Dear colleagues:
My comments to reviewers' objections to image processing:
Image processing occurs when producing microscopic images either
through digital hardware or more prosaic film based systems.  It is
absolutely essential that the author give a full account of how his
images were produced and how they were processed.  This has not
always been true for images taken with "conventional systems".  I
don't think that some reviewers appreciate these facts.
 
Most of us who have been in microscopy for some years know that with
conventional imaging by photography
(a) different films have different spectral sensitivities
(b) contrast can be changed (stretched) by both the film choice,
the paper used for printing
Furthermore, background in am image is influenced by both the
biological background for the reaction (non-specific binding etc.)
and the optical filter combination of the microscope.
 
Quantitation of signals or relative quantitation in  wide-field
microscopy is an important capability of digital systems. The
extrapolation of this to confocal images is a very complex subject.
Even colocalization measurements by confocal systems are only valid
when the user really knows that his multi-wavelength CLSM is giving
exactly the same focal plane for all excitation and emission
wavelengths.  These problems have been addressed in a number of
technical papers but I don't think are widely appreciated by the
community of users.  One has to spend some personal effort to
determine the characteristics of your particular CLSM and to know what
you are really measuring at all times.
 
Pretty pictures are easy to produce with CLSMs but valid and relevant
statements about the biological significance of the data require
more sophisticated understanding of the hardware as well as the
software.
 
Donna Arndt-Jovin
MPI f. Biophys. Chem.
Dept. Mol. Biol.
Goettingen, Germany