penetration depth
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Hello all!
I was wondering for which one penetration depth is higher: NA:1.2 60x lens or NA : 0.3 10x lens?
thanks
Sarah
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Sarah, The penetration depth is generally related with the working distance, and of course, the NA 0.3 10X has much longer penetration depth than that of NA1.2 60x. In general, NA1.2 60x has a working distance of ~100 um, and NA0.3 10x has longer than 1 mm. But the penetration depth is also related with the scattering coefficient of the sample. If you are observing semi-transparent sample, then it is almost equal to working distance if omitted the aberration; but if you are observing highly scattering sample, then it can be the same because most light are simply scattered along the way. Thank you! -- Best regards, Peng Xi Associate Professor Institute for Laser Medicine and Biophotonics Shanghai Jiao Tong University 800 Dongchuan Rd. Shanghai 200240, China Tel: (86) 21-3420-4076 http://biophotonics.sjtu.edu.cn/ On Feb 5, 2008 9:31 AM, Sarah Kefayati <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Hello all! > > I was wondering for which one penetration depth is higher: NA:1.2 60x lens > or NA : 0.3 10x lens? > > thanks > Sarah |
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Search the CONFOCAL archive at
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Thank you so much for your great info,totally helpful!
Sarah
On Feb 4, 2008 8:53 PM, Peng Xi <[hidden email]> wrote: Search the CONFOCAL archive at |
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Search the CONFOCAL archive at
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Dear Sarah,
For best results, you actually should use a long working distance dipping lens for thick live (or hydrated specimens).
Cheers Steve Stephen H. Cody Tip: Learn how to receive reminders about you microscope
booking: -----Original Message-----
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Thank you so much for your great info,totally helpful!
Sarah On Feb 4, 2008 8:53 PM, Peng Xi <[hidden email]> wrote: Search the CONFOCAL archive at > Hello all!
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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In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal ultimately the lower NA with its longer working distance wins if the sample is clear enough.. At 08:31 PM 2/4/2008 -0500, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >Hello all! > >I was wondering for which one penetration depth is higher: NA:1.2 >60x lens or NA : 0.3 10x lens? > >thanks >Sarah |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal What's the best method of making a sample more transparent to reduce scattering, without changing its chemistry too much? (other than methyl salicylate and glycerol). Thanks. Judy Judy Trogadis Bio-Imaging Coordinator St. Michael's Hospital, 7Queen 30 Bond St. Toronto, ON M5B 1W8, Canada ph: 416-864-6060 x6337 pager: 416-685-9219 fax: 416-864-6043 [hidden email] >>> Bill Miller <[hidden email]> 02/04/08 9:16 PM >>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal ultimately the lower NA with its longer working distance wins if the sample is clear enough.. At 08:31 PM 2/4/2008 -0500, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >Hello all! > >I was wondering for which one penetration depth is higher: NA:1.2 >60x lens or NA : 0.3 10x lens? > >thanks >Sarah |
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In reply to this post by Stephen Cody
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Does anyone in either Sydney or Perth have any of the high-index water-miscible mounting medium TDE (2,2`-thiodiethanol)? We need some urgently for a STED session later this month but unfortunately Sigma Australia have no stock and because it's toxic shipping from Germany will take several weeks (which we don't have). 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 +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net <http://www.guycox.net/> No virus found in this outgoing message. Checked by AVG Free Edition. Version: 7.5.516 / Virus Database: 269.19.19/1258 - Release Date: 4/02/2008 10:10 AM |
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In reply to this post by Bill Miller-3
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Just a heads-up: Emails with titles like "Penetration Depth" generally end up in my trash along with other "male enhancement" solicitations. I am just saying! Jeff Travis Bill Miller wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > ultimately the lower NA with its longer working distance wins if the > sample is clear enough.. > > > At 08:31 PM 2/4/2008 -0500, you wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> Hello all! >> >> I was wondering for which one penetration depth is higher: NA:1.2 60x >> lens or NA : 0.3 10x lens? >> >> thanks >> Sarah > |
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In reply to this post by Judy Trogadis
