Imaging organotypic brain slice cultures with an inverted scope?
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lechristophe |
Imaging organotypic brain slice cultures with an inverted scope?
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi, A colleague is using organotypic cultures of rat hippocampal slices, the type that is kept at the interface between medium and air, resting on a Millicell filter. He would like to observe them live (fluorescence with a decent magnification and NA), but we only have access to inverted microscopes. Does anyone know a way to do that ? Thanks for your advices, Christophe -- Christophe Leterrier INSERM UMR641 // Ionic channels Lab IFR Jean Roche, Mediterranée University Marseille, France http://www.cleterrier.net |
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We have one of these and it works well. They custom cut the tube length for us too.
http://www.lsmtech.com/product_objective_inverter.html ________________________________________________________ Michael Cammer, Assistant Research Scientist Skirball Institute of Biomolecular Medicine Lab: (212) 263-3208 Cell: (914) 309-3270 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier Sent: Tuesday, December 13, 2011 9:59 AM To: [hidden email] Subject: Imaging organotypic brain slice cultures with an inverted scope? Hi, A colleague is using organotypic cultures of rat hippocampal slices, the type that is kept at the interface between medium and air, resting on a Millicell filter. He would like to observe them live (fluorescence with a decent magnification and NA), but we only have access to inverted microscopes. Does anyone know a way to do that ? Thanks for your advices, Christophe -- Christophe Leterrier INSERM UMR641 // Ionic channels Lab IFR Jean Roche, Mediterranée University Marseille, France http://www.cleterrier.net ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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Kelly Rogers-2 |
Re: Imaging organotypic brain slice cultures with an inverted scope?
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In reply to this post by lechristophe
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Assuming the filter is optically clear, you could cut it out with a scapel blade and carefully transfer it to a coverglass bottom perfusion chamber (making sure the tissue stays flat and avoiding any mechanical stress). In the past we used chambers from harvard aparatus (http://www.warneronline.com/product_info.cfm?name=Open%20Diamond%20Bath%20%28RC-22%20and%20RC-22C%29&id=733). The tissue slice anchor allows you to keep the membrane/ tissue in place. A simple perfusion set up based on gravity flow can be used to supply the tissue with oxygen or fresh media throughout the experiment (being careful not to get any liquid overflow...). It's not perfect for supplying the underside of the tissue, but it may not be a major problem if you are imaging thin sections or just for very short periods. The tissue tends to thin out a bit in culture. As for the objective, we worked with a relatively low mag objective 20x/0.8 which worked well because we wanted a larger field of view. Not sure how you would go with a higher mag/ NA, but worth giving it a try. From memory the membranes were pretty thin. Kelly > ***** > To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi, > > A colleague is using organotypic cultures of rat hippocampal slices, the type that is kept at the interface between medium and air, resting on a Millicell filter. He would like to observe them live (fluorescence with a > decent magnification and NA), but we only have access to inverted microscopes. Does anyone know a way to do that ? > > Thanks for your advices, > > Christophe > > -- > Christophe Leterrier > INSERM UMR641 // Ionic channels Lab > IFR Jean Roche, Mediterranée University > Marseille, France > http://www.cleterrier.net > Kelly Rogers The Walter & Eliza Hall Institute 1G Royal Parade, Parkville Victoria 3052, Australia ph: +61_3_9345 2450 ______________________________________________________________________ The information in this email is confidential and intended solely for the addressee. You must not disclose, forward, print or use it without the permission of the sender. ______________________________________________________________________ |
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Vladimir Ghukasyan-2 |
Re: Imaging organotypic brain slice cultures with an inverted scope?
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In reply to this post by lechristophe
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** We are doing it with Olympus LUCPLFLN 40x / 0.6. It has working distance of 2.7 - 4 mm adjustable with the correction collar. The objective allows us to acquire decent images. However, if your goal is aiming to resolve intracellular structures, then this approach may not be suitable. Vladimir On Tue, Dec 13, 2011 at 9:59 AM, Christophe Leterrier <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi, > > A colleague is using organotypic cultures of rat hippocampal slices, the type that is kept at the interface between medium and air, resting on a Millicell filter. He would like to observe them live (fluorescence with a decent magnification and NA), but we only have access to inverted microscopes. Does anyone know a way to do that ? > > Thanks for your advices, > > Christophe > > -- > Christophe Leterrier > INSERM UMR641 // Ionic channels Lab > IFR Jean Roche, Mediterranée University > Marseille, France > http://www.cleterrier.net |
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William E. Grever |
Re: Imaging organotypic brain slice cultures with an inverted scope?
