Adaptive-modulated fast fluctuation super-resolution microscopy.

Abstract

Fluorescence microscopy has significantly advanced biological imaging at the nanoscale, particularly with the advent of super-resolution microscopy (SRM), which transcends the Abbe diffraction limit. Most cutting-edge SR methods require high-precision optical setups, which constrain the widespread adoption of SRM. Fluorescence fluctuation-based SRM (FF-SRM) can break the diffraction limit without complex optical components, making it particularly well-suited for biological imaging. However, conventional FF-SRM methods, such as super-resolution optical fluctuation imaging (SOFI), still require specific fluorescent molecular blinking properties. Instead of enhancing the intrinsic blinking characteristics by finding specific fluorescent markers, employing optical methods such as spatial light modulation to adjust the excitation light field allows for easier and more flexible matching of the on-time ratio with the analysis of temporal stochastic intensity fluctuations. Nevertheless, the specific parameters of the modulation patterns have not been thoroughly explored, despite their crucial influence on the reconstruction quality. Herein, we propose adaptive-modulated fast fluctuation super-resolution microscopy. Our method demonstrates theoretically and experimentally that restricting the size of modulation units in a certain range ensures better image quality with fewer artifacts and signal losses. We find it still significantly effective when applied to other FF-SRM. Overall, the further development of the adaptive modulation technique has made it more stable in behavior and maintained high-quality imaging, presenting broader prospects for super resolution imaging based on statistical analysis.