Light Sheet Fluorescence Microscopy Research Articles

Abstract 12907: Aging Drives Cerebrovascular Network Remodeling and Functional Changes in the Mouse Brain

Introduction: Cerebrovascular dysfunction has been implicated in age-related cognitive decline and dementia, but the underlying vascular mechanisms are not well understood. An improved understanding of the nature of normal cerebrovascular aging is needed to help to establish the role that vascular dysfunction might play in cognitive decline and dementia. Methods: Here, we asked how normal aging differentially impacts the vascular structure and function in different brain areas in mice. We investigated structural changes in aged cerebrovascular networks and pericytes utilizing serial two-photon tomography (STPT). To further investigate potential remodeling of different vascular compartments and pericyte subtypes, we utilized tissue clearing, 3D immunolabeling, and light sheet fluorescence microscopy (LSFM) imaging. We also assessed how healthy aging impacts brain hemodynamics in response to voluntary locomotion and whisker stimulation in awake, head-fixed mice using wide field optical signal imaging and two-photon imaging. We tested mice of both sexes at 2-month, 18-month (early aging), and 24-month (late aging) of age. Results: Whole-brain vascular tracing using STPT showed an overall ~10% decrease in vascular length and branching density, and LSFM imaging with 3D immunolabeling further revealed increased arteriole tortuosity in aged brains. We also uncovered a selective vascular and pericyte loss in deep cortical layers, basal forebrain regions, and the hippocampal network. This may contribute to their regional vulnerabilities in neurodegenerative disorders. Moreover, our in vivo imaging in awake, head-fixed mice identified delayed neurovascular coupling response and inefficient oxygen delivery in aged brains. Conclusions: Our study reveals aging-related brain-wide changes in vascular and mural cell types that can explain vulnerability and resilience of different brain areas in normal aging. Moreover, we identified an age-related decrease in brain oxygenation and delayed neurovascular coupling responses which can be linked with cognitive impairment in aged brains. These aging-related changes will serve as a common factor to understand many neurodegenerative disorders and cognition decline in the elderly population.

Abstract 13022: Myocardial Restoration and Functional Recovery Through Transplantation of Vascularized Cardiac Microtissues Derived From Human Induced Pluripotent Stem Cells in a Rat Model of Myocardial Infarction

Introduction: Cardiac regenerative therapy is acknowledged as a promising strategy for treating individuals suffering from severe heart failure. To address the current constraints of stem cell therapy, which primarily relies on indirect paracrine effects for functional improvement, the ideal goal would be to restore the damaged myocardium in the diseased heart. To achieve this, the transplantation of biomimetic heart tissues that closely resemble the natural structure of the heart is highly anticipated. Methods: We successfully generated 3-dimensional vascularized cardiac microtissues (VCMs) using human induced pluripotent stem cells (hiPSCs) through dynamic culture on thin cardiac tissue sheets (CTSs) composed of hiPSC-derived cardiomyocytes and vascular cells. These VCMs exhibited a robust vascular network comprising human vascular cells. Subsequently, we transplanted CTSs (n=6) or VCMs (n=6) onto an athymic rat model of myocardial infarction (MI), while a sham operation was performed on another group (n=6). To assess cardiac function, we employed cardiac magnetic resonance imaging. The survival of grafts, extension of fibrotic scars, neovascularization in the MI border zone, and the formation of a human vascular network within the engrafted region were evaluated through histopathology and light sheet fluorescence microscopy with tissue clearing techniques. Results: Functional analyses demonstrated that the VCM group exhibited the highest left ventricular ejection fraction (Sham: 36.5±0.9%, CTS: 43.5±2.9%, VCM: 55.2±2.5%, p < 0.01). Histopathological evaluation revealed that the VCM group had a larger engraftment area, along with the smallest scar extension and the highest vascular density in the myocardial infarction border zone. Furthermore, LSFM revealed the presence of vascular networks originating from human cells within the engrafted VCMs. Conclusions: The transplantation of hiPSC-derived VCMs demonstrated remarkable therapeutic potential in a rat model of MI, leading to the formation of a vascular network by human cells. This approach might hold great promise as an effective strategy for efficiently restoring the damaged myocardium in hearts afflicted with disease.

MHC II-EGFP Knock-in Mouse Model.

