FilamentID reveals the composition and function of metabolic enzyme polymers during gametogenesis.

Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a "filament identification" (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of partially lysed cells or organelles. FilamentID identified the mitochondrial filaments as being composed of the conserved aldehyde dehydrogenase Ald4ALDH2 and the nucleoplasmic/cytoplasmic filaments as consisting of acetyl-coenzyme A (CoA) synthetase Acs1ACSS2. Structural characterization further revealed the mechanism underlying polymerization and enabled us to genetically perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores and, more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts.

Multiscale drainage dynamics with Haines jumps monitored by stroboscopic 4D X-ray microscopy.

Slow multiphase flow in porous media is intriguing because its underlying dynamics is almost deterministic, yet depends on a hierarchy of spatiotemporal processes. There has been great progress in the experimental study of such multiphase flows, but three-dimensional (3D) microscopy methods probing the pore-scale fluid dynamics with millisecond resolution have been lacking. Yet, it is precisely at these length and time scales that the crucial pore-filling events known as Haines jumps take place. Here, we report four-dimensional (4D) (3D + time) observations of multiphase flow in a consolidated porous medium as captured in situ by stroboscopic X-ray micro-tomography. With a total duration of 6.5 s and 2 kHz frame rate, our experiments provide unprecedented access to the multiscale liquid dynamics. Our tomography strategy relies on the fact that Haines jumps, although irregularly spaced in time, are almost deterministic, and therefore repeatable during imbibition-drainage cycling. We studied the time-dependent flow pattern in a porous medium consisting of sintered glass shards. Exploiting the repeatability, we could combine the radiographic projections recorded under different angles during successive cycles into a 3D movie, allowing us to reconstruct pore-scale events, such as Haines jumps, with a spatiotemporal resolution that is two orders of magnitude higher than was hitherto possible. This high resolution allows us to explore the detailed interfacial dynamics during drainage, including fluid-front displacements and velocities. Our experimental approach opens the way to the study of fast, yet deterministic mesoscopic processes also other than flow in porous media.

Three-dimensional virtual histology of the human hippocampus based on phase-contrast computed tomography.

We have studied the three-dimensional (3D) cytoarchitecture of the human hippocampus in neuropathologically healthy and Alzheimer's disease (AD) individuals, based on phase-contrast X-ray computed tomography of postmortem human tissue punch biopsies. In view of recent findings suggesting a nuclear origin of AD, we target in particular the nuclear structure of the dentate gyrus (DG) granule cells. Tissue samples of 20 individuals were scanned and evaluated using a highly automated approach of measurement and analysis, combining multiscale recordings, optimized phase retrieval, segmentation by machine learning, representation of structural properties in a feature space, and classification based on the theory of optimal transport. Accordingly, we find that the prototypical transformation between a structure representing healthy granule cells and the pathological state involves a decrease in the volume of granule cell nuclei, as well as an increase in the electron density and its spatial heterogeneity. The latter can be explained by a higher ratio of heterochromatin to euchromatin. Similarly, many other structural properties can be derived from the data, reflecting both the natural polydispersity of the hippocampal cytoarchitecture between different individuals in the physiological context and the structural effects associated with AD pathology.

Indirect Correlative Light and Electron Microscopy (iCLEM): A Novel Pipeline for Multiscale Quantification of Structure From Molecules to Organs.

Correlative light and electron microscopy (CLEM) methods are powerful methods that combine molecular organization (from light microscopy) with ultrastructure (from electron microscopy). However, CLEM methods pose high cost/difficulty barriers to entry and have very low experimental throughput. Therefore, we have developed an indirect correlative light and electron microscopy (iCLEM) pipeline to sidestep the rate-limiting steps of CLEM (i.e., preparing and imaging the same samples on multiple microscopes) and correlate multiscale structural data gleaned from separate samples imaged using different modalities by exploiting biological structures identifiable by both light and electron microscopy as intrinsic fiducials. We demonstrate here an application of iCLEM, where we utilized gap junctions and mechanical junctions between muscle cells in the heart as intrinsic fiducials to correlate ultrastructural measurements from transmission electron microscopy (TEM), and focused ion beam scanning electron microscopy (FIB-SEM) with molecular organization from confocal microscopy and single molecule localization microscopy (SMLM). We further demonstrate how iCLEM can be integrated with computational modeling to discover structure-function relationships. Thus, we present iCLEM as a novel approach that complements existing CLEM methods and provides a generalizable framework that can be applied to any set of imaging modalities, provided suitable intrinsic fiducials can be identified.