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Judy, try the BABB after Dent's fixative. BABB or Murray's clear stands for 1:2 mixture of benzyl alcohol and benzyl benzoate both RI around 1.5 and available from Sigma. Let me offline should you have problem or success with this protocol. Thanks Shiv Mayandi Sivaguru PhD, PhD Microscopy Facility Manager Institute for Genomic Biology Unviersity of Illinois at Urbaba-Champaign Urbana 61801 IL USA [hidden email] http://core.igb.uiuc.edu > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > What's the best method of making a sample more transparent to reduce > scattering, without changing its chemistry too much? (other than methyl > salicylate and glycerol). > > Thanks. > Judy > > Judy Trogadis > Bio-Imaging Coordinator > St. Michael's Hospital, 7Queen > 30 Bond St. > Toronto, ON M5B 1W8, Canada > ph: 416-864-6060 x6337 > pager: 416-685-9219 > fax: 416-864-6043 > [hidden email] > > >>>> Bill Miller <[hidden email]> 02/04/08 9:16 PM >>> > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > ultimately the lower NA with its longer working distance wins if the > sample is clear enough.. > > > At 08:31 PM 2/4/2008 -0500, you wrote: >>Search the CONFOCAL archive at >>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>Hello all! >> >>I was wondering for which one penetration depth is higher: NA:1.2 >>60x lens or NA : 0.3 10x lens? >> >>thanks >>Sarah > |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal But, it will depend upon the highest RI component of the sample. you might have to go higher or lower by blending. I've used Arnie Voie's mixture of 5 parts methyl salicylate:3 parts benzyl benzoate to obtain an RI that calculates to about 1.556. It works well for dense connective tissue of bone. Regards, 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] ************************************************************************ ****** The box said "Requires WindowsXP or better", so I bought a Macintosh. ************************************************************************ ****** On Feb 6, 2008, at 7:11 AM, Mayandi Sivaguru wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Judy, try the BABB after Dent's fixative. BABB or Murray's clear > stands > for 1:2 mixture of benzyl alcohol and benzyl benzoate both RI > around 1.5 > and available from Sigma. Let me offline should you have problem or > success with this protocol. > Thanks > Shiv > > Mayandi Sivaguru PhD, PhD > Microscopy Facility Manager > Institute for Genomic Biology > Unviersity of Illinois at Urbaba-Champaign > Urbana 61801 IL USA > [hidden email] > http://core.igb.uiuc.edu > > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> What's the best method of making a sample more transparent to reduce >> scattering, without changing its chemistry too much? (other than >> methyl >> salicylate and glycerol). >> >> Thanks. >> Judy >> >> Judy Trogadis >> Bio-Imaging Coordinator >> St. Michael's Hospital, 7Queen >> 30 Bond St. >> Toronto, ON M5B 1W8, Canada >> ph: 416-864-6060 x6337 >> pager: 416-685-9219 >> fax: 416-864-6043 >> [hidden email] >> >> >>>>> Bill Miller <[hidden email]> 02/04/08 9:16 PM >>> >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> ultimately the lower NA with its longer working distance wins if the >> sample is clear enough.. >> >> >> At 08:31 PM 2/4/2008 -0500, you wrote: >>> Search the CONFOCAL archive at >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> Hello all! >>> >>> I was wondering for which one penetration depth is higher: NA:1.2 >>> 60x lens or NA : 0.3 10x lens? >>> >>> thanks >>> Sarah >> |
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In reply to this post by Judy Trogadis
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Judy, I was impressed with 2,2'-thiodiethanol, used neat (R.I. 1.52; Sigma-Aldrich). Completely black background (N=2 slides, one confocal session). See Staudt, Hell et al 2007 Microsc Res Tech for original reference. They used RI 1.515 by adding 3% H2O. I figured no reason to pollute the TDE with any additives. One oddity was that by eye, both blood vessel elastin fiber autofluorescence was decreased (488 nm excitation, green emission) and the DAPI nuclear counterstain was quenched. Both came through nicely in the confocal. Robert Zucker has several confocal papers on BABB - benzyl alcohol/benzyl benzoate as a clearing agent (see another responder's message for other names). Bob told me the key is to completely dehydrate the specimen through EtOH steps into xylene, before going to BABB. See his papers for exact steps. best wishes, George At 11:09 AM 2/5/2008, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >What's the best method of making a sample more transparent to reduce >scattering, without changing its chemistry too much? (other than >methyl salicylate and glycerol). > >Thanks. >Judy > >Judy Trogadis >Bio-Imaging Coordinator >St. Michael's Hospital, 7Queen >30 Bond St. >Toronto, ON M5B 1W8, Canada >ph: 416-864-6060 x6337 >pager: 416-685-9219 >fax: 416-864-6043 >[hidden email] > > > >>> Bill Miller <[hidden email]> 02/04/08 9:16 PM >>> >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >ultimately