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.

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.

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.

Cryo-correlative light and electron microscopy (cryo-CLEM) is a powerful technique that combines fluorescence imaging for specific localization with electron microscopy for detailed structural analysis, enabling high-resolution exploration of synaptic structures in neurons. In this study, we employed a cryo-CLEM approach using three independent alignment markers to precisely correlate electron microscopy (EM) images with light microscopy (LM) images of neuronal synapses under cryogenic conditions. This methodology revealed a distinctive pattern of electron densities in the synaptic clefts. Additionally, we were able to capture high-resolution images of presynaptic vesicles in various states, underscoring the potential of cryo-CLEM in advancing synaptic research.

Fluorescence microscopy and electron microscopy complement each other as the former provides labelling and localisation of specific molecules and target structures while the latter possesses excellent revolving power of fine structure in context. These two techniques can combine as correlative light and electron microscopy (CLEM) to reveal the organisation of materials within the cell. Frozen hydrated sections allow microscopic observations of cellular components in situ in a near-native state and are compatible with superresolution fluorescence microscopy and electron tomography if sufficient hardware and software support is available and a well-designed protocol is followed. The development of superresolution fluorescence microscopy greatly increases the precision of fluorescence annotation of electron tomograms. Here, we provide detailed instructions on how to perform cryogenic superresolution CLEM on vitreous sections. From fluorescence-labelled cells to high pressure freezing, cryo-ultramicrotomy, cryogenic single-molecule localisation microscopy, cryogenic electron tomography and image registration, electron tomograms with features of interest highlighted by superresolution fluorescence signals are expected to be obtained.

Super-resolution imaging based on single-molecule localization has been developed for more than a decade. These techniques can break through diffraction limit of fluorescent microscopy and initially improve the resolution by an order of magnitude to ~20 nm, by introducing photoactivatable/photoswitching probes and centroid fitting method. As the demand of biological research, the localization precision of single-molecules was further improved by several state-of-the-art methods in the past several years. This review focuses on the latest developed techniques which have greatly improved the performance of single-molecule localization microscopy, from measurement principle to hardware design. These methods are essential for the study of nanostructures and biomacromolecule dynamics inside of cells.