Super-resolution microscopy based on the inherent fluctuations of dye molecules

Abstract

Fluorescence microscopy is a critical tool across various disciplines, from materials science to biomedical research, yet it is limited by the diffraction limit of resolution. Advanced super-resolution techniques such as localization microscopy and stimulated-emission-depletion microscopy often demand considerable resources. These methods depend heavily on elaborate sample-staining, complex optical systems, or prolonged acquisition periods, and their application in 3D and multicolor imaging presents significant experimental challenges. In the current work, we provide a complete demonstration of a widely accessible super-resolution imaging approach capable of 3D and multicolor imaging based on super-resolution optical fluctuation imaging (SOFI). We replace the confocal pinhole with an array of single-photon avalanche diodes and use the microsecond-scale fluctuations of dye molecules as a contrast mechanism. This contrast is transformed into a super-resolved image using a robust and deterministic algorithm. Our technique utilizes natural fluctuations inherent to organic dyes, thereby it does not require engineering of the blinking statistics. Our robust, versatile super-resolution method opens the way to next-generation multimodal imaging and facilitates on-demand super-resolution within a confocal architecture.