the lower NA with its longer working distance wins if the >sample is clear enough.. > > >At 08:31 PM 2/4/2008 -0500, you wrote: > >Search the CONFOCAL archive at > >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hello all! > > > >I was wondering for which one penetration depth is higher: NA:1.2 > >60x lens or NA : 0.3 10x lens? > > > >thanks > >Sarah George McNamara, Ph.D. University of Miami, Miller School of Medicine Image Core Miami, FL 33010 [hidden email] [hidden email] 305-243-8436 office http://home.earthlink.net/~pubspectra/ http://home.earthlink.net/~geomcnamara/ http://www.sylvester.org/research/SR_lab_analytical.asp?ana=desc (Analytical Imaging Core Facility) |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear George, Did Bob say why the xylene was important? I would expect that BABB would be as alcohol-soluble as methyl salicylate:benzyl benzoate. Maybe the xylene was ensuring the last of the water is out, but thorough dehydration should be sufficient. Many mixtures of solvents are given in this book: Spalteholz, W., 1914. Über das Durchsichtigmachen von menschlichen und tierischen Präparaten und seine theoretischen Bedingungen. Hirzel, Leipzig. Glad to hear someone else has tried TDE. Do you notice any tissue shrinkage? How are you storing it? I've haven't yet opened my bottle. One post doc is using my mixture of MSBB on 1200 µm brain slices, but I'm going to try TDE on brain slices <400 µm with grad student. Thinner slices tend to curl after dehydration. Regards, 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] ************************************************************************ ****** The box said "Requires WindowsXP or better", so I bought a Macintosh. ************************************************************************ ****** On Feb 7, 2008, at 6:58 PM, George McNamara wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi Judy, > > I was impressed with 2,2'-thiodiethanol, used neat (R.I. 1.52; > Sigma-Aldrich). Completely black background (N=2 slides, one > confocal session). See Staudt, Hell et al 2007 Microsc Res Tech for > original reference. They used RI 1.515 by adding 3% H2O. I figured > no reason to pollute the TDE with any additives. One oddity was > that by eye, both blood vessel elastin fiber autofluorescence was > decreased (488 nm excitation, green emission) and the DAPI nuclear > counterstain was quenched. Both came through nicely in the confocal. > > Robert Zucker has several confocal papers on BABB - benzyl alcohol/ > benzyl benzoate as a clearing agent (see another responder's > message for other names). Bob told me the key is to completely > dehydrate the specimen through EtOH steps into xylene, before going > to BABB. See his papers for exact steps. > > best wishes, > > George > > > At 11:09 AM 2/5/2008, you wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> What's the best method of making a sample more transparent to >> reduce scattering, without changing its chemistry too much? (other >> than methyl salicylate and glycerol). >> >> Thanks. >> Judy >> >> Judy Trogadis >> Bio-Imaging Coordinator >> St. Michael's Hospital, 7Queen >> 30 Bond St. >> Toronto, ON M5B 1W8, Canada >> ph: 416-864-6060 x6337 >> pager: 416-685-9219 >> fax: 416-864-6043 >> [hidden email] >> >> >> >>> Bill Miller <[hidden email]> 02/04/08 9:16 PM >>> >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> ultimately the lower NA with its longer working distance wins if the >> sample is clear enough.. >> >> >> At 08:31 PM 2/4/2008 -0500, you wrote: >> >Search the CONFOCAL archive at >> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >Hello all! >> > >> >I was wondering for which one penetration depth is higher: NA:1.2 >> >60x lens or NA : 0.3 10x lens? >> > >> >thanks >> >Sarah > > > > > > > George McNamara, Ph.D. > University of Miami, Miller School of Medicine > Image Core > Miami, FL 33010 > [hidden email] > [hidden email] > 305-243-8436 office > http://home.earthlink.net/~pubspectra/ > http://home.earthlink.net/~geomcnamara/ > http://www.sylvester.org/research/SR_lab_analytical.asp?ana=desc > (Analytical Imaging Core Facility) |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Glen, Bob Zucker told me that if the tissue still has any water in it, the BABB will turn cloudy. Another item to be aware of with BABB is that it can solubilize some glues, so using it in an imaging chamber could results in leaks. Bob also emphasized that BABB is a very bad thing to have land on an objective lens or any other optic. One last item about BABB - I found some chemical company web sites that listed the refractive indices of several benzyl X compounds. Some are at or below RI=1.515. So, it may be feasible to find a mixture of two that is right at 1.515. That said, I'm excited about TDE because even neat, its RI is close to that of immersion oil and glass as to not matter except maybe hyper-critical deconvolution. I am currently storing the TDE (25 mL bottle, smallest that Sigma-Aldrich sells) in the chemical cabinet. I will probably aliquot it out into 0.5 or 1.0 mL