Wide-field Imaging Research Articles

Bi-Plane Multicolor Scanning Illumination Microscopy with Multispot Excitation and a Distorted Diffraction Grating

Multifocus microscopy has previously been demonstrated to provide volumetric information from a single shot. However, the practical application of this method is challenging due to its weak optical sectioning and limited spatial resolution. Here, we report on the combination of a distorted diffraction grating and multifocal scanning illumination microscopy to improve spatial resolution and contrast. DG is introduced in the emission path of the multifocal scanning illumination microscopy, which splits the fluorescence signal from different sample layers into different diffraction orders. After postprocessing, super-resolution wide-field images of different sample layers can be reconstructed from single 2D scanning.

Exploring the Interplay of Wavelength, Quantum Yield, and Penetration Depth in In Vivo Fluorescence Imaging.

The intricate interplay between the irradiation wavelength, the fluorophore quantum yield (QY) and penetration depth profoundly influences the efficacy of in vivo fluorescence imaging in various applications. Understanding the complex behavior of fluorescence in vivo, specifically how variations in wavelength affect the QY of commonly used dyes and the depth of imaging is crucial for optimizing fluorescence imaging techniques, as it directly impacts the accuracy and efficiency of imaging in biological tissues. In our study, we explore these dynamics through Monte Carlo simulations conducted under conditions reflective of wide-field fluorescence imaging, examining how variations in wavelength impact the dye's QY and depth of imaging, and consequently, the fluorescence behavior. A transition in the exponential decay of the emission depth exponent is observed around the 500-600nm range, indicating varying degrees of influence of depth on the fluorescence emission. The analysis of the fluorophore's QY reveals wavelength-dependent variations, with the most significant impact observed in the 600-700nm range. Moreover, we continued our investigation to explore multiplexing, unveiling insights into the spacing between identical spots in multiplexing images across various depths and wavelengths.

Translational Tomography with the Wide-field Imager for Parker Solar Probe (WISPR). II. Refinements to the Method

We present progress on the translational tomography technique for measuring the three-dimensional structure of the corona from near-perihelion Wide-field Imager for Parker Solar Probe (WISPR) image sequences. Translational tomography makes use of noncircular motion of a camera to extract three-dimensional information from an optically thin subject. Parker Solar Probe (PSP) presents a special case both because of the particular structure of the corona and because of the nonlinear motion of the vantage point. We show improvements to a previous direct analytic method (described in Paper I of this series) and an alternative inversion pathway using a synthetic sequence of WISPR images. The newer method successfully reconstructs the correct locations of modeled coronal rays in a synthetic WISPR image sequence, with curvilinear camera motion modeled on the PSP orbit. We present the refined methodology and validation study, and show that the technique is ready for application to actual WISPR data.

VEGAS-SSS: Tracing globular cluster populations in the interacting NGC 3640 galaxy group

Context. Globular clusters (GCs) are among the oldest stellar systems in the universe. As such, GC populations are valuable fossil tracers of galaxy formation and interaction history. This paper is part of the VEGAS-SSS series, which focuses on studying the properties of small stellar systems (SSSs) in and around bright galaxies. Aims. We used the multiband wide-field images obtained with the VST to study the properties of the GC population in an interacting pair of galaxies. Methods. We derived ugri photometry over 1.5 × 1.5 sq. degrees centered on the galaxy group composed of two elliptical galaxies: NGC 3640 and its fainter companion NGC 3641. We studied the GC system properties from both the ugri and gri matched catalogs. GC candidates were identified based on a combination of photometric properties (colors and magnitudes) and morphometric criteria (concentration index, elongation, FWHM, etc.), using sources with well-defined classifications from spectroscopic or imaging data available in the literature and numerical simulations as references. The selection criteria were also applied to empty fields to determine a statistical background correction for the number of identified GC candidates. Results. The 2D density maps of GCs appear to align with the diffuse light patches resulting from the merging events of the galaxies. The highest density peak of GCs is observed to be on NGC 3641 rather than NGC 3640, despite the latter being the more massive galaxy. The azimuthal averaged radial density profiles in both galaxies reveal that the GC population extends beyond the galaxy light profile and this indicates the likely presence of an intra-group GC component. A color bimodality in (u − r) and (g − i) is observed for NGC 3641, whereas NGC 3640 shows a broad unimodal distribution. Analysis of the GC luminosity function indicates that both galaxies are roughly located at the same distance (∼27 Mpc). We provide an estimate of the total number of GCs, and determine the specific frequency for NGC 3640, SN = 2.0 ± 0.6, which aligns with expectations, while for NGC 3641 we find a large SN = 4.5 ± 1.6.

