Quantitative 3D imaging of whole,unstained cells by using X-ray diffraction microscopy

Microscopy has greatly advanced our understanding of biology. Although significant progress has recently been made in optical microscopy to break the diffraction-limit barrier, reliance of such techniques on fluorescent labeling technologies prohibits quantitative 3D imaging of the entire contents of cells. Cryoelectron microscopy can image pleomorphic structures at a resolution of 3–5 nm, but is only applicable to thin or sectioned specimens. Here, we report quantitative 3D imaging of a whole, unstained cell at a resolution of 50–60 nm by X-ray diffraction microscopy. We identified the 3D morphology and structure of cellular organelles including cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, nucleus, and nucleolus inside a yeast spore cell. Furthermore, we observed a 3D structure protruding from the reconstructed yeast spore, suggesting the spore germination process. Using cryogenic technologies, a 3D resolution of 5–10 nm should be achievable by X-ray diffraction microscopy. This work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy.     

Biological imaging by soft x-ray diffraction microscopy

We have used the method of x-ray diffraction microscopy to image the complex-valued exit wave of an intact and unstained yeast cell. The images of the freeze-dried cell, obtained by using 750-eV x-rays from different angular orientations, portray several of the cell's major internal components to 30-nm resolution. The good agreement among the independently recovered structures demonstrates the accuracy of the imaging technique. To obtain the best possible reconstructions, we have implemented procedures for handling noisy and incomplete diffraction data, and we propose a method for determining the reconstructed resolution. This work represents a previously uncharacterized application of x-ray diffraction microscopy to a specimen of this complexity and provides confidence in the feasibility of the ultimate goal of imaging biological specimens at 10-nm resolution in three dimensions.     

Compressive fluorescence microscopy for biological and hyperspectral imaging

The mathematical theory of compressed sensing (CS) asserts that one can acquire signals from measurements whose rate is much lower than the total bandwidth. Whereas the CS theory is now well developed, challenges concerning hardware implementations of CS-based acquisition devices--especially in optics--have only started being addressed. This paper presents an implementation of compressive sensing in fluorescence microscopy and its applications to biomedical imaging. Our CS microscope combines a dynamic structured wide-field illumination and a fast and sensitive single-point fluorescence detection to enable reconstructions of images of fluorescent beads, cells, and tissues with undersampling ratios (between the number of pixels and number of measurements) up to 32. We further demonstrate a hyperspectral mode and record images with 128 spectral channels and undersampling ratios up to 64, illustrating the potential benefits of CS acquisition for higher-dimensional signals, which typically exhibits extreme redundancy. Altogether, our results emphasize the interest of CS schemes for acquisition at a significantly reduced rate and point to some remaining challenges for CS fluorescence microscopy.     

High numerical aperture tabletop soft x-ray diffraction microscopy with 70-nm resolution

Light microscopy has greatly advanced our understanding of nature. The achievable resolution, however, is limited by optical wavelengths to approximately 200 nm. By using imaging and labeling technologies, resolutions beyond the diffraction limit can be achieved for specialized specimens with techniques such as near-field scanning optical microscopy, stimulated emission depletion microscopy, and photoactivated localization microscopy. Here, we report a versatile soft x-ray diffraction microscope with 70- to 90-nm resolution by using two different tabletop coherent soft x-ray sources-a soft x-ray laser and a high-harmonic source. We also use field curvature correction that allows high numerical aperture imaging and near-diffraction-limited resolution of 1.5lambda. A tabletop soft x-ray diffraction microscope should find broad applications in biology, nanoscience, and materials science because of its simple optical design, high resolution, large depth of field, 3D imaging capability, scalability to shorter wavelengths, and ultrafast temporal resolution.     

The ePetri dish, an on-chip cell imaging platform based on subpixel perspective sweeping microscopy (SPSM)

We report a chip-scale lensless wide-field-of-view microscopy imaging technique, subpixel perspective sweeping microscopy, which can render microscopy images of growing or confluent cell cultures autonomously. We demonstrate that this technology can be used to build smart Petri dish platforms, termed ePetri, for cell culture experiments. This technique leverages the recent broad and cheap availability of high performance image sensor chips to provide a low-cost and automated microscopy solution. Unlike the two major classes of lensless microscopy methods, optofluidic microscopy and digital in-line holography microscopy, this new approach is fully capable of working with cell cultures or any samples in which cells may be contiguously connected. With our prototype, we demonstrate the ability to image samples of area 6 mm × 4 mm at 660-nm resolution. As a further demonstration, we showed that the method can be applied to image color stained cell culture sample and to image and track cell culture growth directly within an incubator. Finally, we showed that this method can track embryonic stem cell differentiations over the entire sensor surface. Smart Petri dish based on this technology can significantly streamline and improve cell culture experiments by cutting down on human labor and contamination risks.     

