In vivo NIR-II structured-illumination light-sheet microscopy

Noninvasive optical imaging with deep tissue penetration depth and high spatiotemporal resolution is important to longitudinally studying the biology at the single-cell level in live mammals, but has been challenging due to light scattering. Here, we developed near-infrared II (NIR-II) (1,000 to 1,700 nm) structured-illumination light-sheet microscopy (NIR-II SIM) with ultralong excitation and emission wavelengths up to ∼1,540 and ∼1,700 nm, respectively, suppressing light scattering to afford large volumetric three-dimensional (3D) imaging of tissues with deep-axial penetration depths. Integrating structured illumination into NIR-II light-sheet microscopy further diminished background and improved spatial resolution by approximately twofold. In vivo oblique NIR-II SIM was performed noninvasively for 3D volumetric multiplexed molecular imaging of the CT26 tumor microenvironment in mice, longitudinally mapping out CD4, CD8, and OX40 at the single-cell level in response to immunotherapy by cytosine-phosphate-guanine (CpG), a Toll-like receptor 9 (TLR-9) agonist combined with OX40 antibody treatment. NIR-II SIM affords an additional tool for noninvasive volumetric molecular imaging of immune cells in live mammals.

In vivo imaging and monitoring of cells through large volumes of tissues by high-resolution optical microscopy can afford details of complex biological structures and processes in live mammals, facilitating understanding of disease onset, progression, and response to therapy at the single-cell level (1, 2). Using planar illumination and orthogonal wide-field detection, light-sheet microscopy (LSM) is a powerful tool capable of fast three-dimensional (3D) imaging with low phototoxicity (3). Tremendous progress in several generations of LSM in the visible-light region has led to wide applications of fluorescence-based 3D biological imaging (2–15), for chemically cleared mammalian tissues with impressively large field of view (FOV) (10) and superresolution (11). High spatiotemporal 3D longitudinal imaging and tracking of small living organisms have also been realized (2, 16). Nevertheless, the penetration depth and resolution of visible-light LSM for imaging mammal tissues are limited by light scattering that causes light-sheet thickening/spreading as it propagates deeper into tissue, leading to reduction in axial resolution and FOV. Scattering of emitted fluorescence light depletes ballistic photons from deeper imaging planes, which also deteriorates signal-to-background ratio (SBR) and lateral resolution (17). Additionally, indigenous tissue autofluorescence in the visible region could further worsen the image contrast.For mammalian imaging in vivo, currently visible-light LSM of mouse brain using one-photon excitation is limited to ∼200 μm depth after craniotomy to remove skull and scalp skin (10, 15). Two-photon (1,040 nm) light-sheet microscopy utilizing nonlinear excitation achieved deeper penetration up to ∼300 μm (for in vivo mouse brain imaging postcraniotomy) with high resolution owing to reduced scattering of the excitation light (6, 18). The penetration depth can be further increased using Bessel (19, 20) or Airy beams (21). Structured illumination has been used to modulate the excitation of Gaussian beam (22), Bessel beam (23–25), or lattice pattern (2, 14), providing a different approach to remove scattered fluorescence light, reject out-of-focus background, and enable thinner optical sectioning. Superresolution can be realized using structured illumination microscopy (SIM) (2, 12–14) by extracting high-frequency details embedded in low-resolution moiré fringes imaged under an illumination pattern at several shifted phases (26).Recently we developed near-infrared II (NIR-II) LSM employing one-photon excitation and fluorescence emission detection in the near-infrared II window (1,000 to 1,700 nm) using fluorescent or luminescent organic dyes, quantum dots, and rare-earth down-conversion nanoparticles (17, 27–42). The suppressed light scattering and reduced tissue autofluorescence allowed noninvasive in vivo NIR-II LSM imaging through the scalp and skull of intact mouse head without craniotomy and through skin imaging of tumors without installing invasive optical windows, with a penetration depth up to ∼750 μm (17). Nevertheless, since the wavelength in the NIR-II window was approximately two to three times longer than visible light, the spatial resolution of NIR-II LSM was lower than that of visible LSM. Also, NIR-II imaging at deep tissues still experienced light scattering, causing background increase/decrease in the SBR and reduced spatial resolution.Here, we developed oblique mode NIR-II SIM by integrating structured illumination into NIR-II LSM using the approach of digitally scanned laser beams (12, 13). We also employed PbS/CdS core/shell quantum dot (CSQD) probes (28) to extend excitation and emission up to 1,540 nm and 1,600 to 1,700 nm, respectively. The benefits of suppressed scattering at longer wavelength, diminished out-of-focus background, and improved lateral resolution by structured illumination significantly enhanced the imaging capability of NIR-II SIM for both ex vivo and in vivo volumetric imaging.     

