As a widely utilized tool in the field of biological in vivo imaging, light-sheet fluorescence microscopy (LSFM) has become a mainstream tool among biologists due to its rapid imaging speed, high resolution, low phototoxicity, and reduced photobleaching effects. This paper introduces the operational principles of LSFM while calculating its imaging resolution to guide the selection of objective lenses and cameras. It reviews the developmental history of LSFM and classifies existing LSFM systems, particularly detailing implementations based on Gaussian beams and diffraction-free beams, along with field-of-view expansion methodologies. Specialized variants optimized for biological specimens including swept confocally aligned planar excitation (SCAPE) microscopy and open-top light-sheet (OTLS) microscopy are systematically introduced. The review further summarizes recent advancements enabled by LSFM in biological research domains such as pathological analysis, neuroscience, and embryonic development, demonstrating its exceptional capabilities in tumor pathology assessment, human brain visualization, and developmental studies of model organisms' nervous systems and embryogenesis. Finally, potential future directions for LSFM development are envisioned, highlighting that integration with artificial intelligence, advanced image processing, and optogenetic techniques may yield groundbreaking scientific discoveries.

Fluorescence microscopy has become an indispensable tool for visualizing subcellular structures and dynamic biological processes. Light sheet fluorescence microscopy (LSFM), characterized by selective plane illumination and detection, has attracted increasing attention in recent years owing to its low phototoxicity, high spatiotemporal resolution, and large field of view. The emergence of single-objective light sheet systems has further enhanced sample compatibility and system flexibility, opening new avenues for multiscale, long-term, and three-dimensional dynamic imaging. Here, we review the development and diverse configurations of LSFM, with a particular focus on the technical principles, representative applications, and performance advantages of single-objective systems. We also discuss their current bottlenecks in terms of field of view, optical path complexity, and signal transmission efficiency, and explore the potential of zoom-free multiscale single-objective platforms that integrate structural innovations, multimodal imaging, and intelligent control, aiming to provide a reference for the creation of high-performance, bio-friendly, and broadly applicable imaging systems.

This paper first compares point scanning confocal microscopy with spinning disk confocal microscopy in fluorescence confocal microscopy. The former is a serial imaging with single point excitation-detection and has a slower speed; the latter is a parallel imaging with multi-point excitation-detection, which is fast. Furthermore, by comparing the technical principles of single-disk and dual-disk confocal focusing, the limited pinhole size, spacing, and distance between the two disks caused by the micro lens disk in the dual-disk setup were revealed, resulting in limited field-of-view, imaging quality, rotation speed, and the need for precise registration of the disk system. The technical characteristics of ceiling-less single disk confocal system were explained, including multi-color, fast (fast rotation speed, large field-of-view), easy-to-use (high optical quality, customizable pinhole size and spacing), stabilization (high stability, low cost), and its performance was demonstrated through resolution testing (xy direction resolution 230 nm, z direction resolution 470 nm). Finally, the characteristics of the three technologies in terms of resolution and penetration depth, photobleaching and phototoxicity, signal-to-noise ratio, imaging rate, system complexity and imaging field of view were compared. The comparison showed that the single-disk technology outperforms the point scanning and dual-disk technologies in terms of imaging field of view, imaging rate, phototoxicity and system complexity, thus demonstrating its outstanding application value in biomedical research.

Optical microscopes are core tools driving the continuous development of life sciences. Label-free phase imaging, which utilizes refractive index as an endogenous contrast mechanism, avoids phototoxicity and photobleaching caused by fluorescent labeling, and is increasingly becoming a crucial approach for analyzing cellular structures and dynamics. Since Zernike invented the phase-contrast microscope in 1932, phase imaging technology has advanced from two-dimensional qualitative observation to three-dimensional quantitative analysis, with digital holographic microscopy and optical diffraction tomography being two representative milestone methods. However, the reliance of such interferometric techniques on highly coherent light sources and complex interference optical paths leads to fundamental bottlenecks in terms of system stability, imaging noise control, and engineering applications. As a result, they have not yet replaced phase-contrast microscopes in terms of widespread use in life science laboratories. In recent years, the development of computational optical quantitative phase imaging technology and the rise of intensity diffraction tomography have broken through the inherent limitations of traditional interferometry, marking the entry of label-free quantitative phase imaging into a new stage of development. Against this backdrop, relying on more than a decade of theoretical accumulation and methodological innovation, our team has deeply integrated advanced computational imaging frameworks with engineering design, and taken the lead in developing a new generation of label-free microscopic imaging instruments based on intensity diffraction tomography internationally. This instrument combines high resolution, high stability, and high throughput, enabling long-term, quantitative, and multi-scale observation of living cell structures and dynamic processes under label-free conditions. This paper systematically reviews the development history of label-free phase imaging, focuses on summarizing the latest progress of our team in computational optical imaging and instrument development, and looks forward to its broad prospects in disease mechanism analysis, drug screening, clinical diagnosis, and the future scientific instrument system.

