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Background correction can be applied while acquiring images or subsequent to acquisition. The difference between these is that a priori correction uses additional correction images obtained at the time of image capture at the same time as in a posteriori correction the correction images are not available and therefore an ideal illumination model is assumed. The a priori methods are therefore the preferred option. In digitizing images, the camera noise, background illumination intensity and color temperature are the sources of image degradation to consider. The camera noise include random noise that is due to uncorrelated fluctuations above and below; fixed pattern noise that us characterized by pixel intensities that are consistently above random noise fluctuations and it is due to faulty CCD or pixel differences in charge leakage rate; and banding noise that may arise during the process of reading the data from the digital sensor or by interference with other electronic equipment.
The background illumination intensity provided by the microscope light source optics is commonly not homogeneous throughout the view field. The color temperature of the light source also affects image quality. Light sources have a characteristic radiation spectrum. With most filament light bulbs this spectrum varies depends the temperature of the filament. Therefore, images taken at different times may exhibit backgrounds with slightly different hues. This makes difficult to standardize procedures such as color segmentation, color separation, hue quantification, etc. Some microscopes have a button which sets a fixed voltage to the bulb to deliver fixed colour temperatures. If this is used, then the intensity of the light can controlled with neutral density filters in the light path. The camera and microscope settings includes make sure has an infrared filter; switch on all your equipment and leave it warm up for some time (the warming time depends on room temperature, how sensitive the equipment is to temperature and how long it takes to stabilize.
You can find out this by taking a time series of background shots over a period of time and see whether the light distribution drifts over time. You are likely to observe a plateau where the results become more stable. You may want to do image capture during the plateau period.); switch off the camera auto gain; put the specimen on the stage, focus the objects, adjust the light/neutral density filters and condenser, set an appropriate camera exposure time; if the camera has a white balance function, you can apply it now on the illuminated bright field: take out the specimen and apply the white balance; reposition the specimen and check again that the image histogram is not saturated and that the grey scale values span most of the grey scale space; and from now on, the settings of the camera and the microscope light should not be adjusted anymore. When you capture the bright field, you open the light path, remove the specimen and capture a shot. Save it as “bright field” it will be used to compensate the background illumination.
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