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.     

From the Cover: Molecular diffusion in the human nail measured by stimulated Raman scattering microscopy

The effective treatment of diseases of the nail remains an important unmet medical need, primarily because of poor drug delivery. To address this challenge, the diffusion, in real time, of topically applied chemicals into the human nail has been visualized and characterized using stimulated Raman scattering (SRS) microscopy. Deuterated water (D₂O), propylene glycol (PG-d₈), and dimethyl sulphoxide (DMSO-d₆) were separately applied to the dorsal surface of human nail samples. SRS microscopy was used to image D₂O, PG-d₈/DMSO-d₆, and the nail through the O-D, -CD₂, and -CH₂ bond stretching Raman signals, respectively. Signal intensities obtained were measured as functions of time and of depth into the nail. It was observed that the diffusion of D₂O was more than an order of magnitude faster than that of PG-d₈ and DMSO-d₆. Normalization of the Raman signals, to correct in part for scattering and absorption, permitted semiquantitative analysis of the permeation profiles and strongly suggested that solvent diffusion diverged from classical behavior and that derived diffusivities may be concentration dependent. It appeared that the uptake of solvent progressively undermined the integrity of the nail. This previously unreported application of SRS has permitted, therefore, direct visualization and semiquantitation of solvent penetration into the human nail. The kinetics of uptake of the three chemicals studied demonstrated that each altered its own diffusion in the nail in an apparently concentration-dependent fashion. The scale of the unexpected behavior observed may prove beneficial in the design and optimization of drug formulations to treat recalcitrant nail disease.The effective treatment of nail disease requires efficient drug delivery into and through the barrier. However, the tightly woven keratin network of the nail plate means that poor drug uptake following topical administration is common. Despite considerable effort to improve formulations and to enhance drug delivery to the nail, progress has been slow at best. In general, the approaches adopted have failed to elucidate the complex interplay between drug, formulation components (including solvents), and the nail. For example, although it is quite clear that drug uptake from typical “lacquer” formulations (comprising the active, a film-forming polymer, and a volatile organic solvent) is intimately linked to the disposition of the solvent and effectively stops once the solvent has gone, there has been little effort to characterize the transport of these key vehicle components into and across the nail. Only the diffusion of water has received attention, its overall time-dependent uptake having been measured by various techniques (1–3); otherwise, apart from some information on the concentration-depth profiles of water and dimethyl sulphoxide (DMSO) in the very superficial, outermost 20 μm of the nail, there are essentially no time- and position-dependent data on the movement of chemicals into the nail.Stimulated Raman scattering (SRS) microscopy is a label-free imaging technique that offers a solution to this challenge. This method has been applied in a range of biomedical and pharmaceutical studies involving, for example, visualization in living cells (4), characterization of cortical vasculature morphology (5), imaging the constituents of solid, oral dosage forms (6), and tracking the pharmacokinetics of drugs and excipients in mammalian skin (7–9). In this paper, the first application to our knowledge of SRS microscopy to trace and visualize the diffusion of three pharmaceutically relevant solvents, water, propylene glycol (PG), and DMSO, as a function of depth and in real time in human nail is presented. The use of deuterated solvents provides unique Raman-active molecular vibrations that are easily distinguished spectroscopically from those originating in the nail, resulting in excellent, and label-free, image contrast. Because of the linear relationship between the SRS signal and the concentration of the chemical, the spectroscopic signature of which is being monitored, a semiquantitative analysis of solvent diffusion across the nail is possible and offers heretofore-unknown insight into the transport process.     

From the Cover: Unraveling the structure of DNA during overstretching by using multicolor,single-molecule fluorescence imaging

Single-molecule manipulation studies have revealed that double-stranded DNA undergoes a structural transition when subjected to tension. At forces that depend on the attachment geometry of the DNA (65 pN or 110 pN), it elongates ≈1.7-fold and its elastic properties change dramatically. The nature of this overstretched DNA has been under debate. In one model, the DNA cooperatively unwinds, while base pairing remains intact. In a competing model, the hydrogen bonds between base pairs break and two single DNA strands are formed, comparable to thermal DNA melting. Here, we resolve the structural basis of DNA overstretching using a combination of fluorescence microscopy, optical tweezers, and microfluidics. In DNA molecules undergoing the transition, we visualize double- and single-stranded segments using specific fluorescent labels. Our data directly demonstrate that overstretching comprises a gradual conversion from double-stranded to single-stranded DNA, irrespective of the attachment geometry. We found that these conversions favorably initiate from nicks or free DNA ends. These discontinuities in the phosphodiester backbone serve as energetically favorable nucleation points for melting. When both DNA strands are intact and no nicks or free ends are present, the overstretching force increases from 65 to 110 pN and melting initiates throughout the molecule, comparable to thermal melting. These results provide unique insights in the thermodynamics of DNA and DNA-protein interactions.     