Multiscale pink-beam microCT imaging at the ESRF-ID17 biomedical beamline.

Recent trends in hard X-ray micro-computed tomography (microCT) aim at increasing both spatial and temporal resolutions. These challenges require intense photon beams. Filtered synchrotron radiation beams, also referred to as `pink beams', which are emitted by wigglers or bending magnets, meet this need, owing to their broad energy range. In this work, the new microCT station installed at the biomedical beamline ID17 of the European Synchrotron is described and an overview of the preliminary results obtained for different biomedical-imaging applications is given. This new instrument expands the capabilities of the beamline towards sub-micrometre voxel size scale and simultaneous multi-resolution imaging. The current setup allows the acquisition of tomographic datasets more than one order of magnitude faster than with a monochromatic beam configuration.

Advanced X-ray CT scanning can boost tree ring research for earth system sciences.

BACKGROUND AND AIMS: Tree rings, as archives of the past and biosensors of the present, offer unique opportunities to study influences of the fluctuating environment over decades to centuries. As such, tree-ring-based wood traits are capital input for global vegetation models. To contribute to earth system sciences, however, sufficient spatial coverage is required of detailed individual-based measurements, necessitating large amounts of data. X-ray computed tomography (CT) scanning is one of the few techniques that can deliver such data sets. METHODS: Increment cores of four different temperate tree species were scanned with a state-of-the-art X-ray CT system at resolutions ranging from 60 µm down to 4.5 µm, with an additional scan at a resolution of 0.8 µm of a splinter-sized sample using a second X-ray CT system to highlight the potential of cell-level scanning. Calibration-free densitometry, based on full scanner simulation of a third X-ray CT system, is illustrated on increment cores of a tropical tree species. KEY RESULTS: We show how multiscale scanning offers unprecedented potential for mapping tree rings and wood traits without sample manipulation and with limited operator intervention. Custom-designed sample holders enable simultaneous scanning of multiple increment cores at resolutions sufficient for tree ring analysis and densitometry as well as single core scanning enabling quantitative wood anatomy, thereby approaching the conventional thin section approach. Standardized X-ray CT volumes are, furthermore, ideal input imagery for automated pipelines with neural-based learning for tree ring detection and measurements of wood traits. CONCLUSIONS: Advanced X-ray CT scanning for high-throughput processing of increment cores is within reach, generating pith-to-bark ring width series, density profiles and wood trait data. This would allow contribution to large-scale monitoring and modelling efforts with sufficient global coverage.

Multiscale Functional and Molecular Photoacoustic Tomography.

Photoacoustic tomography (PAT) combines rich optical absorption contrast with the high spatial resolution of ultrasound at depths in tissue. The high scalability of PAT has enabled anatomical imaging of biological structures ranging from organelles to organs. The inherent functional and molecular imaging capabilities of PAT have further allowed it to measure important physiological parameters and track critical cellular activities. Integration of PAT with other imaging technologies provides complementary capabilities and can potentially accelerate the clinical translation of PAT.

Imaging Drosophila brain by combining cryo-soft X-ray microscopy of thick vitreous sections and cryo-electron microscopy of ultrathin vitreous sections.

Cryo-soft X-ray microscopy is an emerging imaging tool complementary to cryo-electron microscopy, allowing to image frozen hydrated specimens ten to hundred times thicker, but at lower resolution. We describe how the method, so far restricted to isolated small cells or cell monolayers, can be extended to large cells and tissue. We image the synapses of the Kenyon cells in frozen hydrated Drosophila brains combining cryo-soft X-ray microscopy of thick vitreous sections, and cryo-electron microscopy of ultrathin vitreous sections. We show how to obtain frozen hydrated sections of thicknesses ranging from 40 nm up to 2.5 µm, by tuning the sectioning speed of the cryo-microtome. A fluorescent stereo-microscope mounted on the cryo-microtome allowed us to target the regions of interest after GFP-labeling of synapses. Thick cryo-sections were imaged by cryo-soft X-ray microscopy at a resolution better than 25 nm, while ultrathin cryo-sections of the same regions were explored in parallel at the nanometre level of resolution by cryo-electron microscopy.

Coupling Nanoscale Precision with Multiscale Imaging: A Multifunctional Near-Infrared Dye for the Brain.