eppendorf tubes to make it easier to distribute to users who want to try. My first user had ~5 um lung tissue sections. No way for me to tell whether they had shrinkage. There was an excellent article in one of the trade magazines (Microscopy Today?) a year or so on paraformaldehyde concentration in perfusion fixation. That, and other articles, lead me to expect that shrinkage comes from the PFA step, not from the later tissue clearing and dehydration. George At 11:25 AM 2/8/2008, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Dear George, >Did Bob say why the xylene was important? I would expect that BABB >would be as alcohol-soluble as methyl salicylate:benzyl benzoate. >Maybe the xylene was ensuring the last of the water is out, but >thorough dehydration should be sufficient. Many mixtures of solvents >are given in this book: Spalteholz, W., 1914. Über das >Durchsichtigmachen von menschlichen und tierischen Präparaten und >seine theoretischen Bedingungen. Hirzel, Leipzig. > >Glad to hear someone else has tried TDE. Do you notice any tissue >shrinkage? How are you storing it? I've haven't yet opened my >bottle. One post doc is using my mixture of MSBB on 1200 µm brain >slices, but I'm going to try TDE on brain slices <400 µm with grad >student. Thinner slices tend to curl after dehydration. > >Regards, >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] > >************************************************************************ >****** >The box said "Requires WindowsXP or better", so I bought a Macintosh. >************************************************************************ >****** > > >On Feb 7, 2008, at 6:58 PM, George McNamara wrote: > >>Search the CONFOCAL archive at >>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >>Hi Judy, >> >>I was impressed with 2,2'-thiodiethanol, used neat (R.I. 1.52; >>Sigma-Aldrich). Completely black background (N=2 slides, one >>confocal session). See Staudt, Hell et al 2007 Microsc Res Tech for >>original reference. They used RI 1.515 by adding 3% H2O. I figured >>no reason to pollute the TDE with any additives. One oddity was >>that by eye, both blood vessel elastin fiber autofluorescence was >>decreased (488 nm excitation, green emission) and the DAPI nuclear >>counterstain was quenched. Both came through nicely in the confocal. >> >>Robert Zucker has several confocal papers on >>BABB - benzyl alcohol/ benzyl benzoate as a >>clearing agent (see another responder's >>message for other names). Bob told me the key is to completely >>dehydrate the specimen through EtOH steps into xylene, before going >>to BABB. See his papers for exact steps. >> >>best wishes, >> >>George >> >> >>At 11:09 AM 2/5/2008, you wrote: >>>Search the CONFOCAL archive at >>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>>What's the best method of making a sample more transparent to >>>reduce scattering, without changing its chemistry too much? (other >>>than methyl salicylate and glycerol). >>> >>>Thanks. >>>Judy >>> >>>Judy Trogadis >>>Bio-Imaging Coordinator >>>St. Michael's Hospital, 7Queen >>>30 Bond St. >>>Toronto, ON M5B 1W8, Canada >>>ph: 416-864-6060 x6337 >>>pager: 416-685-9219 >>>fax: 416-864-6043 >>>[hidden email] >>> >>> >>> >>> Bill Miller <[hidden email]> 02/04/08 9:16 PM >>> >>>Search the CONFOCAL archive at >>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>>ultimately the lower NA with its longer working distance wins if the >>>sample is clear enough.. >>> >>> >>>At 08:31 PM 2/4/2008 -0500, you wrote: >>> >Search the CONFOCAL archive at >>> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >Hello all! >>> > >>> >I was wondering for which one penetration depth is higher: NA:1.2 >>> >60x lens or NA : 0.3 10x lens? >>> > >>> >thanks >>> >Sarah >> >> >> >> >> >> >>George McNamara, Ph.D. >>University of Miami, Miller School of Medicine >>Image Core >>Miami, FL 33010 >>[hidden email] >>[hidden email] >>305-243-8436 office >>http://home.earthlink.net/~pubspectra/ >>http://home.earthlink.net/~geomcnamara/ >>http://www.sylvester.org/research/SR_lab_analytical.asp?ana=desc >>(Analytical Imaging Core Facility) George McNamara, Ph.D. University of Miami, Miller School of Medicine Image Core Miami, FL 33010 [hidden email] [hidden email] 305-243-8436 office http://home.earthlink.net/~pubspectra/ http://home.earthlink.net/~geomcnamara/ http://www.sylvester.org/research/SR_lab_analytical.asp?ana=desc (Analytical Imaging Core Facility) |
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In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Search the CONFOCAL archive at George Hi George,
On your last point: shrinkage
In the late 1970s Alan Boyde and Elaine Maconnachie published a
number of papers (some noted below) describing experiments where they
followed the size of various specimens (fetal rat limb buds etc.)
during all the transitions between being alive and being an SEM
specimen (i.e., fixation, dehydration, replacement with intermediate
and transition liquids, actual drying). These images where then
analyzed quantitatively "on-line" with a Quantimet
image analyzing computer.