From the Cover: Encoded multisite two-photon microscopy

The advent of scanning two-photon microscopy (2PM) has created a fertile new avenue for noninvasive investigation of brain activity in depth. One principal weakness of this method, however, lies with the limit of scanning speed, which makes optical interrogation of action potential-like activity in a neuronal network problematic. Encoded multisite two-photon microscopy (eMS2PM), a scanless method that allows simultaneous imaging of multiple targets in depth with high temporal resolution, addresses this drawback. eMS2PM uses a liquid crystal spatial light modulator to split a high-power femto-laser beam into multiple subbeams. To distinguish them, a digital micromirror device encodes each subbeam with a specific binary amplitude modulation sequence. Fluorescence signals from all independently targeted sites are then collected simultaneously onto a single photodetector and site-specifically decoded. We demonstrate that eMS2PM can be used to image spike-like voltage transients in cultured cells and fluorescence transients (calcium signals in neurons and red blood cells in capillaries from the cortex) in depth in vivo. These results establish eMS2PM as a unique method for simultaneous acquisition of neuronal network activity.     

Quantitative biological imaging by ptychographic x-ray diffraction microscopy

Recent advances in coherent x-ray diffractive imaging have paved the way to reliable and quantitative imaging of noncompact specimens at the nanometer scale. Introduced a year ago, an advanced implementation of ptychographic coherent diffractive imaging has removed much of the previous limitations regarding sample preparation and illumination conditions. Here, we apply this recent approach toward structure determination at the nanoscale to biological microscopy. We show that the projected electron density of unstained and unsliced freeze-dried cells of the bacterium Deinococcus radiodurans can be derived from the reconstructed phase in a straightforward and reproducible way, with quantified and small errors. Thus, the approach may contribute in the future to the understanding of the highly disputed nucleoid structure of bacterial cells. In the present study, the estimated resolution for the cells was 85 nm (half-period length), whereas 50-nm resolution was demonstrated for lithographic test structures. With respect to the diameter of the pinhole used to illuminate the samples, a superresolution of about 15 was achieved for the cells and 30 for the test structures, respectively. These values should be assessed in view of the low dose applied on the order of ≃1.3·10⁵ Gy, and were shown to scale with photon fluence.     

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30–60 nm spatial resolution at temporal resolutions down to 1–2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.     

Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins

Fluorescence microscopy is indispensable in many areas of science, but until recently, diffraction has limited the resolution of its lens-based variant. The diffraction barrier has been broken by a saturated depletion of the marker's fluorescent state by stimulated emission, but this approach requires picosecond laser pulses of GW/cm2 intensity. Here, we demonstrate the surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller. The subdiffraction resolution results from reversible photoswitching of a marker protein between a fluorescence-activated and a nonactivated state, whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. After characterizing the switching kinetics of the used marker protein asFP595, we demonstrate the current capability of this RESOLFT (reversible saturable optical fluorescence transitions) type of concept to resolve 50-100 nm in the focal plane. The observed resolution is limited only by the photokinetics of the protein and the perfection of the zero. Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization.     

From the Cover: Femtosecond pump-probe microscopy generates virtual cross-sections in historic artwork

