Le jeudi 22 mars 2012 16:43:29 UTC+1, Zachary Pincus a écrit :

Jean-Pat,
> To perform "perfect" spectral unmixing, I suppose it's necessary to know the integration time, the characteristic of the microscope bandpass filters, the emission spectrum of each fluorochrome; but would it be possible to make some kind of "estimated" unmixing from only the images themselves?
Wait, are you trying to perform spectral unmixing (5 channels -> 25 channels, one per chromosome), or false coloring (5 channels -> 3 channel RGB)? The first email made it sound like the latter, but this one seems more on the former...

Sorry, you're right, my first question was about the 5 ->3 channels issue.

If you want to do unmixing, there's a large literature about the MFISH case specifically, and I think there are some pretty well-known methods at this point. Presumably it would help to know the integration times and spectral characteristics of the instrument / fluorophores, especially given the table you appended to the second email about the spectral mixing issues.
False-coloring is an easier subject. You'd just need to choose "target" RGB colors for each of the 5 raw images, colorize each image by that color, and screen them together. Most of the art is in choosing good "target" colors. You could choose based on the original colors ("aqua" etc.),

I met that problem when I chose the matrix coef (with Stefan's code). The matrix was doing the job with:

   'R': [0, 0, 0.05, 0.15, 0.8],
   'V': [0.1, 0.8, 0.05, 0.05, 0],
   'B': [0.8, 0.15, 0.05, 0, 0]}

or choose equidistant points on the hue-circle (with equivalent saturation, and brightness determined by the original grayscale pixel intensity).

The steps should be for a given image (ex s-aqua):
*calculate saturation and brightness from gray scale s-aqua. (s1,b1)
*choose hue (a1)
Do the same for each fluo
Use the 5x3 hsb values to calculate a matrix to do the convertion 5 channels->3 RGB

Is it correct?

Jean-Pat

Zach

On Mar 22, 2012, at 6:26 AM, jip wrote:
> Each image is a raw image of a given fluorochrome.
>
> A given fluorochrome targets a subset of chromosomes, more informations can be found in:
>
> Automatic Segmentation and Classification of
> Multiplex-Fluorescence In-Situ Hybridization
> Chromosome Images
> by
> Hyo Hun Choi, B.S., M.S.
> Choi gives an example of CCD integration times:
>
> An example of integration times is [DAPI, Aqua,
> Green, Gold, Red, Far Red] = [0.14, 6, 0.76, 6, 2.96, 1.4] seconds
>
> He also gives an example of spectral mixing in the raw images:
>
>
> With the vysis data set (example:https://github.com/jeanpat/MFISH/tree/master/MFISH_TIFF/V13__40), a given fluorochrome corresponds to several chromosomes, in the spectrumAqua image the chromosomes 3,10,11,13,15,20,21,22,X,Y are visibles.
>
> A: S. Aqua
> D DAPI
> F: Far Red
> G: S. Green
> R: S. Red
> Y: S. Gold
>
> To perform "perfect" spectral unmixing, I suppose it's necessary to know the integration time, the characteristic of the microscope bandpass filters, the emission spectrum of each fluorochrome; but would it be possible to make some kind of "estimated" unmixing from only the images themselves?
>
> Best regards
> Jean-Pat
>
> Le mercredi 21 mars 2012 12:10:16 UTC+1, jip a écrit :
> Dear all,
> Some one asked me how I choose the matrix coef to convert a five multispectral image to a three (RGB) multispectral image. I feel that there is a best way to do that by maximising some distance in a color space.
> How would you do that?
> Examples of multispectral images are available here
>
> Best Regard
>
> Jean-Patrick Pommier
>
> Le mercredi 21 mars 2012 12:10:16 UTC+1, jip a écrit :
> Dear all,
> Some one asked me how I choose the matrix coef to convert a five multispectral image to a three (RGB) multispectral image. I feel that there is a best way to do that by maximising some distance in a color space.
> How would you do that?
> Examples of multispectral images are available here
>
> Best Regard
>
> Jean-Patrick Pommier