The MHC II-EGFP knock-in mouse model enables us to visualize and track MHC-II-expressing cells in vivo by expressing enhanced green fluorescent protein (EGFP) fused to the MHC class II molecule under the MHC II beta chain promoter. Using this model, we can easily identify MHC-II-expressing cells, including dendritic cells, B cells, macrophages, and ILC3s, which play a key role as antigen-presenting cells (APCs) for CD4+ T cells. In addition, we can also precisely identify and analyze APC-containing tissues and organs. Even after fixation, EGFP retains its fluorescence, so this model is suitable for immunofluorescence studies, facilitating an unbiased characterization of the histological context, especially with techniques such as light-sheet fluorescence microscopy. Furthermore, the MHC II-EGFP knock-in mouse model is valuable for studying the molecular mechanisms of MHC II gene regulation and expression by making it possible to correlate MHC II expression (MHC II-EGFP) with surface fraction through antibody detection, thereby shedding light on the intricate regulation of MHC II expression. Overall, this model is an essential asset for quantitative and systems immunological research, providing insights into immune cell dynamics and localization, with a tool for precise cell identification and with the ability to study MHC II gene regulation, thus furthering the understanding of immune responses and underlying mechanisms © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Characterization of antigen-specific MHC II loading compartment tubulation toward the immunological synapse Basic Protocol 2: Characterization of overall versus surface MHC II expression Basic Protocol 3: Identification and preparation of the lymphoid organs Basic Protocol 4: Quantification of APC content in lymphoid organs by fluorescence stereomicroscopy Basic Protocol 5: Quantification and measurement of intestinal lymphoid tissue by light-sheet fluorescence stereomicroscopy Basic Protocol 6: Visualization of corneal APCs Basic Protocol 7: Quantification of MHC II+ cells in maternal milk by flow cytometry Support Protocol 1: Cell surface staining and flow cytometry analysis of spleen mononuclear cells.

Successful 3D imaging of cleared biological samples with light sheet fluorescence microscopy.

In parallel with the development of tissue-clearing methods, over the last decade, light sheet fluorescence microscopy has contributed to major advances in various fields, such as cell and developmental biology and neuroscience. While biologists are increasingly integrating three-dimensional imaging into their research projects, their experience with the technique is not always up to their expectations. In response to a survey of specific challenges associated with sample clearing and labeling, image acquisition, and data analysis, we have critically assessed the recent literature to characterize the difficulties inherent to light sheet fluorescence microscopy applied to cleared biological samples and to propose solutions to overcome them. This review aims to provide biologists interested in light sheet fluorescence microscopy with a primer for the development of their imaging pipeline, from sample preparation to image analysis. Importantly, we believe that issues could be avoided with better anticipation of image analysis requirements, which should be kept in mind while optimizing sample preparation and acquisition parameters.

Anatomy of the complete mouse eye vasculature explored by light-sheet fluorescence microscopy exposes subvascular-specific remodeling in development and pathology

Eye development and function rely on precise establishment, regression and maintenance of its many sub-vasculatures. These crucial vascular properties have been extensively investigated in eye development and disease utilizing genetic and experimental mouse models. However, due to technical limitations, individual studies have often restricted their focus to one specific sub-vasculature. Here, we apply a workflow that allows for visualization of complete vasculatures of mouse eyes of various developmental stages. Through tissue depigmentation, immunostaining, clearing and light-sheet fluorescence microscopy (LSFM) entire vasculatures of the retina, vitreous (hyaloids) and uvea were simultaneously imaged at high resolution. In silico dissection provided detailed information on their 3D architecture and interconnections. By this method we describe successive remodeling of the postnatal iris vasculature, involving sprouting and pruning, following its disconnection from the embryonic feeding hyaloid vasculature. In addition, we demonstrate examples of conventional and LSFM-mediated analysis of choroidal neovascularization after laser-induced wounding, showing added value of the presented workflow in analysis of modelled eye disease. These advancements in visualization and analysis of the respective eye vasculatures in development and complex eye disease open for novel observations of their functional interplay at a whole-organ level.

A cellular resolution atlas of Broca's area.

