Imaging without medium?
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, I am wondering what happen if I don't use any medium between cover and slide glasses. No water or anything in between. Thank you for your response in advance. -- Ohkyung Kwon, Ph D Postdoctoral Research Associate Sustainable Engineered Materials Institute Virginia Tech |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal What would happen? Generic answer...poor images. You would still have "something" in between the coverslip and the glass slide, Air, with its resultant refractive index mismatch between the two pieces of glass. It would be much preferable to have mounting medium of some kind, depending on your sample, ideally with a refractive index close to glass. What is your sample, and what are you trying to image? -- Samuel A. Connell Director of Light Microscopy Cell & Tissue Imaging Center St. Jude Children's Research Hospital 332 North Lauderdale St., E7061 Memphis, TN 38105-2794 (901) 495-2536 [hidden email] -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Oky Sent: Thursday, November 08, 2007 12:33 PM To: [hidden email] Subject: Imaging without medium?. . Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, I am wondering what happen if I don't use any medium between cover and slide glasses. No water or anything in between. Thank you for your response in advance. -- Ohkyung Kwon, Ph D Postdoctoral Research Associate Sustainable Engineered Materials Institute Virginia Tech |
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In reply to this post by Oky
Search the CONFOCAL archive at
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Dear Ohkyung,
Not a good idea. This is an optical system which follows Snell's law (see "Refraction" in any simple physics text). Important information necessary for image formation will be refracted out of the collecting angle of the objective. You also run the risk of spherical aberration in which the image will seem "soft" or fuzzy. Hope this was helpful, Barbara Foster, President We've moved! Microscopy/Microscopy Education 7101 Royal Glen Trail, Suite A McKinney TX 75070 P: (972)924-5310 Skype: fostermme W: www.MicroscopyEducation.com MME is now scheduling customized, on-site courses through December. Call us today for details. P. S. Need a good general reference or light microscopy text for next semester? Call us today to learn more about "Optimizing LIght Microscopy". Copies still available through MME... even for class-room lots ... and we give quantity discounts. Just call us here in the MME office for details. At 12:40 PM 11/8/2007, you wrote: Search the CONFOCAL archive at |
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In reply to this post by Oky
Search the CONFOCAL archive at
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Dear Ohkyung, As was pointed out, unless you are creating a vacuum between your slide and coverslip, there will be some medium: air. This being said, and depending on your specific experiment, you could consider avoiding the coverslip altogether, and using an objective designed to work without coverslip... (that is, designed to be used with air as the medium). Those would have the "-" sign, instead of the standard "0.17". It seems that these exist mostly in the lower end of the magnification range, so for confocal work, your choice may be limited. On the other hand, you can get such lenses in higher power versions for stereomicroscopes. For instance, we have high NA 10x and 20x objectives on a Zeiss stereomicroscope, which would generate up to 100x magnification in combination with the additional zoom. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 On Nov 8, 2007, at 10:33 AM, Oky wrote:
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In reply to this post by Oky
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Julio,
There are
plenty of high NA dry objectives - used for
petrology and metallurgy mostly - NA 0.8 is quite
routine
since with no coverslip SA is less of a problem.
All the
makers have them though with Nikon they are a
different
thread so you need an adaptor. Other makers seem
to
use the same thread for both. They are often
called
'Epi' lenses - Epiplan or similar designation. One of
these should do the job for Ohkyung.
Guy
Optical Imaging Techniques in Cell Biology From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julio Vazquez Sent: Friday, 9 November 2007 8:15 AM To: [hidden email] Subject: Re: Imaging without medium? Dear Ohkyung,
As was pointed out, unless you are creating a vacuum between your slide and
coverslip, there will be some medium: air.
This being said, and depending on your specific experiment, you could
consider avoiding the coverslip altogether, and using an objective designed to
work without coverslip... (that is, designed to be used with air as the medium).
Those would have the "-" sign, instead of the standard "0.17". It
seems that these exist mostly in the lower end of the magnification range, so
for confocal work, your choice may be limited. On the other hand, you can get
such lenses in higher power versions for stereomicroscopes. For instance, we
have high NA 10x and 20x objectives on a Zeiss stereomicroscope, which would
generate up to 100x magnification in combination with the additional
zoom.
