Ultra-high spatio-temporal resolution imaging with parallel acquisition-readout structured illumination microscopy (PAR-SIM)

Structured illumination microscopy (SIM) has emerged as a promising super-resolution fluorescence imaging technique, offering diverse configurations and computational strategies to mitigate phototoxicity during real-time imaging of biological specimens. Traditional efforts to enhance system frame rates have concentrated on processing algorithms, like rolling reconstruction or reduced frame reconstruction, or on investments in costly sCMOS cameras with accelerated row readout rates. In this article, we introduce an approach to elevate SIM frame rates and region of interest (ROI) coverage at the hardware level, without necessitating an upsurge in camera expenses or intricate algorithms. Here, parallel acquisition-readout SIM (PAR-SIM) achieves the highest imaging speed for fluorescence imaging at currently available detector sensitivity. By using the full frame-width of the detector through synchronizing the pattern generation and image exposure-readout process, we have achieved a fundamentally stupendous information spatial-temporal flux of 132.9 MPixels s−1, 9.6-fold that of the latest techniques, with the lowest SNR of −2.11 dB and 100 nm resolution. PAR-SIM demonstrates its proficiency in successfully reconstructing diverse cellular organelles in dual excitations, even under conditions of low signal due to ultra-short exposure times. Notably, mitochondrial dynamic tubulation and ongoing membrane fusion processes have been captured in live COS-7 cell, recorded with PAR-SIM at an impressive 408 Hz. We posit that this novel parallel exposure-readout mode not only augments SIM pattern modulation for superior frame rates but also holds the potential to benefit other complex imaging systems with a strategic controlling approach.