From the Cover: Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT

We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5–12-fold compared with their conventional diffraction-limited LS analogs.Far-field nanoscopy (1, 2) methods discern features within subdiffraction distances by briefly forcing their molecules to two distinguishable states for the time period of detection. Typically, fluorophores are switched between a signaling “on” and a nonsignaling (i.e., dark) “off” state. Depending on the switching and fluorescence registration strategy used, these superresolution techniques can be categorized into coordinate-stochastic and coordinate-targeted approaches (2). The latter group of methods, comprising the so-called RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] (1, 3–7) approaches, have been realized using patterns of switch-off light with one or more zero-intensity points or lines, to single out target point (zero-dimensional) or line (1D) coordinates in space where the fluorophores are allowed to assume the on state. The RESOLFT idea can also be implemented in the inverse mode, by using switch-on light and confining the off state. In any case, probing the presence of molecules in new sets of points or lines at every scanning step produces images.Owing to the nature of the on and off states involved––first excited electronic and ground state––stimulated emission depletion (STED) (3) and saturated structured illumination microscopy (SSIM) (8), which both qualify as variants of the RESOLFT principle, typically apply light intensities in the range of MW/cm² and above. Especially when imaging sensitive samples where photoinduced changes must be avoided, RESOLFT is preferably realized with fluorophores which lead to the same factor of resolution improvement at much lower intensities of state-switching light. Reversibly switchable fluorescent proteins (RSFPs) are highly suitable for this purpose (4–7, 9), as transitions between their metastable on and off states require 5 orders of magnitude lower threshold intensities than STED/SSIM to guarantee switch-off. Suitable spectral properties, relatively fast millisecond switching kinetics, and high photostability of recently developed yellow-green-emitting RSFPs like rsEGFP (5), rsEGFP2 (7), and rsEGFP(N205S) (10) compared with early RSFPs have indeed enabled RESOLFT nanoscopy in living cells and tissues. To date, RSFP-based RESOLFT has achieved resolution improvements by factors of 4–5 in rsEGFP2-labeled samples (7). To further reduce the imaging time, massive parallelization of scanning has been reported (10). However, the diffraction-limited axial resolution and lack of background suppression restrict applications to thin samples.Imaging applications typically require careful tuning of imaging parameters including speed, contrast, photosensitivity, and spatial resolution, depending on the information that is sought. Light-sheet fluorescence microscopy (LSFM) (11–15) stands out by its ability to balance most of these parameters for 3D imaging of living specimens. Recently reenacted as the selective plane illumination microscope (13), this microscopy mode has sparked increasing interest notably because of its short acquisition times in 3D imaging and low phototoxicity in living specimens. It excites fluorophores only in a thin diffraction-limited slice of the sample, perpendicular to the direction of fluorescence detection. The LS is generated by a cylindrical lens which focuses an expanded laser beam in only one direction onto the specimen or into the back-focal plane of an illumination objective. Alternatively, a single beam is quickly moved as a “virtual” LS (16) across a specimen section.In such conventional LSFM imaging, the lateral resolution is determined by the numerical aperture (N.A.) of the detection objective (17), whereas axial resolution is given by the LS thickness, provided the latter is thinner than the axial extent of the point-spread function describing the imaging process from the focal plane of the detecting lens to the camera. In a previous study, the axial resolution of LSFM was pushed to the diffraction limit by using the full aperture of the illumination objective with Gaussian beams; this was carried out for practically useful combinations of N.A. (e.g., 0.8 for both illumination and detection objectives) permissible in light of the geometrical constraints given by the objective lens dimensions (18). High-N.A. illumination comes with short Rayleigh ranges of Gaussian beams, which inherently limit the field of view (FOV) along the direction of illumination. Scanned Bessel beams for diffraction-limited excitation with a virtual LS (19–21) typically offer larger FOVs (22), but side lobes broaden the scanned LS in the axial direction and contribute to phototoxicity outside of the focal plane of detection (20). A more complex approach has used Bessel-beam excitation in combination with structured illumination to obtain near-isotropic (but still diffraction-limited) resolution as measured on fluorescent beads (20), albeit at the cost of acquisition time and reduced contrast due to fluorescence generated by the side lobes. In different work, axial resolution has also been improved about fourfold by acquiring two complementary orthogonal views of the sample using two alternating LSs, followed by computationally fusing image information with a deconvolution incorporating both views (23). LS approaches have also helped suppress out-of-focus background for single-molecule imaging in biological situations (e.g., in ref. 24), including at superresolution (25–27).Slight axial resolution improvement beyond the diffraction barrier has been demonstrated by overlapping a Gaussian excitation LS with a STED LS featuring a zero-intensity plane (28). Due to scattering and possibly additional aberrations caused by the wavelength difference between excitation and STED light, the maximal achievable resolution in biological specimens was severely limited. This was the case even in fixed samples. A successful application of LS-STED to living cells or organisms has not been reported. The relatively high average STED laser power required for high resolution gains calls for developing a coordinate-targeted superresolution LS approach with low-power operation, meaning a concept that does not solely rely on changing the way the light is directed to––or collected from––the sample, but a concept that harnesses an “on–off” transition for improved feature separation.     

