Cryo-electron tomography (cryo-ET) is an essential tool for resolving cellular structures in their native state. However, achieving site-specific sample preparation remains a significant challenge, particularly for deeply buried or rare cellular targets. Focused ion beam (FIB) milling is commonly used to prepare thin lamellae from vitrified samples, but traditional FIB methods often lack the abilities to target specific regions of interest. Correlative light and electron microscopy (CLEM) overcomes this limitation by combining light microscopy (LM) with scanning electron microscopy (SEM), enabling the identification and localization of structures of interest within the specimen. This targeted approach enhances the accuracy and efficiency of FIB milling by ensuring that lamellae are thinned at precisely the right locations. Recent advances in integrated cryo-CLEM workflows have streamlined this process, offering enhanced precision and reproducibility in sample preparation for cryo-ET. Here, we present an optimized protocol that utilizes this integrated approach to identify and target specific cellular structures, such as the contact sites between lipid droplets (LD) and mitochondria. This protocol facilitates the precise preparation of cryo-lamellae and enhances the efficiency of data acquisition in cryo-ET, offering a promising strategy for high-resolution structural biology studies.

Cryo-electron tomography (cryo-ET) can provide invaluable insights into subcellular structures in their native environment at molecular resolution. Cryo-focused ion beam (cryo-FIB) has been widely adopted to obtain thin lamellae of cells suitable for tomographic data acquisition. However, its application to tissue samples faces significant limitations. The larger sample size extends milling time, reducing sample preparation throughput. Furthermore, the need for repeated, intricate lift-out procedures adds considerable time and complexity to the workflow. The serial lift-out technique, which can prepare multiple lamellae, increases throughput and better preserves the structural integrity of the tissue sample. Here we outline the workflow of the optimized serial lift-out method and provide a detailed protocol for its implementation, exemplified by a vitrified mouse liver tissue sample.

This mini-review provides a concise overview of the latest advances in fluorescence lifetime imaging microscopy (FLIM). It discusses both time-domain and frequency-domain techniques, analysis methods − including phasor approaches and deep learning, and highlights applications in multiplexed imaging and quantitative biosensing. Furthermore, FLIM-empowered multimodal imaging approaches aimed at enhancing spatial and temporal resolution are discussed. Persistent challenges, including photon efficiency, probe sensitivity, and achieving high-speed imaging in live-cell environments, are critically assessed, outlining pathways toward future innovations.

High-resolution and long-term dynamic imaging are essential for visualizing the spatial distribution and interaction networks of organelles within living cells. Although traditional super-resolution fluorescence microscopy achieves impressive resolutions of 20–100 nm, it faces significant challenges, including phototoxicity, photobleaching, and limited suitability for prolonged live-cell observation. These issues have driven the development of label-free imaging technologies that aim to minimize disruption to cellular physiology while providing high-resolution, non-destructive imaging. Among label-free approaches, Quantitative Phase Imaging (QPI) has emerged as a promising alternative for live-cell research by reconstructing cellular structures based on phase changes in transmitted light. In particular, Fourier ptychographic microscopy (FPM) achieves resolutions as fine as 150 nm while maintaining a large field of view, making it highly suitable for high-resolution, label-free imaging. Since its introduction in 2013, FPM has rapidly advanced, offering computational imaging capabilities that surpass conventional resolution limits. However, current systems are constrained by slow imaging speeds due to the sequential illumination of hundreds of LEDs. Here, we review the collective advancements in FPM that have transformed its capabilities over recent years. While numerous research groups have contributed to this progress, key innovations include the development of two-dimensional super-resolution FPM techniques that overcome the diffraction limit through iterative pattern optimization. Building upon these efforts, our group has introduced three-dimensional fast high-resolution Fourier microscopy, achieving 3D dynamic imaging at sub-micron resolution through computational refocusing algorithms. Collectively, these advancements establish FPM as a groundbreaking tool for real-time, high-resolution imaging of living cells, facilitating comprehensive analysis of organelle interactions and providing valuable insights into cellular functions and disease mechanisms.

