An open-source photobleacher for fluorescence imaging of large pigment-rich tissues

Abstract

Fluorescence imaging enables visualization of the specific molecules of interest with high contrast, and the use of multiple fluorophores in a single tissue sample allows visualization of complex relationships between biological molecules, cell types, and anatomy. The utility of fluorescence imaging in human tissue has been limited by endogenous pigments that can block the light path or emit an autofluorescence, thereby interfering with the specific imaging of target molecules. Although photobleachers have been developed to quench endogenous pigments, the lack of customizability limits their utility for a broad range of applications. Here, we present a high luminous-intensity photobleacher that is based on rigorous simulations of illumination patterns, along with the framework to maximize bleaching efficiency. This open-source project is designed to help scientists customize and scale the device according to their research goals. The photobleacher is applicable to both thin tissue slices and large-volume cleared tissue samples to enable serial three-dimensional imaging of postmortem human brain using multiplexed antibody or oligonucleotide probes.