Label-free imaging of Schwann cell myelination by third harmonic generation microscopy

Understanding the dynamic axon–glial cell interaction underlying myelination is hampered by the lack of suitable imaging techniques. Here we demonstrate third harmonic generation microscopy (THGM) for label-free imaging of myelinating Schwann cells in live culture and ex vivo and in vivo tissue. A 3D structure was acquired for a variety of compact and noncompact myelin domains, including juxtaparanodes, Schmidt–Lanterman incisures, and Cajal bands. Other subcellular features of Schwann cells that escape traditional optical microscopies were also visualized. We tested THGM for morphometry of compact myelin. Unlike current methods based on electron microscopy, g-ratio could be determined along an extended length of myelinated fiber in the physiological condition. The precision of THGM-based g-ratio estimation was corroborated in mouse models of hypomyelination. Finally, we demonstrated the feasibility of THGM to monitor morphological changes of myelin during postnatal development and degeneration. The outstanding capabilities of THGM may be useful for elucidation of the mechanism of myelin formation and pathogenesis.Myelin is a multiple-layered membrane sheath surrounding the axon. In myelinated nerves, the conduction of action potentials is much faster and the speed depends strongly on the structure of myelin. The structural integrity must be therefore tightly regulated for proper conduction of neuronal impulses, but the underlying axon–glial cell interaction is not well understood. Since the days of Ramón y Cajal, light microscopy has been widely used to visualize myelin morphology (1). A variety of fluorescent probes specifically binding to myelin components have allowed studies of the interaction between molecules (2–4). However, most such labeling methods are not suitable for unraveling in vivo dynamics of myelination because cell membranes are compromised during staining (especially immunohistochemistry) and/or the procedures are prohibitively time-consuming and invasive. It is thus of great interest to develop label-free methods. Recently, spectral confocal reflectance microscopy has been demonstrated for high-resolution in vivo imaging of myelin (5). There are also techniques of nonlinear optical microscopy, which are generally known to be more advantageous for imaging deep live tissue. Coherence anti-Stokes Raman scattering (CARS) microscopy, which requires two synchronized short pulse lasers for excitation, has been used for imaging in vivo myelin and detecting pathology (6, 7). Third harmonic generation (THG) microscopy is based on another nonlinear optical process of light emission, yielding distinguishable images from CARS. Though it has been demonstrated for imaging the white matter in the brain (8), so far few studies have applied THG microscopy (THGM) for elucidating the mechanism of myelin formation. Moreover, the omission of the peripheral nervous systems (PNS) in the previous studies is not trivial considering the significant departure from the CNS in terms of the myelin-forming glia and molecular subdomains: Schwann cells wrap individual internodes in the PNS, and oligodendrocytes form multiple myelin sheaths in the CNS. The basal lamina and nodal microvilli are unique to Schwann cells, and Schmidt–Lanterman incisures are more pronounced in the PNS (9). Here we demonstrate, to our knowledge for the first time, the utility of THGM as a method for morphological analysis of myelinating Schwann cells in live culture and ex vivo and in vivo tissue.     

Submillisecond second harmonic holographic imaging of biological specimens in three dimensions

