Perfect Focus
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal All, Have new faculty coming aboard and a major application will be 48 hour timelapse studies in 96 well plates using multichannel collections. Therefore my question is how critical is it to have a system that has a perfect focus mechanism to meet this application? Are there decent work arounds if the perfect focus is not used? Have never actually worked with this type of system so would like input on this matter from the list. Thanks. Scott J. Howell, Ph.D. Manager, Imaging Module Visual Sciences Research Center Case Western Reserve University 2085 Adelbert Rd. Institute of Pathology Room 106 Cleveland, Ohio 44106 216-368-2300 http://www.case.edu/med/vsrc/ |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Scott, there are certainly ways to work without a hardware autofocus system. One main thing is to keep the temperature stable, since even small temperature changes can cause big focus drifts. So a climate chamber around your whole system, plus a stable room air condition 24/7 is necessary. This already gives a good and stable base for long term experiments. Such a hardware autofocus system makes things a bit more easy. It keeps the coverslip or the bottom of your well plage in a constant position, even if the temperature changes or the bottom is not even, due to a slightly tilted stage or something like that. There are a couple of limitations to these systems: - speed of correction (some work parallel to the imaging, some don't) - correction range is limited, so they don't correct over millimeters - they don't work with all setups, you need a clear interference between coverslip and water which acts as a mirror If the major application will be long term time lapse, I definitely recommend to have a closer look at the available systems. Maybe this helps. Michael Scott Howell wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > All, > > Have new faculty coming aboard and a major application will be 48 hour > timelapse studies in 96 well plates using multichannel collections. > Therefore my question is how critical is it to have a system that has > a perfect focus mechanism to meet this application? Are there decent > work arounds if the perfect focus is not used? Have never actually > worked with this type of system so would like input on this matter > from the list. Thanks. > > > Scott J. Howell, Ph.D. > Manager, Imaging Module > Visual Sciences Research Center > Case Western Reserve University > 2085 Adelbert Rd. > Institute of Pathology Room 106 > Cleveland, Ohio 44106 > 216-368-2300 > http://www.case.edu/med/vsrc |
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In reply to this post by Scott Howell-3
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Scott, it looks like you have a fully automated system. Personally, I do not rely on autofocus, I would suggest, if you take multi position, multi channels over multiple time point, then instead of one frame position/well also include a small z stack, say 4 images (2 above and 2 below) from the best focus of each position, so latter even if there is a drift, you will have an option to select the best plane out of those four. Shiv At 07:56 AM 9/18/2008, you wrote: Search the CONFOCAL archive at 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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In reply to this post by Scott Howell-3
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Scott, I'd argue that a hardware auto-focus solution is essential. We do a huge amount of such imaging and without AF it would be much more difficult. Olympus/Nikon and Zeiss offers such a system for their microscopes, but you can buy also a dedicated system (several possibilities). Cheers Gabor -- Gabor Csucs Light Microscopy Centre, ETH Zurich Schafmattstrasse 18, HPM F16 CH-8093, Zurich, Switzerland Web: www.lmc.ethz.ch Phone: +41 44 633 6221 Fax: +41 44 632 1298 e-mail: [hidden email] |
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In reply to this post by Mayandi Sivaguru
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Scott,
The perfect focus systems usually rely on a laser that is reflected off of a cover slip and automatically adjusts the focus of the microscope such that the cover glass is always the same distance from the objective. They ARE NOT autofocus devices - you have to set the initial focus and then trigger the system. For long term experiments and especially for experiments with multi well plates perfect focus systems can be VERY useful. While taking a z stack can help, I have seen more than one multi well plate with enough variation from well to well (especially from one end of the plate to the other) to require 11 or more slices to insure you get the infocus plane. (NOTE: I always recommend using an odd number of slices, based on the idea that if you focus on a specific plane and then do say a four slice stack centered on that plane, you will actually miss the plane the you focused on!) The beauty of the perfect focus system is that it gaurantees that you always start at the same relative plane. If the samples are stable, you may be able to get away with a single plane, but if not you can still use a much smaller stack say three or five planes that actaully bracket the sample ratherthan 11 or more in the hope that the few planes you need will fall somewhere in that range. Chris Tully -- Chris Tully Microscopy and Image Analysis Expert [hidden email] 240-888-1021 http://www.linkedin.com/in/christully On Thu, Sep 18, 2008 at 10:10 AM, Mayandi Sivaguru <[hidden email]> wrote:
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In reply to this post by Mayandi Sivaguru
Search the CONFOCAL archive at