The layering structure of a painting contains a wealth of information about the artist''s choice of materials and working methods, but currently, no 3D noninvasive method exists to replace the taking of small paint samples in the study of the stratigraphy. Here, we adapt femtosecond pump-probe imaging, previously shown in tissue, to the case of the color palette in paintings, where chromophores have much greater variety. We show that combining the contrasts of multispectral and multidelay pump-probe spectroscopy permits nondestructive 3D imaging of paintings with molecular and structural contrast, even for pigments with linear absorption spectra that are broad and relatively featureless. We show virtual cross-sectioning capabilities in mockup paintings, with pigment separation and nondestructive imaging on an intact 14th century painting (The Crucifixion by Puccio Capanna). Our approach makes it possible to extract microscopic information for a broad range of applications to cultural heritage.Identifying an artist’s choice of materials (e.g., support, pigments, binders, and varnishes in a painting) and working methods can lead to greater understanding of past cultures and enhance the ability of conservators to preserve that culture. In a painting, this information is contained in its layered structure, and it is generally studied by the physical removal of a small paint sample, which can be characterized by a plethora of analytical techniques (1). The sample needs to be representative of the painting but as small as possible (typically <0.5 mm), and only local information is obtained. Nondestructive analysis by traditional macroscopic methods, such as X-radiography, near-infrared reflectance imaging, and UV-visible fluorescence photography, can provide some information about a painting’s support, compositional paint changes, underdrawings, paint and varnish applications, and restorations (1). Materials can be identified in situ on the microscopic scale using Raman (2–4) or the macroscopic scale with reflectance imaging spectroscopy (5, 6) and X-ray fluorescence intensity mapping (7). Unfortunately, none of these techniques contain quantitative depth-resolved material information. Methods that could offer 3D information are under active research, such as confocal X-ray fluorescence, absorption near-edge structure imaging (8), optical coherence tomography (9), and terahertz imaging (10), but they are not yet widely used in conservation science laboratories because of their limitations: X-ray–based techniques have absorption limited depths, whereas optical coherence tomography and terahertz imaging produce image contrast that is largely based on refractive index mismatches and therefore, only provide structural contrast.In general, conventional (linear) optical imaging into the paint layer of a painting is limited in its depth penetration by absorption and scattering from the pigment particles. In biology and biomedical applications, nonlinear imaging can provide optical sectioning in highly scattering and absorbing samples (11, 12). Traditional nonlinear imaging has found a few applications to cultural heritage; recent research includes the 3D imaging of wood and varnishes in a violin with second harmonic generation and two-photon excited fluorescence (13) and the mapping of oil and varnish interfaces with third harmonic generation (14). However, most inorganic pigments neither fluoresce nor generate appreciable harmonic light, leaving these techniques limited in their scope for cultural heritage.Near-infrared femtosecond pump-probe optical microscopy expands the range of detectable molecular signatures (15) to include signals from excited state absorption, ground state depletion, and stimulated emission (16). This microscopy technique was mainly developed for biomedical imaging and has been used to provide high-resolution images for the biological pigments hemoglobin (17, 18), eumelanin, and pheomelanin (19, 20) that are present in skin (21) or ocular cancer (22).Extension of pump-probe microscopy from biological pigments to samples of artist’s pigments has yielded promising preliminary results (23). However, achieving pump-probe contrast in fine art objects is more challenging than skin imaging, because artist colorants range from organic dyes to inorganic minerals, with colors spanning the entire visible spectrum. In contrast, the pigments in a sample of skin tissue are mainly limited to hemoglobin, eumelanin, and pheomelanin, which all provide image contrast with a single pump-probe wavelength combination (in this case, 720 and 810 nm, respectively). Here, we show that an increased spectral range of both the pump and the probe beams, from near-IR to visible, and a variable time delay of the pump-probe pulses help to address the complexity introduced by the large range of possible pigments in the paint layers and allow for in situ 3D imaging of paintings with molecular specificity. We first show virtual cross-sectioning capabilities in historically relevant mockup paintings and use specific pump-probe signatures to provide pigment separation. We then perform in situ 3D imaging on a 14th century painting (The Crucifixion by Italian artist Puccio Capanna) to highlight our ability to noninvasively image and create virtual cross-sections of complex pigment layers. Although we focus on historic paintings, our approach can be applied to a wide range of cultural heritage objects and provides information extremely relevant to current areas of interest in conservation science.     

Breaking the diffraction barrier outside of the optical near-field with bright, collimated light from nanometric apertures

The optical diffraction limit has been the dominant barrier to achieving higher optical resolution in the fields of microscopy, photolithography, and optical data storage. We present here an approach toward imaging below the diffraction barrier. Through the exposure of photosensitive films placed a finite and known distance away from nanoscale, zero-mode apertures in thin metallic films, we show convincing, physical evidence that the propagating component of light emerging from these apertures shows a very strong degree of collimation well past the maximum extent of the near-field (lambda(0)/4n-lambda(0)/2n). Up to at least 2.5 wavelengths away from the apertures, the transmitted light exhibits subdiffraction limit irradiance patterns. These unexpected results are not explained by standard diffraction theory or nanohole-based "beaming" rationalizations. This method overcomes the diffraction barrier and makes super-resolution fluorescence imaging practical.     

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.)     

