Abstract We present a novel way to enhance the information content of fluorescence imaging by encoding information in the microscope point-spread function (PSF). In contrast to methods that modify the shape of the PSF itself, our approach encodes the additional information via the creation of faithful copies of the original PSF. Our method is compatible with operation over a broad wavelength range, other PSF engineering modalities, and existing analysis tools. We demonstrate this approach by developing the ‘Circulator’, an optical add-on made of off-the-shelf components that encodes the emission band of the fluorophore into the PSF. The device creates four copies of the original PSF at well-defined relative orientations and positions, where the presence or intensity of each spot indicates the emission color of the fluorophore. Using this device, simultaneous multicolor super-resolution and single-molecule microscopy can be performed using the full field of view of a single camera, resulting in a pronounced increase in experimental throughput. We demonstrate our approach in simultaneous three-color super-resolution imaging with single-molecule localization microscopy (SMLM) and super-resolution optical fluctuation imaging (SOFI).

Simultaneous multicolor fluorescence imaging using duplication-based PSF engineering