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In my opinion the realtime autofocus solutions offered today are
absolutely essential, they are incredibly useful for live cell imaging and
allow multiday non-attended timelapse. However for the system to
work properly there generally has to be a significant refractive index change
from the material being imaged (for example from the coverslip 1.51 to media,
1.36). I have never used a plastic substrate( such as a 96 well
plate) on a focussing system, I would be surprised if they work at all. so
make sure this is possible before investing, perhaps some of our commercial
friends can comment on the utility of real time focussing systems for use on
plastic 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: 412-352-2277 Fax:412-648-2797 URL: http://www.cbi.pitt.edu From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of Mayandi
Sivaguru Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Search the CONFOCAL archive at Mayandi Sivaguru, PhD, PhD |
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In reply to this post by Csúcs Gábor
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Scott, I agree with Gabor and Simon that having some sort of "auto focusing" mechanism (whether it be a z-motor based software autofocus or a laser-based perfect focus system) is extremely helpful, and sometimes essential, when doing long-term timelapse imaging. I find that even with temperature and humidity control, z-drift is likely over a long period of time and needs to be corrected for. If you plan to image fluorescence with live cells over time, imaging multiple planes at each time point is not ideal because of the burdens of increased photobleaching and phototoxicity. I have used both software autofocus, which tends to work just fine, and the Nikon Perfect Focus System on the TE2000E. The PFS is easy to use and works very well (and much quickly than traditional autofocus!). If you look into this, make sure that the objective lenses that you plan to use are rated to work with the PFS (I think that many will work, but there are some exceptions). Best, Lara ----------------------------------------------------------------------- Lara Petrak Microscopy Coordinator Nikon Imaging Center @ Harvard Medical School 240 Longwood Avenue Building LHRRB Room 113 Boston, MA 02115 Systems Biology Microscopy Facility 220 Longwood Avenue Goldenson Room 107 Boston, MA 02115 Phone: 617/432.3547 Fax: 617/432.1144 [hidden email] |
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In reply to this post by Watkins, Simon C
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Answering to the question of Simon: I can't comment on all the auto-focus solutions, but many of them (especially the ones on the specialized screening microscopes) do work with plastic bottom plates. So we do a lot of AF based imaging in plastic bottom plates (different brands) using air objectives. Nevertheless the companies don't guarantee that the AF devices work equally well with all objectives. Cheers Gabor -- Gabor Csucs Light Microscopy Centre, ETH Zurich Schafmattstrasse 18, HPM F16 CH-8093, Zurich, Switzerland Web: www.lmc.ethz.ch Phone: +41 44 633 6221 Fax: +41 44 632 1298 e-mail: [hidden email] |
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In reply to this post by Scott Howell-3
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Scott, To reiterate what everyone else has been saying, even relatively small fluctuations in temperature can have a significant impact on focus. To provide an example: I was in an air-conditioned lab over the summer and as the air-conditioning turned off and on, the change in focus due to thermal expansion was on the order of a micron for a 1-1.5 deg. C change in temperature - this happened at a period of about 200 seconds with the specimen going in and out of focus. Because I use an older spinning disk unit (without infinity-corrected optics) that's no longer sold or supported, many of the "autofocus" options available on newer infinity-corrected scopes were not an option for me. Therefore, to compensate for thermal drift, I regularly sampled two areas from the CCD image every ten seconds and used a metric (e.g. contrast) to determine the z-position and then corrected using the piezo. This was not the most elegant solution - most modern "autofocus" options are faster and run parallel to the image acquisition - however, it works reasonably well for my application (imaging of semiconductor layers) and is pretty cheap. With this in mind, how often do you need to capture an image - do you need continuous video or snapshots? Hope this proves useful. Good luck! -Joe On Sep 18, 2008, at 8:56 AM, Scott Howell wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > All, > > Have new faculty coming aboard and a major application will be 48 hour > timelapse studies in 96 well plates using multichannel collections. > Therefore my question is how critical is it to have a system that has > a perfect focus mechanism to meet this application? Are there decent > work arounds if the perfect focus is not used? Have never actually > worked with this type of system so would like input on this matter > from the list. Thanks. > > > Scott J. Howell, Ph.D. > Manager, Imaging Module > Visual Sciences Research Center > Case Western Reserve University > 2085 Adelbert Rd. > Institute of Pathology Room 106 > Cleveland, Ohio 44106 > 216-368-2300 > http://www.case.edu/med/vsrc/ ------------------------------------ Postdoc, Physics Dept. Optoelectronics Group Mt. Holyoke College ph: (413) 538-2263 http://www.mtholyoke.edu/~jsummers/ |
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In reply to this post by Csúcs Gábor
