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We have received a CARV recently and so I will answer your questions to some
extent. We have used it some on real live samples, but not enough to give a
thorough review. So far the results are quite encouraging in terms of
resolution and practical usability.
Q1: The CARV is a 1-sided TSM. I don't know what they do with the
unused/reflected excitation. However, it seems to be contained inside the
CARV without contaminating the emission signal. For those that are familiar
with the Molecular Probes triple-labeled "Fluocells" slide, the confocal
image can be seen through the CARV eyepieces with relative ease.
Q2: The 5-7% figure appears to be accurate, based on subjective visual
observation. We bought the X-Cite 120 light source w/ the CARV. The X-Cite
120 is supposed to be ~50% more powerful overall than a standard HBO 100 Hg
source, but can be more than twice as bright for FITC/GFP excitation. ( for
more info see http://www.exfo.com/en/products/ProductsFamily.asp?Family=218
) So far the setup and performance of the X-Cite 120 are very good, but
can't recommend without long term experience.
Q3: I got some numbers over the phone from Atto's technical support. For a
60x NA=1.4 lens, AU~1.2; for 100x NA=1.4 lens, AU~1.0 (or a little less?). I
think these numbers are for the image plane where the disk is, not the
sample, and perhaps they are specific for the Olympus IX71 microscope.
Hope this helps.
Cheers,
Jeff Reece
Biomedical Engineer
Confocal Microscopy Center
National Institute of Environmental Health Sciences (NIEHS)
P.O. Box 12233, MD F2-02
111 Alexander Drive, Bldg. 101 / Rm. F219
Research Triangle Park, NC 27709 USA
(919) 541-0311
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-----Original Message-----
From: Guy Cox [mailto:[log in to unmask]]
Sent: Saturday, January 03, 2004 4:39 AM
To: [log in to unmask]
Subject: Re: spinning disk instruments
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I would love to know something more about the CARV device. I've
been to their website, downloaded their pdf, and find a small amount
of information and a lot of 'smoke and mirrors'.
Question 1: is it a 2-sided (eg Petran, Tracor-Northern) TSM or a
1-sided (OTSM, K2Bio, Yokogawa) type system? (Yes, believe it or
not, this fundamental information cannot be gleaned from their site).
If it is 1-sided, how do they deal with the huge majority of the
incident light which is reflected by the disk itself?
Question 2: Traditional (Petran, TN etc) Nipkow disk systems
transit only 1% of the incident light. The CARV claims 5-7%,
which is better, but even so I don't know many 'real-world'
cell biology projects which can get useful images in widefield
with only 6% of the mercury lamp output - most projects use
between 25% and 100%. Do they use some sort of high-power lamp
(a 1kW Hg lamp??)
Question 3: How confocal is it? One of the few figures they
do give is their pinhole size - 70?m. Now an NA 0.75 lens
has an Airy disk ~450nm at the specimen plane at 550nm
(0.61 x 550 / 0.75) and so the x20 lens MiiCarter is using
will produce an image Airy disk of ~ 9?m - very much smaller
than the pinhole. A x40 lens of the same NA will give twice the
size - 18?m, and I can't think of any commonly available objective
which will give a spot anywhere near the size of the pinhole.
Maybe they have some intermediate optics enlarging the spot -
but if so what is the point of quoting the pinhole size?
Maybe they are just not very confocal? Can any user image
some fluorescent beads and give us an estimate of Z-resolution?
I'd welcome some answers either from ATTO or from users!
Guy Cox
Quoting [log in to unmask]:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Re CARV v Yokogawa,
>
> I have the CARV's big brother after trying many many Yokogawa demos
without
> success. For our needs it outperforms the Yokogawa in every respect.
> There
> are many fine details in design of a system which can make all the
> difference:-
>
>
> The CARV has larger pinholes, more widely spaced, which makes it more
> effective on thicker or otherwise more difficult samples. We use a 20x
> 0.75
> objective and get beautiful images, whereas the Yokogawa folks were
dismayed
> at the
> thought of not using top NA immersion objectives.
>
> The CARV uses conventional fluorescence filter cubes, so reconfiguring for
> a
> new dye is a simple cube swap.
>
> Longpass dichroics have far sharper cut-on than shortpass has cut-off, so
> you
> are more prone to light leakage in the unconventional shortpass layout
used
> by the microlens systems.
>
> On the CARV, you can flip a switch and be in full field fluorescence mode
> for
> ocular searching of the specimen. They can even throw in a neutral
density
> filter so the brightness is comparable.
>
> You also have the comfort of binocular viewing at head height, rather than
> an
> upwardly pointing monocular lens with very limited field of view and
> insufficient brightness.
>
> Finally, they now offer a thousands of hour light source for those who
> don't
> like changing bulbs every 200 hours. This wasn't suitable for us, but it
> shows how hard they try to suit the needs of a busy lab.
>
> If you are considering a spinning disc system, you have to evaluate the
> CARV
> from Atto Bioscience. Its even cheaper than the microlens systems because
> you
> don't need all the lasers.
>
> Best wishes,
>
> David
> oQQQQQ@
>
> David Carter, M.A., Ph.D.
> Academic Coordinator (microscopy and imaging)
> Center for Plant Cell Biology
> UC-Riverside CA 92521
>
--
Associate Professor Guy Cox
Electron Microscope Unit, F09
University of Sydney NSW 2006
+61 2 9351 3176
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