Re: Zeiss SD

Posted by Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Zeiss-SD-tp2303456p2311333.html

Sure, but that applies to any confocal - optical section thickness

goes as the inverse square of the NA.  By and large if you want

optimal optical sectioning you won't use a spinning disk anyway.

My point was that there will almost always be more than one lens

which matches a given pinhole size.  

 

                                                                           Guy 

 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
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Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
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---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of RICHARD BURRY
Sent: Thursday, 12 February 2009 12:20 AM
To: [hidden email]
Subject: Re: Zeiss SD

Guy
The key is the thickness of the optical section.  While a 100x 1.4 N.A. objective with a 30 um pin hole has an optical section thickness of 0.7 um, a 20x 0.45 N.A. objective has an optical section thickness of 6.4 um.  So you loose a lot with a 20x not only in optical section thickness but also in through put (because of the lower N.A).
Dick
 
Richard W. Burry, Ph.D.
Department of Neuroscience, College of Medicine
Campus Microscopy and Imaging Facility, Director
The Ohio State University
Associate Editor, Journal of Histochemistry and Cytochemistry
277 Biomedical Research Tower
460 West Twelfth Avenue
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Voice 614.292.2814  Cell 614.638.3345  Fax [phone]

----- Original Message -----
From: Guy Cox <[hidden email]>
Date: Wednesday, February 11, 2009 5:44 am
Subject: Re: Zeiss SD
To: [hidden email]

> Presumably the problem is that the Yokogawa is designed for x60 NA 1.4 - > it's the magnification as well as the NA that determines the size of the Airy > disk at the pinhole.  So a x20 NA 0.5, which is a common lens, ought to > work pretty well (one third the mag and ~ one third the NA). >                                                                                 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 [phone]     Fax [phone] > Mobile [phone] > ______________________________________________ >      http://www.guycox.net   > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Watkins, Simon C > Sent: Wednesday, 11 February 2009 7:47 AM > To: [hidden email] > Subject: Re: Zeiss SD > I tend to agree with Dick here,  The pinhole array solutions while less "mature" than the spinning disk confocals work very well.  We have had a Yokagawa head for ages, and the limitation to a 1.4NA objective was significant.  About a year ago we bought a Prairie swept field array scanning confocal.  It allows multiple pinholes or slits sizes (7)  depending on your specimen and optics used.  It works very well indeed, and in our hands is more flexible than the Yokogawa head.  Its important to note that the yokogawa head I have is very old and there have been many advances.  However without rapid and simple changing of pinhole size (as is possible with the Prairie or Visitech solutions) your image will be compromised when NA is changed. > S.   > Simon C. Watkins Ph.D, FRCPath > Professor and Vice Chair, Cell Biology and Physiology > Professor, Immunology > Director, Center for Biologic Imaging > BSTS 225, University of Pittsburgh > 3500 Terrace St. > Pittsburgh PA 15261 > Tel: [phone] > Fax:[phone] > URL: http://www.cbi.pitt.edu   > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of RICHARD BURRY > Sent: Tuesday, February 10, 2009 3:20 PM > To: [hidden email] > Subject: Re: Zeiss SD   > Fred > We have a Visitech Infinity3 2D array scanning confocal.  It is based on the same principle as the spinning disk but use an array of pin holes.  This confocal allows seven different sizes of pin holes with diameter from 10 um to 60 um.  Our experience is that is gives great performance with a Hamamatsu EM13.  With the 100x 1.4 N.A. the optimal pin hole is 30 um but at 60 um the optical section thickness is a little more than a micron but the intensity is great for even weak fluorescence.  > Dick > ----- Original Message ----- > From: Fred Mast <[hidden email]> > Date: Tuesday, February 10, 2009 2:12 pm > Subject: Re: Zeiss SD > To: [hidden email] > > Does the CSU-X1 allow you to change the disks? I'm still waiting for a spinning-disk unit that is actually designed for live-cell imaging. The previous Yokogawa units were optimized for 100x 1,4 NA oil objectives if I'm not mistaken. Even with relatively thin samples (cultured cells), you're still introducing spherical aberration... By comparison, has anyone else looked at the technology being developed in Raphael Yuste's laboratory? They have combined multiphoton LSM with spatial light modulation technology to effectively "scan" an entire field at once. I'm not an expert in this but in my opinion if you could make it fast enough this seems like a better option to me (they report 60Hz calcium ion imaging). You can check it out here (under the SLM Microscopy section): > > http://www.columbia.edu/cu/biology/faculty/yuste/methods.html > > Cheers, > > Fred > > On 10-Feb-09, at 10:55 AM, G. Esteban Fernandez wrote: > I too was intrigued when I saw Zeiss roll out a spinning disk.  We're happy with our 5 LIVE.  Being able to vary the slit aperture size to match different objectives/samples is important in our multi-user facility.  It's also nice to capture two fluorescent channels simultaneously.  I wish the detector on the 5 LIVE were EM-CCD but the regular CCD does the job.  Images sometimes have vertical line artifacts but those are nicely dealt with by Fourier filtering or dividing by a blank image.  Must be lower cost (if that's true) and/or established familiarity of the spinning disk that makes them easy to sell.   > > -Esteban > > On Tue, Feb 10, 2009 at 10:35 AM, Simon Walker <simon.walker@...> wrote: > > I was interested to see that Zeiss have gone into partnership with Yokogawa > > to launch their own flavour of the spinning disk confocal: > > http://www.microscopy-analysis.com/news/carl-zeiss-and-yokogawa-launch- > > spinning-disk-cell-observer-sd-2008 > > Does this represent an acknowledgment that the 5-live doesn't do everything > > it says on the tin, or just that it's too expensive?  Are there any happy 5-live > > users out there? > > -- > > G. Esteban Fernandez, Ph.D. > > Associate Director > > Molecular Cytology Core Facility > > University of Missouri > > 120 Bond Life Sciences Center > > Columbia, MO  65211 > > http://www.biotech.missouri.edu/mcc/ > > 573-882-4895 > > 573-884-9395 fax   > > Fred D. Mast > > Department of Cell Biology > > Medical Sciences Building Room 5-14 > > University of Alberta > > Edmonton, Alberta, T6G 2H7 > > Canada > > Tel: [phone] > > fmast@... > > Spam > > Not spam > > Forget previous vote > Richard W. Burry, Ph.D. > Department of Neuroscience, College of Medicine > Campus Microscopy and Imaging Facility, Director > The Ohio State University > Associate Editor, Journal of Histochemistry and Cytochemistry > 277 Biomedical Research Tower > 460 West Twelfth Avenue > Columbus, Ohio 43210 > Voice 614.292.2814  Cell 614.638.3345  Fax [phone] > No virus found in this incoming message. > Checked by AVG. > Version: 7.5.552 / Virus Database: 270.10.20/1944 - Release Date: 10/02/2009 5:44 PM > Spam > Not spam > Forget previous vote

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