![]() Bit depth: Be sure your image file’s bit depth suits the camera’s pixel intensity range You should NOT see any pixels piled up on the right, at the maximum of the range – the intensity of pixels there will not be accurately recordedģ. When viewing a typical test image’s histogram, the maximum pixel intensity should use ~50-75% of the range (left), leaving a bit of room if some of your samples or fields of view are brighter. This awesome article will tell you more about dynamic range and image saturation: Fluorescence microscopy – avoiding the pitfalls Claire M. Most microscope software allows you to see a histogram of pixel intensities – you want the histogram to fill most of the available pixel intensities (along the X-axis, usually: see Figure), but you do not want any pixels to reside at the highest intensity value of the camera – you do not want a spike at the right-hand side of the histogram. If you expect one sample in your experiment may be brighter than others (a positive control for example), you may want to use it to determine your exposure settings to decrease the likelihood of ending up with saturated images. Aiming for your image maximum to be ~50-75% of the dynamic range is a safe bet, allowing for some images in the set being brighter than average without becoming saturated. Set the exposure time such that the resulting images use as much of the dynamic range of the camera as possible, but without saturating any images.LED light sources tend to be more consistent and are preferred. Be aware that microscope lamps often take time to warm up, so that a 1 second exposure is not guaranteed to yield the same response, depending on how long it has been since the lamp turned on, or how long it has been since the bulb was changed.For example, don’t use automatic exposure times or change the lamp or filter settings part way through collecting a large image set if you aim to quantitatively compare signals across the set of images. Typically, you want to keep image acquisition conditions constant across an experiment.Proper exposure time: avoid saturation and lack of dynamic range Already saved your images in a lossy format? Sorry, but there is no salvaging the loss of data converting them to a lossless format now provides no benefit.Ģ.Wikipedia: Image file formats can tell you more. Uncompressed file formats are also fine for image analysis (e.g., BMP). Other file compression formats retain exactly the original image information but in a smaller file (“lossless”) so they are perfectly acceptable for image analysis (e.g., PNG, TIFF, GIF). Some methods of file compression sacrifice image quality (“lossy”) and should be avoided for automated image analysis if at all possible (e.g., JPG/JPEG).These tips are a bit CellProfiler-centric but generally applicable to any quantification you might do.Ĭorrected image, using a CellProfiler image analysis pipeline for spot detectionġ. Lossless file formats: please, no JPG! Even though you might not notice any problems by eye, the tips outlined here for acquiring and storing images can improve the quality of data derived from digital image analysis. Not every image you capture on your microscope is suited for quantification, no matter how nice they may look.
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