Optical diffraction tomographic microscopy (ODTM) is an advanced label-free three-dimensional optical microscopic imaging technique. It measures the three-dimensional refractive index (RI) distributions of unstained, transparent biological specimens with high resolution from scattered fields based on the diffraction tomography theorem. Both the morphological and biophysical parameters, as well as the internal organelles of the specimen, can be further analyzed from the measured RI values. ODTM has been increasingly employed in the field of biology, yielding numerous promising results. In order to further promote the application and popularization of this technology in biological research, we provide a tutorial on the fundamental principles and instrumentation of ODTM. The distinct characteristics of ODTM using various illumination strategies and reconstruction algorithms are presented. Observation results from single cells, tissues, and small-scale biological objects are shown to demonstrate the superior performance of ODTM. Current trends and future perspectives of ODTM are discussed.

Scanning probe microscopy (SPM), as a nanoscale characterization technique, employs a sharp probe to detect local tip-sample interactions through near-field physical phenomena. This approach achieves atomic-resolution surface imaging while enabling concurrent characterization of multi-parametric properties — electrical, magnetic, and chemical signals. This review offers a cross-disciplinary perspective on the advances in SPM for biological systems, which serves as a practical guide for life scientists to select from the expanding array of SPM techniques. We outline the fundamental principles of scanning tunneling microscopy (STM) and atomic force microscopy (AFM), before discussing a series of advanced SPM techniques: force spectroscopy for nanomechanical characterization, Kelvin probe force microscopy (KPFM) for surface potential imaging, scanning near-field optical microscopy (SNOM) for super-resolution optics, tip-enhanced Raman spectroscopy (TERS) for nanoscale chemical identification, and scanning electrochemical microscopy (SECM) for localized electrochemical activity detection. A systematic comparison of these technologies provides researchers with clear criteria to select the optimal methodology for diverse demands, either characterizing nucleic acids and proteins or analyzing single-cell ultrastructure and biomechanics. In addition, this review explores the transformative integration of SPM and artificial intelligence (AI). This integration is expected to automate SPM workflows. It will also increase the stability of SPM systems and enhance the reproducibility of experimental results. Furthermore, by addressing current challenges and future perspectives of in vivo imaging, this review aims not merely to review the progress but to empower biologists to harness these intelligent multi-modal SPM systems for groundbreaking discoveries.

Cryo-electron tomography (cryo-ET) enables three-dimensional imaging of cellular architecture in a near-native state, but its use in intact mammalian tissues remains technically challenging. Here, using an optimized tissue-level cryo-ET pipeline that integrates high-pressure freezing, cryo-CLEM, and an improved serial lift-out cryo-FIB workflow, we reconstructed brush-border regions of mouse small intestine from 43 tomographic tilt series and analyzed 490 individual microvilli. Microvilli formed a quasi-regular lattice (mean inter-microvillar spacing ≈ 61 nm) with diameters of 72−114 nm and clearly resolved actin core bundles. Each protrusion bore lateral, nanobristle-like projections that were morphologically heterogeneous and, in rare cases, bridged adjacent microvilli. Importantly, we identified 27 branched microvilli (~5.5%), including Y-shaped and multi-branched forms; the organization of actin bundles in these structures is consistent with a tip-to-base division mechanism for generating daughter protrusions. These observations reveal previously underappreciated structural plasticity in the mammalian brush border and support a fission-like pathway for microvillus renewal.

Diabetes remains an incurable disease, and emerging evidence suggests that exosomes may offer promising therapeutic potential for both diabetes and its complications. However, their specific effects on pancreatic islet function are still under investigation. This meta-analysis aimed to evaluate the efficacy of stem cell-derived exosomes (SC-Exs) in preserving islet function in diabetes. A comprehensive search of PubMed, Embase, Cochrane Library, and Web of Science databases was conducted from inception to May 1, 2025, to identify studies assessing the impact of SC-Exs on islet function in diabetic models. Data on insulin levels, islet quantity, and inflammatory markers were extracted and analyzed using Stata 14.0 and Review Manager (RevMan) 5 software. Ten eligible studies were included in the meta-analysis. Compared with controls, SC-Exs significantly increased insulin levels (SMD = 10.09, 95% CI: 7.97–12.21, P < 0.00001) and islet quantity (SMD = 3.33, 95% CI: 1.47–5.20, P = 0.0005). Moreover, SC-Exs consistently promoted islet proliferation, inhibited cell death, and suppressed inflammatory responses in diabetic models. These findings suggest that SC-Ex treatment effectively preserves pancreatic islet structure and function, highlighting its potential as a novel and promising therapeutic approach for diabetes management. Further high-quality studies are warranted to confirm its efficacy, safety, and clinical applicability.