Dear confocalists

I've been spending the last few weeks at the MBL in Woods Hole. During my
stay I had the opportunity (and the pleasure) to use the Yokogawa CSU10
real-time microlens Nipkow-disk confocal (phew!) in the lab of Shinya
Inoue for a few hours. I have been so impressed that I decided to write a
short note to the list.

Let me start by giving some background information. My 'speciality' is
(mainly conventional) fluorescence microscopy of living cells. The
samples that I have observed with the CSU10 were live rat embryo
fibroblasts expressing a tubulin-GFP fusion protein which incorporates
into microtubules. I have done both Z-series and time-lapse in direct
comparison to non-confocal epifluorescence mode. Comparisons to
point-scanner confocals are indirect, based on my experience with Leica
and Zeiss systems.

comparison to non-confocal video fluorescence microscopy:
+ confocal sectioning (obviously)
+ much sharper, clearer images, even in very thin samples! (the reason
for the latter is not clear to me, but the difference is striking).
+ illumination confined the the scanning plane, so there should be less
phototoxicity problems.
+ elimination of problems with culture medium autofluorescence.
- additional cost (even more so, if a UV-laser is required)

advantages over single point-scanning confocals:
+ ease of use: 1. Working with the CSU10 is as 'fast and easy' as with a
normal video microscopy setup. 2. The ability the see the (almost
unbelieveably sharp) confocal image directly through the ocular allows
for the quick inspection of structures hiding in thick or dense specimens
that can't be seen properly by conventional epifluorescence. On a normal
confocal, one has to work on a trial&error basis in these situations.
+ speed:  Acquiring/scanning images of comparable signal to noise ratio
using a similar effective intensity of illumination, judged by the amount
of photobleaching induced, is about 5-20x faster. This of course is
largely dependent on the type and quality/sensitivity of the camera used
(the Hamamatsu chilled integrating video camera mounted on the test
system was not really optimal(ly configured)).
- lower Z-resolution

BTW, the CSU10 is currently only working at a single wavelength, but a
multi-wavelength option should be available soon, according to Yokogawa.

Disclaimer: I have no commercial interest in Yokogawa - other than
helping them to sell tons of CSU10s, so they can lower the price ;-)

Beat


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Dr. Beat Ludin, FMI, Maulbeerstr 66, 4058 Basel, Switzerland
Tel. +41 61 697 6697 / FAX +41 61 697 3976
Internet:[log in to unmask] / Compuserve:100102,1527