Characterization of multiple parallel fluorophores using a spatial light modulator and a SPAD array

Confocal laser scanning microscopy remains a widely used technique for high-resolution fluorescence imaging and fluorophore characterization, though it is inherently limited by sequential scanning and long acquisition times. To overcome these limitations, we developed a novel, to our knowledge, scan-free fluorescence microscopy approach that combines a spatial light modulator with a single-photon avalanche diode array, enabling the simultaneous measurement of fluorescence lifetime, intensity, and emission spectra from multiple fluorophores. We demonstrated this technique by characterizing six microspheres and comparing the results to those obtained with a conventional confocal microscope, finding strong agreement across measurements. Notably, our parallelized approach achieved a 12-fold reduction in total measurement time by eliminating the emitter-by-emitter scanning step required in confocal microscopy, while also reducing photobleaching through localized excitation.