Simultaneously enhancing the resolution and signal-to-background ratio in STED optical nanoscopy via differential depletion.

STED (stimulated emission depletion) far-field optical nanoscopy achieves resolution beyond the diffraction limit by depleting fluorescence at the periphery of excitation with a donut-shaped depletion laser. What is traded off with the superior resolution of STED nanoscopy is the unwanted elevation of structured background noise which hampers the quality of STED images. Here, we alleviate the background noise problem by adopting the differential stimulated emission depletion (diffSTED) approach. In diffSTED nanoscopy, signals obtained with different depletion strengths are compared and properly subtracted to remove two major background noise sources in STED nanoscopy. We show via simulations that by using diffSTED nanoscopy, background noise is significantly decreased, and the image contrast is improved. In addition, we show by simulation and analytical calculation that diffSTED improves resolution simultaneously. We assess the effect of different parameters, such as the STED beam intensity, depletion intensity ratio of two STED beams, and the subtraction factor, on the signal-to-background ratio (SBR) and the resolution of diffSTED nanoscopy. We introduce a logical algorithm to determine the optimal subtraction factor and the depletion intensity ratio. DiffSTED nanoscopy is a versatile technique that can be readily applied to any STED system without requiring any hardware modifications. We predict the wide applicability of diffSTED for its enhanced resolution, improved SBR, and easiness of implementation.

Pushing the Resolution Limit of Stimulated Emission Depletion Optical Nanoscopy.

Optical nanoscopy, also known as super-resolution optical microscopy, has provided scientists with the means to surpass the diffraction limit of light microscopy and attain new insights into nanoscopic structures and processes that were previously inaccessible. In recent decades, numerous studies have endeavored to enhance super-resolution microscopy in terms of its spatial (lateral) resolution, axial resolution, and temporal resolution. In this review, we discuss recent efforts to push the resolution limit of stimulated emission depletion (STED) optical nanoscopy across multiple dimensions, including lateral resolution, axial resolution, temporal resolution, and labeling precision. We introduce promising techniques and methodologies building on the STED concept that have emerged in the field, such as MINSTED, isotropic STED, and event-triggered STED, and evaluate their respective strengths and limitations. Moreover, we discuss trade-off relationships that exist in far-field optical microscopy and how they come about in STED optical nanoscopy. By examining the latest developments addressing these aspects, we aim to provide an updated overview of the current state of STED nanoscopy and its potential for future research.