They used a wide variety of CDP protocols (different
dehydrations, different intermediate and transition liquids) as well
as freeze-drying from water ice and also from "ices" made by
freezing specimens immersed in non-polar liquids.
Although every type of transition could produced changes in
volume, by far the largest changes were always associated with the
final steps of dehydration (say from 80% to 100% ethanol or
acetone).
I should also point out that the total shrinkage was
"massive". Although freeze drying was best (about 40%
VOLUME shrinkage as I recall, most if it occurring as the
"last of the last" of the ice or bound-water was removed as
the specimen warmed up in the vacuum) , the best CPD protocols caused
"only" about 60% VOLUME shrinkage. Others produced 80%
shrinkage.
How could this be one might ask. Surely SEM specimens could not
look so "real" had they shrunk to this degree. Part of the
answer has to do with the fact that many SEM (and LM) specimens
consist of cells adhered to glass or plastic substrates. The glass
does not shrink. The cells attached to it become thinner, rather like
the skin of an animal pegged out on a frame and left in the sun to
dry. As we seldom take the trouble to measure their thickness, its
reduction goes unremarked.
In Bob Bacallao's Chapter 18 in the Handbook, one can see images
of this effect. On the other hand, the limb buds were merely suspended
in liquid, not constrained in any way. In addition, dehydration
protocols often extract lipids and there my be more of these in limb
buds than in the average tissue culture cell. Finally, cells with high
water content shrunk more.
The basic message is the same: The proteins shrivel up as the
last of the water is removed, whether this occurs by by sublimation or
by dehydration.
I very much suspect that the same will be true of specimens
embedded in clearing agents.
Cheers,
Jim P.
Boyde, A., and Maconnachie, E., 1979,
"Volume changes during preparation
of mouse embryonic tissue for scanning
electron microscopy." Scanning
2:149-163. Boyde, A., and Maconnachie, E., 1981, "Morphological correlations with dimensional change during SEM specimen preparation." Scanning Electron Microsc. 4:278-34.
--
**********************************************
Prof. James B. Pawley, Room 223, Zoology Research Building, 1117 Johnson Ave., Madison, WI, 53706 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ |
 
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George McNamara |
Re: penetration depth ... tissue shrinkage
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Jim, The article I referred to is: C.W. Scouten, R. O'Connor, M. Cunningham 2006 Perfusion fixation of research animal. Microscopy Today May 2006: 26-33. MT low resolution issues are posted online somewhere. Two quotes: "Intracellular fluid of living cells usually fluctuates about 330 millMolar (mM) osmolality. By contrast, four percent formaldehyde in water, commonly used in fixation, is about 1400 mM osmolality." "Several lines of evidence ... show that living brain is about 20% extracellular space. In perfused and fixed tissue, using traditional protocols, this space is absent and the brain is reduced in volume by about 20%. ... The brain atlas by Paxinos and Watson (1998) avoided this problem by working only with fresh frozen tissue, and not fixing." The paper goes on to define better perfusion reagents and protocols. I wish it had been published in a peer reviewed journal instead of a "gray literature" trade magazine, and hope the authors publish in a real journal. Sample preparation artifacts, such as the publications you mention, are among the reasons why I encourage users to image their specimens immediate after sacrificing the animal, with careful attention to perfusion fixation issues such as those raised by Scouten et al. Hoechst dyes only take minutes to soak into even an intact mouse brain (multiphoton). DiI, DiO, etc, can be used to label all the endothelial cells (both sides, including angiogenic sprouts) prior to perfusion fixation (Rong Wen, U Miami, in prep). After the tissue has been imaged by confocal and/or multiphoton microscopy (optionally with slicing into confocal penetration depth compatible thickness), it can be brought to the lab and processed for sectioning, immunofluorescence, in situ hybridization, H&E, IHC, etc. George At 12:30 PM 2/10/2008, you wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal George McNamara, Ph.D. University of Miami, Miller School of Medicine Image Core Miami, FL 33010 [hidden email] [hidden email] 305-243-8436 office http://home.earthlink.net/~pubspectra/ http://home.earthlink.net/~geomcnamara/ http://www.sylvester.org/research/SR_lab_analytical.asp?ana=desc (Analytical Imaging Core Facility) |
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Ron Anderson-4 |
