U.S.A. tel: 901-448-4101 FAX: 901-448-7193 <mailto: [hidden email]> -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Claire Brown Sent: Saturday, January 31, 2009 7:34 AM To: [hidden email] Subject: Re: Live cell Imaging with fluoview 1000 I just finished writing a paper on live cell imaging. It will be published in J. Cell Science in early March. I sent Jean-Pierre a copy if anyone is interested please contact me offline. I would be happy to share a copy of the final copy of the manuscript. We went through all the imaging platforms and made suggestions on how to improve light throughput and give suggested laser powers among other things. I hope it will be a useful contribution to the community. Sincerely, Claire |
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Steffen Dietzel |
Re: percentile calculation, ImageJ or otherwise?
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In reply to this post by Shalin Mehta
Dear all,
thanks for all the replies on and off list. The latter included an e-mail by Jerome Mutterer with a short macro that does the job. With his permission, I post it here so that it gets included in the confocal archive together with the question. Cheers Steffen
Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy Mail room (for letters etc.): Marchioninistr. 15, D-81377 München Building location and address for courier, parcel services etc: Marchioninistr. 27, D-81377 München (Großhadern) |
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Monique Vasseur |
Resolution for fluorescence, brightfield and luminescence
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In reply to this post by Claire Brown
Dear all,
My question is: On the same microscope, same objective, same immersion medium and same sample, is there a difference in resolution depending of the microscopy method I am using (fluorescence, luminescence or brightfield) since the lightpath is not the same? Is it correct to consider the following? For brightfield: r = (1.22 * illumination wavelenght)/(NA objective + NA condenser) For fluorescence and confocal: r = (0.61 * excitation (?) wavelenght) / NA objective For luminescence: r = (0.61 * emission (?) wavelenght) / NA objective Thanks in advance, monique |
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Julio Vazquez |
Re: Resolution for fluorescence, brightfield and luminescence
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Hi Monique,
I won't attempt to give you a definite answer on this, but point you to some literature on the topic: plus a couple of points: for fluorescence, I believe the relevant wavelength generally used in the formula would be the average of the peak excitation and peak emission values, although I'm sure the real value would be a more complex one (if your emission as a tail in the longer wavelengths, these will spread your PSF, thus reducing resolution) The second point that I would stress (and some will probably disagree with this) is that the definition of the minimum resolvable distance is a somewhat arbitrary one. Namely, if you are imaging two diffraction limited spots of similar intensity, their images will be two PSFs, and the minimum resolvable distance will be the distance between the center of the two PSFs so that when you do an intensity profile you see a dip in contrast that you deem is sufficient for you to know with a certain degree of certainty that you have two spots (about 75% of max peak values). Obviously, this depends also on the contrast and noise in your image, and also depends on the fact that you have a priori knowledge that you are looking at two diffraction limited spots. In a real situation, you don't necessarily know that, and therefore you criteria for "certainty" may be a bit different... 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 19, 2009, at 8:10 AM, Vasseur Monique wrote:
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Julio Vazquez |
Re: Resolution for fluorescence, brightfield and luminescence
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In reply to this post by Monique Vasseur
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Sorry... one (major) correction: The formulas for widefield and confocal fluorescence will be different, since in single point scanning confocal, the excitation light is also focused into a PSF and therefore affects resolution, while in widefield fluorescence, illumination is uniform across the field of view, and therefore only the emission wavelength should matter. According to the Olympus/FSU web site I listed: r (lateral) = 0.6 lambda (em) /NA for widefield fluorescence r (lateral) = 0.4 lambda / NA for confocal fluorescence (with lambda = average of Ex and Em, or maybe the geometrical average (square root of sum of squares)) and I expect the formula for luminescence to be the same as the one for widefield florescence (because in both cases you are imaging light emitting points) However, in the Zeiss paper, they claim that lateral confocal resolution only depends on the excitation wavelength (and the pinhole aperture), and their formulas are more complex. I think your formula for brightfield is the generally accepted one. My understanding is that these formulas are all approximations based on certain assumptions and definitions. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 On Feb 19, 2009, at 8:10 AM, Vasseur Monique wrote:
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In reply to this post by Julio Vazquez
In widefield fluorescence the emission wavelength is the only one that matters. Using the Rayleigh criterion the resolution (r) will therefore be r = 0.61 x wavelength / NA - though as Julio says this is a somewhat arbitrary criterion.