Wide-field scanning ghost imaging based on a local binary pattern and untrained neural network

Continuous scene imaging is an important research goal in the field of autonomous driving, and the key is to ensure the imaging quality and efficiency. In this paper, we propose a method for information fusion in wide-field scanning ghost imaging using a local binary pattern (LBP) based on deep learning. The initial physical model formed by the LBP integrated into a deep neural network, which effectively enhances the expression of image texture details. Then the collected bucket signals are used as labels for adaptive image reconstruction, enabling the acquisition of images at each scanning position without the need for training on any dataset. Moreover, by employing weighted fusion to combine the image data from each scanning position, which effectively eliminates gaps that arise from direct stitching. Both simulation and experimental results demonstrate that our approach is capable of achieving high-quality detailed imaging with fewer measurements. Additionally, we analyze the impact of the projection beam step length, finding that our method yields significantly better imaging quality with larger steps compared to other methods using smaller steps. Our research also has the application prospect in medical detection, remote sensing and other fields.

The Smallest Scale of Hierarchy Survey (SSH)

We present new deep, wide-field imaging data from the Large Binocular Telescope (LBT) in g and r bands from the Smallest Scale of Hierarchy Survey (SSH) that reveal previously undetected tidal features and stellar streams in the outskirts of six dwarf irregular galaxies (NGC 5238, UGC 6456, UGC 6541, UGC 7605, UGC 8638, and UGC 8760) with stellar masses in the range 1.2 × 107 M⊙ to 1.4 × 108 M⊙. The six dwarfs are located 1-2 Mpc away from large galaxies, which implies that the observed distortions are unlikely to be due to tidal effects from a nearby, massive companion. At the distances of ~3-4 Mpc at which the dwarfs lie, the identified tidal features are all resolved into individual stars in the LBT images and appear to consist of a population older than 1–2 Gyr. This excludes the possibility that they result from irregular and asymmetric star formation episodes that are common in gas-rich dwarf galaxies. The most plausible explanation is that we witness the hierarchical merging assembly of these dwarfs with their satellite populations. This scenario is also supported by the peculiar morphology and disturbed velocity field of their HI component. From the SSH sample, we estimate that a fraction of ~13% of the late-type dwarfs show signs of merging with satellites. This is in agreement with other recent independent studies and theoretical predictions within the ΛCDM cosmological framework.

Cortico-thalamic communication for action coordination in a skilled motor sequence.

The coordination of forelimb and orofacial movements to compose an ethological reach-to-consume behavior likely involves neural communication across brain regions. Leveraging wide-field imaging and photo-inhibition to survey across the cortex, we identified a cortical network and a high-order motor area (MOs-c), which coordinate action progression in a mouse reach-and-withdraw-to-drink (RWD) behavior. Electrophysiology and photo-inhibition across multiple projection neuron types within the MOs-c revealed differential contributions of pyramidal tract and corticothalamic (CT MOs ) output channels to action progression and hand-mouth coordination. Notably, CT MOs display sustained firing throughout RWD sequence and selectively enhance RWD-relevant activity in postsynaptic thalamus neurons, which also contribute to action coordination. CT MOs receive converging monosynaptic inputs from forelimb and orofacial sensorimotor areas and are reciprocally connected to thalamic neurons, which project back to the cortical network. Therefore, motor cortex corticothalamic channel may selectively amplify the thalamic integration of cortical and subcortical sensorimotor streams to coordinate a skilled motor sequence.

