spectral unmixing without "clean" reference signals

[Guenter Giese]

Dear list members,

how to do spectral unmixing without "clean" reference signals? I have stacks
of images recorded with a ccd camera at different excitation / emission
wavelength combinations. There is considerable autofluorescence signal as
well as some crosstalk (bleedthrough" of fluoophores. Unfortunately, I have
no region with single fluorescence signal to be used as a reference, and it
would be very time-consuming for several reasons (different tissue types,
ages, species, tissue preparation methods etc.) to prepare individual
probes.

Of course there are regions in which any type of fluorescence is prominent
(but not isolated), which could be used as reference regions.

Is there a way to determine the matrix values to be used with, e.g., the
spectral unmixing plugin from J. Walter, or for general spectral unmixing
purposes?

The solution I would favour would be interactive (sliders / updated result
windows).

Best,

Guenter Giese

------------------------------------------
Dr. Guenter Giese
Light Microscopy Facility Manager
Dept. of Biomedical Optics
MPI fuer Medizinische Forschung Jahnstr. 29
D-69120 Heidelberg, Germany
Phone [phone] (Fax: -325)
e-mail: [hidden email]