There have been several questions of this sort of late, and I thought
I would respond in a general way from my recent experience.  I have
gone through this dilemma over the last year in trying to upgrade our
BIorad MRC600 which has been a real workhorse.  I did extensive
testing of several systems, especially the Leica and Zeiss, since
they seemed to be the ones most recommended by others.  The Leica has
the clear advantage of wavelength flexibility due to its
spectrophotometer based system and the ability to detect at any
wavelength. The Zeiss is like other systems in being filter based and
so you are at the mercy of the filters you buy, which is no problem
for most of the things most people will do.  Zeiss has an edge in
that their multitracking is more sophisticated and better implemented
than Leica's.  I still cannot get Leica to tell me whether
bidirectional scanning is alternating scan lines or not.  What you
are doing is very clear on the Zeiss. In addition, I did extensive
testing of sensitivity of the two instruments and came to the
conclusion that the Leica was noisier, but overall the two systems
were comparable in sensitivity.  One confusion in determining this is
that the AOTF scale of the Leica goes lower than the Zeiss, so if you
try to compare without actually measuring the laser power delivered
to the stage, you will be mislead about sensitivity.  We choose the
Leica for a variety of reasons.

        Now that I have had it for a few months, there is a factor
that is dramatically different between the two instruments that I was
unaware of nor have I heard discussed previously, but people should
be aware of.  Using front surface mirror measurements, the Zeiss and
our old Biorad allow you to vary the pinhole size and therefore the
optical section depth by about a factor of 3 between 1 Airy and full
open (about 0.5um to 1.5 um for a 100x lens).  On the Leica the
optical section depth only increases by about 30% when you fully open
the pinhole.  For us, this is very signficant.  In 10 years of live
cell imaging, we have never tried to work with the pinhole closed as
the light budget for cells is always on the edge, and you are always
trying to balance killing cells against getting enough photons to
image.  We use the Biorad with the pinhole fully open most of the
time.  We gain little by doing this with the Leica and so imaging is
necessarily worse at the same laser power.  On the Leica, the
alternative is to use their "beam expander" option.  NOrmally you aim
to fill the back focal plane with your laser beam.  In addition to
the normal setting, they have a reduced beam expansion or no beam
expansion setting that result in different extents of underfilling
the back focal plane.  This will decrease the NA of the objective and
so presumably increase the size of the spot in Z, thus increasing the
z section depth.  It does in fact give you a measurably deeper
section depth.  There are two problems with this. First, it also will
reduce your X-Y resolution, which is not true of the Zeiss and Biorad
implementation.  Also, it is presumably also not as good for your
light budget.  If you can get away with opening the pinhole on a
Biorad and increase the Z section depth without increasing the
excitation spot, then you don't need alot more excitiation in Z to
get more photons in Z. Somewhere, the Leica is losing this data. This
gets very complicated as you also change the exitiation power as you
remove the beam expander (less glass in the path).  I don't
understand this completely, and I don't understand why the Leica is
limited in pinhole, but I believe my data is accururate based upon
the mirror tests and my experience with what the cells look like on
the two systems. Hopefully others who are more technical than I will
read this and clarify what is going on.  Also, for many cell types,
the issue may be irrelevant.  My cells are particularly sensitive to
light exposure. I would recommend testing very carefully with the
cells and fluors you plan to use (GFP in particular) and try varying
the pinhole and see if image quality vs. killing is a problem before
buying.  Good Luck- Dave
--

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Dr. David Knecht
Department of Molecular and Cell Biology
University of Connecticut
75 N. Eagleville Rd.   U-125
Storrs, CT 06269-3125
[log in to unmask]
860-486-2200      860-486-4331 (fax)
home page: http://www.sp.uconn.edu/~mcbstaff/knecht/knecht.html
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