Optical microscopy has played a critical role for discovery in biomedical sciences since Hooke’s introduction of the compound microscope. Recent years have witnessed explosive growth in optical microscopy tools and techniques. Information in microscopy is garnered through contrast mechanisms, usually absorption, scattering, or phase shifts introduced by spatial structure in the sample. The emergence of nonlinear optical contrast mechanisms reveals new information from biological specimens. However, the intensity dependence of nonlinear interactions leads to weak signals, preventing the observation of high-speed dynamics in the 3D context of biological samples. Here, we show that for second harmonic generation imaging, we can increase the 3D volume imaging speed from sub-Hertz speeds to rates in excess of 1,500 volumes imaged per second. This transformational capability is possible by exploiting coherent scattering of second harmonic light from an entire specimen volume, enabling new observational capabilities in biological systems.The need for high-speed 3D imaging is driven by open questions regarding the dynamics of biological organisms. Proper functioning of multicellular organisms relies critically on 4D spatiotemporal organization, requiring precise timing and coordination from distinct spatial regions. In the brain, neurons communicate and process information electrically in the form of millisecond-duration action potential (AP) spikes that propagate between regions of highly connected neural circuits. These circuits process information and produce memory and motor commands (1). Although spatial connections and morphology can be observed with conventional imaging methods, there is a dearth of information regarding how these neural connections behave dynamically, which is essential for developing an understanding of the functional behavior of neural circuitry.Neural dynamics are often studied with electrophysiology measurements, which attain submillisecond temporal resolution yet are limited in their spatial resolution, even for multielectrode arrays (2) that admit recording from many sites. A critical need exists for imaging techniques that can map the neural connections and interactions at a spatial resolution sufficient to resolve individual neural cells yet observe a 3D network of potentially hundreds of neurons at a time scale sufficient to resolve APs. Optical microscopy currently achieves part of this imaging requirement with subcellular spatial resolution in three dimensions, both with linear confocal and nonlinear two-photon laser-scanning imaging modalities (3). Advances in high-speed laser-scanning microscopy (LSM) have improved the temporal resolution of 3D imaging, but millisecond time scale volumetric imaging remains challenging (SI Methods, section 2).Current high spatial resolution images of AP behavior use optical reporters of neural activity that lead to changes in fluorescence or second harmonic generation (SHG). Common calcium (Ca²⁺) indicator probes are constrained by diffusion, saturation effects, and fluorescent decay of several hundred milliseconds that can obscure rapid membrane potential firing rates (4–6). Additionally, Ca²⁺ indicators lack the ability to capture subthreshold events, resulting in a scarcity of information on factors that drive a cell to threshold (7). SHG probes that directly report on the membrane potential alleviate many of these technical challenges (8, 9). Rise times and decay of SHG signal can be nearly instantaneous (4), whereas fluorescent signals decay over hundreds of nanoseconds. Compared with fluorescence, SHG probes are background-free because signals only originate from noncentrosymmetrical molecules arranged in an ordered fashion when inserted into the membrane. SHG also provides label-free contrast for structural tissues (10), and high-speed 3D imaging can be vital to answering questions about other biological systems, such as the morphological dynamics of developing hearts (SI Methods, section 1). SHG contrast mechanisms present advantages for high-speed biological imaging, yet the update rate of SHG imaging has lacked sufficient speed to take advantage of these capabilities.Formation of a high-quality 4D SHG image of a biological specimen through nonlinear optical contrast is challenging due to the low rates of signal generation from the nonlinear optical interaction (10). Conventional 3D SHG imaging uses LSM with tightly focused laser pulses to collect enough photons from each image volume element. The rate of 3D SHG LSM image formation is limited to sub-Hertz update rates because each voxel is serially acquired (SI Methods, section 2).In this article, we exploit the coherence of SHG scattering combined with interferometric off-axis holography (11), which allows the capture of an entire 3D volume in a 2D image format (12–14), to increase the 4D SHG imaging speed dramatically. We show high-quality, single-acquisition images with subcellular resolution captured in as little as 20 μs, which is 500-fold faster than our previous work (13). Continuously updated images of 3D volumes on the order of 30 μm³ are captured at update rates exceeding 1,500 volumes per second—a more than 190-fold increase over previous 3D imaging results (14) and more than 8,000-fold faster than conventional LSM SHG microscopy (SI Methods, section 8). We derive and experimentally validate the conditions for optimizing coherent image formation for high update rate 4D imaging. Particle tracking with accurate position and velocity recovery is demonstrated. In continuous imaging mode, we are able to follow a 3D volume with a spatial resolution of ∼0.85 μm at more than 1,500 3D volume images per second. We validate the accuracy of SHG holography by comparing standard 3D LSM and holographic reconstructions of tissue samples. Additionally, we demonstrate imaging through thick-scattering tissue slices, showing the potential to image high-speed dynamics from SHG signals generated by in vitro slice animal models. The unparalleled speed of SHG holography, coupled with its ability to image weak object fields with high resolution over a broad field of view, represents a significant step in filling the high-speed 4D imaging gap that currently exists for nonlinear optical modalities.     

