Abstract: Disclosed herein are systems and methods of calibrating a microscope or an imaging system prior to acquiring image data of a sample. In some embodiments, a method is disclosed including the steps of (a) running a power output calibration module to calibrate an imaging apparatus for repeatability; (b) running an image intensity calibration module to calibrate the imaging apparatus for reproducibility and to mitigate differences in detection efficiency between channels; (c) collecting image data from a microscope or imaging system; (d) optionally running an unmixing module to unmix the collected image data into individual image channel images; and (e) optionally running a contrast agent intensity correction module to calibrate for differences in brightness between different contrast agents.