Scott E Fraser

Fluorescent biosensors revolutionized biomedical science by enabling the direct measurement of signaling activities in living cells, yet the current technology is limited in resolution and dimensionality. Here, we introduce highly sensitive chemigenetic kinase activity biosensors that combine the genetically encodable self-labeling protein tag HaloTag7 with bright far-red-emitting synthetic fluorophores. This technology enables five-color biosensor multiplexing, 4D activity imaging, and functional super-resolution imaging via stimulated emission depletion (STED) microscopy.

Fourier light field microscopy (FLFM) captures a sample 3D volume in a single snapshot, providing game-changing imaging speed for various bio-imaging applications. Existing FLFM platforms have often been designed as single-purpose implementations, with a fixed performance in resolution and field of view, restricting widespread applications. Here, to democratize FLFM toward broader adoption for biomedical research, we describe a multi-purpose implementation of FLFM that enables synchronous volumetric imaging across a wide range of resolution and field of view. With our single instrument, we demonstrate a variety of bio-imaging applications across scales, including sub-cellular dynamics, tissue cellular dynamics, and whole-brain neural activity.

Insulin vesicle heterogeneity within the pancreatic β cell has been previously observed, but the extent of this heterogeneity has not been determined due to limitations in organelle separation techniques. There has previously been a lack of unbiased methods for studying differences in organelles, such as the insulin vesicle. Direct current insulator-based dielectrophoresis (DC-iDEP) separates particles based on various biophysical properties without requiring fluorescent labels, characterizing them by their electrokinetic mobility ratio (EKMr). This method has been applied to insulin vesicles isolated from mammalian insulin-secreting cell lines in order to investigate the degree of insulin vesicle heterogeneity, as well as how these subpopulations fluctuate under various cell conditions. There were significant differences in the distribution of insulin vesicles between conditions, suggesting variations in subpopulation …

Understanding the complexity of life requires that it be studied with as many dimensions as possible in each experiment. STELLARIS 8 DIVE multiphoton system enables flexible spectral detection and the combination of lifetime-based information with deep imaging beyond 1 mm.

Precise control of cell division is essential for proper patterning and growth during the development of multicellular organisms. Coordination of formative divisions that generate new tissue patterns with proliferative divisions that promote growth is poorly understood. SHORTROOT (SHR) and SCARECROW (SCR) are transcription factors that are required for formative divisions in the stem cell niche of Arabidopsis roots 1, 2. Here we show that levels of SHR and SCR early in the cell cycle determine the orientation of the division plane, resulting in either formative or proliferative cell division. We used 4D quantitative, long-term and frequent (every 15 min for up to 48 h) light sheet and confocal microscopy to probe the dynamics of SHR and SCR in tandem within single cells of living roots. Directly controlling their dynamics with an SHR induction system enabled us to challenge an existing bistable model 3 of the SHR …

For generations researchers have been observing the dynamic processes of life through the lens of a microscope. This has offered tremendous insights into biological phenomena that span multiple orders of time- and length-scales ranging from the pure magic of molecular reorganization at the membrane of immune cells, to cell migration and differentiation during development or wound healing. Standard fluorescence microscopy techniques offer glimpses at such processes in vitro, however, when applied in intact systems, they are challenged by reduced signal strengths and signal-to-noise ratios that result from deeper imaging. As a remedy, two-photon excitation (TPE) microscopy takes a special place, because it allows us to investigate processes in vivo, in their natural environment, even in a living animal. Here, we review the fundamental principles underlying TPE aimed at basic and advanced microscopy …

During development, cells in multicellular organisms undergo a series of heavily regulated steps that lead to their differentiation into specific cell types. When this process goes wrong–it can lead to diseases like cancer and developmental malformations. Unlike animals, where differentiation stops primarily after embryogenesis, plants grow continuously and have differentiating cells throughout their lifespan. One major differentiation process occurs when a specific stem cell divides formatively into the endodermis and cortex. In the model system-Arabidopsis thaliana, SHORTROOT (SHR) and SCARECROW (SCR) are transcription factors (TFs) that regulate the decision-making process of whether to divide formatively or proliferatively. Using an inducible SHR system, we followed the dynamics of these TFs and observed the cell outcome in long timelapses with high temporal resolution of more than 1000 cells. We …

This invention relates to a hyperspectral imaging system for denoising and/or color unmixing multiple overlapping spectra in a low signal-to-noise regime with a fast analysis time. This system may carry out Hyper-Spectral Phasors (HySP) calculations to effectively analyze hyper-spectral time-lapse data. For example, this system may carry out Hyper-Spectral Phasors (HySP) calculations to effectively analyze five-dimensional (5D) hyper-spectral time-lapse data. Advantages of this imaging system may include:(a) fast computational speed,(b) the ease of phasor analysis, and (c) a denoising algorithm to obtain the minimally-acceptable signal-to-noise ratio (SNR). An unmixed color image of a target may be generated. These images may be used in diagnosis of a health condition, which may enhance a patient's clinical outcome and evolution of the patient's health.

Tuesday, February 13, 2024 411a extend an ODE model of the PRLR signaling network, and to update the model structure. This modeling-imaging pipeline demonstrates the utility and robustness of FCS for capturing the protein dynamics needed for integration into the computational model and provides a powerful, generalizable approach to quantitative biology and cellular signaling networks.

Purpose: This study aims to develop a human retinoblastoma organoid (RBRO) model that recapitulates the cell-of-origin and multi-step retinoblastoma genesis. Background: Retinoblastomas originate from maturing cone photoreceptor precursors with biallelic RB1 inactivation. Rb1 mutant animal models fail to recapitulate retinoblastomagenesis with a cone precursor cell-of-origin, likely due to human-specific cone development features. In explanted fetal retina, pRB-depleted post-mitotic cone precursors proliferate, followed by a 3-5 month premalignant indolence phase before retinoblastoma-like masses emerge at tissue ages similar to retinoblastomas in vivo. However, tissue availability limits research with this disease model. RB1−/- retinal organoids (ROs) provide a potential alternative, as they demonstrate cone proliferation, but they deteriorate before forming indolent premalignant lesions or malignant …

One of the calcium-binding proteins more important in the CNS of vertebrates is the parvalbumin (PV). It regulates the calcium dynamics and its associated functions in the neurons. It is of special interest to study the distribution of PV in this particular group of fishes, the cladistians, since they represent a very interesting evolutionary model at the base of the tree of actinopterygian fish, very close to the common osteichthyan ancestor. In this work we have studied the neuroanatomical distribution of PV in the CNS of polypteriform fishes Erpetoichthys calabaricus and Polypterus senegalus. This approach was carried out using immunohistochemistry techniques. The presence of PV was remarkable in multiple brain areas, such as the olfactory bulb, pallial and subpallial regions, preoptic area, alar and basal hypothalamic regions, prethalamus and thalamus, optic tectum, cerebellum, rhombencephalic tegmentum, and …

Systems and methods are provided for increasing photon collection during imaging with a light-sheet fluorescence microscope. In one example, one or more adaptive optical elements may be positioned in a detection light path between a detection objective and an imaging sensor. A depth of field of the detection objective is adjusted as a function of a thickness of an excitation light-sheet used to illuminate a sample during imaging. As a result, the detection objective captures more fluorescence photons generated by light-sheet excitation.

