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.

Analysis of Phase Heterogeneity in Lipid Membranes Using Single-Molecule Tracking in Live Cells.

In live cells, the plasma membrane is composed of lipid domains separated by hundreds of nanometers in dynamic equilibrium. Lipid phase separation regulates the trafficking and spatiotemporal organization of membrane molecules that promote signal transduction. However, visualizing domains with adequate spatiotemporal accuracy remains challenging because of their subdiffraction limit size and highly dynamic properties. Here, we present a single lipid-molecular motion analysis pipeline (lipid-MAP) for analyzing the phase heterogeneity of lipid membranes by detecting the instantaneous velocity change of a single lipid molecule using the excellent optical properties of nanoparticles, high spatial localization accuracy of single-molecule localization microscopy, and separation capability of the diffusion state of the hidden Markov model algorithm. Using lipid-MAP, individual lipid molecules were found to be in dynamic equilibrium between two statistically distinguishable phases, leading to the formation of small (∼170 nm), viscous (2.5× more viscous than surrounding areas), and transient domains in live cells. Moreover, our findings provide an understanding of how membrane compositional changes, i.e., cholesterol and phospholipids, affect domain formation. This imaging method can contribute to an improved understanding of spatiotemporal-controlled membrane dynamics at the molecular level.

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.

Fluorescent Probes for STED Optical Nanoscopy.

Progress in developing fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, is inseparable from the advancement in optical fluorescence microscopy. Super-resolution microscopy, or optical nanoscopy, overcame the far-field optical resolution limit, known as Abbe's diffraction limit, by taking advantage of the photophysical properties of fluorescent probes. Therefore, fluorescent probes for super-resolution microscopy should meet the new requirements in the probes' photophysical and photochemical properties. STED optical nanoscopy achieves super-resolution by depleting excited fluorophores at the periphery of an excitation laser beam using a depletion beam with a hollow core. An ideal fluorescent probe for STED nanoscopy must meet specific photophysical and photochemical properties, including high photostability, depletability at the depletion wavelength, low adverse excitability, and biocompatibility. This review introduces the requirements of fluorescent probes for STED nanoscopy and discusses the recent progress in the development of fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, for the STED nanoscopy. The strengths and the limitations of the fluorescent probes are analyzed in detail.