Single-molecule localization microscopy (SMLM), as a groundbreaking technology surpassing the optical diffraction limit, achieves nanoscale spatial resolution (～20 nm) and has revolutionized biological imaging. However, its widespread application faces critical challenges including reliance on imported hardware systems, complex sample preparation protocols, lack of dual-color imaging fluorescent probes, and insufficient data analysis and mining, leading to underutilization of existing equipment. This review proposes a comprehensive full-chain solution addressing nine critical elements of SMLM, which include three “hard skills” — hardware systems, software control, and image reconstruction; and six “soft skills” — precise sample preparation, blinking probes, data analysis and mining, specialized reagents and consumables, specific antibodies, and practical applications. The “hard skills” can be rapidly deployed through the procurement and integration of mature commercial components, whereas the “soft skills” rely on long-term technological accumulation and deep domain knowledge. This solution aims to promote the transformation of SMLM from a “high-end instrument” into an “efficient tool”, facilitating the broader application of super-resolution imaging technologies and offering new perspectives for the use of advanced microscopy systems.

Fluorescence polarization analysis requires rotating the excitation beam's polarization direction, while conventional structured illumination microscopy (SIM) relies on three illumination orientations to achieve super-resolution. Polarized structured illumination microscopy (Polar-SIM), as an integrated approach combining these two techniques, enables high-precision investigation of the spatial orientation and structure of various subcellular organelles. This study theoretically investigates the relationship between the contrast of interferometric fringes and polarization modulation, compares the advantages and disadvantages of employing a pizza waveplate, circular polarization, and a variable liquid crystal phase retarder for polarization modulation, and further simulates the accuracy of polarization analysis under different modulation conditions. Finally, our experimental results demonstrate Polar-SIM's unique capability for super-resolution directional analysis of molecular dipoles, highlighting its broad potential for addressing diverse biological questions in future research.

As a pivotal tool in life science research, intravital microscopy facilitates real-time monitoring of cellular and molecular dynamics within their native physiological milieu. This review delves into the imaging mechanisms, strengths, and constraints of confocal microscopy, multi-photon microscopy, light-sheet microscopy, and scanning light-field microscopy. By analyzing representative application cases across disciplines such as oncology, immunology, neuroscience, and developmental biology, it offers a comparative overview of the ideal usage scenarios for these technologies. Furthermore, it addresses current technical hurdles and speculates on future advancements in the field. To provide technical reference for related research.

Super-resolution imaging technology is an important technical means for demonstrating related physiological and pathological molecular phenomena, change processes and functional regulation in the field of neuroscience research. It breaks through the diffraction limit of traditional optical microscopes, enabling scientists to study biological structures and material details at the nanoscale. Various super-resolution technologies assist researchers in better exploring neuroscience from different aspects such as spatiotemporal resolution, three-dimensional tomography capabilities, and in vivo imaging capabilities, thereby analyzing the nanoscale distribution and dynamic changes of synaptic structures and neurotransmitter receptors, and providing key details for further research on neurodegenerative diseases. This article summarizes the phased development of super-resolution imaging technology in recent years, as well as the advantages and limitations of different technologies. It particularly introduces the application of super-resolution imaging technology in ultra-microstructure imaging of the nervous system, imaging of living animals, deep imaging of the brain, and related research on neurodegenerative diseases and drug development. It aims to provide references for their efficient and reasonable application in neuroscience research.