Study question To classify embryos according to their metabolic fingerprint with a robust, non-invasive methodology Summary answer Our Hyperspectral (HS) phasor analysis coupled to artificial intelligence simultaneously detects 6+ metabolites to profile and classify competent embryos and oocytes safely and consistently. What is known already Metabolism plays a key role in oocyte and embryo developmental competence. However, current methods to measure oocyte and embryo metabolism are either invasive, phototoxic, too slow or based on indirect analysis in cumulus cells or culture media. Some intracellular metabolites are autofluorescent and their concentration can be quantified using HS imaging when using the correct light excitation. HS imaging is an optical technique whereby the full emission spectrum is obtained for each pixel of an image …

Described herein are methods and compositions for genomic editing. Endonucleases for genomic editing involve inducing breaks in double stranded DNA, for which knock-ins are notoriously inefficient for relying on random integration of homologous DNA sequences into the break site by repair proteins. To address these issues, described herein are novel recombinant fusion proteins that actively recruit linear DNA inserts in closer proximity to the genomic cleavage site, increasing integration efficiency of large DNA fragments into the genome. Such improvements to genomic editing technology allow one to use lower linear DNA concentrations without sacrificing efficiency and can be further combined with other features, such as fluorescent protein reporting systems.

BAZ1B is a ubiquitously expressed nuclear protein with roles in chromatin remodeling, DNA replication and repair, and transcription. Reduced BAZ1B expression disrupts neuronal and neural crest development. Variation in the activity of BAZ1B has been proposed to underly morphological and behavioral aspects of domestication through disruption of neural crest development. Knockdown of baz1b in Xenopus embryos and Baz1b loss-of-function (LoF) in mice leads to craniofacial defects consistent with this hypothesis. We generated baz1b LoF zebrafish using CRISPR/Cas9 gene editing to test the hypothesis that baz1b regulates behavioral phenotypes associated with domestication in addition to craniofacial features. Zebrafish with baz1b LoF show mild underdevelopment at larval stages and distinctive craniofacial features later in life. Mutant zebrafish show reduced anxiety-associated phenotypes and an …

Dual-comb interferometry harnesses the interference of two laser frequency combs to provide unprecedented capability in spectroscopy applications. In the past decade, the state-of-the-art systems have reached a point where the signal-to-noise ratio per unit acquisition time is fundamentally limited by shot noise from vacuum fluctuations. To address the issue, we propose an entanglement-enhanced dual-comb spectroscopy protocol that leverages quantum resources to significantly improve the signal-to-noise ratio performance. To analyze the performance of real systems, we develop a quantum model of dual-comb spectroscopy that takes practical noises into consideration. Based on this model, we propose quantum combs with side-band entanglement around each comb lines to suppress the shot noise in heterodyne detection. Our results show significant quantum advantages in the uW to mW power range …

Signal transduction is challenging to investigate because it depends upon both spatial organization of signaling and receptor molecules and alterations of gene expression spanning multiple time and space scales. We argue that mathematical modeling provides the tools to build upon the results from quantitative imaging to analyze and interpret cell signaling across these scales. Quantitative imaging tools such as fluorescence fluctuation spectroscopy methods provide many of the key parameters needed for predictive modeling, including measurements of: protein mobility, oligomerization, and concentrations, as functions of time and space. Comprehensive predictive models can integrate such multi-dimensional and multi-scale data into frameworks that can be used to expand our understanding of biological signaling networks.Here, we present our efforts to combine quantitative imaging and computational …

Summary In functional imaging studies, accurately synchronizing the time course of experimental manipulations and stimulus presentations with resulting imaging data is crucial for analysis. Current software tools lack such functionality, requiring manual processing of the experimental and imaging data, which is error-prone and potentially non-reproducible. We present VoDEx, an open-source Python library that streamlines the data management and analysis of functional imaging data. VoDEx synchronizes the experimental timeline and events (e.g. presented stimuli, recorded behavior) with imaging data. VoDEx provides tools for logging and storing the timeline annotation, and enables retrieval of imaging data based on specific time-based and manipulation-based experimental conditions. Availability and implementation VoDEx is an open-source Python library and can be …

1Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain, 2Molecular Mechanics of the Cardiovascular System, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain. Single-molecule force spectroscopy (SMFS) has provided multiple advancements in the understanding of the free energy landscape of proteins under force. Specific covalent tethering of proteins to surfaces has improved both the yield and the quality of data acquisition in Atomic Force Spectroscopy (AFS) and Magnetic Tweezers (MT). Given the complementary information obtained by AFS and MT, we propose a modular polyprotein system with HaloTag specific covalent tethering to surfaces, in order to enable testing of the same protein preparation by both techniques. The modular design allows to easily include the proteins of interest in the system via SnoopTag/SnoopCatcher and Spy-Tag/SpyCatcher orthogonal …

Fluorescent reporter pluripotent stem cell-derived retinal organoids are powerful tools to investigate cell type-specific development and disease phenotypes. When combined with live imaging, they enable direct and repeated observation of cell behaviors within a developing retinal tissue. Here, we generated a human cone photoreceptor reporter line by CRISPR/Cas9 genome editing of WTC11-mTagRFPT-LMNB1 human induced pluripotent stem cells (iPSCs) by inserting enhanced green fluorescent protein (EGFP) coding sequences and a 2A self-cleaving peptide at the N-terminus of guanine nucleotide-binding protein subunit alpha transducin 2 (GNAT2). In retinal organoids generated from these iPSCs, the GNAT2-EGFP alleles robustly and exclusively labeled immature and mature cones. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of the morphological maturation …

Hyperspectral fluorescence imaging improves multiplexed observations of biological samples by utilizing multiple color channels across the spectral range to compensate for spectral overlap between labels. Typically, spectral resolution comes at a cost of decreased detection efficiency, which both hampers imaging speed and increases photo-toxicity to the samples. Here, we present a high-speed, high-efficiency snapshot spectral acquisition method, based on optical compression of the fluorescence spectra via Fourier transform, that overcomes the challenges of discrete spectral sampling: single-shot hyperspectral phasor camera (SHy-Cam). SHy-Cam captures fluorescence spatial and spectral information in a single exposure with a standard scientific CMOS camera, with photon efficiency of over 80%, easily and with acquisition rates exceeding 30 datasets per second, making it a powerful tool for multi-color in …

METHOD TO GENERATE BIOCOMPATIBLE DENDRITIC POLYMERS FOR ANALYTE DETECTION WITH MULTIMODAL LABELING Ahttps://dav. uspto. gov/prex/index. html# domesticContinuityND SIGNAL AMPLIFICATION

Objective To articulate how Aboriginal community‐controlled art centres support the role of Elders and older people within an ontologically situated, intergenerational model of care. Methods In this paper, we draw on stories (data) generated through interviews involving 75 people associated with three Aboriginal community‐controlled art centres and field notes taken during a Participatory Action Research (PAR) study. The study was undertaken in collaboration with three community‐controlled art centres and two aged care providers over almost 4 years, in diverse Indigenous sovereignties, all located in geographically remote Australian locations. Results Engaging with decolonising and Indigenous theoretical frameworks, our analysis identified three interwoven meta‐themes. These include connection to law and culture; purpose; and healing. Each theme had important subthemes, and all were central to …

Glucose stimulated insulin secretion is mediated by glucose metabolism via oxidative phosphorylation generating ATP that triggers membrane depolarization and exocytosis of insulin. In stressed beta cells, glucose metabolism is remodeled, with enhanced glycolysis uncoupled from oxidative phosphorylation, resulting in the impaired glucose-mediated insulin secretion characteristic of diabetes. Relative changes in glycolysis and oxidative phosphorylation can be monitored in living cells using the 3-component fitting approach of fluorescence lifetime imaging microscopy (FLIM). We engrafted pancreatic islets onto the iris to permit in vivo FLIM monitoring of the trajectory of glucose metabolism. The results show increased oxidative phosphorylation of islet cells (∼90% beta cells) in response to hyperglycemia; in contrast red blood cells traversing the islets maintained exclusive glycolysis as expected in the absence of …

The breaking of bilateral symmetry in most vertebrates is critically dependent upon the motile cilia of the embryonic left-right organizer (LRO), which generate a directional fluid flow; however, it remains unclear how this flow is sensed. Here, we demonstrated that immotile LRO cilia are mechanosensors for shear force using a methodological pipeline that combines optical tweezers, light sheet microscopy, and deep learning to permit in vivo analyses in zebrafish. Mechanical manipulation of immotile LRO cilia activated intraciliary calcium transients that required the cation channel Polycystin-2. Furthermore, mechanical force applied to LRO cilia was sufficient to rescue and reverse cardiac situs in zebrafish that lack motile cilia. Thus, LRO cilia are mechanosensitive cellular levers that convert biomechanical forces into calcium signals to instruct left-right asymmetry.

