High-throughput snapshot hyperspectral imager for multiplexed fluorescence microscopy - high-multiplex tissue imaging (hmti) is aiding biomedical research by enabling scientists to map dozens of…

The National Institute of General Medical Sciences awarded Third Floor Materials, Inc. (3FM) $298,111 on September 2, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to develop a high-throughput snapshot hyperspectral imager for multiplexed fluorescence microscopy.

The system combines proprietary plasmonic filter arrays with backside-illuminated CMOS sensors to enable capture of complete hyperspectral datacubes—containing both spectral and spatial information—within a single exposure of a widefield fluorescent microscope. By recording full emission spectra at every pixel, the technology eliminates the need for confocal scanning optics or repeated staining and imaging cycles, allowing fluorophore concentrations to be unmixed from spectral data. In Phase I, 3FM will design and fabricate a filter array optimized for fluorescence unmixing, adapt compressive sensing algorithms for accurate spectral recovery of fluorescence signals, and demonstrate system-level feasibility by imaging mixtures of fluorescent microspheres with known emission spectra, followed by characterization of commercially prepared and stained samples against commercial widefield imaging benchmarks.

The award supports basic research that advances high-multiplex tissue imaging capabilities central to spatial omics and precision medicine. Performance occurs in Research Triangle Park, Durham, North Carolina, with completion targeted for August 31, 2027.

High-throughput snapshot hyperspectral imager for multiplexed fluorescence microscopy - high-multiplex tissue imaging (hmti) is aiding biomedical research by enabling scientists to map dozens of molecular markers within intact tissues while preserving spatial context. This ability is increasingly important to understanding cell biology, and advancing spatial omics and precision medicine. Currently, however, barriers remain due to the optical properties of fluorophores, the time-consuming cyclic protocols involving sequential rounds of staining/illumination/imaging and incompatibility with in-vivo imaging. Advancing the state-of-the-art in high-multiplex, widefield fluorescence microscopy will further high-throughput, high-accuracy biomarker discovery, tissue mapping, and mechanistic biology - areas central to the NIH and nigms missions. Snapshot hyperspectral imaging (shsi) enables the capture of a complete hyperspectral datacube - containing both spectral and spatial information of the fluorescent sample - within a single exposure of a widefield fluorescent microscope. By recording a full emission spectrum at every pixel, shsi eliminates the need for confocal scanning optics or repeated staining/imaging cycles since emission spectra can be used to unmix local fluorophore concentrations. In combination with a custom filter cube, the proposed system is a drop-in replacement for standard scientific cameras, leveraging proprietary plasmonic filter arrays integrated with modern, backside illuminated cmos sensors. In phase i, we will (1) design and fabricate a filter array optimized for fluorescence unmixing, (2) adapt compressive sensing algorithms for accurate spectral recovery of fluorescence signals, and (3) demonstrate system-level feasibility and quantify performance by imaging mixtures of fluorescent microspheres with known emission spectra. Finally, a commercially prepared and stained sample will be characterized and the results compared with a commercial widefield microscope. Completion of these aims will establish the technical foundation for the use of shsi in widefield fluorescence microscopy.