Known for its non-invasive and non-destructive nature, optical microscopy can provide structural and functional insights into biological specimens, thus driving progress in fields such as biology, medicine, and related disciplines. Over the past four centuries, optical microscopy has witnessed significant developments. These have been particularly accelerated in the last century by technological advancements in lasers and computational methods. These advancements have led to revolutionary changes, making optical microscopy an essential tool in critical sectors such as healthcare, education, and food safety. With the increasing exploration in cellular biology and biomedicine, a growing need has arisen for optical microscopes with molecular or nanoscale spatial resolution, as exemplified by super-resolution optical microscopy (SRM). Of the various SRM techniques, stimulated emission depletion (STED) microscopy stands out because it achieves resolution enhancement by modulating the depletion power relative to the redshift in the excitation wavelength in the imaging setup. However, excessive power depletion poses challenges, including photobleaching of fluorophores and phototoxicity to biological specimens, which constrain the utility of STED in live-cell imaging scenarios. In recent years, researchers worldwide have collaborated to advance the field of STED microscopy with a particular focus on developing strategies to reduce depletion power. This effectively decreases the amount of power required for imaging while maintaining resolution accuracy. These studies are crucial for understanding the intricate details and underlying mechanisms in living organisms.

In this review, we discuss the basic principles of STED microscopy and emphasize its crucial role in achieving super-resolution imaging of biological samples. Achieving super-resolution imaging using STED microscopy requires precise control over the spatial, temporal, and spectral aspects (Fig. 1). By applying the theoretical framework that governs the resolution calculations in STED microscopy, we outline methods for achieving low-power STED microscopy from four key perspectives: optimizing STED probes, using single-molecule localization techniques, employing advanced image processing methods, and utilizing time-resolved detection approaches.

We then provide a brief summary of the current nanoprobes designed for low-power STED imaging that encompass organic molecule dyes and organic and inorganic nanomaterials. Based on a comparative analysis of their performance parameters and imaging outcomes, we highlight the essential criteria for nanoprobes suitable for STED imaging, with a focus on attributes such as photobleaching resistance, low saturation intensity, and favorable biocompatibility. We also summarize and compare the imaging capabilities of STED microscopy and its derivative technologies. Noteworthy examples include MINFLUX, LocSTED, and MINSTED, which synergistically combine the strengths of STED and single-molecule localization microscopy (SMLM) to achieve substantial enhancements in imaging resolution (Fig. 2). In terms of image processing, we expound on the principles of differential image processing, explaining its effectiveness in modulating fluorescence signals across the spatial, temporal, and frequency domains to facilitate low-power STED imaging (Fig. 3). Moreover, by leveraging the insights into the relationship between the stimulated emission effect and fluorescence lifetimes, we advocate for the adoption of time-resolved detection modules to discern fluorescence photons with long lifetimes. Through techniques such as time-gated detection, phasor plot analysis, and ratiometric photon reassignment, we demonstrate the potential for enhanced resolution by selectively isolating photons with prolonged lifetimes (Fig. 4). Finally, we evaluate the prevailing challenges impeding the widespread adoption of low-power STED microscopy, emphasizing the need for future research endeavors that optimize image quality and enhance both imaging depth and the intelligence and automation of imaging systems. Our primary objective is to advance the application of STED microscopy, particularly in demanding domains such as thick-tissue imaging and in vivo investigations.

In the field of super-resolution imaging, STED microscopy is a pioneering far-field technique distinguished by its real-time capabilities, ultra-high resolution, and three-dimensional layer-slicing capabilities. These attributes make STED microscopy highly promising for bioimaging applications. To extend the utility of STED microscopy to in vivo imaging scenarios, a primary objective is to effectively reduce the depletion power, which is a major focus for future advancements in STED microscopy. With continuing advancements in scientific technology and the increasing demand for various applications, low-power STED microscopy enhancements are anticipated to progress further. For example, tailoring imaging parameters to diverse experimental conditions can be facilitated by integrating artificial intelligence and machine learning methodologies. This facilitates automatic parameter matching and the identification and tracking of target structures, thereby mitigating the complexity associated with experimental operations and enhancing both imaging efficiency and accuracy. In addition, the integration of STED microscopy with complementary advanced technologies holds promise for realizing expanded capabilities, including large-depth, multicolor, and three-dimensional imaging. These advancements are expected to provide researchers in the fields of biology and medicine with powerful tools for understanding complex biological processes.