Wide-field Imaging Research Articles

A fully automated morphological analysis of yeast mitochondria from wide-field fluorescence images

Mitochondrial morphology is an important parameter of cellular fitness. Although many approaches are available for assessing mitochondrial morphology in mammalian cells, only a few technically demanding and laborious methods are available for yeast cells. A robust, fully automated and user-friendly approach that would allow (1) segmentation of tubular and spherical mitochondria in the yeast Saccharomyces cerevisiae from conventional wide-field fluorescence images and (2) quantitative assessment of mitochondrial morphology is lacking. To address this, we compared Global thresholding segmentation with deep learning MitoSegNet segmentation, which we retrained on yeast cells. The deep learning model outperformed the Global thresholding segmentation. We applied it to segment mitochondria in strain lacking the MMI1/TMA19 gene encoding an ortholog of the human TCTP protein. Next, we performed a quantitative evaluation of segmented mitochondria by analyses available in ImageJ/Fiji and by MitoA analysis available in the MitoSegNet toolbox. By monitoring a wide range of morphological parameters, we described a novel mitochondrial phenotype of the mmi1Δ strain after its exposure to oxidative stress compared to that of the wild-type strain. The retrained deep learning model, all macros applied to run the analyses, as well as the detailed procedure are now available at https://github.com/LMCF-IMG/Morphology_Yeast_Mitochondria.

Noninvasive megapixel fluorescence microscopy through scattering layers by a virtual incoherent reflection matrix.

Optical-resolution fluorescence imaging through and within complex samples presents a major challenge due to random light scattering, with substantial implications across multiple fields. While considerable advancements in coherent imaging through severe multiple scattering have been recently introduced by reflection matrix processing, approaches that tackle scattering in incoherent fluorescence imaging have been limited to sparse targets, require high-resolution control of the illumination or detection wavefronts, or require a very large number of measurements. Here, we present an approach that allows the adaptation of well-established reflection matrix techniques to scattering compensation in incoherent fluorescence imaging. We experimentally demonstrate that a small number of conventional wide-field fluorescence microscope images acquired under unknown random illuminations can effectively be used to construct a virtual fluorescence-based reflection matrix. Processing this matrix by an adapted matrix-based scattering compensation algorithm allows reconstructing megapixel-scale images from <150 acquired frames, without any spatial light modulators or computationally intensive processing.

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 behaviour 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 travelled across the majority of the dorsal cortex within approximately 30s. Correspondingly, two-photon calcium imaging in the primary somatosensory cortex (S1) found strong activation of neuropil and neuronal populations during the CCI-induced travelling wave. A depression of calcium signals followed the wave, during which we observed the 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 behavioural measures. Neural and behavioural 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 unanaesthetized mice describe novel spatial and temporal neural adaptations that occur at cortical sites remote to a focal brain injury.

Dual-Wavelength Confocal Laser Speckle Contrast Imaging Using a Deep Learning Approach

This study developed a novel dual-wavelength confocal laser speckle imaging platform. The system includes both visible and near-infrared lasers and two imaging modes: confocal and wide-field laser speckle contrast imaging. The experimental results confirm that the proposed system can be used to measure not only blood flow but also blood oxygen saturation. Additionally, we proposed a blood flow perfusion imaging method called BlingNet (a blood flow imaging CNN) based on the laser speckle contrast imaging technique and deep learning approach. Compared to the traditional nonlinear fitting method, this method has superior accuracy and robustness with higher imaging speed, making real-time blood flow imaging possible.

Combined dual-channel fluorescence depth sensing of indocyanine green and protoporphyrin IX kinetics in subcutaneous murine tumors.

Fluorescence sensing within tissue is an effective tool for tissue characterization; however, the modality and geometry of the image acquisition can alter the observed signal. We introduce a novel optical fiber-based system capable of measuring two fluorescent contrast agents through 2cm of tissue with simple passive electronic switching between the excitation light, simultaneously acquiring fluorescence and excitation data. The goal was to quantify indocyanine green (ICG) and protoporphyrin IX (PpIX) within tissue, and the sampling method was compared with wide-field surface imaging to contrast the value of deep sensing versus surface imaging. This was achieved by choosing filters for specific wavelengths that were mutually exclusive between ICG and PpIX and coupling these filters to two separate detectors, which allows for direct swapping of the excitation and emission channels by switching the on-time of each excitation laser between 780- and 633-nm wavelengths. This system was compared with two non-contact surface imaging systems for both ICG and PpIX, which revealed that the fluorescence depth sensing system was superior in its ability to resolve kinetics differences in deeper tissues that would normally be dominated by strong signals from skin and other surface tissues. Specifically, the system was tested using pancreatic adenocarcinoma tumors injected into murine models, which were imaged at several time points throughout tumor growth to its diameter. This demonstrated the system's capability to track longitudinal changes in ICG and PpIX kinetics that result from tumor growth and development, with larger tumors showing sluggish uptake and clearance of ICG, which was not observable with surface imaging. Similarly, PpIX was quantified, which showed slower kinetics over different time points, and was further compared with the wide-filed imager. These results were further validated through depth measurements in tissue phantoms and model-based interpretation. This fluorescence depth sensing system can be used to sample the interior blood flow characteristics by ICG sensing of tissue as deep as 20mm into the tissue with sensitivity to kinetics that are superior to surface imaging and may be combined with other imaging modalities such as ultrasound to provide guided deep fluorescence measurements.

