Continuing the discussion on z res, I get an elongation of Fluor beads mounted
in oil. I'm assuming that it's because the ref index of latex beads is around
1.6 . I've calibrated my stepping motor by using 100 micron glass beads in oil
with an oil lens in reflection mode. I seem to get no problems that I can
perceive, although I seem to get a smaller focus step calibration factor than
the manufacturer says ( Zeiss). I've tried to get flourescent methacrylate
beads which would have a ref index closer to oil but I can't find any. With a
cell in aq media and water lens, the images look pretty good until you go into
the specimen a bit. I try to capture full range and threshold out values below
the half max for the reconstruction. I figure that cutting the gain or
screwing around with the black level isn't reality. I've been setting the step
size equal to the FWHM, (my Optical sx) and interpolating out in the
reconstructions. At z values much greater than x and y I get elongation
problems simply from interpolation, and I may also have trouble from using some
of the lower values. I don't see how you can have equal resolution in the XYZ
by any method other than throwing away data that's real. In any case, I
am well aware that Nyquist is turning in his grave,but I've been busy with
other things. What's the deal?? should I be overlaping sections and scrunching
them in the reconstruction? should I throw away my Z-res and not overlap?
I'd like to use that .7 or.8 resolution that we work so hard for. What's a
biologist to do? Any of our resident heavies care to comment on any of these
issues?
                              All the best,
                                            BJ