Live imaging of primary neural cells is crucial for monitoring neuronal activity, especially multiscale and multifunctional imaging that offers excellent biocompatibility. Multiscale imaging can provide insights into cellular structure and function from the nanoscale to the millimeter scale. Multifunctional imaging can monitor different activities in the brain. However, this remains a challenge because of the lack of dyes with a high signal-to-background ratio, water solubility, and multiscale and multifunctional imaging capabilities. In this study, we present a neural dye with near-infrared (NIR) emissions (>700 nm) that enables ultrafast staining (in less than 1 min) for the imaging of primary neurons. This dye not only enables multiscale neural live-cell imaging from vesicles in neurites, neural membranes, and single neurons to the whole brain but also facilitates multifunctional imaging, such as the monitoring and quantifying of synaptic vesicles and the changes in membrane potential. We also explore the potential of this NIR neural dye for staining brain slices and live brains. The NIR neural dye exhibits superior binding with neural membranes compared to commercial dyes, thereby achieving multiscale and multifunctional brain neuroimaging. In conclusion, our findings introduce a significant breakthrough in neuroimaging dyes by developing a category of small molecular dyes.

Multi-scale cellular imaging of DNA double strand break repair.

Live-cell and high-resolution fluorescence microscopy are powerful tools to study the organization and dynamics of DNA double-strand break repair foci and specific repair proteins in single cells. This requires specific induction of DNA double-strand breaks and fluorescent markers to follow the DNA lesions in living cells. In this review, where we focused on mammalian cell studies, we discuss different methods to induce DNA double-strand breaks, how to visualize and quantify repair foci in living cells., We describe different (live-cell) imaging modalities that can reveal details of the DNA double-strand break repair process across multiple time and spatial scales. In addition, recent developments are discussed in super-resolution imaging and single-molecule tracking, and how these technologies can be applied to elucidate details on structural compositions or dynamics of DNA double-strand break repair.

Multiscale Label-Free Imaging of Fibrillar Collagen in the Tumor Microenvironment.

With recent advances in cancer therapeutics, there is a great need for improved imaging methods for characterizing cancer onset and progression in a quantitative and actionable way. Collagen, the most abundant extracellular matrix protein in the tumor microenvironment (and the body in general), plays a multifaceted role, both hindering and promoting cancer invasion and progression. Collagen deposition can defend the tumor with immunosuppressive effects, while aligned collagen fiber structures can enable tumor cell migration, aiding invasion and metastasis. Given the complex role of collagen fiber organization and topology, imaging has been a tool of choice to characterize these changes on multiple spatial scales, from the organ and tumor scale to cellular and subcellular level. Macroscale density already aids in the detection and diagnosis of solid cancers, but progress is being made to integrate finer microscale features into the process. Here we review imaging modalities ranging from optical methods of second harmonic generation (SHG), polarized light microscopy (PLM), and optical coherence tomography (OCT) to the medical imaging approaches of ultrasound and magnetic resonance imaging (MRI). These methods have enabled scientists and clinicians to better understand the impact collagen structure has on the tumor environment, at both the bulk scale (density) and microscale (fibrillar structure) levels. We focus on imaging methods with the potential to both examine the collagen structure in as natural a state as possible and still be clinically amenable, with an emphasis on label-free strategies, exploiting intrinsic optical properties of collagen fibers.

Multiscale imaging reveals the presence of autophagic vacuoles in developing maize endosperm.

Cereal endosperm is solely devoted to the storage of proteins and starch that will be used by the embryo upon germination. The high degree of specialization of this tissue is reflected in its endomembrane system, in which ER derived protein bodies and protein storage vacuoles (PSVs) are of particular interest. In maize seeds, the main storage proteins are zeins, that form transport incompetent aggregates within the ER lumen and finally build protein bodies that bud from the ER. In contrast to the zeins, the maize globulins are not very abundant and the vacuolar storage compartment of maize endosperm is not fully described. Whereas in other cereals, including wheat and barley, the PSV serves as the main protein storage compartment, only small, globulin-containing PSVs have been identified in maize so far. We present here a multi-scale set of data, ranging from live-cell imaging to more sophisticated 3D electron microscopy techniques (SBF-SEM), that has allowed us to investigate in detail the vacuoles in maize endosperm cells, including a novel, autophagic vacuole that is present in early developmental stages.

Multiscale photoacoustic imaging without motion using single-pixel imaging.