In confocal both wavelengths count, and if the pinhole is infinitely small the Rayleigh resolution r becomes r / sqrt2, so is somewhat better, though most microscopists work with a pinhole size equal to the Airy disk, which will not give any resolution improvement. However the contribution of the shorter excitation wavelength still means you do slightly better than in widefield (for a simple calculation just assume a wavelength midway between Ex & Em). In multiphoton fluorescence only the excitation wavelength counts, but the resolution becomes r / sqrt2 without the need for any pinhole - this helps to counteract the loss from the longer wavelength. For more details see Guy Cox & Colin Sheppard, 2004. Practical limits of resolution in confocal and non-linear microscopy. Microscopy Research & Technique, 63, 18-22 In transmitted light microscopy resolution is determined by diffraction at the sample (which cannot occur in fluorescence) and this is the famous relation given by Abbe: r = wavelength / 2xNA. This assumes that the condenser NA equals or exceeds the objective NA, and so long as this is the case condenser NA doesn't come into it. With a very small condenser NA (parallel illumination) the resolution is worse: r = wavelength / NA. Anything in between will give you resolution in between these extremes. Thus while condenser NA does affect the result it has much less effect than objective NA. Unlike Rayleigh, Abbe is a 'hard' criterion - it describes the limiting case which a lens can possibly resolve, since rays from smaller detail will not enter the objective. I hope this helps, Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net <http://www.guycox.net/> ________________________________ From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julio Vazquez Sent: Friday, 20 February 2009 4:23 AM To: [hidden email] Subject: Re: Resolution for fluorescence, brightfield and luminescence Hi Monique, I won't attempt to give you a definite answer on this, but point you to some literature on the topic: http://micro.magnet.fsu.edu/primer/anatomy/numaperture.html http://www.olympusfluoview.com/theory/resolutionintro.html http://zeiss-campus.magnet.fsu.edu/referencelibrary/pdfs/ZeissConfocalPrinciples.pdf plus a couple of points: for fluorescence, I believe the relevant wavelength generally used in the formula would be the average of the peak excitation and peak emission values, although I'm sure the real value would be a more complex one (if your emission as a tail in the longer wavelengths, these will spread your PSF, thus reducing resolution) The second point that I would stress (and some will probably disagree with this) is that the definition of the minimum resolvable distance is a somewhat arbitrary one. Namely, if you are imaging two diffraction limited spots of similar intensity, their images will be two PSFs, and the minimum resolvable distance will be the distance between the center of the two PSFs so that when you do an intensity profile you see a dip in contrast that you deem is sufficient for you to know with a certain degree of certainty that you have two spots (about 75% of max peak values). Obviously, this depends also on the contrast and noise in your image, and also depends on the fact that you have a priori knowledge that you are looking at two diffraction limited spots. In a real situation, you don't necessarily know that, and therefore you criteria for "certainty" may be a bit different... 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 19, 2009, at 8:10 AM, Vasseur Monique wrote: Dear all, My question is: On the same microscope, same objective, same immersion medium and same sample, is there a difference in resolution depending of the microscopy method I am using (fluorescence, luminescence or brightfield) since the lightpath is not the same? Is it correct to consider the following? For brightfield: r = (1.22 * illumination wavelenght)/(NA objective + NA condenser) For fluorescence and confocal: r = (0.61 * excitation (?) wavelenght) / NA objective For luminescence: r = (0.61 * emission (?) wavelenght) / NA objective Thanks in advance, monique No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.10.25/1958 - Release Date: 18/02/2009 8:57 AM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.11.1/1961 - Release Date: 19/02/2009 6:45 PM |
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Arwed Weigel |
Re: Resolution for fluorescence, brightfield and luminescence
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In reply to this post by Monique Vasseur
All these formulas for confocal microscope resolution assume a
homogeneously illuminated back-aperture, which is not always the case in commercially available microscopes due to the Gaussian intensity profile of the excitation laser beam. Therefore, the lateral resolution of a confocal microscope can even be worse than of a widefield fluorescence microscope with arc-lamp illumination. -- arwed weigel abteilung fuer neurophysiology und zellulaere biophysik universitaet goettingen humboldtallee 23 37073 goettingen phon +49 (0)551 3912201 fax +49 (0)551 398399 |
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In reply to this post by Guy Cox
The formula for resolution in bright field is not exact. The problem was solved by Hopkins and Barham, Proc Phys Soc B, 63, 737-744, 1950. The effect of condenser on resolution is also shown on the Olympus site, http://www.olympusfluoview.com/java/resolution3d/index.html