Widefield optical coherence tomography by electro-optical modulation

Optical coherence tomography (OCT) is a unique imaging modality capable of axial sectioning with a resolution of only a few microns. Its ability to image with high resolution deep within tissue makes it ideal for material inspection, dentistry, and, in particular, ophthalmology. Widefield retinal imaging has garnered increasing clinical interest for the detection of numerous retinal diseases. However, real-time applications in clinical practice demand the contrast of swept-source OCT at scan speeds that limit their depth range. The curvature of typical samples, such as teeth, corneas, or retinas, thus restricts the field-of-view of fast OCT systems. Novel high-speed swept sources are expected to further improve the scan rate; however, not without exacerbating the already severe trade-off in depth range. Here, we show how, without the need for mechanical repositioning, harmonic images can be rapidly synthesized at any depth. This is achieved by opto-electronic modulation of a single-frequency swept source laser in tandem with tailored numerical dispersion compensation. We demonstrate experimentally how real-time imaging of highly-curved samples is enabled by extending the effective depth-range 8-fold. Even at the scan speed of a 400 kHz swept source, harmonic OCT enables widefield retinal imaging.

All-in-one quantum diamond microscope for sensor characterization

Ensembles of nitrogen-vacancy (NV) centers in diamond are a leading platform for sensing and imaging magnetic fields at room temperature, in part due to advances in diamond growth. An essential step to improving diamond material involves the characterization of crystal and NV-related properties, such as strain and paramagnetic impurities, which can shift and broaden the NV resonances used for sensing. Full sample characterization through wide-field imaging enables both fast and detailed feedback for growers, along with the estimation of sensing performance before use. We present a quantum diamond microscope tailored for millimeter-scale wide-field mapping of key quantum properties of NV-diamond chips, including NV ensemble photoluminescence intensity, spin-lattice relaxation time (T1), and spin-coherence lifetimes (T2 and T2*). Our design also allows for lattice stress/strain and birefringence magnitude/angle mapping, and their in situ correlation with NV properties.

Topography of putative bidirectional interaction between hippocampal sharp wave ripples and neocortical slow oscillations.

Systems consolidation relies on coordination between hippocampal sharp-wave ripples (SWRs) and neocortical UP/DOWN states during sleep. However, whether this coupling exists across neocortex and the mechanisms enabling it remain unknown. By combining electrophysiology in mouse hippocampus (HPC) and retrosplenial cortex (RSC) with widefield imaging of dorsal neocortex, we found spatially and temporally precise bidirectional hippocampo-neocortical interaction. HPC multi-unit activity and SWR probability was correlated with UP/DOWN states in mouse default mode network, with highest modulation by RSC in deep sleep. Further, some SWRs were preceded by the high rebound excitation accompanying DMN DOWN→UP transitions, while large-amplitude SWRs were often followed by DOWN states originating in RSC. We explain these electrophysiological results with a model in which HPC and RSC are weakly coupled excitable systems capable of bi-directional perturbation and suggest RSC may act as a gateway through which SWRs can perturb downstream cortical regions via cortico-cortical propagation of DOWN states.

Atomic force microscopy wide-field scanning imaging using homography matrix optimization

During the offline combination of multiple atomic force microscopy (AFM) images, changes in probe displacement can be affected by dynamic noise, leading to dislocation and tearing in the stitching. To overcome this, we designed a homography matrix optimization method to describe the relative positional relationship between images by constructing the original image matrix and applying singular value decomposition to denoise the homography matrix. Additionally, we implemented a scale-invariant feature transform (SIFT) to extract feature points. To verify the effectiveness of this method, the SIFT + RANSAC (R), SIFT + affine transformation (AT), and oriented FAST and rotated BRIEF (ORB) algorithms were compared with the proposed algorithm. The experimental results demonstrate that the proposed method precisely computes the transformation matrix, thereby guaranteeing the geometric consistency of mosaic imaging. The proposed method preserves the intricate details of the original image and enhances the stitching quality of wide-field images.

Reduced Non-Specific Binding of Super-Resolution DNA-PAINT Markers by Shielded DNA-PAINT Labeling Protocols.

