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Key Points Fluorescence nanoscopy (also known as super-resolution microscopy) methods have expanded optical imaging to reach the nanometre resolution range, typically 20–50 nm and even down to the 1 nm level. Diffraction-unlimited nanoscopy methods, which neutralize the resolution-limiting role of diffraction, separate fluorophores by transiently transferring them between (at least) two discernible states, typically an 'on' and an 'off' state of fluorescence. The counting of molecules in nanoscale settings such as within organelles is a crucially important development, along with labelling strategies to reliably pinpoint the locations and spatial proximities of all the molecules investigated in an imaging experiment. Dynamic nanoscopy and extensions of nanoscopy imaging to tissue and in vivo contexts are further frontiers. Examples taken from mitochondrial biology and neurobiology illustrate the capabilities and discovery potential of nanoscale molecule-specific imaging with focused light. Fluorescence nanoscopy enables the optical imaging of cellular components with resolutions at the nanometre scale. With the growing availability of super-resolution microscopes, nanoscopy methods are being increasingly applied. Quantitative, multicolour, live-cell nanoscopy and the corresponding labelling strategies are under continuous development. Fluorescence nanoscopy uniquely combines minimally invasive optical access to the internal nanoscale structure and dynamics of cells and tissues with molecular detection specificity. While the basic physical principles of 'super-resolution' imaging were discovered in the 1990s, with initial experimental demonstrations following in 2000, the broad application of super-resolution imaging to address cell-biological questions has only more recently emerged. Nanoscopy approaches have begun to facilitate discoveries in cell biology and to add new knowledge. One current direction for method improvement is the ambition to quantitatively account for each molecule under investigation and assess true molecular colocalization patterns via multi-colour analyses. In pursuing this goal, the labelling of individual molecules to enable their visualization has emerged as a central challenge. Extending nanoscale imaging into (sliced) tissue and whole-animal contexts is a further goal. In this Review we describe the successes to date and discuss current obstacles and possibilities for further development.

The high spatial resolution of secondary ion mass spectrometry and the high resolving power of the Orbitrap mass spectrometer are combined in a single imaging platform, the 3D OrbiSIMS. The instrument's capabilities for resolving lipids and neurotransmitters in the brain with subcellular spatial resolution, and a drug in a single cell in three dimensions is demonstrated. We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 μm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmitters—gamma-aminobutyric acid, dopamine and serotonin—with high spectroscopic confidence in the mouse hippocampus. We also putatively annotated and mapped the subcellular localization of 29 sulfoglycosphingolipids and 45 glycerophospholipids, and we confirmed lipid identities with tandem mass spectrometry. We demonstrated single-cell metabolomic profiling using rat alveolar macrophage cells incubated with different concentrations of the drug amiodarone, and we observed that the upregulation of phospholipid species and cholesterol is correlated with the accumulation of amiodarone.

in vivo bioluminescence imaging (BLi) is an optical molecular imaging technique used to visualize molecular and cellular processes in health and diseases and to follow the fate of cells with high sensitivity using luciferase-based gene reporters. The high sensitivity of this technique arises from efficient photon production, followed by the reaction between luciferase enzymes and luciferin substrates. Novel discoveries and developments of luciferase reporters, substrates, and gene-editing techniques, and emerging fields of applications, promise a new era of deeper and more sensitive molecular imaging. BLi is now a standard technique for in vivo imaging of gene expression and to follow cells and their fate. However, many applications are limited by the use of a single reporter and limited sensitivity in deep tissue. Novel far-red and near-infrared emitting systems for enhanced sensitivity and resolution in deep tissue and multicolor applications have recently become available. Caged bioluminescent substrates for analyzing specific enzyme activity or detecting bioactive small molecules are under development. Opportunities in technical improvements of signal acquisition and processing are emerging. Newly available bioluminescent tools and recent applications are altering the practice of BLI.

A 194-cm-long total-body positron emission tomography/computed tomography (PET/CT) scanner (uEXPLORER), has been constructed to offer a transformative platform for human radiotracer imaging in clinical research and healthcare. Its total-body coverage and exceptional sensitivity provide opportunities for innovative studies of physiology, biochemistry, and pharmacology. The objective of this study is to develop a method to perform ultrahigh (100 ms) temporal resolution dynamic PET imaging by combining advanced dynamic image reconstruction paradigms with the uEXPLORER scanner. We aim to capture the fast dynamics of initial radiotracer distribution, as well as cardiac motion, in the human body. The results show that we can visualize radiotracer transport in the body on timescales of 100 ms and obtain motion-frozen images with superior image quality compared to conventional methods. The proposed method has applications in studying fast tracer dynamics, such as blood flow and the dynamic response to neural modulation, as well as performing real-time motion tracking (e.g., cardiac and respiratory motion, and gross body motion) without any external monitoring device (e.g., electrocardiogram, breathing belt, or optical trackers).

For over two decades photoacoustic imaging has been tested clinically, but successful human trials have been limited. To enable quantitative clinical spectroscopy, the fundamental issues of wavelength-dependent fluence variations and inter-wavelength motion must be overcome. Here we propose a real-time, spectroscopic photoacoustic/ultrasound (PAUS) imaging approach using a compact, 1-kHz rate wavelength-tunable laser. Instead of illuminating tissue over a large area, the fiber-optic delivery system surrounding an US array sequentially scans a narrow laser beam, with partial PA image reconstruction for each laser pulse. The final image is then formed by coherently summing partial images. This scheme enables (i) automatic compensation for wavelength-dependent fluence variations in spectroscopic PA imaging and (ii) motion correction of spectroscopic PA frames using US speckle tracking in real-time systems. The 50-Hz video rate PAUS system is demonstrated in vivo using a murine model of labelled drug delivery. Spectroscopic photoacoustic imaging has been limited by wavelength-dependent fluence variations and tissue motion. Here, the authors achieve real-time fluence and motion correction in vivo by using a wavelength-tunable laser, sequential scan of a narrow beam and partial image reconstruction for each pulse.