Machine learning has been used to classify melanoma from 2D dermoscopic images, with diagnosis accuracy comparable to that of dermatologists. However, there are few approaches for reconstructing the Breslow depth of the melanoma, which is a crucial metric for skin cancer diagnosis and stage identification. To estimate the depth of tissue lesions, we propose a multiplexed Diffused Optical Imaging Generative Adversarial Network (mDOI-GAN) solution. mDOI-GAN is composed of two main parts: the reconstruction and the projection sections. The reconstruction section estimates the optimal structure of 3D tissue volume depending on the presented 2D reflectance images. The projection section simulates the light-tissue interaction, generating 2D diffusion reflectance images corresponding to the given composition of 3D tissue volume. These two sections work together to form an optimization loop. The network …

Styrene dyes are useful imaging probes and fluorescent sensors due to their strong fluorogenic responses to environmental changes or binding macromolecules. Previously, indole-containing styrene dyes have been reported to selectively bind RNA in the nucleolus and cytoplasm. However, the application of these indole-based dyes in cell imaging is limited by their moderate fluorescence enhancement and quantum yields, as well as relatively high background associated with these green-emitting dyes. In this work, we have investigated the positional and electronic effects of the electron donor by generating regioisomeric and isosteric analogues of the indole ring. Select probes exhibited large Stokes shifts, enhanced molar extinction coefficients, and bathochromic shifts in their absorption and fluorescence wavelengths. In particular, the indolizine analogues displayed high membrane permeability, strong …

The expansion of fluorescence bioimaging toward more complex systems and geometries requires analytical tools capable of spanning widely varying timescales and length scales, cleanly separating multiple fluorescent labels and distinguishing these labels from background autofluorescence. Here we meet these challenging objectives for multispectral fluorescence microscopy, combining hyperspectral phasors and linear unmixing to create Hybrid Unmixing (HyU). HyU is efficient and robust, capable of quantitative signal separation even at low illumination levels. In dynamic imaging of developing zebrafish embryos and in mouse tissue, HyU was able to cleanly and efficiently unmix multiple fluorescent labels, even in demanding volumetric timelapse imaging settings. HyU permits high dynamic range imaging, allowing simultaneous imaging of bright exogenous labels and dim endogenous labels. This enables …

Systems and methods are provided for multi-spectral or hyper-spectral fluorescence imaging. In one example, a spectral encoding device may be positioned in a detection light path between a detection objective and an imaging sensor of a microscope. In one example, the spectral encoding device includes a first dichroic mirror having a sine transmittance profile and a second dichroic mirror having a cosine transmittance profile. In addition to collecting transmitted light, reflected light from each dichroic mirror is collected and used for total intensity normalization and image analysis.

Convergent results suggest that lateral habenula (LHb) activity reduces reward value and enhances aversive learning. Electrical stimulation of LHb neurons reduces sucrose intake and cocaine/morphine seeking, whereas LHb lesions attenuate taste aversion learning and avoidance of predator odor, retard appetitive extinction, and interfere with appetitive conditioned inhibition training. However, the role of the LHb in consummatory successive negative contrast (cSNC), an animal model of acute anxiety/frustration induced by reward loss, remains unknown. We hypothesized that a surprising reward downshift would enhance activity in the LHb. Three groups of rats received access to sucrose during eleven 5-min sessions. Group 32-2 had access to 32% sucrose for 10 sessions followed by a downshift to 2% sucrose on session 11. Groups 2-2 and 32-32 (unshifted controls) had access to 2% and 32% sucrose …

Developmental studies have revealed the importance of the transcription factor Hand2 in cardiac development. Hand2 promotes cardiac progenitor differentiation and epithelial maturation, while repressing other tissue types. The mechanisms underlying the promotion of cardiac fates are far better understood than those underlying the repression of alternative fates. Here, we assess Hand2-dependent changes in gene expression and chromatin remodeling in cardiac progenitors of zebrafish embryos. Cell-type specific transcriptome analysis shows a dual function for Hand2 in activation of cardiac differentiation genes and repression of pronephric pathways. We identify functional cis-regulatory elements whose chromatin accessibility are increased in hand2 mutant cells. These regulatory elements associate with non-cardiac gene expression, and drive reporter gene expression in tissues associated with Hand2-repressed genes. We find that functional Hand2 is sufficient to reduce non-cardiac reporter expression in cardiac lineages. Taken together, our data support a model of Hand2-dependent coordination of transcriptional programs, not only through transcriptional activation of cardiac and epithelial maturation genes, but also through repressive chromatin remodeling at the DNA regulatory elements of non-cardiac genes.

A new DNA knock-in approach is provided based on the usage of three single guide RNA (sgRNA) to increase the integration efficiency of donor DNA based on the CRISRP-Cas system. The approach uses a pair of universal sgRNAs complementary to the donor DNA and a single sgRNA that targets the locus of interest. In various embodiments, targeting is achieved by pre-forming a DNA: RNA: protein (DNA: RNP) complex in vitro and introducing the complex into the embryo or cells of interest either by microinjection or transfection.

The broad sharing of research data is widely viewed as critical for the speed, quality, accessibility, and integrity of science. Despite increasing efforts to encourage data sharing, both the quality of shared data and the frequency of data reuse remain stubbornly low. We argue here that a significant reason for this unfortunate state of affairs is that the organization of research results in the findable, accessible, interoperable, and reusable (FAIR) form required for reuse is too often deferred to the end of a research project when preparing publications–by which time essential details are no longer accessible. Thus, we propose an approach to research informatics in which FAIR principles are applied continuously, from the inception of a research project and ubiquitously, to every data asset produced by experiment or computation. We suggest that this seemingly challenging task can be made feasible by the adoption of …

Defining the structural and functional changes in the nervous system underlying learning and memory represents a major challenge for modern neuroscience. Although changes in neuronal activity following memory formation have been studied [B. F. Grewe et al., Nature 543, 670–675 (2017); M. T. Rogan, U. V. Stäubli, J. E. LeDoux, Nature 390, 604–607 (1997)], the underlying structural changes at the synapse level remain poorly understood. Here, we capture synaptic changes in the midlarval zebrafish brain that occur during associative memory formation by imaging excitatory synapses labeled with recombinant probes using selective plane illumination microscopy. Imaging the same subjects before and after classical conditioning at single-synapse resolution provides an unbiased mapping of synaptic changes accompanying memory formation. In control animals and animals that failed to learn the task, there …

Light field microscopy uses plen-optic detection and computational reconstruction to record 3D volume in a single snapshot [1]. Recently-proposed Fourier light field microscopy (FLFM) achieves this by placing a lens array at the Fourier plane of the detection path, generating a set of simultaneous multi-view projections that can be efficiently reconstructed to the original 3D sample [2]. Here, we improve the speed and accuracy of the currently available reconstruction package [3] with GPU acceleration and a simple calibration process. We build a FLFM based on a commercial microscope and validate the results using a variety of fixed and live biological samples, toward applications in cancer, cardiac, and neuronal functional imaging.

