Tissue autofluorescence images used for overlaying with MALDI images were captured prior to MALDI matrix application. Autofluorescence images were acquired using a Zeiss 710 confocal microscope and a W plain-Apochromat 20× objective. Tissue sections were excited with 405 and 490 nm laser wavelengths and corresponding emission wavelengths were collected from 404 to 488 nm and 495–720 nm, respectively, for analysis.23 (link) Using the tile scan function in ZEN 2.3 SP1 software (Zeiss), several 303.6 μm × 303.6 μm images were stitched over different areas on the slides to acquire mosaic images of whole tissue sections. Since, such large area mosaic acquisition takes time, z stack imaging of the 10 μm thick tissue sections was avoided by adjusting the pinhole size to an equivalent of 4.63 airy units. The pinhole adjustments allowed acquisition of image information above and below the focal plane. All microscopy images were analyzed using the ZEN image analysis software (Zeiss).