Live-cell Raman imaging based on bioorthogonal Raman probes with distinct signals in the cellular Raman-silent region (1800–2800 cm −1 ) has attracted great interest in recent years. We report here a class of water-soluble and biocompatible polydiacetylenes with intrinsic ultrastrong alkyne Raman signals that locate in this region for organelle-targeting live-cell Raman imaging. Using a host-guest topochemical polymerization strategy, we have synthesized a water-soluble and functionalizable master polydiacetylene, namely poly(deca-4,6-diynedioic acid) (PDDA), which possesses significantly enhanced (up to ~10 4 fold) alkyne vibration compared to conventional alkyne Raman probes. In addition, PDDA can be used as a general platform for multi-functional ultrastrong Raman probes. We achieve high quality live-cell stimulated Raman scattering imaging on the basis of modified PDDA. The polydiacetylene-based Raman probes represent ultrastrong intrinsic Raman imaging agents in the Raman-silent region (without any Raman enhancer), and the flexible functionalization of this material holds great promise for its potential diverse applications. Raman probes which operate in the cellular silent region are of interest for live cell imaging. Here, the authors report on the development of a water soluble polydiacetylene Raman probe with enhanced Raman signal in the silent region which can be functionalised for organelle targeting and demonstrate application.

Algorithms that account for and overcome the intrinsic pixel-dependent readout noise from scientific complementary metal-oxide semiconductor (sCMOS) cameras prevent localization artifacts and avoid substantial compromises in localization uncertainty in single-molecule imaging. When combined with multi-emitter fitting, sCMOS cameras allow video-rate single-molecule switching nanoscopy. Newly developed scientific complementary metal-oxide semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition, enlarge the field of view and increase the effective quantum efficiency in single-molecule switching nanoscopy. However, sCMOS-intrinsic pixel-dependent readout noise substantially lowers the localization precision and introduces localization artifacts. We present algorithms that overcome these limitations and that provide unbiased, precise localization of single molecules at the theoretical limit. Using these in combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at rates of up to 32 reconstructed images per second in fixed and living cells.

Photoacoustic (PA) computed tomography (PACT) benefits from genetically encoded probes with photochromic behavior, which dramatically increase detection sensitivity and specificity through photoswitching and differential imaging. Starting with a DrBphP bacterial phytochrome, we have engineered a near-infrared photochromic probe, DrBphP-PCM, which is superior to the full-length RpBphP1 phytochrome previously used in differential PACT. DrBphP-PCM has a smaller size, better folding, and higher photoswitching contrast. We have imaged both DrBphP-PCM and RpBphP1 simultaneously on the basis of their unique signal decay characteristics, using a reversibly switchable single-impulse panoramic PACT (RS-SIP-PACT) with a single wavelength excitation. The simple structural organization of DrBphP-PCM allows engineering a bimolecular PA complementation reporter, a split version of DrBphP-PCM, termed DrSplit. DrSplit enables PA detection of protein–protein interactions in deep-seated mouse tumors and livers, achieving 125-µm spatial resolution and 530-cell sensitivity in vivo. The combination of RS-SIP-PACT with DrBphP-PCM and DrSplit holds great potential for noninvasive multi-contrast deep-tissue functional imaging. Bacterial phytochrome-based probes improved sensitivity in photoacoustic computed tomography. Here the authors engineer a small near-infrared switchable photochromic probe that allows multi-contrast imaging at depths and can be adapted to study protein–protein interactions in deep-seated tumors.

We demonstrate the use of cryogenic super-resolution correlative light and electron microscopy (csCLEM) to precisely determine the spatial relationship between proteins and their native cellular structures. Several fluorescent proteins (FPs) were found to be photoswitchable and emitted far more photons under our cryogenic imaging condition, resulting in higher localization precision which is comparable to ambient super-resolution imaging. Vitrified specimens were prepared by high pressure freezing and cryo-sectioning to maintain a near-native state with better fluorescence preservation. A 2-3-fold improvement of resolution over the recent reports was achieved due to the photon budget performance of screening out Dronpa and optimized imaging conditions, even with thin sections which is at a disadvantage when calculate the structure resolution from label density. We extended csCLEM to mammalian cells by introducing cryo-sectioning and observed good correlation of a mitochondrial protein with the mitochondrial outer membrane at nanometer resolution in three dimensions.

This report describes an imaging and analysis platform enabling high-throughput single-molecule fluorescence investigations of fast, transient molecular recognition events. Single-molecule fluorescence microscopy is uniquely suited for detecting transient molecular recognition events, yet achieving the time resolution and statistics needed to realize this potential has proven challenging. Here we present a single-molecule imaging and analysis platform using scientific complementary metal-oxide semiconductor (sCMOS) detectors that enables imaging of 15,000 individual molecules simultaneously at millisecond rates. This system enabled the detection of previously obscured processes relevant to the fidelity mechanism in protein synthesis.