Fluorescence lifetime imaging microscopy (FLIM) evaluates the metabolic state of tissue based on reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD). Fluorescence lifetime imaging ophthalmoscopy (FLIO) can image the fundus of the eyes, but cannot detect NAD(P)H. We used multiphoton FLIM to study the metabolic state of the retina in fixed eyes of wild-type mice C57BL6/J. We sectioned the eye using a polyacrylamide gel-embedding technique and estimated the percentage of bound NAD(P)H. We found that oxidative phosphorylation was the predominant metabolic state, particularly in the inner retina, when a fixed retina was used. We also demonstrated the feasibility of FAD imaging of the retina. In addition, we demonstrated that autofluorescence and various FLIM channels, such as hemoglobin, melanin and collagen, can be used to evaluate the structure of the retina and other parts of the eye without any special staining.

466a Wednesday, February 23, 2022 on brightness analysis. Our approach was validated with computer simulations and in vitro experiments on artificial oligomerisation of fluorescent proteins. This revealed a surprising effect of anti-GFP nanobody binding on (E) GFP mobility at the plasma membrane, best explained by preferential nanobody binding to a dark oligomer pool. Exploiting the power of a reconstituted supported lipid bilayer system, we investigated the interaction of fluorescently labelled CD40-His and CD40 ligand (CD40L) at the surface of primary T-cells. The combination of sFCS-based brightness-diffusion analysis, in combination with a reconstituted bilayer system, allows dissection of bio-molecular reorganisation and paves the way for future investigations of protein and lipid interactions in more complex settings such as the full physiological context in vivo.

Hyperspectral fluorescence imaging improves multiplexed observations of biological samples by utilizing multiple color channels across the spectral range to compensate for spectral overlap between labels. Typically, spectral resolution comes at a cost of decreased detection efficiency, which both hampers imaging speed and increases photo-toxicity to the samples. Here, we present a high-speed, high-efficiency snapshot spectral acquisition method, based on optical compression of the fluorescence spectra via Fourier transform, that overcomes the challenges of discrete spectral sampling: Singleshot Hyperspectral Phasor Camera (SHy-Cam). SHy-Cam captures fluorescence spatial and spectral information in a single exposure with a standard scientific CMOS camera, with photon efficiency of over 80%, easily and acquisition rates exceeding 30 datasets per second, making it a powerful tool for multi-color in-vivo imaging. Its simple design, using readily available optical components, and its easy integration, provide a low-cost solution for multi-color fluorescence imaging with increased efficiency and speed.

Precise control of cell division is essential for proper patterning and growth during the development of multicellular organisms. Coordination of formative (asymmetric) divisions that generate new tissue patterns with proliferative (symmetric) divisions that promote growth is poorly understood. Here, we employed quantitative 4D light sheet and confocal microscopy to probe in vivo the dynamics of two transcription factors, SHORTROOT (SHR) and SCARECROW (SCR), which are required for asymmetric division in the stem cell niche of Arabidopsis roots [,]. Long-term (up to 48 hours), frequent (every 15 minutes) imaging of the two regulators in tandem in single cells, in conjunction with a SHR induction system, enabled us to challenge an existing bistable model of the SHR/SCR gene regulatory network. By directly controlling SHR and SCR expression dynamics, we were able to identify key features that are essential for rescue of asymmetric division in shr mutants. We show that instead of high stable levels of nuclear SHR and SCR, only low transient levels of expression are required. Nuclear SHR kinetics do not follow predictions of the bistable model, and the regulatory relationship between SHR and SCR can be modeled by monostable alternatives. Furthermore, expression of these two regulators early in the cell cycle determines the orientation of the division plane, resulting in either formative or proliferative cell division. Our findings provide evidence for an uncharacterized mechanism by which developmental regulators directly coordinate patterning and growth.

Publisher Correction: Author Correction: High-contrast, synchronous volumetric imaging with selective volume illumination microscopy - PMC Back to Top Skip to main content NIH NLM Logo Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation Search PMC Full-Text Archive Search in PMC Advanced Search User Guide Journal List Commun Biol v.5; 2022 PMC9142577 Other Formats PDF (311K) Actions Cite Collections Share Permalink Copy RESOURCES Similar articles Cited by other articles Links to NCBI Databases Journal List Commun Biol v.5; 2022 PMC9142577 As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Logo of commbiol Commun Biol. 2022; 5: 533. Published online 2022 …

Interfacial solar-driven steam generation as a cost-effective green technology has shown a great potential in realizing water desalination and purification. To achieve high solar steam generation efficiency, it is important to simultaneously realize high solar absorption and effective water transportation. Among various solar absorbers, Janus structures composed of different layered materials for multiple functions have been demonstrated to be promising for high efficiency. However, the complex material composition and the corresponding multistep fabrication process become a barrier for high efficiency realization and large-scale applications. Developing a simple, low-cost, and fast fabrication process holds the key to producing Janus absorbers with high efficiency. Herein, we propose and demonstrate a monolithic Janus absorber fabricated by a single shot flash reduction of an ultrathin (370 nm) graphene oxide film …

The broad sharing of research data is widely viewed as of critical importance for the speed, quality, accessibility, and integrity of science. Despite increasing efforts to encourage data sharing, both the quality of shared data, and the frequency of data reuse, remain stubbornly low. We argue here that a major reason for this unfortunate state of affairs is that the organization of research results in the findable, accessible, interoperable, and reusable (FAIR) form required for reuse is too often deferred to the end of a research project, when preparing publications, by which time essential details are no longer accessible. Thus, we propose an approach to research informatics that applies FAIR principles continuously, from the very inception of a research project, and ubiquitously, to every data asset produced by experiment or computation. We suggest that this seemingly challenging task can be made feasible by the adoption of simple tools, such as lightweight identifiers (to ensure that every data asset is findable), packaging methods (to facilitate understanding of data contents), data access methods, and metadata organization and structuring tools (to support schema development and evolution). We use an example from experimental neuroscience to illustrate how these methods can work in practice.

A sense of non-symbolic numerical magnitudes is widespread in the animal kingdom and has been documented in adult zebrafish. Here, we investigated the ontogeny of this ability using a group size preference (GSP) task in juvenile zebrafish. Fish showed GSP from 21 days post-fertilization and reliably chose the larger group when presented with discriminations of between 1 versus 3, 2 versus 5 and 2 versus 3 conspecifics but not 2 versus 4 conspecifics. When the ratio between the number of conspecifics in each group was maintained at 1 : 2, fish could discriminate between 1 versus 2 individuals and 3 versus 6, but again, not when given a choice between 2 versus 4 individuals. These findings are in agreement with studies in other species, suggesting the systems involved in quantity representation do not operate separately from other cognitive mechanisms. Rather they suggest quantity processing in fishes …

Fluorescence fluctuation spectroscopy (FFS) offers a window to observe and investigate dynamics of bio-molecular organisation. Furthermore, FFS acquisitions uniquely allow obtaining insights into other properties of the sample such as concentration and brightness (counts per molecule) of the fluorescently labelled species simultaneously. Here, we exploit the large number of measurements and statistics obtained from scanning fluorescence fluctuation spectroscopy (sFFS) acquisitions to elucidate bio-molecular organisation. We demonstrate the versatility and sensitivity of the approach using computer simulations, synthetic biological membranes and living cells.

