Newly developed scientific complementary metal–oxide–semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition in single-molecule switching nanoscopy (SMSN) while simultaneously increasing the effective quantum efficiency. However, sCMOS-intrinsic pixel-dependent readout noise substantially reduces the localization precision and introduces localization artifacts. Here we present algorithms that overcome these limitations and provide unbiased, precise localization of single molecules at the theoretical limit. In combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at up to 32 reconstructed images/second (recorded at 1,600–3,200 camera frames/second) in both fixed and living cells.

Single-molecule switching nanoscopy overcomes the diffraction limit of light by stochastically switching single fluorescent molecules on and off, and then localizing their positions individually. Recent advances in this technique have greatly accelerated the data acquisition speed and improved the temporal resolution of super-resolution imaging. However, it has not been quantified whether this speed increase comes at the cost of compromised image quality. The spatial and temporal resolution depends on many factors, among which laser intensity and camera speed are the two most critical parameters. Here we quantitatively compare the image quality achieved when imaging Alexa Fluor 647-immunolabeled microtubules over an extended range of laser intensities and camera speeds using three criteria – localization precision, density of localized molecules, and resolution of reconstructed images based on Fourier Ring Correlation. We found that, with optimized parameters, single-molecule switching nanoscopy at high speeds can achieve the same image quality as imaging at conventional speeds in a 5–25 times shorter time period. Furthermore, we measured the photoswitching kinetics of Alexa Fluor 647 from single-molecule experiments, and, based on this kinetic data, we developed algorithms to simulate single-molecule switching nanoscopy images. We used this software tool to demonstrate how laser intensity and camera speed affect the density of active fluorophores and influence the achievable resolution. Our study provides guidelines for choosing appropriate laser intensities for imaging Alexa Fluor 647 at different speeds and a quantification protocol for future evaluations of other probes and imaging parameters.

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construct a binary molecular gate which can be controlled by the effector molecule lactose. The device is constructed of three DNA strands which form an elongated hairpin structure of two dsDNA arms with a short ssDNA hinge at one end, and two ssDNA regions on the opposite end. A fourth ssDNA molecule (the 'locking' strand), complementary to the two ssDNA regions, binds by complementary base pairing to seal the gate in the closed configuration. Addition of lac repressor protein to the device causes a displacement of the locking strand by binding to two lac operators embedded in the double-stranded hairpin structure. Binding of the proteins is rapid and sufficiently strong to cause a displacement of the locking strand, switching the gate into the open conformation. Addition of lactose quickly causes a release of the lac repressor protein from the operators allowing the gate to be closed by the binding of the locking strand. The opening and closing of this binary gate was detected by gel shift assays and by fluorescence resonance energy transfer spectroscopy of two dyes located near the opening of this gate. The gate could be cycled repeatedly, responded uniquely to lactose, and may be useful as a device for moving or holding structures on a molecular scale.