Brain cells are arranged in laminar, nuclear, or columnar structures, spanning a range of scales. Here, we construct a reliable cell census in the frontal lobe of human cerebral cortex at micrometer resolution in a magnetic resonance imaging (MRI)-referenced system using innovative imaging and analysis methodologies. MRI establishes a macroscopic reference coordinate system of laminar and cytoarchitectural boundaries. Cell counting is obtained with a digital stereological approach on the 3D reconstruction at cellular resolution from a custom-made inverted confocal light-sheet fluorescence microscope (LSFM). Mesoscale optical coherence tomography enables the registration of the distorted histological cell typing obtained with LSFM to the MRI-based atlas coordinate system. The outcome is an integrated high-resolution cellular census of Broca's area in a human postmortem specimen, within a whole-brain reference space atlas.

Light sheet fluorescence microscopy of cleared human eyes

We provide here a procedure enabling light sheet fluorescence microscopy (LSFM) of entire human eyes after iDISCO + -based clearing (ClearEye) and immunolabeling. Demonstrated here in four eyes, post-processing of LSFM stacks enables three-dimensional (3D) navigation and customized display, including en face viewing of the fundus similarly to clinical imaging, with resolution of retinal capillaries. This method overcomes several limitations of traditional histology of the eyes. Tracing of spatially complex structures such as anterior ciliary vessels and Schlemm’s canal was achieved. We conclude that LSFM of immunolabeled human eyes after iDISCO + -based clearing is a powerful tool for 3D histology of large human ocular samples, including entire eyes, which will be useful in both anatomopathology and in research.

CD47 blockade enhances phagocytosis of cardiac cell debris by neutrophils.

CD47 is a cell surface protein controlling phagocytotic activity of innate immune cells. CD47 blockade was investigated as an immune checkpoint therapy in cancer treatment, enhancing phagocytosis of tumor cells by macrophages. Anti-CD47 treatment also reduced injury size during reperfused acute myocardial infarction (repAMI) by enhancing phagocytotic acitivity of macrophages. Little is known about the impact of CD47 blockade on neutrophils, representing the main portion of early infiltrating immune cells after repAMI. Therefore, we performed 45min of cardiac ischemia followed by 24h of reperfusion, observing a decreased cardiac injury size measured by triphenyl tetrazolium chloride (TTC) Evan's blue staining. We were able to detect this effect with an innovative three-dimensional method based on light sheet fluorescence microscopy (LSFM). This further allowed us a simultaneous analysis of neutrophil infiltration, showing an unaltered amount of injury-associated neutrophils with reduced cardiac injury volume from repAMI. This observation suggests modulated phagocytosis of cell debris by neutrophils. Therefore, we performed flow cytometry analysis, revealing an increased phagocytotic activity of neutrophils in vitro. These findings highlight that CD47 blockade also enhances phagocytosis of cardiac cell debris by neutrophils, which might be an additional protective effect of anti-CD47 treatment after repAMI.

D-LMBmap: a fully automated deep-learning pipeline for whole-brain profiling of neural circuitry

Recent proliferation and integration of tissue-clearing methods and light-sheet fluorescence microscopy has created new opportunities to achieve mesoscale three-dimensional whole-brain connectivity mapping with exceptionally high throughput. With the rapid generation of large, high-quality imaging datasets, downstream analysis is becoming the major technical bottleneck for mesoscale connectomics. Current computational solutions are labor intensive with limited applications because of the exhaustive manual annotation and heavily customized training. Meanwhile, whole-brain data analysis always requires combining multiple packages and secondary development by users. To address these challenges, we developed D-LMBmap, an end-to-end package providing an integrated workflow containing three modules based on deep-learning algorithms for whole-brain connectivity mapping: axon segmentation, brain region segmentation and whole-brain registration. D-LMBmap does not require manual annotation for axon segmentation and achieves quantitative analysis of whole-brain projectome in a single workflow with superior accuracy for multiple cell types in all of the modalities tested.

Large field of view aberrations correction with deformable lenses and multi conjugate adaptive optics.

Optical microscopes can have limited resolution due to aberrations caused by samples and sample holders. Using deformable mirrors and wavefront sensorless optimization algorithms can correct these aberrations, but the correction is limited to a small area of the field of view. This study presents an adaptive optics method that uses a series of plug-and-play deformable lenses for large field of view wavefront correction. A direct wavefront measurement method using the spinning sub-pupil aberration measurement technique is combined with correction based on the deformable lenses. Experimental results using fluorescence microscopy with a wide field and a light sheet fluorescence microscope show that the proposed method can achieve detection and correction over an extended field of view with a compact transmissive module placed in the detection path of the microscope. This method could improve the resolution and accuracy of imaging in a variety of fields, including biology and materials science.