The avian egg is a closed system that protects the growing embryo from external factors but prevents direct observation of embryo development. Various culture systems exist in the literature to study the development of the embryo for short periods of incubation (from 12 h up to a maximum of 60 h of egg incubation). A common flaw to these culture techniques is the inability to culture the unincubated avian blastoderm with intact tissue tensions on its native yolk. The goal of this work is to create a unique novel egg-in-cube system that can be used for long-term quail embryo culture initiated from its unincubated blastoderm stage. The egg-in-cube acts as an artificial transparent eggshell system that holds the growing embryo, making it amenable to microscopy. With the egg-in-cube system, quail embryos can be grown up to 9 days from the unincubated blastoderm (incubated in air, 20.9% O2), which improves to 15 days on switching to a hyperoxic environment of 60% O2. Using transgenic fluorescent quail embryos in the egg-in-cube system, cell movements in the unincubated blastoderm are imaged dynamically using inverted confocal microscopy, which has been challenging to achieve with other culture systems. Apart from these observations, several other imaging applications of the system are described in this work using transgenic fluorescent quail embryos with upright confocal or epifluorescence microscopy. To demonstrate the usefulness of the egg-in-cube system in perturbation experiments, the quail neural tube is electroporated with fluorescent mRNA “in cubo”, followed by the incubation of the electroporated embryo and microscopy of the …

Genetic mutations have long been recognized as drivers of cancer drug resistance, but recent work has defined additional non-genetic mechanisms of plasticity, wherein cancer cells assume a drug resistant phenotype marked by altered epigenetic and transcriptional states. Currently, little is known about the real-time, dynamic nature of this phenotypic shift. Using a bladder cancer model of nongenetic plasticity, we discovered that rapid transition to drug resistance entails upregulation of mitochondrial gene expression and a corresponding metabolic shift towards the tricarboxylic acid cycle and oxidative phosphorylation. Based on this distinction, we were able to track cancer cell metabolic profiles in real time using fluorescence lifetime microscopy (FLIM). We observed single cells transitioning spontaneously to an oxidative phosphorylation state over hours to days, a trend that intensified with exposure to cisplatin …

An ability to estimate quantities, such as the number of conspecifics or the size of a predator, has been reported in vertebrates. Fish, in particular zebrafish, may be instrumental in advancing understanding of magnitudes cognition. We review here the behavioural studies that have described the ecological relevance of quantity estimation in fish and the current status of the research aimed at investigating the neurobiological bases of these abilities. By combining behavioural methods with molecular genetics and calcium imaging an involvement of the retina and optic tectum has been documented for estimation of continuous quantities in the larval and adult zebrafish brain, and a contribution of the thalamus and dorso-central pallium for discrete magnitude estimation in the adult zebrafish brain. Evidence for basic circuitry can now be complemented and extended to research that make use of transgenic lines to deepen our understanding of quantity cognition at genetic and molecular levels.

Visualizing cell shapes and interactions of differentiating cells is instrumental for understanding organ development and repair. Across species, strategies for stochastic multicolour labelling have greatly facilitated in vivo cell tracking and mapping neuronal connectivity. Yet integrating multi-fluorophore information into the context of developing zebrafish tissues is challenging given their cytoplasmic localization and spectral incompatibility with common fluorescent markers. Inspired by Drosophila Raeppli, we developed FRaeppli (Fish-Raeppli) by expressing bright membrane- or nuclear-targeted fluorescent proteins for efficient cell shape analysis and tracking. High spatiotemporal activation flexibility is provided by the Gal4/UAS system together with Cre/lox and/or PhiC31 integrase. The distinct spectra of the FRaeppli fluorescent proteins allow simultaneous imaging with GFP and infrared subcellular reporters or …

Author Correction: High-contrast, synchronous volumetric imaging with selective volume illumination microscopy - PMC Back to Top Skip to main content NIH NLM Logo Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation Search PMC Full-Text Archive Search in PMC Advanced Search User Guide Journal List Commun Biol v.5; 2022 PMC9001636 Other Formats PubReader PDF (312K) Actions Cite Collections Share Permalink Copy RESOURCES Similar articles Cited by other articles Links to NCBI Databases Journal List Commun Biol v.5; 2022 PMC9001636 As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Logo of commbiol Commun Biol. 2022; 5: 363. Published online 2022 Apr 11. doi…

The sensitivity of the αβ T cell receptor (TCR) is enhanced by the coreceptors CD4 and CD8αβ, which are expressed primarily by cells of the helper and cytotoxic T cell lineages, respectively. The coreceptors bind to major histocompatibility complex (MHC) molecules and associate intracellularly with the Src-family kinase Lck, which catalyzes TCR phosphorylation during receptor triggering. Although coreceptor/kinase occupancy was initially believed to be high, a recent study suggested that most coreceptors exist in an Lck-free state, and that this low occupancy helps to effect TCR antigen discrimination. Here, using the same method, we found instead that the CD4/Lck interaction was stoichiometric (~100%) and that the CD8αβ/Lck interaction was substantial (~60%). We confirmed our findings in live cells using fluorescence cross-correlation spectroscopy (FCCS) to measure coreceptor/Lck codiffusion in situ. After …

Purpose: To study retinal mouse metabolic states ex-vivo using novel tissue preparation and fluorescent lifetime imaging microscopy (FLIM).Methods: Wild type C57BL/6J mice were included in the study. Following euthanasia and enucleation, corneas were removed, and eyecups were fixed overnight in a 4% Paraformaldehyde (PFA) solution. The next day, eyecups were embedded in polyacrylamide, and 200 μm thick retinal sections were made using a vibratome. Images were taken using the Leica SP8 DIVE FALCON for multiphoton FLIM measurements of metabolic states and tissue structure, using the phasor approach to FLIM analysis. Tissue excitation was performed with 740 nm light from our ultrafast tunable laser at~ 500 μW power. FLIM was collected simultaneously on two hybrid detectors from~ 425-475 nm for NADH and~ 530-650 nm for FAD, retinoids and hemoglobin.Results: The percentage of bound …

Light-sheet microscopes must compromise among field of view, optical sectioning, resolution, and detection efficiency. High-numericalaperture (NA) detection objective lenses provide higher resolution, but their narrow depth of field inefficiently captures the fluorescence signal generated throughout the thickness of the illumination light sheet when imaging large volumes. Here, we present ExD-SPIM (extended depth-of-field selective-plane illumination microscopy), an improved light-sheet microscopy strategy that solves this limitation by extending the depth of field (DOF) of high-NA detection objectives to match the thickness of the illumination light sheet. This extension of the DOF uses a phase mask to axially stretch the point-spread function of the objective lens while largely preserving lateral resolution. This matching of the detection DOF to the illumination-sheet thickness increases the total fluorescence collection …

We found a region of the zebrafish pallium that shows selective activation upon change in the numerosity of visual stimuli. Zebrafish were habituated to sets of small dots that changed in individual size, position, and density, while maintaining their numerousness and overall surface. During dishabituation tests, zebrafish faced a change in number (with the same overall surface), in shape (with the same overall surface and number), or in size (with the same shape and number) of the dots, whereas, in a control group, zebrafish faced the same stimuli as during the habituation. Modulation of the expression of the immediate early genes c-fos and egr-1 and in situ hybridization revealed a selective activation of the caudal part of the dorso-central division of the zebrafish pallium upon change in numerosity. These findings support the existence of an evolutionarily conserved mechanism for approximate magnitude and …

Domestication is associated with both morphological and behavioural phenotypic changes that differentiate domesticated species from their wild counterparts. Some of the traits are those purposely targeted by the selection process, whilst others co-occur as a result of selection. The combination of traits is referred to as the ‘domestication syndrome’ and their shared characteristics has given rise to the neural crest domestication syndrome (NCDS) hypothesis. According to this hypothesis, the phenotypic changes are a consequence of a selection towards animals with mild underdevelopment of the neural crest. A similar mechanism is suggested to affect some species of “self-domesticated” animals, including humans. Recent research supports a role for BAZ1B, one of the haplo-insufficient genes in Williams syndrome (WS), in the evolution of craniofacial features of modern humans. Interestingly, WS recapitulates some of the traits observed in the domestication syndrome, including hypersociability and reduced facial bones. However, the evidence linking BAZ1B to behavioural phenotypes associated with domestication is presumptive. Here, we use zebrafish as a model to test the hypothesis that baz1b loss-of-function leads to both morphological and behavioural phenotypes associated with the domestication syndrome by influencing the development of the neural crest. Our research provides further evidence supporting the NCDS hypothesis and bazlb’s role in this process.

