Quantifying and Optimizing Single-Molecule Switching Nanoscopy at High Speeds

Abstract

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.

The Role of Probe Photophysics in Localization-Based Superresolution Microscopy

“…The finding of an optimum in resolution as a function of excitation intensity is consistent with experimental findings by Lin et al (48). Even with an indefinitely long acquisition time, there will still be a best possible resolution as long as the number of emitters that can be localized within the sample is finite.…”

“…At even lower intensities, both the fluorophore and the background will emit proportional to excitation intensity, and signal-to-background will not be further improved by lowering intensity. These phenomena have been discussed previously in Lin et al (48).…”

PALM and STORM: Into large fields and high-throughput microscopy with sCMOS detectors

“…While reducing the exposure time reduces the number of collected photons per molecule per frame, it also reduces the number of background photons collected, reducing the impact of low exposure times on resolution. This has been recently demonstrated and quantified by Lin et al [32] for Alexa 647, which show speeds of 1600 FPS (for a cropped sCMOS FOV) can achieve resolutions comparable to slower acquisition speeds and reaching satisfactory localization densities in a matter of seconds. This resulted in a 16-fold reduction in acquisition time when compared to the full FOV speed at 100 FPS (Table 2).…”

“…If fast dSTORM imaging is to be performed, the 405 nm laser can be continuously present with the excitation laser, making fast modulation unnecessary. However, fast dSTORM imaging requires powerful lasers (laser intensity at the sample in the order of 1-100 kW/cm 2 [32]) since excitation intensity is inversely proportional to ''on-times'' [16]. For example, Huang et al [46] use a 500 mW 642 nm laser to illuminate relatively small FOV's to be able to achieve power densities of 5-18 kW/cm 2 .…”

“…This will yield the expected on-sample laser power, which must be divided by the area to image; depending on the laser power, it may be necessary to reduce the FOV to reach power densities at least in the few kW/cm 2 . Lin et al [32] provide useful guidelines to determine the necessary powers given a maximum camera frame rate (determined by the size of the FOV), keeping in mind that for HT it is always useful to maximize the FOV. The entire laser launching and homogenization assembly ( Fig.…”