Search the CONFOCAL archive at
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For an academic core facility that is moving into automated microscopy, scopes that can focus on higher mag/higher NA lenses are likely the most useful/versatile; if you will never “screen” at 40x or higher, then the systems that only go to 20x, or a low NA 40x, should be fine. Just depends what you need..... And here is a quick head’s up for anyone using glass or plastic bottom multi-well plate: keep in mind some of the vendors put out plates that have considerable amounts of intrinsic estrogenic activity (from the various plasticizers (e.g., bisphenol A, etc) and/or glue used to manufacture the dish, or adfix the bottoms)...... Some endocrine studies could be considerably influenced by such “noise” in the system. Mike On 9/18/08 9:51 AM, "Gabor Csucs" <[hidden email]> wrote: Search the CONFOCAL archive at |
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In reply to this post by Chris Tully
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Chris Tully <[hidden email]> writes: > well plates perfect focus systems can be VERY useful. While taking a z stack > can help, I have seen more than one multi well plate with enough variation > from well to well (especially from one end of the plate to the other) to One important factor is to have a setup that allows position definition in 3D, ie X,Y and Z. This saves problems from tilted, or non-flat plates. I have done this with 72h time courses on 24 well plates without any automatic focus adjustment, but with computer control of x,y and z to visit the defined positions in 3D. With good temperature stability this is not difficult at relatively low mag, say 10-20x. At high mag, or more importantly high NA, your depth of focus is much smaller and therefore this is much more demanding. Ian |
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In reply to this post by Mancini, Michael A
Dear confocalists,
Today I came across a new aqueous-based mounting medium recently introduced by EM sciences (in the USA) and Citifluor (in the UK). They claim it has a refractive index of 1.52, i.e., perfectly matched to oil/glass. It is being sold in 3 flavors, CFM-1, CFM-2, CFM-3. These vary in pH and antifade content. Sounds promising. Has anybody tried these? Mike Michael J. Schell Dept. Pharmacology USUHS 4301 Jones Bridge Rd. Bethesda, MD 20814 Tel: 301-295-3249 |
 
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Jerry Sedgewick-2 |
plasmid for CFP-YFP tandem FRET
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Hello All,
A person using our lab is interested in the following: We want to know if there's a commercially available plasmid to express a CFP-YFP tandem FRET, with a linker protein that can be cleaved by introducing a protease (that is non membrane-permeable). If one is not commercially available, a lab that has produced this and would be willing to share is also an option. If anyone on the list has any experience with this/would be willing to share, please let me know. Thanks! Jerry Sedgewick --- Get FREE High Speed Internet from USFamily.Net! -- http://www.usfamily.net/mkt-freepromo.html --- |
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Miller, Jason |
Re: plasmid for CFP-YFP tandem FRET
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Just such a plasmid has been published by the Daugherty Group from UCSC, I believe (http://www.ncbi.nlm.nih.gov/pubmed/15696158). It was published in Nature Biotechnology and they now have both bacterially optimized and mammalian optimized codon plasmids. The construct contains CyPet and Ypet (the evolutionarily optimized CFP and YFP derivatives that are supposed to FRET with each other with greater efficiency) with a Caspase 3 cleavage site in the middle.
-Jason ------------------- Medical Scientist Training Program (MD/PhD) University of California- San Francisco Home Address: 1434 Lakeshore Ave., Apt #8 Oakland, CA 94606 Home: (510) 625-1334 Cell: (415) 225-2134 E-mail: [hidden email] A Few of Dave Barry's Pearls for Living: 1. Never, under any circumstances, take a sleeping pill and a laxative on the same night. 2. If you had to identify, in one word, the reason why the human race has not achieved and never will achieve its full potential, that word would be "meetings." 3. There is a very fine line between "hobby" and "mental illness." 4. The one thing that unites all human beings, regardless of age, gender, religion, economic status or ethnic background, is that, deep down inside, we ALL believe that we are above average drivers. 5. Never be afraid to try something new. Remember that a lone amateur built the Ark. A large group of professionals built the Titanic. 6. Men are like fine wine. They start out as grapes, and it's up to the women to stomp the crap out of them until they turn into something acceptable to have dinner with. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jerry Sedgewick Sent: Monday, June 22, 2009 9:11 AM To: [hidden email] Subject: plasmid for CFP-YFP tandem FRET Hello All, A person using our lab is interested in the following: We want to know if there's a commercially available plasmid to express a CFP-YFP tandem FRET, with a linker protein that can be cleaved by introducing a protease (that is non membrane-permeable). If one is not commercially available, a lab that has produced this and would be willing to share is also an option. If anyone on the list has any experience with this/would be willing to share, please let me know. Thanks! Jerry Sedgewick --- Get FREE High Speed Internet from USFamily.Net! -- http://www.usfamily.net/mkt-freepromo.html --- |
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