Hyperpsectral fluorescence imaging has been gaining its popularity in life-science field for its simultaneous multiplexing capability of multiple fluorescent labels. Traditional diffraction grating-based hyperspectral acquisition has limited photon-throughput due to the loss at the diffractive optics. The uniform spectral sampling using multiple narrow spectral bands also limits the detectable photons for each channel, which limits the imaging speed as longer exposure is required to achieve sufficient signal to noise ratios. Here we present a Fourier transform-based spectral sampling strategy based on high efficiency dichroic mirrors, enabling video-speed snapshot acquisition with the capability of multiplexing more than five fluorescent signatures.

The availability and the cost of 3D imaging systems are still a problem nowadays, which brings us to the need and urgency of a new way to democratize optical biopsy. Inspired by structural illumination and diffuse optical imaging, we propose a 3D-multiplexed diffused optical imaging (3D-mDOI) solution, a technique to reconstruct the 3D optical properties of the tissue from 2D diffuse images and estimate the depth of tissue lesions. 3D-mDOI uses a low-cost and contact-free design of the imaging acquisition platform, integrating a digital micromirror device (DMD) and an infrared-enhanced CCD camera. The imaging setup that creates custom sampling patterns for tissue photon migration enables spatial multiplexing to overcome low photon signals. We design a hybrid reconstruction pipeline for harvesting the benefits from existing mathematical solutions. The analytical solution of the steady-state radiation transfer …

Introduction There are approximately 90 Aboriginal and Torres Strait Islander community controlled art centres across Australia, the majority in geographically remote locations. This survey explored how these centres are supporting older people, including people living with dementia, if and how they are collaborating with aged care services and what challenges and opportunities they identify in these arrangements. Methods An online survey was developed by a team of Aboriginal and non-Aboriginal researchers, art centre staff and art centre consultants. The survey was distributed in 2018 to art centres across Australia via their four art centre peak bodies: Desart; the Association of Northern, Kimberley and Arnhem Aboriginal Artists; the Indigenous Art Centre Alliance - Far North Queensland and Torres Strait Islands; and the Aboriginal Art Centre Hub - Western Australia. The survey was also conducted face-toface …

Volumetric imaging with selective volume illumination (SVI) using light field detection is provided using various systems and techniques. A volumetric imaging apparatus includes a light source configured to emit an illumination light that propagates via an illumination light path to illu minate a three-dimensional (3D) sample; and an optical system arranged with respect to the light source to receive a light field, which comes from the illuminated 3D sample.The light field propagates via a detection light path, and the light source, the optical system, or both; are configurable to perform SVI, which selects a volume of a 3D-confined illumination of the 3D sample based on the 3D sample to be illuminated and a light field detection (LFD) process to be applied. Further, the volume of the 3D-confined illumination is a selected 3D volume of the 3D sample to be particularly

Pancreatic β-cells respond to increased extracellular glucose levels by initiating a metabolic shift. That change in metabolism is part of the process of glucose-stimulated insulin secretion and is of particular interest in the context of diabetes. However, we do not fully understand how the coordinated changes in metabolic pathways and metabolite products influence insulin secretion. In this work, we apply systems biology approaches to develop a detailed kinetic model of the intracellular central carbon metabolic pathways in pancreatic β-cells upon stimulation with high levels of glucose. The model is calibrated to published metabolomics datasets for the INS1 823/13 cell line, accurately capturing the measured metabolite fold-changes. We first employed the calibrated mechanistic model to estimate the stimulated cell’s fluxome. We then used the predicted network fluxes in a data-driven approach to build a partial least squares regression model. By developing the combined kinetic and data-driven modeling framework, we gain insights into the link between β-cell metabolism and glucose-stimulated insulin secretion. The combined modeling framework was used to predict the effects of common anti-diabetic pharmacological interventions on metabolite levels, flux through the metabolic network, and insulin secretion. Our simulations reveal targets that can be modulated to enhance insulin secretion. The model is a promising tool to contextualize and extend the usefulness of metabolomics data and to predict dynamics and metabolite levels that are difficult to measure in vitro. In addition, the modeling framework can be applied to identify, explain, and …

In utero MR microscopy of developing mouse embryos is complicated by maternal respiratory motion and by the general lack of tissue contrast between embryonic tissues, particularly in the CNS. We explore here the use of volumetric diffusion-weighted MR microscopy to visualize the embryonic brain in utero at later development stages.

Implementations of the present disclosure include methods, systems, and computer-readable storage mediums for image reconstruction. Actions include receiving an image acquired by an endoscopic system including an optical fiber bundle with multiple optical fibers, each optical fiber being sur rounded by cladding, determining in the image fiber core locations corresponding to the optical fibers, reconstructing missing information from the image using interpolation performed in accordance with the fiber core locations, the missing information corresponding to artifacts in the acquired image that result from the cladding, and providing a fiber-pattern removed image, in which the artifacts in the acquired image are removed using the missing information.

Fluorescence lifetime imaging microscopy (FLIM) with phasor analysis provides easy visualization and analysis of fluorophores’ lifetimes which is valuable for multiple applications including metabolic imaging, STED imaging, FRET imaging and functional imaging. However, FLIM imaging typically suffers from low photon budgets, leading to unfavorable signal to noise ratios which in many cases prevent extraction of information from the data. Traditionally, median filters are applied in phasor analysis to tackle this problem. This unfortunately degrades high spatial frequency FLIM information in the phasor analysis. These high spatial frequency components are typically edges of features and puncta, which applies to membranes, mitochondria, granules and small organelles in a biological sample. To tackle this problem, we propose a filtering strategy with complex wavelet filtering and Anscombe transform for FLIM …

Increasing evidence of new-onset diabetes during the COVID19 pandemic indicates that the SARS-CoV2 virus may drive beta-cell dysfunction leading to diabetes, but it is unclear if it is a primary or secondary effect. Here, we present evidence of SARS-CoV-2 infection of pancreatic beta cells in vivo using a robust and reproducible non-human primates model of mild to moderate COVID19 pathogenesis. Pancreas from SARS-CoV-2 infected subjects were positive for the SARS-CoV2 spike protein by immunohistochemistry and structures indicative of viral replication were evident by electron microscopy. Total beta cell area was decreased in SARS-CoV-2-infected pancreas, attributable to beta cell atrophy. Beta cell granularity was decreased. These histologic phenotypes persisted beyond the duration of the clinical disease course. Detailed electron microscopy of SARS-CoV-2 infected beta-cells revealed …

Art Centres Keep our Elders Connected — Charles Darwin University Skip to main navigation Skip to search Skip to main content Charles Darwin University Home Charles Darwin University Logo Home Profiles Research Outputs Datasets Organisational Units Student theses Projects Search by expertise, name or affiliation Art Centres Keep our Elders Connected Paulene Mackell, Melissa Lindeman, Roslyn Malay, Maree Meredith, Scott Fraser, Jessica Cecil, Kathryn Squires, Lynley Nargoodah, Chrischona Schmidt, Michelle Young, Margaret Smith, Briony Dow Social Work Nursing Research output: Other contribution - including Text-based ERA-eligible Creative Works › Other types of outputs Overview Original language English Type Short Film Media of output Film Publication status Published - 2021 Access to Document https://www.sbs.com.au/ondemand/tv-program/arts-centres-keep-our-elders-connected/…

Type 2 diabetes is characterized by β and α cell dysfunction. We used phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) to monitor oxidative phosphorylation and glycolysis in living islet cells before and after glucose stimulation. In healthy cells, glucose enhanced oxidative phosphorylation in β cells and suppressed oxidative phosphorylation in α cells. In Type 2 diabetes, glucose increased glycolysis in β cells, and only partially suppressed oxidative phosphorylation in α cells. FLIM uncovers key perturbations in glucose induced metabolism in living islet cells and provides a sensitive tool for drug discovery in diabetes.