From the Cover: PNAS Plus: Thermodynamic origin of surface melting on ice crystals

Since the pioneering prediction of surface melting by Michael Faraday, it has been widely accepted that thin water layers, called quasi-liquid layers (QLLs), homogeneously and completely wet ice surfaces. Contrary to this conventional wisdom, here we both theoretically and experimentally demonstrate that QLLs have more than two wetting states and that there is a first-order wetting transition between them. Furthermore, we find that QLLs are born not only under supersaturated conditions, as recently reported, but also at undersaturation, but QLLs are absent at equilibrium. This means that QLLs are a metastable transient state formed through vapor growth and sublimation of ice, casting a serious doubt on the conventional understanding presupposing the spontaneous formation of QLLs in ice–vapor equilibrium. We propose a simple but general physical model that consistently explains these aspects of surface melting and QLLs. Our model shows that a unique interfacial potential solely controls both the wetting and thermodynamic behavior of QLLs.In general, surfaces and interfaces yield unique phase transitions absent in the bulk (1–5). Surface melting (or premelting) of ice (3, 4) is one typical and classical example that has been known since the first prediction by Michael Faraday in 1842 (6). He hypothesized that thin water layers, now called quasi-liquid layers (QLLs), wet ice crystal surfaces even at a temperature below the melting point. Since then, this phenomenon has attracted considerable attention not only because of its importance in the fundamental understanding of melting (a solid-to-liquid transition) itself but also as a link to a diverse set of natural phenomena in subzero environments: making snowballs, slippage on ice surfaces, frost heave, recrystallization and coarsening of ice grains, morphological change of snow crystals, electrification of thunderclouds, and ozone-depleting reactions (3, 4, 7). Furthermore, it is now recognized that surface melting is not specific to ice but rather is universally seen in a wide range of crystalline surfaces such as metals, semiconductors, ceramics, rare gases, and organic and colloidal systems (8–12). Its underlying physics is therefore also inseparable from material science and technology.Although the origin of surface melting, including the nature of QLLs themselves, is still far from completely understood and a matter of active debate (13–18), it is at least phenomenologically believed that surface melting is driven by the reduction of the surface free energy by the presence of intervening liquid between the solid and gas phases (3, 4, 13, 19). More sophisticated approaches have also been proposed in terms of surface phase transitions (1, 3, 4, 20). In contrast to such theoretical speculations, however, the direct observation and the accurate characterization of QLLs by experiments are still highly challenging because of their thinness, assumed to be less than tens of nanometers (21). Experimental efforts in the past have often been bedeviled by large uncertainties depending on the experimental methods and researchers (see table S1 in ref. 22 for details). Even the first convincing evidence for the existence of surface melting of ice was not provided until 1987 (13, 14), more than one century after Faraday’s suggestion. Thus, the conventional theories, although rigorous themselves, have suffered from the lack of reliably experimental support.Recently, we succeeded in making in situ observations of QLLs on ice surfaces using an advanced optical microscope (laser confocal microscopy combined with differential interference contrast microscopy: LCM-DIM), whose resolution in the height direction reaches the order of an angstrom (22, 23). Surprisingly, this work revealed that, contrary to the common belief that QLLs completely and homogeneously wet ice surfaces, they are spatiotemporally heterogeneous and are absent in the equilibrium conditions (22, 24–26). Furthermore, we have observed that QLLs exhibit more than one wetting morphology: droplet type, thin-layer type, and their coexistence (sunny-side-up type) at supersaturation (22, 24–26). This finding fundamentally requires us to recast the conventional understanding based on spatiotemporally averaged equilibrium theories and experiments (e.g., scattering, spectroscopy, and ellipsometry), because of ignorance of the counterintuitive nature of QLLs.In this paper, we present a simple physical model bridging the gap between the conventional interpretation and the above aspects of surface melting based on in situ observations with our advanced optical microscopy combined with a two-beam interferometer. Here we revisit the thermodynamics of wetting (27). The general nature of surface melting suggests the relevance of the phenomenological approach. Starting from the phenomenological interfacial free energy, we robustly determine a full interfacial potential between ice and vapor in the medium of a QLL, governing both the selection and stability of the wetting states, and the thermodynamic condition for the existence of QLLs. As a theoretical consequence, we extend the concept of surface melting into nonequilibrium regimes, more specifically, supersaturation and undersaturation, which has a significant implication for exploring the possible existence of this phenomenon in a wider range of crystalline surfaces. Our model provides not only a clear-cut answer to the long-standing question of the origin of surface melting of ice but also offers a general insight into the origin of surface melting of other solid–gas interfaces.     

