With the rapid revolution in super-resolution microscopy, the resolution of far-field optical microscopy has entered the sub-nanometer era, providing new insights into macromolecules in vitro and in situ.

Wide-field quantum sensing with fluorescent nanodiamonds (FNDs) in biological systems offers significant potential for understanding intracellular dynamics at the nanoscale. However, current wide-field quantum sensing methods are limited to 2D correlated measurements. 3D correlated quantum sensing remains challenging due to the inherent properties of wide-field microscopy. Here, we have developed a multi-plane wide-field microscope platform that achieves an imaging volume of 50 × 50 × 5 μm³. This is accomplished by simultaneously imaging eight focal planes at varying sample depths using a beam-splitting prism. By employing a Fourier-transform-based fluorescent particle positioning method, the platform attains lateral positioning precision of 9 nm and axial precision of 12 nm. Using this platform, we performed correlated 3D positioning of FNDs in mouse cardiomyocytes and conducted optically detected magnetic resonance on nitrogen-vacancy color centers within intracellular FNDs. Our results demonstrate the potential of this platform for single-particle tracking and highlight its capability to achieve correlated 3D quantum sensing.

Light-sheet fluorescence microscopy (LSFM), with its innovative design of selective plane illumination and orthogonal detection optics, significantly reduces phototoxicity and photobleaching inherent in conventional microscopy, providing a revolutionary tool for long-term dynamic imaging of living specimens. This review focuses on throughput enhancement strategies of LSFM, systematically summarizing advancements in optical architecture optimization and multimodal integration. Key technological innovations include: improved sample compatibility, large-field imaging via optimized light-sheet generation, microfluidics-coupled high-throughput automation, and hyperspectral imaging for multiplexed analysis. Through adaptive light-sheet modulation, remote focusing synchronization, and AI-driven algorithmic optimization, LSFM achieves multiscale 3D imaging spanning subcellular structures to centimeter-scale tissues at speeds exceeding hundreds of volumetric frames per second. In biomedical applications, LSFM has successfully resolved complex processes such as cellular lineage dynamics during embryogenesis, whole-brain neuronal activity mapping, and structure-function correlations in cardiovascular systems, while enabling high-throughput drug screening and pathological model analysis. These breakthroughs establish LSFM as a cornerstone technology for intravital imaging, offering an integrated solution that combines high spatiotemporal resolution, minimal photodamage, and big-data throughput. By bridging molecular, cellular, and organ-level observations, LSFM drives paradigm shifts in developmental biology, neuroscience, and translational medicine, empowering unprecedented exploration of living systems across scales.

Intravital mesoscale imaging plays a crucial role in bridging the gap between cellular and organ-level investigations by enabling high-resolution visualization across large fields of view. Continuous advancements in optical microscopy have significantly improved imaging performance, yet fundamental challenges remain. Effective intravital mesoscale imaging requires a balance between spatial resolution, imaging speed, field of view, and while overcoming limitations such as scattering, aberrations, phototoxicity and photobleaching. This review summarizes key challenges in achieving high-performance intravital mesoscale optical imaging and provides an overview of advanced optical imaging techniques, including wide field, laser scanning, as well as computational imaging approaches. Despite these advancements, further improvements are necessary to address existing limitations and unlock new possibilities. Future developments will focus on enhancing imaging depth, further improving space bandwidth products, and integrating computational methods for real-time processing and large-scale data analysis, further advancing mesoscale imaging for biological research.

We describe the statistical characteristics of optical speckle patterns formed by illuminating biological tissues, commonly called biospeckles. The predominant techniques used to gather information from the movement of speckle patterns are detailed. Using vegetable tissues, we monitored the senescence process and created vascularization maps of leaf tissues. The Fujii method, which has been modified, has emerged as the most effective approach for highlighting the biological activity across leaf tissues. This technique relies on the presence of fluid flow to create highly detailed maps of tissue microcirculation. The method of temporal contrast evaluation produced a significant spectral activity map, which allowed for the detection of both instant and invisible bruised tissue. The evaluation revealed that biological specimens can exhibit a unique time history of speckle pattern (THSP) patterns, which may serve as a biological signature for the sample. Additionally, an activity index was calculated to define the assay’s activity under different biological conditions, and the results were tested and verified across multiple samples.