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In reply to this post by mcammer
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Michael, I did something similar for my dissertation work when I was at AECOM 15 years ago. The only trouble is that the slice culture will need to be sacrificed for imaging using this method. I assume the brain slice is free floating. If so, you might try something like what I did. I used cover slips that had an adhesive gasket from Grace Bio-labs. After labeling, I would equilibrate the slice in an anti-fade/glycerol solution. I then placed the slice on a large cover slip and covered the slice with one of the adhesive cover slips. This would allow imaging from both sides of the slice. I did a quick search for the product I used and found this link that may be helpful. There are many more products available then there was 15 years ago. http://www.gracebio.com/products/imaging-microscopy/imaging-spacers.html Will Grever ----- Original Message ----- From: "Michael Cammer" <[hidden email]> To: [hidden email] Sent: Tuesday, December 13, 2011 10:23:23 AM Subject: Re: Imaging organotypic brain slice cultures with an inverted scope? We have one of these and it works well. They custom cut the tube length for us too. http://www.lsmtech.com/product_objective_inverter.html ________________________________________________________ Michael Cammer, Assistant Research Scientist Skirball Institute of Biomolecular Medicine Lab: (212) 263-3208 Cell: (914) 309-3270 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier Sent: Tuesday, December 13, 2011 9:59 AM To: [hidden email] Subject: Imaging organotypic brain slice cultures with an inverted scope? Hi, A colleague is using organotypic cultures of rat hippocampal slices, the type that is kept at the interface between medium and air, resting on a Millicell filter. He would like to observe them live (fluorescence with a decent magnification and NA), but we only have access to inverted microscopes. Does anyone know a way to do that ? Thanks for your advices, Christophe -- Christophe Leterrier INSERM UMR641 // Ionic channels Lab IFR Jean Roche, Mediterranée University Marseille, France http://www.cleterrier.net ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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In reply to this post by lechristophe
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Christophe The following link takes you to the Bioptechs Micro-Environmental accessories page where at the top of the page is a picture of a Costar Snapwell™ in a Delta T Dish. This is an ideal combination when used with the LSM tech objective lens inverter. The Delta T dish provides fluid containment and temperature control and the Snapwell "Cap" fits right in nicely. http://www.bioptechs.com/Products/Delta_T/Options/options.html#snapwell Just below the Snapwell™ is our Atmospheric Barrier Ring. This is a Pyrex ring precision cut to fit around your objective to prevent ambient contamination and can be fitted with a gas port if necessary to trap and allow control of gas concentrations in the dish. This is very useful if you are using water dipping objectives to get the higher resolution and N.A. Below that on the page is our Brain Slice Adapter for Delta T™ dishes made for inverted microscopes. You might find it useful also. Dan On Dec 13, 2011, at 9:59 AM, Christophe Leterrier wrote: ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi, A colleague is using organotypic cultures of rat hippocampal slices, the type that is kept at the interface between medium and air, resting on a Millicell filter. He would like to observe them live (fluorescence with a decent magnification and NA), but we only have access to inverted microscopes. Does anyone know a way to do that ? Thanks for your advices, Christophe -- Christophe Leterrier INSERM UMR641 // Ionic channels Lab IFR Jean Roche, Mediterranée University Marseille, France http://www.cleterrier.net Dan Focht Bioptechs, Inc. 3560 Beck Rd. Butler, PA 16002 www.bioptechs.com P: (724)282-7145 F: (724)282-0745 [hidden email] |
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Glen MacDonald-2 |
Re: Imaging organotypic brain slice cultures with an inverted scope?
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In reply to this post by lechristophe
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** We've done this with slices of avian and mouse auditory brainstem imaged on inverted microscopes. With long working distance lenses, the entire millicel filter insert can be transferred to suitable coverglass bottom chamber, Some filters have 3 feet that can be cut down to still allow media circulation yet bring the sample closer to the lens. For high NA air lenses and water immersion lenses, we have also cut out the filter with the slice then transferred the brain slice to either a coverslip culture chamber or a Warner oocyte chamber. If the slice is well adhered, you can usually flip it over to avoid the slight optical problems from imaging through the membrane. If you are reasonably clean, the slice can be placed on a new filter insert (membrane side down) and returned to the incubator for future imaging. Do avoid folding, the brain slice will stick to itself. Good luck 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] On Dec 13, 2011, at 6:59 AM, Christophe Leterrier wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi, > > A colleague is using organotypic cultures of rat hippocampal slices, the type that is kept at the interface between medium and air, resting on a Millicell filter. He would like to observe them live (fluorescence with a decent magnification and NA), but we only have access to inverted microscopes. Does anyone know a way to do that ? > > Thanks for your advices, > > Christophe > > -- > Christophe Leterrier > INSERM UMR641 // Ionic channels Lab > IFR Jean Roche, Mediterranée University > Marseille, France > http://www.cleterrier.net |
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Pascal Weber |
Re: Imaging organotypic brain slice cultures with an inverted scope?
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In reply to this post by lechristophe
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I realized a few years ago, an assembly that can work on those boxes with a lens with high numerical aperture and short distance work. During several hours of acquisition. I always use a water immersion objective. But it was better with the Zeiss W Plan-Apochromat 20x/1,0. It was on Zeiss (AXIO IMAGER) inverted microscope and this objective. This setup is not recommended by Zeiss but I assure you it works very well. |
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