Roadmap to a large area multispectral fluorescence imaging system

Significance: Multispectral fluorescence imaging (MSFI) is a technique that measures endogenous and exogenous tissue fluorescence to reveal crucial insights into underlying biological mechanisms. Developing well-characterized high-performance fluorescence imaging equipment is crucial to any clinical or research application. Considering the diverse range of clinical scenarios and ongoing research areas in biomedicine leveraging MSFI instrumentation, there is an evident and pressing need for comprehensive resources that detail the development of such tools. Aim: This study provides a template for developing an MSFI instrument by highlighting the development and verification of our instrumentation. We present the design roadmap alongside the development of a large-area MSFI instrument to measure tissue fluorescence and diffuse reflectance properties. Approach: We divide our design approach into four subsections, highlighting the important physical milestones: illumination, imaging, detection, and computation. Each subsection includes design considerations, the methods used to validate the performance, and finally, the results and discussion of the validation process. Results: We present the validation of the instrument across the illumination, imaging, detection, and computation subsystems. Two fluorophores are used to validate the instrument through serial dilution to establish a detection threshold and spectral capabilities limit. A mouse model expressing multi-colored fluorescent proteins is used to verify the multispectral performance. Conclusions: Our study lays out the groundwork for researchers to design and validate their own MSFI instrument. We present this alongside our instrument design to study fluorescence and diffuse reflectance properties of large-area tissues, such as murine or resected surgical specimens. Findings from the application of MSFI instrumentation can be translated to motivate and guide the design of fluorescence-based medical tools for the clinic or research environment. (c) 2024 Society of Photo-Optical Instrumentation Engineers (SPIE)