The DNA-based single molecule super-resolution imaging approach, DNA-PAINT, can achieve nanometer resolution of single targets. However, the approach can suffer from significant non-specific background signals originating from non-specifically bound DNA-conjugated DNA-PAINT secondary antibodies as shown here. Using dye-modified oligonucleotides the location of DNA-PAINT secondary antibody probes can easily be observed with widefield imaging prior to beginning a super-resolution measurement. This reveals that a substantial proportion of DNA probes can accumulate, non-specifically, within the nucleus, as well as across the cytoplasm, of cells. Here, Shielded DNA-PAINT labeling is introduced, a method using partially or fully double-stranded docking strand sequences, prior to labeling, in buffers with increased ionic strength to greatly reduce non-specific interactions in the nucleus as well as the cytoplasm. This new labeling approach is evaluated against various conditions and it is shown that applying Shielded DNA-PAINT can reduce non-specific events approximately five-fold within the nucleus. This marked reduction in non-specific binding of probes during the labeling procedure is comparable to results obtained with unnatural left-handed DNA albeit at a fraction of the cost. Shielded DNA-PAINT is a straightforward adaption of current DNA-PAINT protocols and enables nanometer precision imaging of nuclear targets with low non-specific backgrounds.

Rastermap: a discovery method for neural population recordings.

Neurophysiology has long progressed through exploratory experiments and chance discoveries. Anecdotes abound of researchers listening to spikes in real time and noticing patterns of activity related to ongoing stimuli or behaviors. With the advent of large-scale recordings, such close observation of data has become difficult. To find patterns in large-scale neural data, we developed 'Rastermap', a visualization method that displays neurons as a raster plot after sorting them along a one-dimensional axis based on their activity patterns. We benchmarked Rastermap on realistic simulations and then used it to explore recordings of tens of thousands of neurons from mouse cortex during spontaneous, stimulus-evoked and task-evoked epochs. We also applied Rastermap to whole-brain zebrafish recordings; to wide-field imaging data; to electrophysiological recordings in rat hippocampus, monkey frontal cortex and various cortical and subcortical regions in mice; and to artificial neural networks. Finally, we illustrate high-dimensional scenarios where Rastermap and similar algorithms cannot be used effectively.

Weak-Lensing Shear-Selected Galaxy Clusters from the Hyper Suprime-Cam Subaru Strategic Program: II. Cosmological Constraints from the Cluster Abundance

We present cosmological constraints using the abundance of weak-lensing shear-selected galaxy clusters in the Hyper Suprime-Cam (HSC) Subaru Strategic Program. The clusters are selected on the aperture-mass maps constructed using the three-year (Y3) weak-lensing data with an area of ≈500deg 2, resulting in a sample size of 129 clusters with high signal-to-noise ratios ν≥4.7. Owing to the deep, wide-field, and uniform imaging of the HSC survey, this is by far the largest sample of shear-selected clusters, for which the selection solely depends on gravity and is free from any assumptions about the dynamical state and complex baryon physics. Informed by the optical counterparts, the shear-selected clusters span a redshift range of z≲0.7 with a median of z≈0.3. The lensing sources are securely selected at z≳0.7 with a median of z≈1.3, leading to nearly zero cluster member contamination. We carefully account for (1) the bias in the photometric redshift of sources, (2) the bias and scatter in the weak-lensing mass using a simulation-based calibration, and (3) the measurement uncertainty that is directly estimated on the aperture-mass maps using an injection-based method developed in a companion paper (Chen et al. submitted). In a blind analysis, the fully marginalized posteriors of the cosmological parameters are obtained as Ωm=0.50−0.24+0.28, σ8=0.685−0.088+0.161, Ŝ8≡σ8(Ωm/0.3)0.25=0.835−0.044+0.041, and σ8Ωm/0.3=0.993−0.126+0.084 in a flat ΛCDM model. We compare our cosmological constraints with other studies, including those based on cluster abundances, galaxy-galaxy lensing and clustering, and Cosmic Microwave Background observed by Planck, and find good agreement at levels of ≲2σ. [abridged]

Cell class-specific long-range axonal projections of neurons in mouse whisker-related somatosensory cortices.