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024
On Nov 8, 2007, at 10:33 AM, Oky wrote:
No virus found in this incoming message. No virus found in this outgoing message. |
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In reply to this post by Oky
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Sending this again as it didn't seem to get through -
apologies
if it appears twice.
Julio,
There are
plenty of high NA dry objectives - used for
petrology and metallurgy mostly - NA 0.8 is quite
routine
since with no coverslip SA is less of a problem.
All the
makers have them though with Nikon they are a
different
thread so you need an adaptor. Other makers seem
to
use the same thread for both. They are often
called
'Epi' lenses - Epiplan or similar designation. One of
these should do the job for Ohkyung.
Guy
Optical Imaging Techniques in Cell Biology From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julio Vazquez Sent: Friday, 9 November 2007 8:15 AM To: [hidden email] Subject: Re: Imaging without medium? Dear Ohkyung,
As was pointed out, unless you are creating a vacuum between your slide and
coverslip, there will be some medium: air.
This being said, and depending on your specific experiment, you could
consider avoiding the coverslip altogether, and using an objective designed to
work without coverslip... (that is, designed to be used with air as the medium).
Those would have the "-" sign, instead of the standard "0.17". It
seems that these exist mostly in the lower end of the magnification range, so
for confocal work, your choice may be limited. On the other hand, you can get
such lenses in higher power versions for stereomicroscopes. For instance, we
have high NA 10x and 20x objectives on a Zeiss stereomicroscope, which would
generate up to 100x magnification in combination with the additional
zoom.
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024
On Nov 8, 2007, at 10:33 AM, Oky wrote:
No virus found in this incoming message. No virus found in this outgoing message. |
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In reply to this post by Barbara Foster
Search the CONFOCAL archive at
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Hi
all,
somehow related with this, I often observe a phenomenon I have no
explanation for. Sometimes users come with slides in wich they have put too
small amount of mounting media and/or they have pressed the coverslip too strong
to eliminate the excess, resulting in areas of different size (from small
bubbles to big areas i.e. next to cell aggregates) without mounting media (and
therefore with air instead) along the preparation. In this situation I would
expect fluorescence in that areas to be less intense and/or fuzzy due to the
refraction/aberration issues all of you have mentioned, but surprisingly it
appears brighter at the ocular. If I image these brighter,
mounting-media-lacking areas with the confocal (with laser and PMT set for the
fluorescence in the normal -with mounting medium- areas of the slide),
they appear totally saturated on the screen.
Anybody has an explanation for this? Is reflection related with
this?
Thanks
in advance,
Xavi.
___________________________________
Xavier Sanjuan
Servei de Microscòpia Confocal Departament de Ciències Experimentals i de la Salut Universitat Pompeu Fabra Parc de Recerca Biomèdica de Barcelona Doctor Aiguader, 88 - Sala 309 08003 Barcelona - Spain Tel.: + 34 93 316 08 64 Fax: + 34 93 316 09 01 E-mail: [hidden email] Web: http://www.upf.edu/cexs/sct
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McDonald, David L |
Re: Imaging without medium?
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Search the CONFOCAL archive at
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I have noticed this as well, primarily with DAPI. Is it possible the free DAPI in the mounting media is quenching the target signal and in the areas lacking the media the signals aren't quenched thus brighter? One thing I have also noticed is the signals in the lacking areas, although often brighter, usually bleach much quicker. Dave Dave McDonald Scientific Imaging Lab Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North, DE-512 Seattle, WA 98109 206-667-4205 http://www.fhcrc.org At 01:49 AM 11/9/2007, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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Search the CONFOCAL archive at
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In the multiphoton imaging case, the medium breakdown by Ti:sapphire laser (visible spectra from ions instead molecules) may contribute a lot to the final fluorescence brightness. Due to bubbles etc, the focused laser will likely generate hot spots inside the medium and cause atoms to ionize. Just a thought.
Lingqing Lingqing Zhang
Cell and Tisuue Imaging Center
St Jude Children's Research Hospital
Memphis, TN
On 11/9/07, Dave McDonald <[hidden email]> wrote:
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Reply about burn produced in 2-photon imaging
On Nov 12, 2007 11:55 PM, Lingqing Zhang <[hidden email]> wrote:
-- My co-ordinates: Shalin Mehta, Graduate student Graduate Programme in Bioengineering, NUS, Singapore Mobile: +65 90694182 |
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Search the CONFOCAL archive at
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Sorry everyone that the 'reminder' that I was setting for myself got sent by mistake....
