Fig. 1.

Histograms and images. Confocal microscopy image of a mollusk embryo at the 4-cell stage, showing the cytoskeleton of a single cell. The image is courtesy of James Cooley and Lisa Nagy, University of Arizona. Unprocessed image—The original 8-bit (256 shades of grey) image. No post-acquisition image processing was performed. The intensity of this image ranges from the darkest pixel value of 11 to the brightest pixel value of 186. The intensity histogram scale, by convention, runs from darkest on the left, to brightest on the right. There are no true black or white pixels in this image. Appropriately processed image—The same image, after an appropriate contrast/histogram stretch. Using the Photoshop levels tool, the value of 11 from the original image was re-mapped to black (=0) and the value of 186 was remapped to white (=255). Note that the shape of the histogram is essentially the same as in the unprocessed image. The gaps in the histogram are a result of the contrast/histogram stretch. This is generally considered an acceptable image processing step. With color images that will be used for illustrative purposes, it can be useful to apply the levels tool in this way to each of the red, green, and blue channels. If the color images are for quantitative use, or if the relationships of the intensities or colors to one another will be interpreted in any way, this is not recommended. Over-processed image—The same image, this time with a contrast/histogram stretch that was too aggressive. Using the Photoshop levels tool, the value of 20 from the original image was re-mapped to black and the value of 145 was re-mapped to white. Compare the shape of the intensity histogram with the original. Note that the data at each end of the histogram have changed. The data at the ends of the original histogram have been truncated, creating the spikes at black and white (arrows). Nothing scientific can be inferred about these white and black pixels, as their relationship to the rest of the data has been lost. This is a common image processing mistake, arrived at by a number of different techniques, as users try to create striking, “contrasty” images. Boxes—50 × 50 pixel areas from the same area in the un-processed and overprocessed images above. The areas have been enlarged using the Photoshop CS2 nearest neighbor algorithm. Box 1—Note the loss of information in the darkest pixels. The loss is easier to see in the intensity histogram than in the image (arrow). Scientists are often not interested in this end of the histogram; however, backgrounds that are too “clean” do not accurately represent real biology. Box 2—Note the over-saturation of many of the brightest pixels in this image (arrow). Since many journals are using on-line images as the “journal of record”, the data of record are missing some of the fine detail that may be of more interest to the reader than they were to the authors