The next steps in our preprocessing routine involve subtracting the contributions from autofluorescence and correcting for heterogeneities in fiber core efficiency. We follow a modified procedure laid out by Perchant and Zhong.20,21 Three calibration images are acquired before imaging begins. The first, , is acquired with the laser turned off while imaging a nonfluorescent blank solution, such as water, in a dark compartment. This provides a measurement of camera bias and diffuse room light that passes the emission filter. The second, , is acquired under the same conditions but with the laser turned on, providing an estimate of the autofluorescence of the fiber core and cladding material. Since the autofluorescence is a function of laser power while the bias and room light are not, the two images need to be acquired separately. The third image, , is acquired while imaging a homogeneously labeled fluorescent fluid. When camera bias, room light, and autofluorescence signals are subtracted out, this third image provides a mapping of the core transmission and collection efficiencies. In practice, each calibration image is derived from averaging many () raw images to improve signal-to-noise ratio. Calibration images are reacquired each time the fiber bundle probe is connected and aligned to the system.