I wanted to mention something related to what Lingging said:Local heating during 2-photon imaging. Julio also mentioned having seen this earlier.. But I was not sure if it is to be attributed to heating of mounting medium or ablation of cells. But from looks of images it appears most likely it is medium.. I have posted the images on my blog at http://shalin.wordpress.com/2007/04/26/be-careful-when-you-use-scan-zoom-on-confocal/ On Nov 13, 2007 11:26 AM, Shalin Mehta <[hidden email]> wrote: Reply about burn produced in 2-photon imaging -- My co-ordinates: Shalin Mehta, Graduate student Graduate Programme in Bioengineering, NUS, Singapore Mobile: +65 90694182 |
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Steffen Dietzel |
New Bioimaging seminar series for Munich
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Search the CONFOCAL archive at
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Dear confocalists,
I hope nobody objects when I use the list to to introduce a local event in Munich, Germany. Don't worry, although a seminar series is a recurring event, this will be the only posting to the confocal list. For those interested, you can download the pdf-anouncement from http://www.bin.lmu.de/bimug/ There you will also find a possibility to subscribe to a mailing list to receive future anouncements. Best regards Steffen Dietzel New Bioimaging seminar series Our new bioimaging seminar series is founded by the bioimaging network of the LMU-Innovativ process to foster connections, collaborations and exchange of knowledge between research groups using bioimaging techniques in and around Munich. Respective techniques include all types of microscopy (light, electron, force), X-ray crystallography and image analysis. One seminar session will include two talks. A focus of the talks will be on technical aspects of bioimaging, including advantages, limitations and potential pitfalls of applied techniques. More information about the seminar series can be found at http://www.bin.lmu.de/bimug/ The first session will be held on Tuesday, December 4th, 2007. Speakers will be Patrick Cramer and Jürgen Plitzko: Patrick Cramer, Professor at the Gene Center, works on the structural details of gene transcription by RNA polymerases. He uses X-ray crystallography as a primary experimental tool. Last year, he received the Leibniz Award of the Deutsche Forschungsgemeinschaft. For more information on his research please see http://www.lmb.uni-muenchen.de/cramer/research/index.htm. Jürgen Plitzko is head of transmission EM at the Dept. of Molecular Structural Biology (Baumeister Group) at the MPI of Biochemistry. He uses Cryo-Electron Tomography to generate 3D reconstructions e.g. of Viruses, parasites and cells. For more information on his research please see http://www.biochem.mpg.de/en/rd/baumeister/research/ContentCET/ The second session will be held on Tuesday, January 8th, 2008. Speakers will be Heinrich Leonhardt and Rainer Hillenbrand. Heinrich Leonhardt, Professor at the Biocenter, will talk about a new development that allows to target antigens in live cells with antibody-like detection. Chromobodies are fluorescently labeled, small single chain antibodies derived from Camelidae. See here for his Web-page: http://www.biologie.uni-muenchen.de/ou/epigenetics/ and here for a related publication: http://www.nature.com/nmeth/journal/v3/n11/abs/nmeth953.html Rainer Hillenbrand, leader of the Nano-Photonics group at the MPI of Biochemistry, develops near-field optical microscopy, based on atomic force microscopy (AFM). "Scattering-type Scanning Near-field Optical Microscopy" (s-SNOM) thus couples AFM with optics. His Web page is here: http://www.biochem.mpg.de/en/rg/hillenbrand/ If you wish to receive future announcements for our bioimaging seminar series, please subscribe to our mailing list: http://www.bin.lmu.de/bimug/list.html Please find details about time and location in the pdf-files at our web site at http://www.bin.lmu.de/bimug/. If you have questions or if you would like to speak in the seminar series please contact me. The third session is planned for February 12, 2008. Best regards Steffen Dietzel 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-81366 München Building location and address for courier, parcel services etc: Marchioninistr. 27, D-81366 München (Großhadern) Phone: +49/89/2180-76509 Fax: +49/89/2180-76503 (please anounce incoming fax by e-mail) skype: steffendietzel |
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