If you’re acquiring images on a microscope or gel-documentation system, the hard part already is done for you, because these instruments typically show you the histogram of the image you’ve just acquired. If you’ve hit the limit on either end of the histogram, the detector won’t be able to tell you if a band or a cell feature is two times or 20 times more intense than a neighboring band or cell. Why? From the point of view of the detector, i.e., the camera or film, once it has recorded the maximum amount of signal, it cannot register any more. On the other hand, oversaturation leads to loss of fine details and makes it impossible to quantify the signal. For example, aggressively adjusting the black levels of an immunofluorescence image to reduce the background eliminates hallmarks of a true experimental image. If the pixels are clustered at either end, you’ve likely oversaturated or underexposed your image. This ensures that the fine details of your images are captured. Ideally, you want the pixels to lie between the two extremes. The histogram will not tell you how these pixels are distributed in space, just the distribution of the pixel intensity. For an eight-bit grayscale image, there are 256 possible intensities ranging from 0 (black) to 255 (white) for each pixel in the image. Here’s a quick overview for those not yet accustomed to viewing them: A histogram of an image displays the distribution of pixels in the image, showing a graph of the number of pixels with a given intensity. If you’re a digital photography aficionado, you probably are very familiar with histograms and the information they contain. Being able to interpret the histogram correctly can tell you if you can move forward with snapping the next picture of your mutant phenotype or if you need to tinker with the acquisition settings. One way to tell if you’ve nailed your image’s acquisition parameters is to look at your image’s histogram. If the data you’ve collected is poor quality from the outset, your figure is already compromised. Once you’ve snapped your picture or exposed your Western blot, that image becomes the version of record for your experiment. In past columns, I’ve made the point that figure preparation begins at data acquisition, but I haven’t really explained my reasoning in depth.
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