During adolescence, the lungs expand while alveoli increase greatly in number. A precise and deep understanding of the biological processes responsible for the many fold increase in alveoli during adolescence could form the foundation for novel treatments to reoptimize lung function after disease or injury. Herein we provide a unique insight into the alveolar growth in the mouse lung during first postnatal 4 weeks herein described as the adolescence phase. Using 3-Dimensionnal (3D) high-resolution large field of view optical imaging coupled with qualitative and quantitative analysis, we suggest the bulk of alveolarization during adolescent lung growth occurs while the surface to volume of the acinar volume remains constant. The most distal 10 or more bifurcations within the acinar saccular region, comprised of small ducts and alveoli, are of similar scale. We explored the connections between the growth of lung and it’s structural and functional parameters, by comparing the correlations between alveolar size, surface area of gas exchange regions, breathing rate, metabolic rate, and oxygen demand across mammalian species which exhibit widely divergent body sizes.

Non-symbolic number cognition based on an approximate sense of magnitude has been 70 documented in adult zebrafish. Here we investigated the ontogeny of this ability using 71 a group size preference task in juvenile zebrafish. Fish showed group size preference 72 from 26 days post fertilization (dpf) and from 27 dpf fish reliably chose the larger group 73 when presented with discrimination ratios from 1: 8 to 2: 3. When the ratio between the 74 number of conspecifics in each group was maintained at 1: 2, fish could discriminate 75 between 1 vs. 2 individuals and 3 vs. 6, but not when given a choice between 2 vs. 4 76 individuals. These findings suggest that the systems involved in numerosity 77 representation in fish do not operate separately from other cognitive mechanisms. 78 Rather they suggest numerosity processing is the result of an interplay between 79 attentional, cognitive and memory-related mechanisms that orchestrate numerical 80 competence both in humans and animals. Our results emphasise the potential of the use 81 of zebrafish to explore the genetic and neural processes underlying the ontogeny of 82 number cognition. 83

Methods for fabricating flexible substrate nanostructured devices are disclosed. The nanostructures comprise nano-pillars and metallic bulbs or nano-apertures. The nanostructures can be functionalized to detect biological entities. The flexible substrates can be rolled into cylindrical tubes for detection of fluidic samples.

The performance of light-field microscopy is improved by selectively illuminating the relevant subvolume of the specimen with a second objective lens. Here we advance this approach to a single-objective geometry, using an oblique one-photon illumination path or two-photon illumination to accomplish selective-volume excitation. The elimination of the second orthogonally oriented objective to selectively excite the volume of interest simplifies specimen mounting; yet, this single-objective approach still reduces the out-of-volume background, resulting in improvements in image contrast, effective resolution, and volume reconstruction quality. We validate our new, to the best of our knowledge, approach through imaging live developing zebrafish, demonstrating the technology’s ability to capture imaging data from large volumes synchronously with high contrast while remaining compatible with standard microscope …

Organelle heterogeneity and inter-organelle associations within a single cell contribute to the limited sensitivity of current organelle separation techniques, thus hindering organelle subpopulation characterization. Here we use direct current insulator-based dielectrophoresis (DC-iDEP) as an unbiased separation method and demonstrate its capability by identifying distinct distribution patterns of insulin vesicles from pancreatic β-cells. A multiple voltage DC-iDEP strategy with increased range and sensitivity has been applied, and a differentiation factor (ratio of electrokinetic to dielectrophoretic mobility) has been used to characterize features of insulin vesicle distribution patterns. We observed a significant difference in the distribution pattern of insulin vesicles isolated from glucose-stimulated cells relative to unstimulated cells, in accordance with functional maturation of vesicles upon glucose stimulation, and interpret this to be indicative of high-resolution separation of vesicle subpopulation. DC-iDEP provides a path for future characterization of subtle biochemical differences of organelle subpopulations within any biological system.

The gold standard of molecular pathogen detection is the quantitative polymerase chain reaction (qPCR). Modern qPCR instruments are capable of detecting 4–6 analytes in a single sample: one per optical detection channel. However, many clinical applications require multiplexing beyond this traditional single-well capacity, including the task of simultaneously testing for SARS-CoV-2 and other respiratory pathogens. This can be addressed by dividing a sample across multiple wells, or using technologies such as genomic sequencing and spatial arrays, but at the expense of significantly higher cost and lower throughput compared with single-well qPCR. These trade-offs represent unacceptable compromises in high-throughput screening scenarios such as SARS-CoV-2 testing. We demonstrate a novel method of detecting up to 20 targets per well with standard qPCR instrumentation: high-definition PCR (HDPCR …

Canonical roles for macrophages in mediating the fibrotic response after a heart attack include extracellular matrix turnover and activation of cardiac fibroblasts to initiate collagen deposition. Here we reveal that macrophages directly contribute collagen to the forming post-injury scar. Unbiased transcriptomics shows an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. Adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse GFPtpz-collagen donors, enhances scar formation via cell autonomous production of collagen. In zebrafish, the majority of tagged collagen localises proximal to the injury, within the overlying epicardial region, suggesting a possible distinction between macrophage-deposited collagen and that predominantly laid-down by myofibroblasts. Macrophage-specific targeting of col4a3bpa and cognate col4a1 in zebrafish …

Metabolism is a key regulator of hematopoietic stem cell (HSC) functions. There is a lack of real-time, non-invasive approaches to evaluate metabolism in single HSCs. Using fluorescence lifetime imaging microscopy, we developed a set of metabolic optical biomarkers (MOBs) from the auto-fluorescent properties of metabolic coenzymes NAD(P)H and FAD. The MOBs revealed the enhanced glycolysis, low oxidative metabolism, and distinct mitochondrial localization of HSCs. Importantly, the fluorescence lifetime of enzyme-bound NAD(P)H (τbound) can non-invasively monitor the glycolytic/lactate dehydrogenase activity in single HSCs. As a proof of concept for metabolism-based cell sorting, we further identified HSCs within the Lineage-cKit+Sca1+ (KLS) hematopoietic stem/progenitor population using MOBs and a machine-learning algorithm. Moreover, we revealed the dynamic changes of MOBs, and the …

Light-sheet microscopy offers faster imaging and reduced phototoxicity in comparison to conventional point-scanning microscopy, making it a preferred technique for imaging biological dynamics for durations of hours or days. Such extended imaging sessions pose a challenge, as it reduces the number of specimens that can be imaged in a given day. Here, we present a versatile light-sheet imaging instrument that combines two independently controlled microscope-twins, built so that they can share an ultrafast near-infrared laser and a bank of continuous-wave visible lasers, increasing the throughput and decreasing the cost. To permit a wide variety of specimens to be imaged, each microscope-twin provides flexible imaging parameters, including (i) operation in one-photon and/or two-photon excitation modes,(ii) delivery of one to three light-sheets via a trio of orthogonal excitation arms,(iii) sub-micron to micron …

This disclosure relates to the field of Optical Coherence Tomography (OCT). This disclosure particularly relates to an OCT system that generates an image with improved quality. In one example, the OCT system may generate an improved Bscan image by using multiple shaping functions to shape the raw A-scans. In another example, the OCT system may generate the improved B-scan image by forming multiple apodization patterns on a detector and acquiring raw A-scans by using the apodization patterns. A better diagnosis of a health condition may be reached by using the improved images generated by the OCT system of this disclosure.

