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Hi all,
Yes, scanning the specimen does make transmission confocal almost
possible: as long as the refractive index of the specimen is entirely
constant. (i.e., the specimen is dead and stained)
focal plane and the coverslip on the "in" direction than they are on
the "out" direction, then even if you initially line up the
illumination optics to coincide with the detection path, you will
find that this overlap will change as the specimen is moved past the
spot.
Consequently, the amount of signal actually passing through the
detection pinhole depends not only on how much absorbing material is
located at the focus of the laser but also (negatively) on the degree
of misalignment. i.e. the output signal is no longer dependent on
events occurring at the focal spot and therefore the resulting data
no longer can be thought of as coming from an optical section.
There have been suggestions that this "dynamic misalignment" problem
could be avoided if the detector were not a fixed pinhole but a
mini-image recording device (say a 5x5 CCD). As this spot was
displaced from the axis by the inhomogeneities of the specimen, it
would be tracked, the centroid of the spot located and its intensity
measured. Sort of a movable pinhole to track the displaced spot to
keep the whole system "confocal".
This would be neat (In fact is was one proposed uses for the
"EM-CCiode" detector proposed by me and others) but it would be
really complicated to sort out the data. Probably for this reason, it
has not been done.
The other reason is that it really isn't clear what useful
information would be obtained from the transmitted light signal that
could not be obtained far more easily by detecting Backscattered
light. Using any laser line, BSL will give you a lovely,
fully-confocal darkfield image that shows all inhomogeneities as
white.
For more information, you might look at the chapter on Transmission
Confocal by Carol Cogswell in the Second edition (but not the Third)
of the Handbook.
Cheers,
Jim P.
--
**********************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building,
FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706
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3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2008
"If it ain't diffraction, it must be statistics." Anon.
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