The conventional photoacoustic microscopy usually uses mechanical raster scanning to obtain three-dimensional information, and the imaging speed is limited. Meanwhile, the conventional photoacoustic microscopy can only be performed at one single scale due to fixed resolution, it cannot make full use of multiscale information for integrated imaging. Here, we proposed a multiscale photoacoustic microscopy based on single-pixel imaging. A sequence of sinusoidal fringes with varying spatial frequencies is used to obtain the Fourier coefficients in the case of a single ultrasonic transducer. By controlling the spatial frequency of fringe, the acquisition of Fourier coefficients can be controlled and multiscale imaging can be achieved. The feasibility of this method is verified by theory and simulation. The results show that the lateral resolution can be tuned from several microns to tens of microns without mechanical scanning. This method will expand the application of photoacoustic imaging in biomedical research.

High-fidelity deconvolution for acoustic-resolution photoacoustic microscopy enabled by convolutional neural networks.

Acoustic-resolution photoacoustic microscopy (AR-PAM) image resolution is determined by the point spread function (PSF) of the imaging system. Previous algorithms, including Richardson-Lucy (R-L) deconvolution and model-based (MB) deconvolution, improve spatial resolution by taking advantage of the PSF as prior knowledge. However, these methods encounter the problems of inaccurate deconvolution, meaning the deconvolved feature size and the original one are not consistent (e.g., the former can be smaller than the latter). We present a novel deep convolution neural network (CNN)-based algorithm featuring high-fidelity recovery of multiscale feature size to improve lateral resolution of AR-PAM. The CNN is trained with simulated image pairs of line patterns, which is to mimic blood vessels. To investigate the suitable CNN model structure and elaborate on the effectiveness of CNN methods compared with non-learning methods, we select five different CNN models, while R-L and directional MB methods are also applied for comparison. Besides simulated data, experimental data including tungsten wires, leaf veins, and in vivo blood vessels are also evaluated. A custom-defined metric of relative size error (RSE) is used to quantify the multiscale feature recovery ability of different methods. Compared to other methods, enhanced deep super resolution (EDSR) network and residual in residual dense block network (RRDBNet) model show better recovery in terms of RSE for tungsten wires with diameters ranging from 30 µ m to 120 µ m . Moreover, AR-PAM images of leaf veins are tested to demonstrate the effectiveness of the optimized CNN methods (by EDSR and RRDBNet) for complex patterns. Finally, in vivo images of mouse ear blood vessels and rat ear blood vessels are acquired and then deconvolved, and the results show that the proposed CNN method (notably RRDBNet) enables accurate deconvolution of multiscale feature size and thus good fidelity.

Novel Multimodal, Multiscale Imaging System with Augmented Reality.

A novel multimodal, multiscale imaging system with augmented reality capability were developed and characterized. The system offers 3D color reflectance imaging, 3D fluorescence imaging, and augmented reality in real time. Multiscale fluorescence imaging was enabled by developing and integrating an in vivo fiber-optic microscope. Real-time ultrasound-fluorescence multimodal imaging used optically tracked fiducial markers for registration. Tomographical data are also incorporated using optically tracked fiducial markers for registration. Furthermore, we characterized system performance and registration accuracy in a benchtop setting. The multiscale fluorescence imaging facilitated assessing the functional status of tissues, extending the minimal resolution of fluorescence imaging to ~17.5 µm. The system achieved a mean of Target Registration error of less than 2 mm for registering fluorescence images to ultrasound images and MRI-based 3D model, which is within clinically acceptable range. The low latency and high frame rate of the prototype system has shown the promise of applying the reported techniques in clinically relevant settings in the future.

A Review on Multiscale Bone Damage: From the Clinical to the Research Perspective.

The investigation of bone damage processes is a crucial point to understand the mechanisms of age-related bone fractures. In order to reduce their impact, early diagnosis is key. The intricate architecture of bone and the complexity of multiscale damage processes make fracture prediction an ambitious goal. This review, supported by a detailed analysis of bone damage physical principles, aims at presenting a critical overview of how multiscale imaging techniques could be used to implement reliable and validated numerical tools for the study and prediction of bone fractures. While macro- and meso-scale imaging find applications in clinical practice, micro- and nano-scale imaging are commonly used only for research purposes, with the objective to extract fragility indexes. Those images are used as a source for multiscale computational damage models. As an example, micro-computed tomography (micro-CT) images in combination with micro-finite element models could shed some light on the comprehension of the interaction between micro-cracks and micro-scale bone features. As future insights, the actual state of technology suggests that these models could be a potential substitute for invasive clinical practice for the prediction of age-related bone fractures. However, the translation to clinical practice requires experimental validation, which is still in progress.