A horseshoe mixture model for Bayesian screening with an application to light sheet fluorescence microscopy in brain imaging

A horseshoe mixture model for Bayesian screening with an application to light sheet fluorescence microscopy in brain imaging

Neutrophils for Revascularization Require Activation of CCR6 and CCL20 by TNFα.

Activation of immune-inflammatory pathways involving TNFα (tumor necrosis factor alpha) signaling is critical for revascularization and peripheral muscle tissue repair after ischemic injury. However, mechanisms of TNFα-driven inflammatory cascades directing recruitment of proangiogenic immune cells to sites of ischemia are unknown. Muscle tissue revascularization after permanent femoral artery ligation was monitored in mutant mice by laser Doppler imaging and light sheet fluorescence microscopy. TNFα-mediated signaling and the role of the CCL20 (C-C motif chemokine ligand 20)-CCR6 (C-C chemokine receptor 6) axis for formation of new vessels was studied in vitro and in vivo using bone marrow transplantation, flow cytometry, as well as biochemical and molecular biological techniques. TNFα-mediated activation of TNFR (tumor necrosis factor receptor) 1 but not TNFR2 was found to be required for postischemic muscle tissue revascularization. Bone marrow-derived CCR6+ neutrophil granulocytes were identified as a previously undescribed TNFα-induced population of proangiogenic neutrophils, characterized by increased expression of VEGFA (vascular endothelial growth factor A). Mechanistically, postischemic activation of TNFR1 induced expression of the CCL20 in vascular cells and promoted translocation of the CCL20 receptor CCR6 to the cell surface of neutrophils, essentially conditioning VEGFA-expressing proangiogenic neutrophils for CCL20-dependent recruitment to sites of ischemia. Moreover, impaired revascularization of ischemic peripheral muscle tissue in diabetic mice was associated with reduced numbers of proangiogenic neutrophils and diminished CCL20 expression. Administration of recombinant CCL20 enhanced recruitment of proangiogenic neutrophils and improved revascularization of diabetic ischemic skeletal muscles, which was sustained by sequential treatment with fluvastatin. We demonstrate that site-specific activation of the CCL20-CCR6 axis via TNFα recruits proangiogenic VEGFA-expressing neutrophils to sites of ischemic injury for initiation of muscle tissue revascularization. The findings provide an attractive option for tissue revascularization, particularly under diabetic conditions.

Three-dimensional morphological and fluorescent imaging of zebrafish with a continuous-rotational light-sheet microscope

Light-sheet fluorescence microscopy (LSFM) has been widely used to image the three-dimensional (3D) structures and functions of various millimeter-size bio-specimen such as zebrafish. However, the sample adsorption and scattering cause shading of the light-sheet illumination, preventing the even 3D image of thick samples. Herein, we report a continuous-rotational light-sheet microscope (CR-LSM) that enables simultaneous 3D bright-field and fluorescence imaging. With a high-accuracy rotational stage, CR-LSM records the outline projections and the fluorescent images of the sample at multiple rotation angles. Then, 3D morphology and fluorescent structure were reconstructed with a developed algorithm. Using CR-LSM, zebrafish’s whole-fish contour and blood vessel structures were obtained simultaneously.

Abstract P1180: Multi-scale Imaging And Analysis To Elucidate Cardiac Structure And Contractile Function

Heart failure remains the leading cause of mortality worldwide, largely due to the lack of regenerative capacity of mature cardiomyocytes. The advanced understanding of cardiac architecture and function is critical to uncover the underlying mechanism of cardiac morphogenesis and the remodeling process in response to myocardial infarction. Recent progress has demonstrated that zebrafish and neonatal mice have the capacity to restore cardiac function following the injury, laying the foundation to investigate the cues to structural and functional abnormalities. For this reason, we have developed a light-sheet fluorescence microscope along with the tailored computational methods to investigate the 3-dimensional (3D) architecture of the intact mouse heart, and 4D (3D spatial + 1D temporal) cardiac contractility in zebrafish larvae. This method provides us ~ 2 um isotropic spatial resolution across the atria and ventricles with minimal photo-damage and maximal penetration depth. In combination with established tissue clearing methods, we are able to investigate myocardial trabeculation and compaction in mouse hearts within the 2-minute laser scanning. To unravel the contractile dysfunction following the anticancer therapy, we have coupled our imaging system with retrospective synchronization algorithm for the 4D reconstruction of myocardial deformation from end-systole to end-diastole in zebrafish hearts. To further interpret the deluge of 3D and 4D images, we have also developed a machine learning-based platform for interactive image analysis. Collectively, our multi-scale imaging strategy provides an entry point to assess micro-architecture and cardiac contractile function in the entire heart without physical slicing, holding the great potential to uncover the process of cardiac morphogenesis and regeneration.

