The MATLAB script in this repository is designed to accompany our manuscript submission to Science Advances by Guo et al in 2024. This simple script takes raw "mixed" images and a calibration matrix as input and applies simple linear unmixing to output "unmixed" images for a number of image channels. Two example image datasets and calibration matrixes are included. The first dataset includes files for excitation multiplexing: (a) Brain_mixedsignal_3channel_exmultiplex.tif (b) ExcitationMultiplexCalibrationMatrix.mat This tif file contains the raw image data corresponding to Figure 3 and Figure S7 in the manuscript. Here, a brain slice was stained with 3 polymer dots and imaged in 3 channels. After using the accompanying script, a user can generate the unmixed images shown in the manuscript main figures. The second dataset includes files for emission multiplexing: (a) Brain_mixedsignal_8channel_emmultiplex.tif (b) EmissionMultiplexCalibrationMatrix.mat This tif file contains the raw data corresponding to Figure 2 and Figure S3 in the manuscript. Here, a brain slice was stained with 8 polymer dots and imaged in 8 channels. After using the accompanying script, a user can generate the unmixed images shown in the manuscript main figures. A calibration matrix is also included for each dataset. This matrix contains normalized contributions of each stain in the individual image channels. This calibration of bleedthrough and cross-excitation was performed by imaging polymer dots in solution.