interactive fluorescence crosstalk correction ?

[Guenter Giese]

Dear list members,

how can one perform the following task with ImageJ ( interactive math with
two greyscale stacks):

--------
I would like to do:
- some simple fluorescence crosstalk correction

--------
I have:
- two greyscale (8 bit or 16 bit) image stacks (two fluorescence channels)

--------
I would like to have in ImageJ:
- a first slider acting on both stacks to navigate through corresponding
image planes

- a new display (the resulting scatterplot (2D histogram with dedicated LUT)
of the actual image plane combination)

- a second slider to enter the factor for multiplication of the second
channel (float value (range: 0-1, default 0)

- "on the fly" calculation (subtract result from first image)

- update of first channel image display abd the scatterplot

- an "apply to stack" button (option: generate new stack or overwrite actual
stack)


Is there a way to implement this / via a macro?
Maybe someone did such type of interactive calculations / display with
ImageJ before.
(I did last century using AVS software, but do not have access to it
anymore.)
Guy Cox made similar suggestions concerning implementation.

This would be a simple way to correct for fluorescence crosstalk (a quick
and dirty method; I know that for sound results one should use dedicated
spectral unmixing plugins, which, however, need a matrix to be generated
from "clean" reference signals.)

The best solution would be to have two sliders in a spectral unmixing plugin
(to enter fluorescence bleedthrough values for a two channel stack
combination) and to see the result of the unmixing calculation on the fly
....



Best,

Guenter



My question above is related to my previous posting repeated here:

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]