A Zeiss LSM 710 confocal microscope (Carl Zeiss) was used to acquire spectral images. All images were acquired with Plan-Apochromat 20x 0.8 NA objective in lambda mode with 29.1 nm channel bandwidth resulting in 9 channels detected over the visible spectrum. Simultaneous excitation with 405 nm, 488 nm and 561 nm lasers was used. 3-D z-stack images were acquired as tile scans with 9 z-planes centered in the middle of the gut spanning 8.3 μm total in z dimension. Linear unmixing was performed on the spectrally acquired images after stitching the tiles together in ZEN software v 3.4. We extracted the reference spectra for DAPI, EUB 338 Alexa flour 594, WGA Oregon green 488 and autofluorescence from the labelled zebrafish and applied them for the linear unmixing.