From the Cover: Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy

Fluorescent molecular switches have widespread potential for use as sensors, material applications in electro-optical data storages and displays, and superresolution fluorescence microscopy. We demonstrate that adjustment of fluorophore properties and environmental conditions allows the use of ordinary fluorescent dyes as efficient single-molecule switches that report sensitively on their local redox condition. Adding or removing reductant or oxidant, switches the fluorescence of oxazine dyes between stable fluorescent and nonfluorescent states. At low oxygen concentrations, the off-state that we ascribe to a radical anion is thermally stable with a lifetime in the minutes range. The molecular switches show a remarkable reliability with intriguing fatigue resistance at the single-molecule level: Depending on the switching rate, between 400 and 3,000 switching cycles are observed before irreversible photodestruction occurs. A detailed picture of the underlying photoinduced and redox reactions is elaborated. In the presence of both reductant and oxidant, continuous switching is manifested by “blinking” with independently controllable on- and off-state lifetimes in both deoxygenated and oxygenated environments. This “continuous switching mode” is advantageously used for imaging actin filament and actin filament bundles in fixed cells with subdiffraction-limited resolution.     

From the Cover: The Ndc80 kinetochore complex directly modulates microtubule dynamics

The conserved Ndc80 complex is an essential microtubule-binding component of the kinetochore. Recent findings suggest that the Ndc80 complex influences microtubule dynamics at kinetochores in vivo. However, it was unclear if the Ndc80 complex mediates these effects directly, or by affecting other factors localized at the kinetochore. Using a reconstituted system in vitro, we show that the human Ndc80 complex directly stabilizes the tips of disassembling microtubules and promotes rescue (the transition from microtubule shortening to growth). In vivo, an N-terminal domain in the Ndc80 complex is phosphorylated by the Aurora B kinase. Mutations that mimic phosphorylation of the Ndc80 complex prevent stable kinetochore-microtubule attachment, and mutations that block phosphorylation damp kinetochore oscillations. We find that the Ndc80 complex with Aurora B phosphomimetic mutations is defective at promoting microtubule rescue, even when robustly coupled to disassembling microtubule tips. This impaired ability to affect dynamics is not simply because of weakened microtubule binding, as an N-terminally truncated complex with similar binding affinity is able to promote rescue. Taken together, these results suggest that in addition to regulating attachment stability, Aurora B controls microtubule dynamics through phosphorylation of the Ndc80 complex.     

From the Cover: PNAS Plus: Quantitative optical coherence tomography angiography of vascular abnormalities in the living human eye

Retinal vascular diseases are important causes of vision loss. A detailed evaluation of the vascular abnormalities facilitates diagnosis and treatment in these diseases. Optical coherence tomography (OCT) angiography using the highly efficient split-spectrum amplitude decorrelation angiography algorithm offers an alternative to conventional dye-based retinal angiography. OCT angiography has several advantages, including 3D visualization of retinal and choroidal circulations (including the choriocapillaris) and avoidance of dye injection-related complications. Results from six illustrative cases are reported. In diabetic retinopathy, OCT angiography can detect neovascularization and quantify ischemia. In age-related macular degeneration, choroidal neovascularization can be observed without the obscuration of details caused by dye leakage in conventional angiography. Choriocapillaris dysfunction can be detected in the nonneovascular form of the disease, furthering our understanding of pathogenesis. In choroideremia, OCT''s ability to show choroidal and retinal vascular dysfunction separately may be valuable in predicting progression and assessing treatment response. OCT angiography shows promise as a noninvasive alternative to dye-based angiography for highly detailed, in vivo, 3D, quantitative evaluation of retinal vascular abnormalities.Optical coherence tomography (OCT) has become the most commonly used imaging modality in ophthalmology. It provides cross-sectional and 3D imaging of the retina and optic nerve head with micrometer-scale depth resolution. Structural OCT enhances the clinician’s ability to detect and monitor fluid exudation associated with retinal vascular diseases. Whereas anatomical alterations that impact vision are readily visible, structural OCT has a limited ability to image the retinal or choroidal vasculatures. Furthermore, it is unable to directly detect capillary dropout or pathologic new vessel growth (neovascularization) that are the major vascular changes associated with two of the leading causes of blindness, age-related macular degeneration (AMD) and diabetic retinopathy (1). To visualize these changes, traditional i.v. contrast dye-based angiography techniques are currently used.Fluorescein dye is primarily used to visualize the retinal vasculature. A separate dye, indocyanine green (ICG), is necessary to evaluate the choroidal vasculature. Both fluorescein angiography (FA) and ICG angiography require i.v. injection, which is time consuming, and which can cause nausea, vomiting, and, rarely, anaphylaxis (2). Dye leakage or staining provides information regarding vascular incompetence (e.g., from abnormal capillary growth), but it also obscures the image and blurs the boundaries of neovascularization. Additionally, conventional angiography is 2D, which makes it difficult to distinguish vascular abnormalities within different layers. Therefore, it is desirable to develop a no-injection, dye-free method for 3D visualization of ocular circulation.In recent years, several OCT angiography methods have been developed to detect changes in the OCT signal caused by flowing red blood cells in blood vessels. Initially, Doppler OCT angiography methods were investigated for the visualization and measurement of blood flow (3–8). Because Doppler OCT is only sensitive to motion parallel to the OCT probe beam, it is limited in its ability to image retinal and choroidal circulations, which are predominantly perpendicular to the OCT beam. More recent approaches, based on detecting variation in the speckle pattern over time, are sensitive to both transverse and axial flow. Several types of speckle-based techniques have been described, including amplitude-based (9–11), phase-based (12), or a combination of both amplitude and phase (13) variance methods.We developed an amplitude-based method called split-spectrum amplitude-decorrelation angiography (SSADA). The SSADA algorithm detects motion in the blood vessel lumen by measuring the variation in reflected OCT signal amplitude between consecutive cross-sectional scans. The novelty of SSADA lies in how the OCT signal is processed to enhance flow detection and reject axial bulk motion noise. Compared with the full-spectrum amplitude method, SSADA using fourfold spectral splits improved the signal-to-noise ratio (SNR) by a factor of two, which is equivalent to reducing the scan time by a factor of four (14). More recent SSADA implementations use even more than a fourfold split to further enhance the SNR of flow detection. This highly efficient algorithm generates high-quality angiograms of both the retina and choroid. The angiograms have capillary-level detail and can be obtained with currently available commercial OCT systems.This article uses six illustrative cases and highlights the various types of vascular pathologies that can be detected and measured using SSADA and an OCT angiography system of visualization. Additionally, we describe techniques designed to help clinicians rapidly interpret OCT angiograms and to easily identify pathological vascular features. These techniques include (i) separation of the 3D angiogram into individual vascular beds via segmentation algorithms, (ii) presentation of en face OCT angiograms, analogous to traditional angiography, (iii) creation of cross-sectional structural OCT images with superimposed OCT angiograms to help correlate anatomical alterations with vascular abnormalities, and (iv) quantification of neovascularization and capillary dropout in both the retinal and choroidal circulation.     

