Interestingly, the problem was answered by my next email (see below), but you still want to know just where various classes of pixels are distributed spatially--i.e., where the pixels are associated with cells and their particular subcellular structures, or where within a cell are there regions of antigen expression or coexpression. Dave dcoder@u.washington.edu Date: Sun, 16 Apr 2000 22:56:35 +0100 From: Richard Herd <rahe@unixa.nerc-keyworth.ac.uk> To: cyto-inbox Subject: Re: ratio - 2 colour Message-Id: <l03130300b51fe62c94b4@[209.88.233.113]> Content-Type: text/plain; charset="us-ascii" >> In addition, I'm >>interested in plotting the intensities of a given region of pixels, to >>compare the Red and green channels pixel by pixel (ie creating a >>'colocalization graph' if plotted on the x and y axes). Im interested in >>doing this either on binary (thresholded) images, or on 256 grey scale. I did this a while ago and it was available on the ftp site [http://rsb.info.nih.gov/nih-image/]. I don't think it's still there, but I can prepare you a version of NIH-Image which will let you plot the XY scatter of pixel intensities for a pair of images in a stack. Alternatively, the source code if you want to put it in your version. Regards - Richard Richard Herd Montserrat Volcano Observatory St Johns Montserrat West Indies email : rahe@ua.nkw.ac.uk phone : (1) 664 491 5647 FAX : (1) 664 491 2324 ----- Original Message ----- From: Michael Dustin <dustin@pathbox.wustl.edu> To: cyto-inbox Sent: Friday, April 14, 2000 4:23 PM Subject: analysis of overlap/segregation betwee two colors in confocal image data We are doing multiple color imaging of molecules involved in T cell activation. We need to develop or employ an existing index for the degree of overlap (or segregation) between two different molecules that are labeled with different dyes and imaged with a confocal microscope. One idea would be to start with a scatter plot (FL1 vs FL2) of the type used for two color flow cytometry where individual pixels would be plotted to define regions with high overlap (on diagonal lower left to upper right) or areas with low overlap (off diagonal). We can generate lists of pixel values, but need to translate the text files to a form readable by flow cytometry software like Cell-quest to get the plots. Is there software to go from Excel files to the FCS format? Has this problem been dealt with differently by others? Thanks for any advice. Michael L. Dustin, Ph.D. Associate Professor of Pathology Washington University School of Medicine 660 S. Euclid Ave Campus Box 8118 St. Louis, MO 63110 Office- (314) 362-9618 Lab- (314) 362-8719 Fax- (314) 362-8888 Jerri Smith, Admin Assist. (314) 362-8740
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