Long-range axonal projections of diverse classes of neocortical excitatory neurons likely contribute to brain-wide interactions processing sensory, cognitive and motor signals. Here, we performed light-sheet imaging of fluorescently labeled axons from genetically defined neurons located in posterior primary somatosensory barrel cortex and supplemental somatosensory cortex. We used convolutional networks to segment axon-containing voxels and quantified their distribution within the Allen Mouse Brain Atlas Common Coordinate Framework. Axonal density was analyzed for different classes of glutamatergic neurons using transgenic mouse lines selectively expressing Cre recombinase in layer 2/3 intratelencephalic projection neurons (Rasgrf2-dCre), layer 4 intratelencephalic projection neurons (Scnn1a-Cre), layer 5 intratelencephalic projection neurons (Tlx3-Cre), layer 5 pyramidal tract projection neurons (Sim1-Cre), layer 5 projection neurons (Rbp4-Cre), and layer 6 corticothalamic neurons (Ntsr1-Cre). We found distinct axonal projections from the different neuronal classes to many downstream brain areas, which were largely similar for primary and supplementary somatosensory cortices. Functional connectivity maps obtained from optogenetic activation of sensory cortex and wide-field imaging revealed topographically organized evoked activity in frontal cortex with neurons located more laterally in somatosensory cortex signaling to more anteriorly located regions in motor cortex, consistent with the anatomical projections. The current methodology therefore appears to quantify brain-wide axonal innervation patterns supporting brain-wide signaling.

Cell class-specific long-range axonal projections of neurons in mouse whisker-related somatosensory cortices

Long-range axonal projections of diverse classes of neocortical excitatory neurons likely contribute to brain-wide interactions processing sensory, cognitive and motor signals. Here, we performed light-sheet imaging of fluorescently labeled axons from genetically defined neurons located in posterior primary somatosensory barrel cortex and supplemental somatosensory cortex. We used convolutional networks to segment axon-containing voxels and quantified their distribution within the Allen Mouse Brain Atlas Common Coordinate Framework. Axonal density was analyzed for different classes of glutamatergic neurons using transgenic mouse lines selectively expressing Cre recombinase in layer 2/3 intratelencephalic projection neurons (Rasgrf2-dCre), layer 4 intratelencephalic projection neurons (Scnn1a-Cre), layer 5 intratelencephalic projection neurons (Tlx3-Cre), layer 5 pyramidal tract projection neurons (Sim1-Cre), layer 5 projection neurons (Rbp4-Cre), and layer 6 corticothalamic neurons (Ntsr1-Cre). We found distinct axonal projections from the different neuronal classes to many downstream brain areas, which were largely similar for primary and supplementary somatosensory cortices. Functional connectivity maps obtained from optogenetic activation of sensory cortex and wide-field imaging revealed topographically organized evoked activity in frontal cortex with neurons located more laterally in somatosensory cortex signaling to more anteriorly located regions in motor cortex, consistent with the anatomical projections. The current methodology therefore appears to quantify brain-wide axonal innervation patterns supporting brain-wide signaling.

Imaging the large-scale and cellular response to focal traumatic brain injury in mouse neocortex.

Traumatic brain injury (TBI) affects neural function at the local injury site and also at distant, connected brain areas. However, the real-time neural dynamics in response to injury and subsequent effects on sensory processing and behavior are not fully resolved, especially across a range of spatial scales. We used in vivo calcium imaging in awake, head-restrained male and female mice to measure large-scale and cellular resolution neuronal activation, respectively, in response to a mild/moderate TBI induced by focal controlled cortical impact (CCI) injury of the motor cortex (M1). Widefield imaging revealed an immediate CCI-induced activation at the injury site, followed by a massive slow wave of calcium signal activation that traveled across the majority of the dorsal cortex within approximately 30 s. Correspondingly, two-photon calcium imaging in primary somatosensory cortex (S1) found strong activation of neuropil and neuronal populations during the CCI-induced traveling wave. A depression of calcium signals followed the wave, during which we observed atypical activity of a sparse population of S1 neurons. Longitudinal imaging in the hours and days after CCI revealed increases in the area of whisker-evoked sensory maps at early time points, in parallel to decreases in cortical functional connectivity and behavioral measures. Neural and behavioral changes mostly recovered over hours to days in our M1-TBI model, with a more lasting decrease in the number of active S1 neurons. Our results in unanesthetized mice describe novel spatial and temporal neural adaptations that occur at cortical sites remote to a focal brain injury.