Brain-Wide Projections and Differential Encoding of Prefrontal Neuronal Classes Underlying Learned and Innate Threat Avoidance.

To understand how the brain produces behavior, we must elucidate the relationships between neuronal connectivity and function. The medial prefrontal cortex (mPFC) is critical for complex functions including decision-making and mood. mPFC projection neurons collateralize extensively, but the relationships between mPFC neuronal activity and brain-wide connectivity are poorly understood. We performed whole-brain connectivity mapping and fiber photometry to better understand the mPFC circuits that control threat avoidance in male and female mice. Using tissue clearing and light sheet fluorescence microscopy (LSFM), we mapped the brain-wide axon collaterals of populations of mPFC neurons that project to nucleus accumbens (NAc), ventral tegmental area (VTA), or contralateral mPFC (cmPFC). We present DeepTraCE (deep learning-based tracing with combined enhancement), for quantifying bulk-labeled axonal projections in images of cleared tissue, and DeepCOUNT (deep-learning based counting of objects via 3D U-net pixel tagging), for quantifying cell bodies. Anatomical maps produced with DeepTraCE aligned with known axonal projection patterns and revealed class-specific topographic projections within regions. Using TRAP2 mice and DeepCOUNT, we analyzed whole-brain functional connectivity underlying threat avoidance. PL was the most highly connected node with functional connections to subsets of PL-cPL, PL-NAc, and PL-VTA target sites. Using fiber photometry, we found that during threat avoidance, cmPFC and NAc-projectors encoded conditioned stimuli, but only when action was required to avoid threats. mPFC-VTA neurons encoded learned but not innate avoidance behaviors. Together our results present new and optimized approaches for quantitative whole-brain analysis and indicate that anatomically defined classes of mPFC neurons have specialized roles in threat avoidance.SIGNIFICANCE STATEMENT Understanding how the brain produces complex behaviors requires detailed knowledge of the relationships between neuronal connectivity and function. The medial prefrontal cortex (mPFC) plays a key role in learning, mood, and decision-making, including evaluating and responding to threats. mPFC dysfunction is strongly linked to fear, anxiety and mood disorders. Although mPFC circuits are clear therapeutic targets, gaps in our understanding of how they produce cognitive and emotional behaviors prevent us from designing effective interventions. To address this, we developed a high-throughput analysis pipeline for quantifying bulk-labeled fluorescent axons [DeepTraCE (deep learning-based tracing with combined enhancement)] or cell bodies [DeepCOUNT (deep-learning based counting of objects via 3D U-net pixel tagging)] in intact cleared brains. Using DeepTraCE, DeepCOUNT, and fiber photometry, we performed detailed anatomic and functional mapping of mPFC neuronal classes, identifying specialized roles in threat avoidance.

Uncovering CNS access of lipidated exendin-4 analogues by quantitative whole-brain 3D light sheet imaging

Peptide-based drug development for CNS disorders is challenged by poor blood-brain barrier (BBB) penetrability of peptides. While acylation protractions (lipidation) have been successfully applied to increase circulating half-life of therapeutic peptides, little is known about the CNS accessibility of lipidated peptide drugs. Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful method to visualize whole-brain 3D distribution of fluorescently labelled therapeutic peptides at single-cell resolution. Here, we applied LSFM to map CNS distribution of the clinically relevant GLP-1 receptor agonist (GLP-1RA) exendin-4 (Ex4) and lipidated analogues following peripheral administration. Mice received an intravenous dose (100 nmol/kg) of IR800 fluorophore-labelled Ex4 (Ex4), Ex4 acylated with a C16-monoacid (Ex4_C16MA) or C18-diacid (Ex4_C18DA). Other mice were administered C16MA-acylated exendin 9-39 (Ex9-39_C16MA), a selective GLP-1R antagonist, serving as negative control for GLP-1R mediated agonist internalization. Two hours post-dosing, brain distribution of Ex4 and analogues was predominantly restricted to the circumventricular organs, notably area postrema and nucleus of the solitary tract. However, Ex4_C16MA and Ex9-39_C16MA also distributed to the paraventricular hypothalamic nucleus and medial habenula. Notably, Ex4_C18DA was detected in deeper-lying brain structures such as dorsomedial/ventromedial hypothalamic nuclei and the dentate gyrus. Similar CNS distribution maps of Ex4_C16MA and Ex9-39_C16MA suggest that brain access of lipidated Ex4 analogues is independent on GLP-1 receptor internalization. The cerebrovasculature was devoid of specific labelling, hence not supporting a direct role of GLP-1 RAs in BBB function. In conclusion, peptide lipidation increases CNS accessibility of Ex4. Our fully automated LSFM pipeline is suitable for mapping whole-brain distribution of fluorescently labelled drugs.

