Acquisition and Post-Processing of Immunohistochemical Images

Abstract

Augmentation of digital images is almost always a necessity in order to obtain a reproduction that matches the appearance of the original. However, that augmentation can mislead if it is done incorrectly and not within reasonable limits. When procedures are in place for insuring that originals are archived, and image manipulation steps reported, scientists not only follow good laboratory practices, but avoid ethical issues associated with post processing, and protect their labs from any future allegations of scientific misconduct. Also, when procedures are in place for correct acquisition of images, the extent of post processing is minimized or eliminated. These procedures include white balancing (for brightfield images), keeping tonal values within the dynamic range of the detector, frame averaging to eliminate noise (typically in fluorescence imaging), use of the highest bit depth when a choice is available, flatfield correction, and archiving of the image in a non-lossy format (not JPEG).

When post-processing is necessary, the commonly used applications for correction include Photoshop, and ImageJ, but a free program (GIMP) can also be used. Corrections to images include scaling the bit depth to higher and lower ranges, removing color casts from brightfield images, setting brightness and contrast, reducing color noise, reducing “grainy” noise, conversion of pure colors to grayscale, conversion of grayscale to colors typically used in fluorescence imaging, correction of uneven illumination (flatfield correction), merging color images (fluorescence), and extending the depth of focus. These corrections are explained in step-by-step procedures in the chapter that follows.