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells.Biological processes are inherently driven by the molecular-scale organization of biomolecular assemblies, which arrange in precise structures that are essential for biological functions in cells and tissues. The extent to which the biological function depends on the underlying molecular-scale organization is particularly evident in filamentous assemblies, such as DNA filaments and cytoskeletal protein filaments. Changes in the local higher-order organization of DNA filaments is tightly linked to essential DNA-mediated processes including control of gene expression, DNA replication, and DNA repair. However, how specific DNA-binding proteins affect DNA filament architecture and thus DNA-mediated functions is poorly understood (1). Similarly, the spatial organization of cytoskeletal filaments in cells and tissues is also weakly explored, despite their central role in generating forces and driving cell motility, cell division, and tissue morphogenesis (2). Electron microscopy has been widely used to provide molecular-scale images of the structure of such filament assemblies; however, it typically involves several daylong sample preparation and ultrathin sectioning of the biological material, thus limiting investigations in whole cells and tissues.Polarized fluorescence imaging is a powerful approach for elucidating the structural organization of filament assemblies because it is compatible with a wide variety of microscopy techniques, thus enabling studies across multiple spatial and temporal scales. Polarized fluorescence imaging allows not only retrieval of structural information from molecular orientation measurements (3, 4) but also quantification of depolarization-induced processes such as in homo-fluorescence resonant energy transfer, which serve as indicators for biomolecular clustering or polymerization (5, 6). Molecules act as oriented absorption/emission dipoles, whose pointing direction can be monitored, exploiting the polarized nature of light. This property has been used to determine the organization of molecular assemblies whose orientation is constrained, in particular in the cell membrane (7–11). Recent works using polarized microscopy have also evidenced microscopic-scale organization of septin filaments in budding yeast (12, 13) and actin filaments in Drosophila tissues (14), opening a path for in vivo structural imaging. Mapping the local organization of complex filament assemblies using polarized fluorescence imaging thus appears as a key approach to understand fundamental biological functions as diverse as DNA-mediated processes and animal cell mechanics. Although significant advances have been made toward orientational order imaging using polarized fluorescence, investigations are still limited to the diffraction-scale resolution, which hampers single filament-scale observation that is required for a quantitative structural analysis of local disorder. Moreover, the retrieved information does not directly report the orientational organization of protein filaments, but rather the mixture between filament orientations and the intrinsic rotational mobility of the attached fluorescent probes (wobbling), which depends on the rigidity of their linker (10, 15).It is possible to extract information on orientational mobility using single-molecule detection, which can reveal processes that are often missed in ensemble averaging. Seminal single-molecule studies have used light polarization to measure single-molecule orientations, using excitation polarization modulation (16), analyzed direction (17), or more refined schemes to access out-of-plane tilt information (for a review, see ref. 18). Another great advantage of single-molecule imaging is the possibility to achieve superresolution imaging, which relies on single-molecule localization to reconstruct images at nanometer-scale precision, providing that emitters emit temporally independently (19–21). Combining superresolution imaging with single-molecule orientation measurements would provide an ultimate way to image the structural organization of filamentous assemblies at high spatial resolution in vivo. Although a similar combination has been applied to probe the rotational mobility of single molecules in isotropic environments (22), its use to quantify orientations in ordered systems (e.g., filamentous structures) presents several challenges. First, molecular orientation can itself affect localization properties (23–25), and therefore the quality of image reconstruction. This effect has been shown, however, to be less dramatic when rotational mobility occurs in 2D in the sample plane, or in a large angular range (26). Second, the measurement of molecular orientation involves splitting the signal into polarized detection/excitation channels, which can decrease localization precision if not properly processed. At last, although in-plane components of single-molecule orientations are relatively simple to extract, the measurement of out-of-plane orientations of molecules requires more sophisticated experimental schemes and detection algorithms (18, 27). Several signal analysis solutions have been proposed for both orientation and localization monitoring (25, 28–30); however, there is no report yet on quantitative superresolution imaging of molecular orientations in ordered systems.In this work, we present a quantitative method combining steady-state in-plane single-molecule orientation measurements and superresolution imaging, using polarization-resolved direct stochastic optical reconstruction microscopy (polar-dSTORM). We propose a simple experimental scheme, which is compatible with high signal-to-noise ratio conditions and which provides structural information in filamentous assemblies in 2D, which is sufficient for order interpretation. We present a dedicated algorithm for polarized single-molecule localization and quantification, and show that the imaging of orientational behaviors can be achieved with high accuracy, providing that stringent signal analysis is performed. We further show how to exploit the measured polarized signals to retrieve information on both the fluorescence label wobbling and the local disorder of biological filaments, two parameters that are not discernible in diffraction-limited ensemble methods. We illustrate the use of polar-dSTORM in microtubule networks in fixed cells and double-stranded DNA (dsDNA) in vitro, and evidence the effect of the fluorophore structure on its angular wobbling when linked to actin stress fibers in fixed cells via phalloidin conjugates. These results reveal that ensemble polarization-resolved methods generally overestimate molecular order, illustrating that single-molecule approaches are needed for determining the structural organization of biomolecular assemblies in an unbiased manner.     

X-ray linear dichroic ptychography

Biominerals such as seashells, coral skeletons, bone, and tooth enamel are optically anisotropic crystalline materials with unique nanoscale and microscale organization that translates into exceptional macroscopic mechanical properties, providing inspiration for engineering new and superior biomimetic structures. Using Seriatopora aculeata coral skeleton as a model, here, we experimentally demonstrate X-ray linear dichroic ptychography and map the c-axis orientations of the aragonite (CaCO₃) crystals. Linear dichroic phase imaging at the oxygen K-edge energy shows strong polarization-dependent contrast and reveals the presence of both narrow (<35°) and wide (>35°) c-axis angular spread in the coral samples. These X-ray ptychography results are corroborated by four-dimensional (4D) scanning transmission electron microscopy (STEM) on the same samples. Evidence of co-oriented, but disconnected, corallite subdomains indicates jagged crystal boundaries consistent with formation by amorphous nanoparticle attachment. We expect that the combination of X-ray linear dichroic ptychography and 4D STEM could be an important multimodal tool to study nano-crystallites, interfaces, nucleation, and mineral growth of optically anisotropic materials at multiple length scales.