A more complete understanding of biological processes at cellular and molecular levels requires the ability to study them in time and space. Such observation became increasingly easy with the use of genetically encoded fluorescent markers, such as GFP, and the latest developments in optical microscopy. This combination of tools permits the structure and function of biological tissues to be imaged in zebrafish non-invasively. For example, it has become possible to image cardiac structure when GFP is fused to cytoskeletal elements, such as alpha-catenin; similarly, neural activity can be followed when genetically encoded calcium sensors are used to eavesdrop on sets of neurons. Such intravital imaging has furthered our understanding of the relationships between cardiac structure and function, as well as between neuronal activity patterns and complex behaviors such as sleep. Imaging these processes in 3 dimensions–at whole tissue scale and at subcellular resolution–is challenging to perform at sufficient speeds to capture the dynamics under study. Here we review some emerging approaches that combine the speed of Light Sheet microscopy with sets of computational image processing and image analysis tools, offering clear paths to overcome these challenges. We will take advantage of the zebrafish as an excellent system for imaging, and offer examples drawn from recent

The hydraulic conductivity of the vitreous humor has been measured for the bovine eye. The experiment was carried out by placing the vitreous within an upright cylindrical chamber, open at both ends, and letting its liquid content drain out of the bottom opening by gravity through a 20-μm nylon mesh filter. Additional negative pressure was provided at the exit by a hanging drainage tube. The diminishing vitreous volume was measured in terms of the height in the chamber and recorded as a function of time. The reduction in the vitreous liquid content also caused the hydraulic conductivity to reduce, and this parameter was quantified on the basis of previously developed theories of fibrous porous media that have been very well established. A theoretical model with a fully analytical expression for the vitreous volume undergoing drainage was developed and used as a least-squares best fit to deliver the initial hydraulic …

Devices, systems, and methods for detection of an analyte in a sample are disclosed. In some embodiments, an optical sensor can include a metallic layer and a plurality of dielectric pillars extending through the metallic layer. A plurality of regions of concentrated light can be supported in proximity to the ends of the plurality of dielectric pillars when a surface of the metallic layer is illuminated. Concentrated light within one or more of these regions can interact with an analyte molecule, allowing for detection of the analyte.

Microbubbles acting as lenses are interesting for optical and photonic applications such as volumetric displays, optical resonators, integration of photonic components onto chips, high-resolution spectroscopy, lithography, and imaging. However, stable, rationally designed, and uniform microbubbles on substrates such as silicon chips are challenging because of the random nature of microbubble formation. We describe the fabrication of elastic microbubbles with a precise control of volume and curvature based on femtosecond laser irradiated graphene oxide. We demonstrate that the graphene microbubbles possess a near-perfect curvature that allows them to function as reflective microlenses for focusing broadband white light into an ultrahigh aspect ratio diffraction-limited photonic jet without chromatic aberration. Our results provide a pathway for integration of graphene microbubbles as lenses for nanophotonic …

Gold nanoparticles continue to generate interest for use in several biomedical applications. Recently, researchers have been focusing on exploiting their dual diagnostic/therapeutic theranostic capabilities. Before clinical translation can occur, regulatory agencies will require a greater understanding of their biodistribution and safety profiles post administration. Previously, the real-time identification and tracking of gold nanoparticles in free-flowing vasculature had not been possible without extrinsic labels such as fluorophores. Here, we present a label-free imaging approach to examine gold nanoparticle (AuNP) activity within the vasculature by utilizing multiphoton intravital microscopy. This method employs a commercially available multiphoton microscopy system to visualize the intrinsic luminescent signal produced by a multiphoton absorption-induced luminescence effect observed in single gold nanoparticles at frame rates necessary for capturing real-time blood flow. This is the first demonstration of visualizing unlabeled gold nanoparticles in an unperturbed vascular environment with frame rates fast enough to achieve particle tracking. Nanoparticle blood concentration curves were also evaluated by the tracking of gold nanoparticle flow in vasculature and verified against known pre-injection concentrations. Half-lives of these gold nanoparticle injections ranged between 67 and 140 s. This label-free imaging approach could provide important structural and functional information in real time to aid in the development and effective analysis of new metallic nanoparticles for various clinical applications in an unperturbed environment, while …

Hyperspectral fluorescence imaging is gaining popularity for it enables multiplexing of spatio-temporal dynamics across scales for molecules, cells and tissues with multiple fluorescent labels. This is made possible by adding the dimension of wavelength to the dataset. The resulting datasets are high in information density and often require lengthy analyses to separate the overlapping fluorescent spectra. Understanding and visualizing these large multi-dimensional datasets during acquisition and pre-processing can be challenging. Here we present Spectrally Encoded Enhanced Representations (SEER), an approach for improved and computationally efficient simultaneous color visualization of multiple spectral components of hyperspectral fluorescence images. Exploiting the mathematical properties of the phasor method, we transform the wavelength space into information-rich color maps for RGB display …

Evidence has shown that a variety of vertebrates, including fish, can discriminate collections of visual items on the basis of their numerousness using an evolutionarily conserved system for approximating numerical magnitude (the so-called Approximate Number System, ANS). Here we combine a habituation/dishabituation behavioural task with molecular biology assays to start investigating the neural bases of the ANS in zebrafish. Separate groups of zebrafish underwent a habituation phase with a set of 3 or 9 small red dots, associated with a food reward. The dots changed in size, position and density from trial to trial but maintained their numerousness, and the overall areas of the stimuli was kept constant. During the subsequent dishabituation test, zebrafish faced a change (i) in number (from 3 to 9 or vice versa with the same overall surface), or (ii) in shape (with the same overall surface and number), or (iii) in …

We present a novel approach for imaging the beating embryonic heart, based on combining two independent imaging channels to capture the full spatio-temporal information of the moving 3D structure. High-resolution, optically-sectioned image recording is accompanied by simultaneous acquisition of low-resolution, whole-heart recording, allowing the latter to be used in post-acquisition processing to determine the macroscopic spatio-temporal phase of the heart beating cycle. Once determined, or 'stamped', the phase information common to both imaging channels is used to reconstruct the 3D beating heart. We demonstrated our approach in imaging the beating heart of the zebrafish embryo, capturing the entire heart over its full beating cycle, and characterizing cellular dynamic behavior with sub-cellular resolution.

(57) ABSTRACT A sclera or contact lens with an image of an eye that is fenestrated, or uniformly speckled with many sub-millimeter sized transparent regions or holes, is disclosed. The micro fenestrated contact lens can be worn on a strabismic, or misaligned, eye so that its image is aligned with the wearer's dominant eye. The fenestrations allow the wearer to see through the opaque or translucent image printed on the contact lens, thus allowing binocular vision even though his or her strabismic eye is covered.

Cell lineage relationships play a necessary role in the establishment of regional gene activity in sea urchin embryos. Asynchronous unequal cleavages characterize the vegetal half of the sea urchin embryo and the motile nature of these cells renders more difficult analysis of the postgastrular diversification of these vegetal plate cell types which contribute three of the five embryonic tissue territories. A comparison of the unique ectodermal contributions of each macromere with labelled pigment cells and subectodermal strand cells reveals no radial asymmetry to either the origin or final position of these cell types. The contribution of macromere progeny to the coelomic pouches is asymmetrical in that the oral macromere contributes twice the progeny as the other three macromeres and contributes to both pouches as opposed to a single pouch for the other three.