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Hi Jens,
this depends on the combination of objective used, desired imaging depth
etc.
I assume you want to image tissue of 100 micrometer thickness or more,
probably with a higher N.A objective calculated for use with a # 1.5 (0.17
mm) cover slip.
I recommend usage of a medium with a refractive index of around 1.45 (e.g.
for the LEICA 63x glycerol lens or the respective ZEISS 63x immersion lens).
The final refractive index should closely match the refractive index of the
immersion medium the lens is calculated for. Minor aberrations causing
spherical aberration in many cases can be corrected for by an objective
correction collar. But for but best correction across the whole imaging
depth the refractive indices should match precisely. In addition, the
correction collar should be adjusted carefully at the begin of the scanning
session.
We routinely use 80% glycerol in PBS pH 8.9 plus antifade (1% DABCO) or
Citifluor or SlowFade Gold adjusted to a refractive index of 1.45 with
glycerol (adjust pH). The antifade used depends on the fluorochrome
combination used.
For clearing, I recommend to change the final clearing/immersion medium at
least once (total time: at least 5 hours, maybe overnight at room temp.).
Clearing speed is lowered with temperature because of increasing viscosity
of glycerol.
Use cover slip strips as spacers between slide and cover slip and fix coveer
slip with nail hardener.
Such a sample can also be imaged with lower magnification objectives (e.g.
20x, 10x immersion objectives).
You may contact me for more details.
Guenter
------------------------------------------
Dr. Guenter Giese
Light Microscopy Facility Manager
Dept. of Biomedical Optics
MPI fuer Medizinische Forschung Jahnstr. 29
D-69120 Heidelberg, Germany
Phone (+49) 6221-486-360 (Fax: -325)
e-mail: [log in to unmask]
http://lightmicro.mpimf-heidelberg.mpg.de
> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List
> [mailto:[log in to unmask]] Im Auftrag von Rietdorf, Jens
> Gesendet: Donnerstag, 8. März 2007 08:45
> An: [log in to unmask]
> Betreff: How to clear brain tissue?
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear listers,
>
> Could you kindly help me with the following.
>
> Which clearing method/solution is best suited for
> paraformaldehyde-fixed brain tissue? Murrey Clear?
> MethylSalicylate? Others? Why?
>
> Some of the neurons are labeled with fluorescent proteins and
> in some cases the signal is amplified by Alexa488 conjugate
> anti GFP antibody labeling.
>
> Any hints?
>
> Thanks & regards, jens
>
> ---
> Dr. Jens Rietdorf
> head of microscopy unit
> Novartis Foundation
> Friedrich-Miescher-Institute, wro1066.2.32 Maulbeerstr.66,
> CH-4058 Basel, Switzerland phone +41(61)69-75172 mobil +41 798284737
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[log in to unmask]] On Behalf Of Rietdorf, Jens
> Sent: Mittwoch, 7. März 2007 08:48
> To: [log in to unmask]
> Subject: Re: Micron Level Registration
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Gary,
>
> Colombelli and colleagues use a pulsed UV laser to etch the
> cover glass. The marks created here are about 1 mikometer
> thin lines and can reliably be re-loclised in EM (in EM it's
> the replica in the embedding which is easy to spot).
>
> The paper is submitted, so maybe contact Julien directly,
> works is at the EMBL in Heidelberg, Germany.
>
> Colombelli J, Tängemo C, Haselmann U, Antony C, Stelzer EHK,
> Pepperkok R, Reynaud EG. A correlative light and electron
> microscopy method based on laser micropatterning and etching.
> Methods in Molecular Biology. Submitted.
>
> regards, jens
>
> ---
> Dr. Jens Rietdorf
> head of microscopy unit
> Novartis Foundation
> Friedrich-Miescher-Institute, wro1066.2.32 Maulbeerstr.66,
> CH-4058 Basel, Switzerland phone +41(61)69-75172 mobil +41 798284737
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[log in to unmask]] On Behalf Of Gary Laevsky
> Sent: Donnerstag, 1. März 2007 16:23
> To: [log in to unmask]
> Subject: Micron Level Registration
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello All,
>
> We're taking some multimodal images of skin (SHG, 2P (auto),
> reflectance, and Raman), 20-60x.
>
> The SHG, 2P, and reflectance are on one scope and the Raman
> is on another (pretty cool images in all modes).
>
> We've reached a quandary on how to register these images
> (even get near similar fields of view, which are between 50-250 um).
>
> A pen mark would be way too big. Too many hair follicles to
> narrow down to. Latex beads will probably float away (the
> skin needs to stay hydrated). A grid coverslip? Maybe a gene gun?
>
> The scopes are in different buildings, so stability of the
> "mark" IS an issue.
>
> Any and all input is appreciated, again.
> Best,
>
> Gary
>
>
>
> Gary Laevsky, Ph.D.
> Keck Facility Manager, CenSSIS
> Northeastern University
> 302 Stearns
> 360 Huntington Ave.
> Boston, MA 02115
> Office(617) 373 - 2589
> Lab(617) 373 - 7756
> Fax(617) 373 - 7783
>
> http://www.censsis.neu.edu
>
> http://www.ece.neu.edu/groups/osl
>
> http://www.keck3dfm.neu.edu
>
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