Re: penetration depth ... tissue shrinkage
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal You can download the article at http://www.microscopy-today.com click on tables of contents/2006/ and then the May issue. Ron Anderson, MT Editor Subscribe to MT at the same website, free in North America. Only my hair is gray. George McNamara wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi Jim, > > The article I referred to is: > > C.W. Scouten, R. O'Connor, M. Cunningham 2006 Perfusion fixation of > research animal. Microscopy Today May 2006: 26-33. MT low resolution > issues are posted online somewhere. > > Two quotes: > > "Intracellular fluid of living cells usually fluctuates about 330 > millMolar (mM) osmolality. By contrast, four percent formaldehyde in > water, commonly used in fixation, is about 1400 mM osmolality." > > "Several lines of evidence ... show that living brain is about 20% > extracellular space. In perfused and fixed tissue, using traditional > protocols, this space is absent and the brain is reduced in volume by > about 20%. ... The brain atlas by Paxinos and Watson (1998) avoided > this problem by working only with fresh frozen tissue, and not fixing." > > The paper goes on to define better perfusion reagents and protocols. I > wish it had been published in a peer reviewed journal instead of a > "gray literature" trade magazine, and hope the authors publish in a > real journal. > > Sample preparation artifacts, such as the publications you mention, > are among the reasons why I encourage users to image their specimens > immediate after sacrificing the animal, with careful attention to > perfusion fixation issues such as those raised by Scouten et al. > Hoechst dyes only take minutes to soak into even an intact mouse brain > (multiphoton). DiI, DiO, etc, can be used to label all the endothelial > cells (both sides, including angiogenic sprouts) prior to perfusion > fixation (Rong Wen, U Miami, in prep). After the tissue has been > imaged by confocal and/or multiphoton microscopy (optionally with > slicing into confocal penetration depth compatible thickness), it can > be brought to the lab and processed for sectioning, > immunofluorescence, /in situ/ hybridization, H&E, IHC, etc. > > George > > > > > At 12:30 PM 2/10/2008, you wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> Search the CONFOCAL archive at >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>> Hi Glen, >>> >>> Bob Zucker told me that if the tissue still has any water in it, the >>> BABB will turn cloudy. Another item to be aware of with BABB is that >>> it can solubilize some glues, so using it in an imaging chamber >>> could results in leaks. Bob also emphasized that BABB is a very bad >>> thing to have land on an objective lens or any other optic. One last >>> item about BABB - I found some chemical company web sites that >>> listed the refractive indices of several benzyl X compounds. Some >>> are at or below RI=1.515. So, it may be feasible to find a mixture >>> of two that is right at 1.515. That said, I'm excited about TDE >>> because even neat, its RI is close to that of immersion oil and >>> glass as to not matter except maybe hyper-critical deconvolution. >>> >>> I am currently storing the TDE (25 mL bottle, smallest that >>> Sigma-Aldrich sells) in the chemical cabinet. I will probably >>> aliquot it out into 0.5 or 1.0 mL eppendorf tubes to make it easier >>> to distribute to users who want to try. >>> >>> My first user had ~5 um lung tissue sections. No way for me to tell >>> whether they had shrinkage. There was an excellent article in one of >>> the trade magazines (Microscopy Today?) a year or so on >>> paraformaldehyde concentration in perfusion fixation. That, and >>> other articles, lead me to expect that shrinkage comes from the PFA >>> step, not from the later tissue clearing and dehydration. >>> George >> >> >> Hi George, >> >> On your last point: shrinkage >> >> In the late 1970s Alan Boyde and Elaine Maconnachie published a >> number of papers (some noted below) describing experiments where they >> followed the size of various specimens (fetal rat limb buds etc.) >> during all the transitions between being alive and being an SEM >> specimen (i.e., fixation, dehydration, replacement with intermediate >> and transition liquids, actual drying). These images where then >> analyzed quantitatively "on-line" with a Quantimet image analyzing >> computer. >> >> They used a wide variety of CDP protocols (different dehydrations, >> different intermediate and transition liquids) as well as >> freeze-drying from water ice and also from "ices" made by freezing >> specimens immersed in non-polar liquids. >> >> Although every type of transition could produced changes in volume, >> by far the largest changes were always associated with the final >> steps of dehydration (say from 80% to 100% ethanol or acetone). >> >> I should also point out that the total shrinkage was "massive". >> Although freeze drying was best (about 40% VOLUME shrinkage as I >> recall, most if it occurring as the "last of the last" of the ice or >> bound-water was removed as the specimen warmed up in the vacuum) , >> the best CPD protocols caused "only" about 60% VOLUME shrinkage. >> Others produced 80% shrinkage. >> >> How could this be one might ask. Surely SEM specimens could not look >> so "real" had they shrunk to this degree. Part of the answer has to >> do with the fact that many SEM (and LM) specimens consist of cells >> adhered to glass or plastic substrates. The glass does not shrink. >> The cells attached to it become thinner, rather like the skin of an >> animal pegged out on a frame and left in the sun to dry. As we seldom >> take the trouble to measure their thickness, its reduction goes >> unremarked. >> >> In Bob Bacallao's Chapter 18 in the Handbook, one can see images of >> this effect. On the other hand, the limb buds were merely suspended >> in liquid, not constrained in any way. In addition, dehydration >> protocols often extract lipids and there my be more of these in limb >> buds than in the average tissue culture cell. Finally, cells with >> high water content shrunk more. >> >> The basic message is the same: The proteins shrivel up as the last of >> the water is removed, whether this occurs by by sublimation or by >> dehydration. >> >> I very much suspect that the same will be true of specimens embedded >> in clearing agents. >> >> Cheers, >> >> Jim P. >> >> Boyde, A., and Maconnachie, E., 1979, "Volume changes during preparation >> of mouse embryonic tissue for scanning electron microscopy."/ Scanning >> /2:149-163. >> Boyde, A., and Maconnachie, E., 1981, "Morphological correlations with >> dimensional change during SEM specimen preparation."/ Scanning Electron >> Microsc./ 4:278-34. >> >> >> -- >> >> ********************************************** >> Prof. James B. Pawley, Ph. >> 608-263-3147 >> Room 223, Zoology Research Building, >> FAX 608-265-5315 >> 1117 Johnson Ave., Madison, WI, 53706 >> [hidden email] >> 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, >> Vancouver Canada >> Info: http://www.3dcourse.ubc.ca/ Applications due by >> March 15, 2008 >> "If it ain't diffraction, it must be statistics." Anon. > > > > > > > George McNamara, Ph.D. > University of Miami, Miller School of Medicine > Image Core > Miami, FL 33010 > [hidden email] > [hidden email] > 305-243-8436 office > http://home.earthlink.net/~pubspectra/ > <http://home.earthlink.net/%7Epubspectra/> > http://home.earthlink.net/~geomcnamara/ > <http://home.earthlink.net/%7Egeomcnamara/> > http://www.sylvester.org/research/SR_lab_analytical.asp?ana=desc > (Analytical Imaging Core Facility) > > |
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In reply to this post by George McNamara
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi, Ok, so it is a dehydration issue. Incomplete dehydration of tissue passing through xylene will also cause Permount and DPX to become cloudy, a few days following coverslipping. I've been rigorous on dehydration and passing through graded mixtures of absolution EtOH and the MSBB mixture without cloudiness. Xylene tends to make tissues brittle. Yeah, the BB mixtures dissolve almost anything, even makes temporary wells of silicone grease very runny. I've been mounting in aluminum frames used to support laser microdissection films to which I've attached a measured 24x60 mm coverglass using silicone aquarium glue. So far, they've survived a year or so. I wonder if storing TDE at in aliquots at -80 would avoid the need to fill the bottle with inert gas after opening. The class of compounds to which TDE belongs is supposed to have some higher RI members, which would be helpful for high RI samples. thanks, Glen > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hi Glen, > > Bob Zucker told me that if the tissue still has any water in it, > the BABB will turn cloudy. Another item to be aware of with BABB is > that it can solubilize some glues, so using it in an imaging > chamber could results in leaks. Bob also emphasized that BABB is a > very bad thing to have land on an objective lens or any other > optic. One last item about BABB - I found some chemical company web > sites that listed the refractive indices of several benzyl X > compounds. Some are at or below RI=1.515. So, it may be feasible to > find a mixture of two that is right at 1.515. That said, I'm > excited about TDE because even neat, its RI is close to that of > immersion oil and glass as to not matter except maybe hyper- > critical deconvolution. > > I am currently storing the TDE (25 mL bottle, smallest that Sigma- > Aldrich sells) in