Recent advances in intravital microscopy for preclinical research.

Intravital microscopy (IVM) has revolutionized our understanding of single-cell behavior in complex tissues by enabling real-time observation of molecular and cellular processes in their natural environment. In preclinical research, IVM has emerged as a standard tool for mechanistic studies of therapy response and the rational design of new treatment strategies. Technological developments keep expanding the imaging depth and quality that can be achieved in living tissue, and the maturation of imaging modalities such as fluorescence and phosphorescence lifetime imaging facilitates co-registration of individual cell dynamics with metabolic tissue states. Correlation of IVM with mesoscopic and macroscopic imaging modalities further promotes the translation of mechanistic insights gained by IVM into clinically relevant information. This review highlights some of the recent advances in IVM that have made the transition from experimental optical techniques to practical applications in basic and preclinical research.

Imaging and visualizing SARS-CoV-2 in a new era for structural biology.

The SARS-CoV-2 pandemic has had a global impact and has put scientific endeavour in the spotlight, perhaps more than any previous viral outbreak. Fortuitously, the pandemic came at a time when decades of research in multiple scientific fields could be rapidly brought to bear, and a new generation of vaccine platforms was on the cusp of clinical maturity. SARS-CoV-2 also emerged at the inflection point of a technological revolution in macromolecular imaging by cryo-electron microscopy, fuelled by a confluence of major technological advances in sample preparation, optics, detectors and image processing software, that complemented pre-existing techniques. Together, these advances enabled us to visualize SARS-CoV-2 and its components more rapidly, in greater detail, and in a wider variety of biologically relevant contexts than would have been possible even a few years earlier. The resulting ultrastructural information on SARS-CoV-2 and how it interacts with the host cell has played a critical role in the much-needed accelerated development of COVID-19 vaccines and therapeutics. Here, we review key imaging modalities used to visualize SARS-CoV-2 and present select example data, which have provided us with an exceptionally detailed picture of this virus.

Unobstructed Multiscale Imaging of Tissue Sections for Ultrastructural Pathology Analysis by Backscattered Electron Scanning Microscopy.

Ultrastructural analysis of healthy, diseased, or experimental tissues is essential in diagnostic and investigative pathology. Evaluation of large tissue areas with suborganelle resolution is challenging because biological structures ranging from several millimeters to nanometers in size need to be identified and imaged while maintaining context over multiple scales. Imaging with field emission scanning electron microscopes (FE-SEMs) is uniquely suited for this task. We describe an efficient workflow for the preparation and unobstructed multiscale imaging of tissue sections with backscattered electron scanning electron microscopy (BSE-SEM) for applications in ultrastructural pathology. We demonstrate that a diverse range of tissues, processed by conventional electron microscopy protocols and avoiding the use of mordanting agents, can be imaged on standard glass slides over multiple scales, from the histological to the ultrastructural level, without any visual obstructions. Our workflow takes advantage of the very large scan fields possible with modern FE-SEMs that allow for the acquisition of wide-field overview images which can be explored at the ultrastructural level by digitally zooming into the images. Examples from applications in pulmonary research and neuropathology demonstrate the versatility and efficiency of this method. This BSE-SEM-based multiscale imaging procedure promises to substantially simplify and accelerate ultrastructural tissue analysis in pathology.

3-D-Printed Registration Phantom for Combined Ultrasound and Optical Imaging of Biological Tissues.

Efforts to develop quantitative ultrasound biomarkers would benefit from comparisons between ultrasound data and higher-resolution images of the tissue microstructure, such as from optical microscopy. However, only a few studies have used these methods for multiscale imaging because it is difficult to register low-resolution (>100 µm) ultrasound images to high-resolution microscopy images. To address this need, we have designed a 3-D-printed registration phantom that is made of a hard fluorescent resin, fits into a glass-bottom dish and can be used to calculate a coordinate system transform between ultrasound and optical microscopy. We report the phantom design, a registration protocol and an example registration using 18.5-MHz ultrasound and second harmonic generation microscopy. We evaluate the registration precision, achieving standard deviations smaller than the ultrasound resolution across all axes, and illustrate on a mouse mammary gland that this method yields results superior to those of manual landmark registration.