Spatially patterned, spectral single-molecule microscopy

Abstract Multicolour and spectrally resolved single-molecule microscopy can reveal molecular interactions, nanoscale environments and dynamics, but usually depends on experimentally complex detection architectures based on beam splitting, spectral dispersion or engineered point spread functions. Here we show that spatially patterned detectors offer a conceptually simpler route to spectral single-molecule imaging. By replacing a conventional monochrome camera with a commercially available colour CMOS detector and fitting the raw detector response directly, we recover both molecular position and spectral fingerprint from a single image without optical splitting, channel registration or demosaicing. We term this approach spatial-spectral single-molecule microscopy, or S 3 M. We show that S 3 M retains single-molecule sensitivity across the visible spectrum (400–785 nm), enables robust spectral multiplexing, and supports applications spanning multicolour single-molecule tracking, single-molecule Förster resonance energy transfer, multicolour localisation microscopy and spectral PAINT. Although spatial patterning necessarily trades photon efficiency for spectral information, current low-noise detectors already provide sufficient performance (