Hot and Cold pixels
Hot and cold pixels are defect pixels on the detector that do not respond to incoming light in the same way as the other pixels, creating artifacts in the image and hindering analysis. Defect pixels stand out as unusually bright or dark compared to the surrounding pixels.
Hot and cold pixels are defects that occur in most camera based detection systems. Line scanning microscopes such as confocal microscopes scan every pixel with the same detector, and are thus devoid of the hot and cold pixel issue. Luckily, the Huygens Hot & Cold pixel remover can detect and correct these pixels after acquisition.
Exaggerated depiction of how hot and cold pixels affect the image. The object under study is green fluorescent, hot pixels will give higher then usual values (bright green) and hot cold pixels will give lower values (black).
Causes of Hot and cold pixels
Hot pixels
Hot pixels appear as abnormally bright pixels on long exposed images. They can be seen at the same position in every z-slice of a 3D stack, in every time frame, and in multiple channels. In practice this looks like a vertical line in a 3D image, this is the same pixel that always has the same high intensity.The most widely used microscope detector in fluorescence microscopy are CCD (charge-coupled device) cameras. A CCD consists of many photosensitive sensors that translate incoming photons to electrons creating an electrical charge. Most of the sensors have some charge leakage, causing a small electrical charge even when no photons are detected. However, in the case of hot pixels, some individual sensors on the CCD have a higher than normal rate of charge leakage. With long exposure times, the charge leakage can be so severe that the maximum charge is reached, resulting in a pixel displaying the maxium intensity value.
The charge leakage is worse at higher temperatures, a shift in 20 degrees can cause a 10 fold increase in charge leakage. Therefore it is adviced to image with minimum background light and under lower temperatures. In addition, altering the gain setting will not solve the problem of defect sensors. Increasing the gain would increase the appearance of hot pixels.
Hot pixels appear as vertical lines in a MIP projection of a 3D image. Image is courtesy of Dr. Rebecca Lee and Genevieve Phillips, Fluorescence Microscopy Shared Resource, University of New Mexico School of Medicine, Albuquerque, USA.
Example of a 2D image that is strongly affected by cold pixels. The cold pixels are clearly visible as they appear much darker than the surrounding pixels.
Cold pixels
Cold pixels appear as abnormally dark pixels in otherwise bright areas of an image. Like hot pixels, cold pixels can be seen at the same position in every z-slice of a 3D stack, in every time frame, and in multiple channels. Cold pixels are caused by individual sensors defects on the detector (usually a CCD camera) that have a lower than normal sensitivity (even as low as zero, a 'dead' pixel). Low sensitivity means that incoming photons are not translated correctly to electrons. Therefore, cold pixels give a lower electrical charge when they are hit by light from the sample, compared to the other pixels in the detector.In the Huygens software, cold pixels are best detected by opening the image in the (Twin) Slicer tool and using the 'Sum' projection mode.
Correcting hot and cold pixels
Unfortunately, defect pixels are present in most camera based systems and they can only be fixed by replacing the detector. However, hot and cold pixels are relatively easy to identify and filter out post acquisition because they have much different intensity values than the neighbouring pixels. Using the Huygens Hot & Cold pixel remover hot and cold pixels can be detected, visualized and removed from your image. As hot and cold pixels are caused by the errors on the detector, every image from the same microscope set-up will have similar hot or cold pixels. You can save the detected hot and cold pixels and reuse the mask for these pixels on all images made with the same microscope.External links
- More information on hot pixels: http://www.imaging-resource.com/ARTS/HOT/HOT.HTM
- More information on imaging detectors: https://www.olympus-lifescience.com/en/microscope-resource/primer/techniques/confocal/detectorsintro/