The AstroSat UV Deep Field South. I. Far- and Near-ultraviolet Source Catalog of the GOODS South Region

We present the AstroSat UV Deep Field south (AUDFs), an imaging survey using the wide-field Ultraviolet Imaging Telescope on board AstroSat. AUDFs cover ∼236 arcmin2 of the sky area, including the Great Observatories Origins Deep Survey (GOODS) South field in F154W and N242W filters. The deep and shallow parts of AUDFs have an exposure time ∼62,000 and ∼31,000 s, respectively, in the F154W filter, while in the N242W filter, they are ∼64,000 and ∼34,000 s. These observations reached a 3σ depth of 27.2 and 27.7 AB mag with a 50% completeness limit of 27 and 27.6 AB mag in the F154W and N242W filters, respectively. With the acquired depth, AUDFs is the deepest far- and near-UV imaging data covering the largest area known to date at 1.″2–1.″6 spatial resolution. Two primary catalogs were constructed for the F154W and N242W filters, each containing 13,495 and 19,374 sources brighter than the 3σ detection limit, respectively. Our galaxy counts of a power-law slope of ∼0.43 dex mag−1 in the N242W filter match well with Hubble Space Telescope/Wide-Field Camera 3/UVIS observations. A wide range of extragalactic science can be achieved with this unique data, such as providing a sample of galaxies emitting ionizing photons in the redshift range of z ∼1–3 and beyond, constraining the UV luminosity function, investigating the extended UV emission around star-forming galaxies and UV morphologies for z < 1. The UV catalog will enhance the legacy value of the existing optical/IR imaging and spectroscopic observations from ground- and space-based telescopes on the GOODS South field.

Psychedelic 5-HT2A receptor agonism: neuronal signatures and altered neurovascular coupling.

Psychedelics hold therapeutic promise for mood disorders due to rapid, sustained results. Human neuroimaging studies have reported dramatic serotonin-2A receptor-(5-HT2AR)-dependent changes in functional brain reorganization that presumably reflect neuromodulation. However, the potent vasoactive effects of serotonin have been overlooked. We found psilocybin-mediated alterations to fMRI-HRFs in humans, suggesting potentially altered NVC. To assess the neuronal, hemodynamic, and neurovascular coupling (NVC) effects of the psychedelic 5-HT2AR agonist, 2,5-Dimethoxy-4-iodoamphetamine (DOI), wide-field optical imaging (WFOI) was used in awake Thy1-jRGECO1a mice during stimulus-evoked and resting-state conditions. While DOI partially altered tasked-based NVC, more pronounced NVC alterations occurred under resting-state conditions and were strongest in association regions. Further, calcium and hemodynamic activity reported different accounts of RSFC changes under DOI. Co-administration of DOI and the 5-HT2AR antagonist, MDL100907, reversed many of these effects. Dissociation between neuronal and hemodynamic signals emphasizes a need to consider neurovascular effects of psychedelics when interpreting blood-oxygenation-dependent neuroimaging measures.

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.

Slicing Network for Wide-Field Fluorescence Image Based on the Improved U-Net Model.

Fluorescence imaging stands as a pivotal component in biomedical research, requiring the elimination of out-of-focus background noise resulting from wide-field volumetric illumination of the whole field-of-view and scattering within thick biological tissues. Traditional methods struggle to effectively address varying degrees of defocusing in fluorescence images. This study introduces the utilization of upU-Net, 3D U-Net, and 3D upU-Net as defocusing networks tailored for 2D and 3D wide-field fluorescence images, yielding notable enhancements. These advancements facilitate more economically viable confocal microscopy, delivering significant advantages to biologists presently utilizing wide-field fluorescence microscopy.

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.

Simultaneous mesoscopic measurement and manipulation of mouse cortical activity.

Dynamics of activity across the cerebral cortex at the mesoscopic scale - coordinated fluctuations of local populations of neurons - are essential to perception and cognition and relevant to computations like sensorimotor integration and goal-directed task engagement. However, understanding direct causal links between population dynamics and behavior requires the ability to manipulate mesoscale activity and observe the effect of manipulation across multiple brain regions simultaneously. Here, we develop a novel system enabling simultaneous recording and manipulation of activity across the dorsal cortex of awake mice, compatible with large-scale electrophysiology from any region across the brain. Transgenic mice expressing the GCaMP calcium sensor are injected systemically with an adeno-associated virus driving expression of the ChrimsonR excitatory opsin. This strategy drives expression of the blue-excited calcium indicator, GCaMP, in excitatory neurons and red-excited Chrimson opsin in inhibitory neurons. We demonstrate widefield single-photon calcium imaging and simultaneous galvo-targeted laser stimulation over the entire dorsal cortical surface. The light channels of the imaging and the opsin do not interfere. We characterize the spatial and temporal resolution of the method, which is suitable for targeting specific cortical regions and specific time windows in behavioral tasks. The preparation is stable over many months and thus well-suited for long-term behavioral experiments. This technique allows for studying the effect of cortical perturbations on cortex-wide activity, on subcortical spiking activity, and on behavior, and for designing systems to control cortical activity in closed-loop.

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 (CTMOs) output channels to action progression and hand-mouth coordination. Notably, CTMOs display sustained firing throughout RWD sequence and selectively enhance RWD-relevant activity in postsynaptic thalamus neurons, which also contribute to action coordination. CTMOs 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.