Mike -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox Sent: Thursday, February 19, 2009 7:49 PM To: [hidden email] Subject: Re: Resolution for fluorescence, brightfield and luminescence In widefield fluorescence the emission wavelength is the only one that matters. Using the Rayleigh criterion the resolution (r) will therefore be r = 0.61 x wavelength / NA - though as Julio says this is a somewhat arbitrary criterion. In confocal both wavelengths count, and if the pinhole is infinitely small the Rayleigh resolution r becomes r / sqrt2, so is somewhat better, though most microscopists work with a pinhole size equal to the Airy disk, which will not give any resolution improvement. However the contribution of the shorter excitation wavelength still means you do slightly better than in widefield (for a simple calculation just assume a wavelength midway between Ex & Em). In multiphoton fluorescence only the excitation wavelength counts, but the resolution becomes r / sqrt2 without the need for any pinhole - this helps to counteract the loss from the longer wavelength. For more details see Guy Cox & Colin Sheppard, 2004. Practical limits of resolution in confocal and non-linear microscopy. Microscopy Research & Technique, 63, 18-22 In transmitted light microscopy resolution is determined by diffraction at the sample (which cannot occur in fluorescence) and this is the famous relation given by Abbe: r = wavelength / 2xNA. This assumes that the condenser NA equals or exceeds the objective NA, and so long as this is the case condenser NA doesn't come into it. With a very small condenser NA (parallel illumination) the resolution is worse: r = wavelength / NA. Anything in between will give you resolution in between these extremes. Thus while condenser NA does affect the result it has much less effect than objective NA. Unlike Rayleigh, Abbe is a 'hard' criterion - it describes the limiting case which a lens can possibly resolve, since rays from smaller detail will not enter the objective. I hope this helps, Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net <http://www.guycox.net/> ________________________________ From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julio Vazquez Sent: Friday, 20 February 2009 4:23 AM To: [hidden email] Subject: Re: Resolution for fluorescence, brightfield and luminescence Hi Monique, I won't attempt to give you a definite answer on this, but point you to some literature on the topic: http://micro.magnet.fsu.edu/primer/anatomy/numaperture.html http://www.olympusfluoview.com/theory/resolutionintro.html http://zeiss-campus.magnet.fsu.edu/referencelibrary/pdfs/ZeissConfocalPrinciples.pdf plus a couple of points: for fluorescence, I believe the relevant wavelength generally used in the formula would be the average of the peak excitation and peak emission values, although I'm sure the real value would be a more complex one (if your emission as a tail in the longer wavelengths, these will spread your PSF, thus reducing resolution) The second point that I would stress (and some will probably disagree with this) is that the definition of the minimum resolvable distance is a somewhat arbitrary one. Namely, if you are imaging two diffraction limited spots of similar intensity, their images will be two PSFs, and the minimum resolvable distance will be the distance between the center of the two PSFs so that when you do an intensity profile you see a dip in contrast that you deem is sufficient for you to know with a certain degree of certainty that you have two spots (about 75% of max peak values). Obviously, this depends also on the contrast and noise in your image, and also depends on the fact that you have a priori knowledge that you are looking at two diffraction limited spots. In a real situation, you don't necessarily know that, and therefore you criteria for "certainty" may be a bit different... 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 19, 2009, at 8:10 AM, Vasseur Monique wrote: Dear all, My question is: On the same microscope, same objective, same immersion medium and same sample, is there a difference in resolution depending of the microscopy method I am using (fluorescence, luminescence or brightfield) since the lightpath is not the same? Is it correct to consider the following? For brightfield: r = (1.22 * illumination wavelenght)/(NA objective + NA condenser) For fluorescence and confocal: r = (0.61 * excitation (?) wavelenght) / NA objective For luminescence: r = (0.61 * emission (?) wavelenght) / NA objective Thanks in advance, monique No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.10.25/1958 - Release Date: 18/02/2009 8:57 AM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.11.1/1961 - Release Date: 19/02/2009 6:45 PM |
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In reply to this post by Arwed Weigel
This is completely true but, while early systems did not provide adjustable beam expansion, most modern ones do. In case of doubt go to maximum expansion - you'll lose some light but there's usually plenty to spare (unless you are using a green He-Ne). Then you get a reasonable approximation to homogeneous illumination with most objectives - close enough, anyway, given all the other potential confounding factors.
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 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Arwed Weigel Sent: Friday, 20 February 2009 10:37 PM To: [hidden email] Subject: Re: Resolution for fluorescence, brightfield and luminescence All these formulas for confocal microscope resolution assume a homogeneously illuminated back-aperture, which is not always the case in commercially available microscopes due to the Gaussian intensity profile of the excitation laser beam. Therefore, the lateral resolution of a confocal microscope can even be worse than of a widefield fluorescence microscope with arc-lamp illumination. -- arwed weigel abteilung fuer neurophysiology und zellulaere biophysik universitaet goettingen humboldtallee 23 37073 goettingen phon +49 (0)551 3912201 fax +49 (0)551 398399 No virus found in this incoming message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.11.1/1961 - Release Date: 19/02/2009 6:45 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.552 / Virus Database: 270.11.1/1961 - Release Date: 19/02/2009 6:45 PM |
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