Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment

For dual channel correlation analysis of double-labeled immunofluorescence, the separate micrographs taken with rhodamine and fluorescein selective filters were scanned with a Perkin-Elmer Micro D i010 at a step size of 25/~m or were taken directly as digital images from the modular confocal microscope using 512 x 0.093 ixm by 512 x 0.93/~m by 20 • 0.55 t~m steps. The image data was processed with a series of networks in the Application Visualization System (AVS, Stardent Computer) on a Stardent 2000 GS. The separate image datasets were aligned by cross correlation and combined. The confocal microscope data could be used without further alignment. The further analysis followed the general procedure introduced by Dr. Jean Davoust (Marseilles) for the treatment of double-labeled confocal images and implemented by us as a series of AVS modules. The reader is directed to his paper (manuscript submitted for publication) for a complete description of the procedure. Briefly, the two vector valued array of pixel data is repiotted as the number of occurrences of pixels with a given ratio of red to green. This second plot reveals the correlation between red and green values for a given pixei and hence the likelihood that the antigen labeled with the fluorescein coupled antibody is found in the same position as the antigen labeled with the rhodamine-coupled antibody. Particular regions of this occurrence graph can then be selected and a new image composed which shows only those pixels corresponding to a given ratio of red to green or a three color image in which the selected pixels are shown in blue and the others are shown in their original red and green color. The AVS modules written by us for this processing are written in C and are available upon request.