Different Z setting per channel - possible on Zeiss/Leica?
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Graham Wright-5 |
Different Z setting per channel - possible on Zeiss/Leica?
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello, Here's question that I've been asked that doesn't seem possible to do, but I wonder if anyone has already done it? One of our users has a fixed sample with 3 fluorophores (for ease of explanation blue, green and red) and would like to image all three channels, but with different z setting depending on the channel. She would like a single medial optical section of the blue and red channels, but a full z-series from the green channel. We have both Zeiss (from the LSM 510 series) and Leica (SPE and SP5) confocals available to us. I know this is easy by doing 2 different scans: 1 2-channel scan of the blue and red, then a second scan of a z-series of the green, BUT is it possible to do this at the single click of a button to save time and user input. It is possible on the SP5 using the live data mode, but this scope is really reserved and heavily used for live cell imaging. Our SPE doesn't have live data mode and therefore applies any Z settings to all sequential scans (generating unnecessary quantities of data and taking too long). Any suggestions welcome, Thanks, Graham |
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RICHARD BURRY |
Re: Different Z setting per channel - possible on Zeiss/Leica?
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Graham While this is not elegant, we collect all three fluorochromes at the same time in a single z stack with the 510. With the single track option (assuming no bleed through) this will take no more time that a single z tack. The advantage is that you can sellect one z section, with one fluorophore and know where that fits with regard to all three fluorophores in the z stack. The only down side is that the data files are larger. In fact we frequently add a DIC image to the Z stack just so we have a historical record of cells. The DIC is not confocal but a single DIC image is sometimes very useful, and it takes no more time to add a DIC. For viewing, the DIC channel can turned off when so only the other channels are seen. Dick Burry ----- Original Message ----- From: "Dr. Graham Wright" <[hidden email]> Date: Wednesday, September 10, 2008 9:27 pm Subject: Different Z setting per channel - possible on Zeiss/Leica? To: [hidden email] > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello, > > Here's question that I've been asked that doesn't seem possible > to do, but I > wonder if anyone has already done it? One of our users has a > fixed sample > with 3 fluorophores (for ease of explanation blue, green and > red) and would > like to image all three channels, but with different z setting > depending on > the channel. She would like a single medial optical section of > the blue and > red channels, but a full z-series from the green channel. > > We have both Zeiss (from the LSM 510 series) and Leica (SPE and > SP5) > confocals available to us. > > I know this is easy by doing 2 different scans: 1 2-channel scan > of the blue > and red, then a second scan of a z-series of the green, BUT is > it possible > to do this at the single click of a button to save time and user > input. It > is possible on the SP5 using the live data mode, but this scope > is really > reserved and heavily used for live cell imaging. Our SPE doesn't > have live > data mode and therefore applies any Z settings to all sequential > scans > (generating unnecessary quantities of data and taking too long). > > Any suggestions welcome, > Thanks, > Graham > > > -- > BEGIN-ANTISPAM-VOTING-LINKS > ------------------------------------------------------ > > Teach CanIt if this mail (ID 678485001) is spam: > Spam: > https://antispam.osu.edu/b.php?c=s&i=678485001&m=c6a1d1b19215Not > spam: https://antispam.osu.edu/b.php?c=n&i=678485001&m=c6a1d1b19215 > Forget vote: > https://antispam.osu.edu/b.php?c=f&i=678485001&m=c6a1d1b19215---- > -------------------------------------------------- > END-ANTISPAM-VOTING-LINKS > |
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Rietdorf, Jens |
Re: Different Z setting per channel - possible on Zeiss/Leica?
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In reply to this post by Graham Wright-5
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello Graham, On the 510 you can do the following: -create 2 tracks (or configurations, depending if you wish to change any parameter but the colors), (a) for the 'blue & red', (b) for the 'green'. -Mark stack top & bottom in the z settings dialog. -press the 'median' button from the z settings dialog and acquire a single plane with condition (a) then -acquire a stack selecting condition (b). You could then project the (b) or a subset of it and merge it with the (a), though this will produce a complicated picture, which needs quite some explanation. You can automate the procedure using the 'multi-time' macro downloadable from the Zeiss microscopy website, but I would not do it for just one set of data, because the macro is quite difficult to handle. The above mentioned procedure does only make sense if the green channel is bleaching too quickly than to allow for taking a stack as well in this channel. Hope that helps, jens. --- dr. jens rietdorf head microscopy novartis research foundation friedrich-miescher-institute, wro1066.2.16 maulbeerstr.66, 4058 basel, switzerland couriel:rietdorf(at)fmi(dot)ch fon: +41616975172 fax: +41616973976 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dr. Graham Wright Sent: Thursday, September 11, 2008 3:31 AM To: [hidden email] Subject: Different Z setting per channel - possible on Zeiss/Leica? Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello, Here's question that I've been asked that doesn't seem possible to do, but I wonder if anyone has already done it? One of our users has a fixed sample with 3 fluorophores (for ease of explanation blue, green and red) and would like to image all three channels, but with different z setting depending on the channel. She would like a single medial optical section of the blue and red channels, but a full z-series from the green channel. We have both Zeiss (from the LSM 510 series) and Leica (SPE and SP5) confocals available to us. I know this is easy by doing 2 different scans: 1 2-channel scan of the blue and red, then a second scan of a z-series of the green, BUT is it possible to do this at the single click of a button to save time and user input. It is possible on the SP5 using the live data mode, but this scope is really reserved and heavily used for live cell imaging. Our SPE doesn't have live data mode and therefore applies any Z settings to all sequential scans (generating unnecessary quantities of data and taking too long). Any suggestions welcome, Thanks, Graham |
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Barbara Foster |
Re: Different Z setting per channel - possible on Zeiss/Leica?