From the Cover: Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG expansion within the huntingtin gene that encodes a polymorphic glutamine tract at the amino terminus of the huntingtin protein. HD is one of nine polyglutamine expansion diseases. The clinical threshold of polyglutamine expansion for HD is near 37 repeats, but the mechanism of this pathogenic length is poorly understood. Using Förster resonance energy transfer, we describe an intramolecular proximity between the N17 domain and the downstream polyproline region that flanks the polyglutamine tract of huntingtin. Our data support the hypothesis that the polyglutamine tract can act as a flexible domain, allowing the flanking domains to come into close spatial proximity. This flexibility is impaired with expanded polyglutamine tracts, and we can detect changes in huntingtin conformation at the pathogenic threshold for HD. Altering the structure of N17, either via phosphomimicry or with small molecules, also affects the proximity between the flanking domains. The structural capacity of N17 to fold back toward distal regions within huntingtin requires an interacting protein, protein kinase C and casein kinase 2 substrate in neurons 1 (PACSIN1). This protein has the ability to bind both N17 and the polyproline region, stabilizing the interaction between these two domains. We also developed an antibody-based FRET assay that can detect conformational changes within endogenous huntingtin in wild-type versus HD fibroblasts. Therefore, we hypothesize that wild-type length polyglutamine tracts within huntingtin can form a flexible domain that is essential for proper functional intramolecular proximity, conformations, and dynamics.     