Optical coherence tomography (OCT) is a key technology for high-resolution biomedical imaging. Intraoperative handheld endoscopic OCT combines optical fiber communication with endoscopic technology. Based on the principle of low coherence interference, it uses a submillimeter miniaturized handheld probe (diameter≤1 mm) and flexible scanning mechanisms such as MEMS actuation and fiber vibration. Overcoming the penetration depth limitation of traditional OCT, it can obtain micron-level high-resolution structural images such as retinal stratification and vascular intima in turbid media such as blood and tissue fluid. Its clinical application widely covers ophthalmology (real-time navigation of vitreoretinal surgery), cardiology (vascular anastomosis evaluation), neurosurgery (deep brain tissue imaging) and breast tumor resection (microscopic margin assessment), etc. Through the integrated surgical instruments such as lateral or forward scanning probes and OCT surgical forceps, the operation and imaging can be synchronized and the blind area of the visual field can be eliminated. The current technology has challenges in narrow lumen adaptability and multimodal information fusion. The future development will focus on the development of submillimeter flexible probes, artificial intelligence-assisted lesion recognition, robot collaborative navigation, and the popularization of low-cost disposable devices, so as to promote its evolution to an intelligent surgical navigation system. This article briefly describes the current development status of intraoperative handheld endoscopic OCT, reviews its representative works in terms of system structure optimization and clinical application, and presents an outlook for its future development.

In vivo small animal imaging technology serves as a powerful tool for investigating tumor biology and evaluating anti-tumor therapeutic efficacy. This non-invasive modality enables real-time, dynamic observation of tumor initiation, progression, metastasis, and treatment response in living organisms, offering distinct advantages including visualizability, high spatiotemporal resolution, and exceptional sensitivity. Widely adopted in oncology research, this technology encompasses multimodal imaging approaches such as optical, nuclear medicine, and magnetic resonance imaging, each demonstrating unique characteristics and application scenarios. This article systematically summarizes three predominant in vivo small animal imaging techniques—optical 3D imaging, near-infrared fluorescence imaging, and 3D tomographic scanning, highlighting their respective technical advantages and application domains. Through presenting cutting-edge case studies in tumor mechanism research, metastasis monitoring, and therapeutic response assessment, this review aims to provide substantive references for advancing the application of in vivo small animal imaging in oncology research and anti-tumor treatment development.

Intravital two-photon imaging technology has the advantage of deep imaging, enabling the direct monitoring of the dynamic processes within living organisms. It has become an important research tool in the field of life sciences. We briefly explain the basic principles of two-photon microscopy and its applications in life science research in recent years. We mainly focuse on analyzing the development trends of intravital two-photon imaging technology and discuss the current challenges and future optimization directions. We look forward to the continuous optimization of intravital two-photon imaging technology, which can promote its wider application.

To meet the demand for portability and low cost to facilitate the widespread application of spectrometers, this paper presents the comparative analysis of different spectrometer types. Taking the crossed Czerny-Turner (C-T) optical path as a reference, we designed and implemented a portable fiber-optic spectrometer based on a crossed C-T structure. An orthogonal crossed dual spherical mirror configuration was adopted to compress the optical path dimensions, and a prototype system was constructed for experimental validation. Through optimized component selection, a low-cost spectrometer with high portability and strong functionality was developed. The prototype system underwent wavelength calibration, and its aberrations were briefly analyzed. Ultimately, the system achieves a spectral resolution of 1 nm within the 381～716 nm wavelength range. This design resolves the traditional trade-off between size and performance in fiber-optic spectrometers, demonstrating significant application value for rapid detection and real-time spectral analysis.

Intravital two-photon imaging technology has become an essential tool in life science research due to its ability to intuitively monitor the in vivo dynamic processes of living organisms. However, sample preparation has always been a major challenge in the application of this technology, and it directly determines the stability and quality of imaging. There is currently no systematic introduction to sample preparation for intravital two-photon imaging. Based on the rich practical experience accumulated in the laboratory over the years in the preparation of samples for intravital imaging, we mainly introduce the sample preparation procedures and key points of operation for tissues such as the lung, intestine, brain, liver, and skin. The aim is to provide a reference for sample preparation and operation before intravital two-photon imaging, assist researchers in mastering the operation of intravital two-photon imaging, and promote the broader application of this technology.