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In reply to this post by Graham Wright-5
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Dear Dick
I applaud your using DIC as a reference for your fluorescence. Also, you've made an interesting comment re: DIC not being confocal. Of all techniques, DIC has the shallowest depth of focus, so it is very nearly confocal. You can test this using a simple cheek cell smear. Set with the condenser aperture wide open and the beam splitters on optimum shear (dove gray background). You can readily focus on the bacteria and folds on the top of a cheek cell, then focus on granules and structures within the cytoplasm, then on the folds where the cell adheres to the slide. For those of you who are interested in more of the details behind how DIC works, there is a scan of an old article from the April 1988 American Lab on our website, <a href="http://www.microscopyeducation.com. /" eudora="autourl"> www.MicroscopyEducation.com. Just go to The Library and Articles. They are listed in chronological order, with the most recent at the top. Good hunting! Best regards, Barbara Foster, President Microscopy/Microscopy Education 7101 Royal Glen Trail, Suite A McKinney TX 75070 P: (972)924-5310 Skype: fostermme W: www.MicroscopyEducation.com NEWS! Visit the NEW and IMPROVED www.MicroscopyEducation.com! And don't forget: MME is now scheduling customized, on-site courses through Dec 2008. Call me for a free assessment and quote. At 09:23 PM 9/10/2008, you wrote:. Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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Rosemary.White |
DIC on confocal - OK with non-oscillating laser polarity....
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DIC is less useful when you use confocal partly as a way to look at the surface of quite thick tissues, e.g. sections of living plant tissues which have to be at least as thick as the cell - up to 200 microns. DIC doesn’t do so well then, and I prefer to use transmitted brightfield. This is especially true for those of us with Leica SP2 confocals because the oscillation in polarity of the laser drives you completely mad if you’re trying to collect a z-stack with DIC – the transmitted DIC varies from very dark to saturated from one end of the stack to the other. cheers, Rosemary Dr Rosemary White [hidden email] CSIRO Plant Industry ph. 02-6246 5475 GPO Box 1600 fax. 02-6246 5334 Canberra, ACT 2601 Australia On 11/9/08 11:51 PM, "Barbara Foster" <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Dick |
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Christian-103 |
Re: DIC on confocal - OK with non-oscillating laser polarity....
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Rosemary,
You are correct. I've have given up trying to explain why an entire leaf will never give a decent DIC image. Funny how those thick cell walls just play all kinds of tricks on our light. I often tell people they'll just have to get a label to an organelle if they want confirmation, as we're not going to be able to do it via the transmitted light detector. Christian |
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George McNamara |
Re: Different Z setting per channel - possible on Zeiss/Leica?
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In reply to this post by Barbara Foster
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An even better reference - for adherent cells - is the confocal mode reflection of the coverglass (or slide, if the cells are on that side). Coverglass is brightest when in focus. With a 63x/1.4 NA lens, 488 nm excitation, optical slice thickness is ~0.7 um. As a bonus, you for free the interference reflection contrast of the cell-substratum adhesion (note to software manufacturers or graduate students who need an open source software project: Sackmann has published absolute distance measurements by dual wavelength IRM, see for example PubMed 14995485, but their software may not be available - would be nice to add this calculation to, say, ImageJ, Zeis LSM and Leica confocal software). Speaking of Barbara's statement that DIC has shallowest depth of field, the only location in Z where the IRM image goes to its minimum is when an object is effectively at 'zero distance' from the coverglass, in cells these are focal adhesion sites or where filopodia touch the glass. The literature suggests this 'zero distance' is <5 nm, so this is much thinner than DIC. In the more general case (no object dependency), TIRF is likely to have a thinner depth of field than DIC. An exception with TIRF is that if an object has high refractive index at the interface, the waves/rays/photons can escape into the second medium (ex. at focal adhesions). In the context of this thread, using DIC to focus is good if the user is trying to image, say, "nucleus", but this is unlikely to result in great reproducibility in acquiring single plane images. With the coverglass reflection method, it is easy to focus consistently on the coverglass, and have the focus motor acquire at that location and at, for example, +2 um. George At 09:51 AM 9/11/2008, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Dick |
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Stephen Cody |
Re: DIC on confocal - OK with non-oscillating laser polarity....
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In reply to this post by Christian-103
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G’day Rosemary and Christian,
I found using Keith Nugent’s algorithm that you can calculate the DIC image from brightfield (BF) images collected with the transmitted light detector (TLD) of a confocal. This technique requires a z-series of BF images and some software (QPm or QPi) to calculate the DIC image even from very thick specimens. Zebrafish embyos that were too thick to obtain DIC images using purely optics, were suitable for this technique. I imagine it will work well with plant tissue too. Also the contribution of heavily absorbing pigments such as melanin could be made to “disappear”.
It should also be immune to oscillations in the polarity of your laser as you don’t require a polariser or analyser.
See: Barone-Nugent, E.D., Barty, A. & Nugent, K.A. (2002) Quantitative phase-amplitude microscopy I: optical microscopy. J. Microsc. 206, 194–203.
and
S. H. Cody, S. D. Xiang, M . J. Layton, E. Handman, M. H. C. Lam, J. E. Layton, E. C. Nice & J. K. Heath. (2005) J. Micros., 217, 265–274 Short Technical Note. A simple method allowing DIC imaging in conjunction with confocal microscopy. (I can send a PDF of this paper upon request).
Also see:
http://www.iatia.com.au/technology/applicationNotes.asp
No commercial interest etc.
Steve Stephen H. Cody Tip: Learn how to receive reminders about you microscope
booking: From:
Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian
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archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Rosemary, This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research does not waiver any rights if you have received this communication in error. The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research. |
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