Miniaturized photoacoustic microscope for multi-segmental spinal cord imaging in freely moving mice.

Long-term and non-narcotic hemodynamic imaging is indispensable for observing factual physiological information of the spinal cord. Unfortunately, achieving label-free, high-resolution, and widefield spinal cord imaging for mice under freely moving conditions is challenging. In this study, we developed a miniaturized photoacoustic microscope along with a corresponding photoacoustic spinal window to realize high-resolution, multi-segmental hemodynamic imaging of the spinal cord for freely moving mice. The microscope has an outer size of 32 mm × 23 mm × 10 mm, a weight of 5.8 g, and a 4.4 µm lateral resolution within an effective field of view (FOV) of 2.6 mm × 1.8 mm. To eliminate the off-focus phenomena during spinal imaging, the microscope is equipped with a miniature motor to adapt the focal plane. Besides, the microscope is slidable along a customized rail on the window to expand the FOV. We evaluated the stability of the microscope and analyzed vascular images of the spinal cord under various physiological states. The results suggest that the microscope is capable of performing stable, multi-segmental spinal cord imaging in freely moving mice, offering new insights into spinal cord hemodynamics and neurovascular coupling research.

Dynamic 3D Fresnel incoherent correlation holography imaging based on single-shot mirrored phase-shifting technology.

Fresnel incoherent correlation holography (FINCH) records coaxial holograms for wide-field 3D imaging with incoherent light, but its temporal phase-shifting strategy makes dynamic imaging challenging. Here, we present a compact, portable single-shot mirrored phase-shifting (SSPMS) module that can be easily integrated into the FINCH system, achieving secondary modulation of self-interference beams to enable the simultaneous acquisition of four phase-shift holograms in a single exposure. Compared with previously reported methods that use diffraction gratings to spatially separate self-interference beams at specific angles, this module duplicates a laterally shifted mirrored beam using a simply modified Michelson interferometer, so the phase-shifting holograms obtained via this module are free from optical aberrations or higher-order diffracted light noises. The feasibility of the proposed method is experimentally demonstrated through imaging dynamic 3D grayscale scenes.

Fourier Ptychographic Coherent Anti-Stokes Raman Scattering Microscopy with Point-Scanning for Super-Resolution Imaging.

Fourier ptychography (FP) is a high resolution wide-field imaging method based on the extended aperture in the Fourier space, which is synthesized from raw images with varying illumination angles. If FP is extended to coherent nonlinear optical imaging, the resolution could be further improved due to the increase of the cutoff frequency of the synthesized coherent optical transfer function (C-OTF) with respect to the order of nonlinear optical processes. However, there is a fundamental conflict between wide-field FP and nonlinear optical imaging, whereby the nonlinear optical imaging typically requires a focused excitation laser beam with high power density. To tackle the problem, in this work, a unique point-scanning FP (PS-FP) method is presented for super-resolution nonlinear optical imaging, in which the nonlinear optical signal is obtained by using focused laser beam, while the conventional FP algorithm can still be used to retrieve the super-resolution image. PS-FP coherent anti-Stokes Raman scattering (PS-FP-CARS) imaging on a variety of samples, where a 1.8-fold expansion of the OTF is achieved experimentally for enhancing vibrational imaging. Further theoretical calculation shows that the C-OTF of PS-FP higher-order CARS (PS-FP-HO-CARS) can be expanded up to ≈4.9-fold, thereby improving the spatial resolution by ≈3-fold in comparison with conventional point-scanning CARS with under tightly focused beams. The generality of PS-FP method developed in this work can be adapted to other coherent nonlinear optical imaging modalities for super-resolution imaging in tissue and cells.