Direct laser manipulation reveals the mechanics of cell contacts in vivo

Cell-generated forces produce a variety of tissue movements and tissue shape changes. The cytoskeletal elements that underlie these dynamics act at cell–cell and cell–ECM contacts to apply local forces on adhesive structures. In epithelia, force imbalance at cell contacts induces cell shape changes, such as apical constriction or polarized junction remodeling, driving tissue morphogenesis. The dynamics of these processes are well-characterized; however, the mechanical basis of cell shape changes is largely unknown because of a lack of mechanical measurements in vivo. We have developed an approach combining optical tweezers with light-sheet microscopy to probe the mechanical properties of epithelial cell junctions in the early Drosophila embryo. We show that optical trapping can efficiently deform cell–cell interfaces and measure tension at cell junctions, which is on the order of 100 pN. We show that tension at cell junctions equilibrates over a few seconds, a short timescale compared with the contractile events that drive morphogenetic movements. We also show that tension increases along cell interfaces during early tissue morphogenesis and becomes anisotropic as cells intercalate during germ-band extension. By performing pull-and-release experiments, we identify time-dependent properties of junctional mechanics consistent with a simple viscoelastic model. Integrating this constitutive law into a tissue-scale model, we predict quantitatively how local deformations propagate throughout the tissue.During the development of an organism, cells change their shape and remodel their contacts to give rise to a variety of tissue shapes. Analysis of tissue kinematics has revealed that epithelial tissue morphogenesis is partly controlled by actomyosin contractility. The spatiotemporal deployment and coordination of actomyosin contractility produce shrinkage and extension of cell surfaces and interfaces, which can drive tissue invagination, tissue folding, or tissue extension (1). Understanding the mechanical nature of these processes requires force measurements in vivo; however, measurements in developing epithelia are limited, and most methods have been indirect. They rely on either force inference from image analysis (2–4) or laser dissection experiments at cell (5, 6) or tissue scales (7, 8), which provide the relative magnitude and direction of stresses from cell or tissue shape changes. In contrast, mechanical approaches have been developed in recent years to impose or measure stresses of cells in contact, including cell monolayer micromanipulation (9), pipette microaspiration on cell doublets (10), and traction force microscopy on migrating epithelia (11) and single-cell doublets (12). Recently, an elegant method using deformable cell-sized oil microdroplets has provided absolute values of stresses at the cell level in cell cultures and embryonic mesenchymes (13) but not yet in live epithelia. In this context, we sought a direct in vivo method for tension measurements and mechanical characterization at cell contacts and developed an experimental approach combining optical tweezers with light-sheet microscopy.To probe epithelial mechanics in a live organism, we chose the early epithelium of the Drosophila embryo as a model system. It consists of a simple sheet of cells that spread over the yolk and are in contact with each other through E-cadherin–based adhesion. During early embryogenesis at the blastula stage just after the end of cellularization, epithelial cells have very similar hexagonal shapes, suggesting that cell junctions have similar mechanical properties and that the internal pressure of these cells is homogeneous. At the later gastrula stage, cells undergo shape changes at distinct regions in the embryo. On the ventral side of the embryo, apical cell constriction of a few rows of cells drives tissue invagination (14), whereas on the ventrolateral side of the embryo, cell intercalation, a process whereby cells exchange neighbors by polarized remodeling of their junctions, drives tissue extension. The latter morphogenetic movement is driven by an anisotropic distribution of Myosin-II (Myo-II), which is more concentrated along junctions aligned with the dorsal/ventral (D/V) axis (15). Laser dissection of cortical actomyosin networks at cell junctions in the ventrolateral tissue has shown that such an anisotropic distribution of Myo-II causes an anisotropic cortical tension (6). However, the absolute values of tensile forces have not yet been measured, and more generally, the mechanics of cell–cell interfaces in vivo are largely unknown. Here, we addressed this issue by analyzing local mechanical measurements at cell junctions during tissue morphogenesis and determining the contribution of Myo-II to tension in this context. We determined the time-dependent response of cell–cell interfaces to forced deflection and delineated a viscoelastic model of junctions. Finally, we explored the propagation of local forces within the epithelial tissue.     

Brief Report: Optical sectioning of unlabeled samples using bright-field microscopy

The bright-field (BF) optical microscope is a traditional bioimaging tool that has been recently tested for depth discrimination during evaluation of specimen morphology; however, existing approaches require dedicated instrumentation or extensive computer modeling. We report a direct method for three-dimensional (3D) imaging in BF microscopy, applicable to label-free samples, where we use Köhler illumination in the coherent regime and conventional digital image processing filters to achieve optical sectioning. By visualizing fungal, animal tissue, and plant samples and comparing with light-sheet fluorescence microscopy imaging, we demonstrate the accuracy and applicability of the method, showing how the standard microscope is an effective 3D imaging device.     

From the Cover: Turbulence-driven instabilities limit insect flight performance

Environmental turbulence is ubiquitous in natural habitats, but its effect on flying animals remains unknown because most flight studies are performed in still air or artificially smooth flow. Here we show that variability in external airflow limits maximum flight speed in wild orchid bees by causing severe instabilities. Bees flying in front of an outdoor, turbulent air jet become increasingly unstable about their roll axis as airspeed and flow variability increase. Bees extend their hindlegs ventrally at higher speeds, improving roll stability but also increasing body drag and associated power requirements by 30%. Despite the energetic cost, we observed this stability-enhancing behavior in 10 euglossine species from 3 different genera, spanning an order of magnitude in body size. A field experiment in which we altered the level of turbulence demonstrates that flight instability and maximum flight speed are directly related to flow variability. The effect of environmental turbulence on flight stability is thus an important and previously unrecognized determinant of flight performance.