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Dear Renato,
Ironically, the big issue here is the NA of the objective. The
larger the NA, the shorter the working distance. The shorter
working distance wil limit how far you can go into the sample without
running into it. It was a lesson I learned the hard way, in my
early days as technical marketing manager and field support for Sarastro,
one of the pioneers in confocal microscopy. We were running high
resolution scans which took a great deal of time. After about the
first 10 sections, the image just began to go out of focus. It didn't
occur to me for over 20 minutes that we were working with a high NA
objective, with a VERY short working distance, and that the rest of the
scans were just pushing the objective into the sample! How
embarrassing! But it was one trial learning.
Please remember that working with a lower NA is a trade-off. While you
will be able to image deeply into the tissue, your individual sections
will not be as thin as usual. If you are interested in the general
structure of the tissue, this will not be an issue. If you are
trying to image very fine particles within the tissue, that might be
another story.
George's comments are well founded. While at Sarastro, I
imaged 300microns into a proto-type of a J&J wet bandage, showing the
gel layer and then the non-woven fabric underneath, as well as 200+
microns into poly-urethane foams and, if memory serves, about 200microns
into central nervous system tissue, all with lower mag/lower NA
objectives.
As with any microscopy technique, now that you know the fundamentals,
give it a try and find out what works best.
Good hunting!
Barbara Foster, President and Sr. Consultant
Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: (972)924-5310 Skype: fostermme
W:
www.MicroscopyEducation.com
NEWS! Visit the NEW and IMPROVED
www.MicroscopyEducation.com! And don't forget: MME is now
scheduling customized, on-site courses for the balance of the year.
Call me for a free assessment and quote.
At 05:24 PM 10/5/2009, you wrote:
Commercial interest:
We have set up several CSU-10s and CSUX systems for similar purposes. One
paper describing this system was published last year.
Disease Models Mech. 1, 155 (2008) cited as an editors Choice
in Science Magazine.
80 to 100 ums into live tissue was obtainable in 4 colors using air lens.
Water or glycerin immersion lens should improve this.
Best regards
George A. Peeters MD, MS
President, Solamere Technology
Group Inc
1427 Perry Ave
Salt Lake City, UT 84103
www.solameretech.com
801 322-2645
office 801 322-2645
fax
801 232-6911 cell
On Oct 5, 2009, at 2:50 PM, Kathryn Spencer wrote:
Hi Renato;
We
routinely image 200 micron mouse brain sections with our Yokogawa CSU-10
spinning disk. Effectively, we can see about 100 microns well. We are
using a 20x LUCPlan FluorN 0.45 dry objective. This is mostly due to
constraints of using Millipore chambers for culturing the mouse brains;
we need the extra working distance. I realize the mismatch between the
pinholes and the objective NA. Our four day, time-lapse images are really
very nice with this objective, although our expression levels need to be
rather high for the cell processes to be seen.
Best,
Kathy
From: Confocal Microscopy List
[[hidden email][hidden email]] On Behalf Of Renato
Mortara
Sent: Monday, October 05, 2009 1:04 PM
To:
[hidden email]
Subject: How thick can samples be to be imaged on a Spinning Disk
Confocal ?
Hello,
I am in the process of deciding the best possible configuration to
assemble a spinning disk confocal with the Yokogawa CSU-X1 scanning
head.
It is common knowledge that imaging 'thick' samples can be tricky or
simply not feasible with spinning disk confocals.
Does anyone there have practical experience for instance, with mouse
brain sections ?
Many thanks for the input,
Best
Renato
Renato A. Mortara
Parasitology Division
UNIFESP - Escola Paulista de Medicina
Rua Botucatu, 862, 6th floor
São Paulo, SP
04023-062
Brazil
Phone: 55 11 5579-8306
Fax: 55 11 5571-1095
email:
[hidden email]
home page:
www.ecb.epm.br/~ramortara
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