We have done some work concerning the instensity fall-off with depth.
We try to compensate for the intensity fall-off but also to use it as
a basis for segmentation, i.e. regions with same intensity fall-off
can be segmented.
Here are some references:

A. Liljeborg, N. Åslund,
"A method for analysis and segmentation of 3-dimensional images
from a confocal laser microscope,"
NeuroProtocols: A Companion to Methods in Neurosciences
Vol. 2, 150 - 159 (1993)
Academic Press.

A. Liljeborg, M. Czader, A. Porwit, "A method to compensate for light
attenuation with depth in 3-D DNA image cytometry using a CSLM,"
Journal of Microscopy, Vol. 177, Pt. 2, pp. 108 - 114 (1995)

A. Liljeborg,
"Simulation of light attenuation within fluorescent microspheres used
for liquid fraction separation recorded by a CSLM,"
Proc. SPIE Symposium on Three-Dimensional Microscopy: Image Acquisition and
Processing,
San Jose, Calif., USA, January 30 - February 1, 1996. SPIE Vol. 2655, 11-17.

The above mentioned work is much based on this publication:
T. D. Visser, F. C. A. Groen, G. J. Brakenhoff,
"Absorption and scattering correction in fluorescence confocal microscopy,"
J. of Micr., Vol. 163, Pt. 2, August 1991, pp. 189-200.

J. P. Rigaut, J. Vassy, P. Herlin, F. Duigou, E. Masson, D. Briane,
J. Foucrier, S. Carvajal-Gonzalez, A. M. Downs, A-M., Mandard,
"Three-dimensional DNA image cytometry by confocal scanning laser
microscopy in thick tissue blocks,"
Cytometry 12:511-524 (1991).

>>>>> "Chris" == Chris Pudney <[log in to unmask]> writes:

    Chris> The reviewers are probably right - you should easily be
    Chris> able to outperform thresholding.  One of the problems with
    Chris> thresholding is that a good threshold at the top of the
    Chris> stack is no good deeper in the stack as the intensity often
    Chris> falls-off with depth along the optical axis.  Is anyone
    Chris> aware of published methods for correcting this?  Then
    Chris> thresholding might do better.

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        Ph.D. Anders Liljeborg
        Biomedical Physics (formerly Physics IV)
        Teknikringen 14
        Royal Inst. of Technology
        S - 100 44 Stockholm, Sweden
        Tel. +46 8 790 7219
        Fax. +46 8 205 609
        E-mail: [log in to unmask]

        Department of Neuroscience
        Doktorsringen 12, 5 tr.
        Karolinska Institutet
        S - 171 77 Stockholm, Sweden
        Phone +46 8 728 7379
        E-mail: [log in to unmask] ([log in to unmask])
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