Super-resolution optical fluctuation imaging

We present a comprehensive review of super-resolution optical fluctuation imaging (SOFI), a robust technique that leverages temporal fluctuations in fluorescence intensity to achieve super-resolution imaging without the need for single-molecule localization. The Review starts with a historical overview of super-resolution microscopy techniques, and then focuses on SOFI’s core principle—the analysis of intensity fluctuations using cumulants to improve spatial resolution. The paper discusses technical challenges, such as photobleaching, blinking kinetics and pixel size limitations, as well as proposing solutions like Fourier upsampling and balanced SOFI to mitigate these issues. Additionally, we discuss potential advancements in the field, including the integration of SOFI with other super-resolution modalities like structured illumination microscopy and image scanning microscopy, and the application of SOFI in cryo-fluorescence microscopy and quantum emitter-based imaging. This paper aims to serve as an essential resource for researchers interested in utilizing SOFI for high-resolution imaging in diverse biological applications. The authors present a review of super-resolution optical fluctuation imaging (SOFI), including its core working principle, recent advances and remaining challenges.