Humans have been using biogenic materials as tools since the dawn of humanity. Biominerals such as bone, teeth, seashells, and coral skeletons exhibit remarkable mechanical properties and complex hierarchical organization (1). Due to these unique characteristics, biominerals often outperform their geologic or synthetic inorganic counterparts, thus attracting significant interest in understanding the mechanisms of the biologically controlled mineralization processes for modern nanotechnology (2). Careful understanding of the three-dimensional (3D) arrangement of biominerals has important engineering implications and has led to bioinspired materials that outperform nonbiomimetic, inorganic synthetic analogs (3).One of the most common natural biominerals is calcium carbonate (CaCO₃), which occurs in bacteria, algae, marine organisms, and humans (4). CaCO₃ absorbs light anisotropically, such that the π-bonded p orbitals of O and C atoms parallel to the crystal c axis exhibit maximum absorption when aligned parallel to linearly polarized light. The absorption intensity changes with a cos² law with respect to the azimuthal orientation of the carbonate groups in the crystal. This information can reveal structural and mechanical properties in CaCO₃ biominerals (5). Coral biomineralization is a subject of intense studies, and the mechanisms of crystal nucleation and growth in coral skeletons are only beginning to be revealed (6–9).The optical anisotropy in CaCO₃ has been leveraged in polarized visible light microscopy to study macroscopic biomineral structure and formation mechanisms (10, 11) and with imaging polarimetry to study crystal orientation uniformity (12, 13). In the shorter-wavelength regime, X-ray absorption near-edge structure spectroscopy has been used to study the orientations of various polymorphs of CaCO₃ (14, 15), and polarization-dependent imaging contrast (PIC) mapping using X-ray photoemission electron microscopy (X-PEEM) has been demonstrated to quantitatively map crystal orientations in CaCO₃ (15–17,). Currently, PIC mapping mostly uses X-PEEM in reflection geometry to achieve tens-of-nanometer resolution. However, PEEM’s limited achievable spatial resolution (∼20 nm) and the confinement to polished two-dimensional surfaces are insurmountable limits. Scanning transmission X-ray microscopy (STXM) has taken advantage of dichroic contrast to study polymer fibers (18) to resolve 30-nm features, but it is limited in achievable spatial resolution by the focusing optics, which also has a low efficiency and a short working-distance constraint.Although macroscopic morphologies in biominerals have been studied extensively, their nanoscopic structures are still not studied routinely in a quantitative fashion, mostly due to the lack of a proper transmission microscope that offers bulk-sensitive information with spatial resolution down to the nanometer scale. With the development of high-brilliance synchrotron radiation facilities worldwide, advancements in high-resolution imaging techniques, and the increasing availability of insertion-device X-ray sources providing polarization control, such as elliptically polarizing undulators (EPUs), new synchrotron-based tools are now becoming available for probing nanoscale crystal orientation in CaCO₃ minerals and biominerals. By taking advantage of brilliant X-ray sources, coherent diffractive imaging (CDI) can directly achieve high-resolution structural information of noncrystalline samples and nanocrystals from their diffraction patterns (19–28). In particular, ptychography, a scanning CDI technique (28), has attracted considerable attention for its general applicability (29–32). Ptychography measures a series of diffraction patterns from spatially overlapping illumination probes on a sample, where phase-retrieval algorithms are used to iteratively recover the incident wave and complex exit wave of the sample. This versatile diffractive imaging technique has been applied to study various biological materials in two and three dimensions with high resolution (33–40).In this work, we present X-ray linear dichroic ptychography of biominerals using the aragonite (CaCO₃) coral skeleton of Seriatopora aculeata as a model. Aragonite is an orthorhombic CaCO₃ polymorph, with all three crystal axes being unequal in length and perpendicular to one another (1). Carbonate crystals grow acicularly with a needle-like habit and with 10 times greater growth rate along the c axis than along the a axis (41), resulting in densely packed bundles of thin crystals in coral skeletons. It has been hypothesized that this elongated growth pattern with crystals growing mostly along the fast c axis but in all directions is the most efficient way for aragonite fill 3D space (6). Consequently, this space-filling strategy may endow a unique evolutionary advantage to host organisms that adhere to the pattern by providing greater resilience to environmental stresses such as ocean acidification (42). Therefore, the exact nanoscopic mechanisms of biomineral growth along various crystal axes are of significant scientific interest in understanding the macroscopic structural changes in coral species around the world.We imaged several coral-skeleton samples on and off the O K-edge π* peak and observed significant contrast differences between absorption and phase images. Using three linear dichroic ptychography images, we performed PIC mapping to quantitatively determine crystal c-axis orientations in the coral with 35-nm spatial resolution. Our dichroic ptychography results were qualitatively validated by correlating the ptychography PIC maps with four-dimensional (4D) scanning transmission electron microscopy (STEM), a scanning nano-electron diffraction technique for probing crystal orientations in crystalline materials (43). We observed that, at the nanoscale, crystallite orientations can be narrowly distributed, as is characteristic of spherulitic crystals, but also randomly distributed in submicrometer particles. Moreover, we verified linear dichroic phase contrast at a pre-edge energy below the absorption resonance. The use of such phase contrast may lead to new dose-efficient dichroic imaging techniques for studying anisotropic biominerals and has important implications for understanding the nanoscale organization of crystallites in biominerals.     

Whole-cell,multicolor superresolution imaging using volumetric multifocus microscopy

Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 µm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼4-µm-deep volume, with lateral and axial localization precisions of ∼20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.Because of its specificity and ability to image live samples, fluorescence microscopy is the most widely used imaging tool for biological studies. In recent years, several methods have been introduced to increase the resolution of fluorescence microscopy beyond the diffraction limit (1, 2). These methods include stimulated emission depletion (3), structured illumination (4), and single-molecule localization microscopy (LM) (5, 6). In the latter approach, precise control over illumination conditions enables sparse activation of individual fluorescent molecules, permitting determination of their positions with an accuracy of a few tens of nanometers. The sequential photoactivation, imaging, and bleaching (or photoswitching) of large numbers of fluorophores then allow the reconstruction of the investigated structure, embodying the principles of photo-activated localization microscopy (PALM) (5), fluorescence photo-activation localization microscopy (FPALM) (7), and stochastic optical reconstruction microscopy (STORM) (6, 8).LM techniques have garnered significant interest in biological studies (9, 10) but are still predominantly implemented using evanescent-wave (or total internal reflection fluorescence) microscopy (11), which limits its application to 2D or thin structures close to the cell membrane. However, in many biological contexts, it is highly desirable to access the 3D intracellular organization of the cell with subdiffraction resolution.For efficient 3D LM in cultured cells, two issues need to be resolved. First, single molecules must be localized with subdiffraction accuracy both laterally and axially. Second, the axial depth over which localizations are made should be comparable with the thickness of the whole cell. Approaches developed to address the former issue usually rely on encrypting axial information into 2D images by engineering the point-spread function (PSF) of the microscope (8, 12–15), single-photon interferometry (16), or biplane imaging (17–19). However, the typical imaging depth of most of these methods does not exceed 1.2 μm and hence, is insufficient for whole-cell imaging. Furthermore, because of the wide-field excitation configuration predominantly used for imaging in 3D LM, the activation and emission of out-of-focus molecules lead to increased background in the fluorescence image and their unnecessary bleaching (Fig. 1 A and B). In other words, information outside the imaging plane is lost, whereas the signal-to-noise ratio in the image is reduced. Selective plane excitation (20) or activation (21, 22) can circumvent this problem but requires sequential scanning to image entire cell volume.Open in a separate windowFig. 1.Comparison between conventional wide-field detection and multifocus detection. (A and B) The whole volume is excited in the wide-field configuration, and fluorescence from out-of-focus molecules constitutes the background of the recorded in-focus signal. (C and D) In the multifocus configuration, the 3D extent of the PSF is used to localize molecules within the volume as information is obtained from multiple focal planes.We report the successful implementation of a volumetric PALM/STORM superresolution method that avoids the aforementioned problems. Our method relies on the recently developed multifocus microscope (MFM) (23), which achieves simultaneous acquisition of nine equally spaced focal planes (Fig. 1 C and D) on a single camera through the combination of a specialized diffractive grating and chromatic correction elements placed in the microscope emission path (Fig. 2). The spacing between consecutive focal planes is ∼440 nm, allowing accurate 3D localization of single fluorescent molecules with 3D Gaussian fitting of resulting PSFs. We show that such volumetric acquisition is compatible with two-color superresolution PALM/STORM imaging of mammalian and yeast cells, with lateral and axial localization precisions of ∼20 and 50 nm, respectively. The imaging depth is ∼4 µm, well beyond the capabilities of other 3D superresolution techniques (review in ref. 24), and notably, it permits complete 3D imaging of many cellular organelles or whole cells (Movies S1 and S2).Open in a separate windowFig. 2.MFM setup. The excitation lasers are combined in a fiber through an acousto-optic tunable filter, collimated, reflected on a dichroic mirror (DM), and focused at the back aperture of a high-N.A. objective to achieve wide-field excitation. The collected emission is transmitted through the DMs and passes through the MFG placed in a plane conjugated to the back pupil plane of the objective. The diffraction orders pass through the chromatic correction module before being separately focused on the detector. To record sample and stage drift, polystyrene beads (4-μm diameter) were immobilized on the cover glass, illuminated by an infrared light-emitting diode (IR LED), and their diffraction pattern was recorded with an IR camera (IR CAM) through an additional beamsplitter inserted into the emission path. The graphs are the recorded positions of the bead along the x and z axes. (Lower Inset) Example of emitting molecules recorded at different z planes corresponding to labeled nucleopores (the raw data are in Movie S1, and a reconstructed image is in Movie S2). (Scale bar: 5 μm.)     

Multilayer three-dimensional super resolution imaging of thick biological samples

Recent advances in optical microscopy have enabled biological imaging beyond the diffraction limit at nanometer resolution. A general feature of most of the techniques based on photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM) has been the use of thin biological samples in combination with total internal reflection, thus limiting the imaging depth to a fraction of an optical wavelength. However, to study whole cells or organelles that are typically up to 15 μm deep into the cell, the extension of these methods to a three-dimensional (3D) super resolution technique is required. Here, we report an advance in optical microscopy that enables imaging of protein distributions in cells with a lateral localization precision better than 50 nm at multiple imaging planes deep in biological samples. The approach is based on combining the lateral super resolution provided by PALM with two-photon temporal focusing that provides optical sectioning. We have generated super-resolution images over an axial range of ≈10 μm in both mitochondrially labeled fixed cells, and in the membranes of living S2 Drosophila cells.     

Quantitative single-molecule imaging by confocal laser scanning microscopy

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.     

Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins

Single-molecule (SM) microscopy allows outstanding insight into biomolecular mechanisms in cells. However, selective detection of single biomolecules in their native environment remains particularly challenging. Here, we introduce an easy methodology that combines specific targeting and nanometer accuracy imaging of individual biomolecules in living cells. In this method, named complementation-activated light microscopy (CALM), proteins are fused to dark split-fluorescent proteins (split-FPs), which are activated into bright FPs by complementation with synthetic peptides. Using CALM, the diffusion dynamics of a controlled subset of extracellular and intracellular proteins are imaged with nanometer precision, and SM tracking can additionally be performed with fluorophores and quantum dots. In cells, site-specific labeling of these probes is verified by coincidence SM detection with the complemented split-FP fusion proteins or intramolecular single-pair Förster resonance energy transfer. CALM is simple and combines advantages from genetically encoded and synthetic fluorescent probes to allow high-accuracy imaging of single biomolecules in living cells, independently of their expression level and at very high probe concentrations.     

Quantitative live-cell imaging of human immunodeficiency virus (HIV-1) assembly

Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.     

Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase

We present results of combined studies of the enzyme human aldose reductase (h-AR, 36 kDa) using single-crystal x-ray data (0.66 A, 100K; 0.80 A, 15K; 1.75 A, 293K), neutron Laue data (2.2 A, 293K), and quantum mechanical modeling. These complementary techniques unveil the internal organization and mobility of the hydrogen bond network that defines the properties of the catalytic engine, explaining how this promiscuous enzyme overcomes the simultaneous requirements of efficiency and promiscuity offering a general mechanistic view for this class of enzymes.     

Improving the applicability of myocardial densitometry and parametric imaging by extended automated densogram analysis

In clinical applications the analysis of X-ray contrast densograms acquired in regions of interest (ROI's) over the myocardium is disturbed by many complex factors. For this reason we acquire redundant densogram information for quality control before extracting densitometric parameters. In our approach, initially some stable measures of quality for densograms are used to lower the influence of poor quality densograms by a quality weighted averaging. For example a shape quality measure, Q₁, is calculated using regions of optimal and minimal acceptable quality defined with respect to a prototype densogram. Not a few myocardial ROI's yield densograms that differ from single-source densograms (SSD's) due to e.g. superposition of different perfusion beds or the position of the ROI relative to the coronary sinus or stenoses. This might result in a densogram shape with oscillating or plateau behavior. For densograms of a such general shape many parameters defined in the usual way do not depend smoothly on the densogram values. The conventional definitions of some parameters (appearance time, rise time) are therefore extended for application to multi-maxima densograms as well as to SSD's. These new methods are evaluated using digitized clinical angiocardiograms and are applied to parametric imaging (pixeldensograms) in a slightly modified way. Taking into account the densogram quality, its shape and its origin results in a considerable improvement both for densitometry and parametric imaging of myocardial perfusion.Abbreviations AP appearance time - Dmax maximum density - LAO 60° left anterior oblique 60° projection - LCA left coronary artery - MIRT mean integrated rise time - MTT mean transit time - RAO 30° right anterior oblique 60° projection - RCA right coronary artery - ROI region of interest - RT rise time - SSD single-source densogram - THM time of half-maximum, tmax - time of maximum     

4D visualization of embryonic, structural crystallization by single-pulse microscopy

In many physical and biological systems the transition from an amorphous to ordered native structure involves complex energy landscapes, and understanding such transformations requires not only their thermodynamics but also the structural dynamics during the process. Here, we extend our 4D visualization method with electron imaging to include the study of irreversible processes with a single pulse in the same ultrafast electron microscope (UEM) as used before in the single-electron mode for the study of reversible processes. With this augmentation, we report on the transformation of amorphous to crystalline structure with silicon as an example. A single heating pulse was used to initiate crystallization from the amorphous phase while a single packet of electrons imaged selectively in space the transformation as the structure continuously changes with time. From the evolution of crystallinity in real time and the changes in morphology, for nanosecond and femtosecond pulse heating, we describe two types of processes, one that occurs at early time and involves a nondiffusive motion and another that takes place on a longer time scale. Similar mechanisms of two distinct time scales may perhaps be important in biomolecular folding.     

Quantitative assessment of aortic regurgitation by magnetic resonance imaging.

Thirty patients with aortic regurgitation and 10 controls were examined using an 0.5 T superconducting magnet with ECG gating. In each case a multislice-multiphase spinecho study in sagittal-coronal double angulated projection (four-chamber equivalent) was performed to assess left and right ventricular volumes, ejection fraction and regurgitation fraction. Additionally, a blood-flow sensitive cine-study (gradient echo, FAME) was performed to visualize direction and area of regurgitant jet. Magnetic resonance imaging (MRI) data were compared with quantitative and qualitative assessment of aortic regurgitation by angiography, Doppler and colour flow mapping. Using the FAME mode MRI, we were able to detect the regurgitant jet as an area of signal loss within the left ventricle in all patients; moderate correlation to jet area was determined by colour flow mapping (R = 0.60, P less than 0.001). Determination of left and right ventricular end-diastolic, end-systolic and stroke volumes by MRI revealed excellent correlation with invasive data (R = 0.94, P = 0.0001). With MRI regurgitant fraction (RF) could be calculated from the difference between right and left ventricular stroke volumes, which showed good correlation with invasively determined RF (R = 0.91, P = 0.001) and with qualitative Sellers' scoring (R = 0.70, P less than 0.001), respectively. Thus MRI provides the basis for noninvasive detection and quantification of aortic regurgitation.