O-006 Novel and scalable non-invasive metabolic imaging of early embryos using a lab-on-a-chip approach

Abstract Study question Is it feasible to safely determine metabolic imaging signatures to measure nicotinamide adenine dinucleotide (NADH) associated auto-fluorescence in early embryos using a scalable lab-on-a-chip approach? Summary answer We developed an optofluidic device capable of non-invasively obtain high-resolution 3D images of the metabolic activity of live mouse embryos using a lab-on-a-chip approach. What is known already Selecting the most suitable embryos for implantation and subsequent healthy live birth is crucial to the success rate of assisted reproduction and offspring health. Thus, developing non-invasive methods that are reliable to assess oocyte and embryo quality has been a significant aim for assisted reproduction. Besides morphological evaluation using optical microscopy, a promising alternative is the non-invasive imaging of live embryos to establish metabolic activity performance. However, metabolic imaging has been only achieved using highly complex advanced microscopy such as Fluorescence-lifetime imaging (FLIM) and hyperspectral microscopy, methods that are costly and challenging, limiting the potential for deployment in fertility clinics. Study design, size, duration The non-invasive nature of the system was investigated by assessing the development and viability of live embryos after embryo culture for 67hrs post metabolic imaging at the 2-cell embryo stage (n = 115), including a control for culture conditions and sham controls (system no-illuminated). Embryo quality of developed blastocysts was assessed by immunocytochemistry to quantify trophectoderm and inner mass cells (n = 75). Furthermore, inhibition of metabolic activity (FK866 inhibitor) during embryo culture was also assessed (n = 18). Participants/materials, setting, methods Optofluidic devices were manufactured by cast-moulding using a negative photoresist (SU8-2075; MicroChemicals GmbH, Ulm, Germany) by a standard UV-photolithography process. The microstructures fabricated of polydimethylsiloxane (PDSM) integrated Light Sheet Fluorescence Microscopy into a microfluidic system, including on-chip micro-lenses to generate a light sheet at the center of a microchannel. Super-ovulated F1 (CBA/C57Bl6) mice were used to produce 2-cell embryos and embryo culture experiments. Blastocyst formation rates and embryo quality (immunocytochemistry) were compared between study groups. Main results and the role of chance The optofluidic device was capable of non-invasively obtaining high-resolution 3D images of the metabolic activity in live mouse embryos. The system’s design allowed continuous tracking of the embryo location, including high control displacement through the light-sheet, fast imaging of the embryos (<2 second) and keeping a low dose of light exposure (16 J cm-2 and 8 J cm-2). Optimum settings for keeping sample viability showed that a modest light dosage was capable to obtain 30 times higher signal-noise-ratio images than images obtained with a confocal system (p < 0.00001; t-test). The results showed no significant differences between the control, illuminated and non-illuminated embryos (sham control) for embryo development as well as embryo quality at the blastocyst stage (p > 0.05; Yate’s chi squared test). Additionally, embryos with inhibited metabolic activity showed decreased blastocyst formation rates as well as 47% reduction in metabolic activity measured by non-invasive metabolic imaging (p < 0.0001; t-test). This study reports an optofluidic device capable of non-invasive metabolic imaging of live embryos using a similar concept as previously reported using FLIM technology and hyperspectral microscopy, but allowing a novel, scalable and affordable system with implementation potential in standard IVF laboratory equipment. Limitations, reasons for caution The study was conducted using a mouse model focused on early embryo development. Further safety studies are required to assess embryonic health by investigating the impact of the use of light during embryo development, live birth, embryo gene expression and epigenome stability. Wider implications of the findings This lab-on-a-chip is novel, scalable and has the potential to be used in fertility clinics as a technology platform to enable real-time monitoring of the metabolic function of embryos prior embryo transfer. Future applications include potential integration into existing IVF laboratory equipment such as bench and time-lapse incubators. Trial registration number N/A

Enhanced neuroimaging with a calcium sensor in ex-vivo Drosophila melanogaster brains using closed-loop adaptive optics light-sheet fluorescence microscopy.

