Simultaneous detection of many genes. (A) A single human colon adenocarcinoma cell (DLD-1) G2 nucleus (by DAPI signal) with a pseudo-colored representation of 17 transcription sites detected in situ. The image is "flattened" such that all 12 0.5-Ám Z-sections are displayed on the background, which is the DAPI counterstain from the middle image of the stack. Gene identity is denoted by color and the Z-location is recorded by the adjoining number. Lower numbers represent closer proximity to the cover slip. Scale bar, 3 Ám. (B) Three G1 DLD-1 nuclei from the same field, which together express all 10 genes assayed. Arrows indicate sites that are shown below magnified from the original data. From left to right, the 10 marked transcription sites are IL-8, MCL-1, DUSP-1, cyclin D1, g-actin, EGR-1, TIEG-1, b-actin, c-myc, and c-jun. Scale bar, 3 Ám. (C) Chart of the 10 genes detected in (B). The "Pseudo" column shows the arbitrary pseudo-color used to denote the gene identity of each transcription site in the renderings above. Columns at right show the actual signal recorded at the appropriate Z-section for the transcription sites shown with an arrow in (B). Each band of data of fluorescein isothiocyanate (FITC), Cy3, Cy3.5, and Cy5 is shown, with the positively scored signals highlighted by surrounding boxes. Each area of the unprocessed image shown is 1 Ám2. Observed misalignment is due to chromatic shift between filter sets.

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