Continuous-Wave SOFISM with SPAD Array Detection

We present a continuous-wave (CW) implementation of super-resolution optical fluctuation imaging with image scanning microscopy (SOFISM) using a pixelated single-photon avalanche diode (SPAD) detector camera. In a scanning-based geometry, SOFISM requires fluorescence fluctuations on timescales compatible with the pixel dwell time while maintaining sufficient signal under confocal excitation. Photoswitchable fluorophores in an aqueous switching buffer containing the glucose oxidase-catalase oxygen-scavenging system (GLOXY) and mercaptoethylamine (MEA) were therefore used to promote blinking and reduce photobleaching. Correlation analysis of sparse-emitter measurements showed that Alexa Fluor 647 (AF647) exhibits microsecond-scale fluctuation dynamics suitable for CW SOFISM and guided the choice of imaging conditions for scanned samples. Frame rate and delay accumulation were optimized from detector-pair subset variability using a mean-to-STD criterion. The optimal binning time was 2.5 $μ$s, underscoring the advantage of SPAD-based detection for resolving the relevant fluctuation dynamics. The method was demonstrated on HeLa cell microtubules labeled with AF647, yielding improved spatial resolution in the constructed SOFISM images compared with the corresponding ISM images. Additional super-resolved images from samples labeled with CF568 and Alexa Fluor 555 (AF555) showed the approach is readily generalizable to other fluorophores. These results establish CW SOFISM as a practical fluctuation-based super-resolution method for confocal microscopy.