Adaptive optics (AO) has been implemented on several microscopy setups and has proven its ability to increase both signal and resolution. However, reported configurations are not suited for fast imaging of live samples or are based on an invasive or complex implementation method. Provide a fast aberration correction method with an easy to implement AO module compatible with light-sheet fluorescence microscopy (LSFM) for enhanced imaging of live samples. Development of an AO add-on module for LSFM based on direct wavefront sensing without requiring a guide star using an extended-scene Shack-Hartmann wavefront sensor. The enhanced setup uses a two-color sample labeling strategy to optimize the photon budget. Fast AO correction of in-depth aberrations in an ex-vivo adult Drosophila brain enables doubling the contrast when imaging with either cell reporters or calcium sensors for functional imaging. We quantify the gain in terms of image quality on different functional domains of sleep neurons in the Drosophila brain at various depths and discuss the optimization of key parameters driving AO. We developed a compact AO module that can be integrated into most of the reported light-sheet microscopy setups, provides significant improvement of image quality and is compatible with fast imaging requirements such as calcium imaging.

Detection of Choroidal Neovascularization Using Optical Tissue Transparency.

Optical tissue transparency (OTT) provides a tool for visualizing the entire tissue block. This study provides insights into the potential value of OTT with light-sheet fluorescence microscopy (LSFM) in detecting choroidal neovascularization (CNV) lesions. OTT with LSFM, hematoxylin and eosin (H&E) staining of paraffin sections, choroidal flatmount immunofluorescence, and optical coherence tomography angiography (OCTA) were used to obtain images of CNV. We determined the rate of change as (Data of week 1 - Data of week 2)/Data of week 1 × 100%. Finally, we compared the rate of change acquired from OTT with LSFM and the other methodologies. We found that OTT with LSFM can realize three-dimensional (3D) visualizations of the entire CNV. The results showed that the decline in the rate of change from week 1 to week 2 after laser photocoagulation was 33.05% with OTT, 53.01% with H&E staining, 48.11% with choroidal flatmount, 24.06% with OCTA (B-scan), 18.08% with OCTA (en face), 10.98% with OCTA (3D reconstruction), and 7.74% with OCTA (vessel diameter index). OTT with LSFM will continue to be an invaluable resource for investigators to detect more visualized and quantified information regarding CNV. OTT with LSFM now serves as a tool for detecting CNV in mice, and it may undergo human clinical trials in the future.

Field dipole interaction and polarization effects in light-sheet optical fluorescence microscopy

Polarization plays a crucial role in understanding the interaction of fluorescent molecules in a light field. We report the study on the effect of a field–dipole interaction under polarization light-sheet fluorescence microscopy using the vectorial theory of light. The molecule is suitably modeled as a radiating electric dipole in a polarized electric field (both linear and random), and the system point spread function (PSF) is determined for different orientations of the dipole (both fixed and random). PSF analysis and contour plots suggest distinct nature of a field distribution in each case, indicating the importance of a field–dipole interaction for high-quality fluorescence imaging. The analysis suggests that the field spreads gradually along the polarization axis at a high numerical aperture (NA) of the objective lens, whereas it is more isotropic and homogeneous at low NA. Moreover, fast changes are not observed at low NA (i.e., far from the central lobe in the field contour plots), suggesting the absence of high-frequency components. However, sidelobes are prominent for linear polarized (along x) light. On the other hand, rapid variations are evident for randomly polarized light, depicting the presence of high spatial frequencies in the system optical transfer function. The other significant observation is the distinct frequency spectrum (both kx and ky) for random and fixed dipoles, indicating the significance of dipole orientation in a light-sheet field. Compared to the point-illumination-based fluorescence microscopy, sheet based polarization technique provides a high signal-to-noise ratio, a uniform field, an order large field of view, and critical information (related to the micro-environment of a dipole and its short-range interactions). The study is expected to facilitate polarization-sensitive investigation of large biological specimens (both fixed and live).