the chemical cabinet. I will probably aliquot it > out into 0.5 or 1.0 mL eppendorf tubes to make it easier to > distribute to users who want to try. > > My first user had ~5 um lung tissue sections. No way for me to tell > whether they had shrinkage. There was an excellent article in one > of the trade magazines (Microscopy Today?) a year or so on > paraformaldehyde concentration in perfusion fixation. That, and > other articles, lead me to expect that shrinkage comes from the PFA > step, not from the later tissue clearing and dehydration. > > George > > > > At 11:25 AM 2/8/2008, you wrote: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Dear George, >> Did Bob say why the xylene was important? I would expect that BABB >> would be as alcohol-soluble as methyl salicylate:benzyl benzoate. >> Maybe the xylene was ensuring the last of the water is out, but >> thorough dehydration should be sufficient. Many mixtures of solvents >> are given in this book: Spalteholz, W., 1914. Über das >> Durchsichtigmachen von menschlichen und tierischen Präparaten und >> seine theoretischen Bedingungen. Hirzel, Leipzig. >> >> Glad to hear someone else has tried TDE. Do you notice any tissue >> shrinkage? How are you storing it? I've haven't yet opened my >> bottle. One post doc is using my mixture of MSBB on 1200 µm brain >> slices, but I'm going to try TDE on brain slices <400 µm with grad >> student. Thinner slices tend to curl after dehydration. >> >> Regards, >> 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] >> >> ********************************************************************* >> *** ****** >> The box said "Requires WindowsXP or better", so I bought a Macintosh. >> ********************************************************************* >> *** ****** >> >> >> On Feb 7, 2008, at 6:58 PM, George McNamara wrote: >> >>> Search the CONFOCAL archive at >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>> Hi Judy, >>> >>> I was impressed with 2,2'-thiodiethanol, used neat (R.I. 1.52; >>> Sigma-Aldrich). Completely black background (N=2 slides, one >>> confocal session). See Staudt, Hell et al 2007 Microsc Res Tech for >>> original reference. They used RI 1.515 by adding 3% H2O. I figured >>> no reason to pollute the TDE with any additives. One oddity was >>> that by eye, both blood vessel elastin fiber autofluorescence was >>> decreased (488 nm excitation, green emission) and the DAPI nuclear >>> counterstain was quenched. Both came through nicely in the confocal. >>> >>> Robert Zucker has several confocal papers on BABB - benzyl >>> alcohol/ benzyl benzoate as a clearing agent (see another >>> responder's >>> message for other names). Bob told me the key is to completely >>> dehydrate the specimen through EtOH steps into xylene, before going >>> to BABB. See his papers for exact steps. >>> >>> best wishes, >>> >>> George >>> >>> >>> At 11:09 AM 2/5/2008, you wrote: >>>> Search the CONFOCAL archive at >>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>>> >>>> What's the best method of making a sample more transparent to >>>> reduce scattering, without changing its chemistry too much? (other >>>> than methyl salicylate and glycerol). >>>> >>>> Thanks. >>>> Judy >>>> >>>> Judy Trogadis >>>> Bio-Imaging Coordinator >>>> St. Michael's Hospital, 7Queen >>>> 30 Bond St. >>>> Toronto, ON M5B 1W8, Canada >>>> ph: 416-864-6060 x6337 >>>> pager: 416-685-9219 >>>> fax: 416-864-6043 >>>> [hidden email] >>>> >>>> >>>> >>> Bill Miller <[hidden email]> 02/04/08 9:16 PM >>> >>>> Search the CONFOCAL archive at >>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>>> >>>> ultimately the lower NA with its longer working distance wins if >>>> the >>>> sample is clear enough.. >>>> >>>> >>>> At 08:31 PM 2/4/2008 -0500, you wrote: >>>> >Search the CONFOCAL archive at >>>> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>>> >Hello all! >>>> > >>>> >I was wondering for which one penetration depth is higher: NA:1.2 >>>> >60x lens or NA : 0.3 10x lens? >>>> > >>>> >thanks >>>> >Sarah >>> >>> >>> >>> >>> >>> >>> George McNamara, Ph.D. >>> University of Miami, Miller School of Medicine >>> Image Core >>> Miami, FL 33010 >>> [hidden email] >>> [hidden email] >>> 305-243-8436 office >>> http://home.earthlink.net/~pubspectra/ >>> http://home.earthlink.net/~geomcnamara/ >>> http://www.sylvester.org/research/SR_lab_analytical.asp?ana=desc >>> (Analytical Imaging Core Facility) > > > > > > > George McNamara, Ph.D. > University of Miami, Miller School of Medicine > Image Core > Miami, FL 33010 > [hidden email] > [hidden email] > 305-243-8436 office > http://home.earthlink.net/~pubspectra/ > http://home.earthlink.net/~geomcnamara/ > http://www.sylvester.org/research/SR_lab_analytical.asp?ana=desc > (Analytical Imaging Core Facility) |
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