Wide-field Fluorescence Microscopy Research Articles

In Situ Immunofluorescence Imaging of Vital Human Pancreatic Tissue Using Fiber-Optic Microscopy.

Surgical resection is the only curative option for pancreatic carcinoma, but disease-free and overall survival times after surgery are limited due to early tumor recurrence, most often originating from local microscopic tumor residues (R1 resection). The intraoperative identification of microscopic tumor residues within the resection margin in situ could improve surgical performance. The aim of this study was to evaluate the effectiveness of fiber-optic microscopy for detecting microscopic residues in vital pancreatic cancer tissues. Experimental Design. Fresh whole-mount human pancreatic tissues, histological tissue slides, cell culture, and chorioallantoic membrane xenografts were analyzed. Specimens were stained with selected fluorophore-conjugated antibodies and studied using conventional wide-field and self-designed multicolor fiber-optic fluorescence microscopy instruments. Whole-mount vital human tissues and xenografts were stained and imaged using an in situ immunofluorescence protocol. Fiber-optic microscopy enabled the detection of epitope-based fluorescence in vital whole-mount tissue using fluorophore-conjugated antibodies and enabled visualization of microvascular, epithelial, and malignant tumor cells. Among the selected antigen-antibody pairs, antibody clones WM59, AY13, and 9C4 were the most promising for fiber-optic imaging in human tissue samples and for endothelial, tumor and epithelial cell detection. Fresh dissected whole-mount tissue can be stained using direct exposure to selected antibody clones. Several antibody clones were identified that provided excellent immunofluorescence imaging of labeled structures, such as endothelial, epithelial, or EGFR-expressing cells. The combination of in situ immunofluorescence staining and fiber-optic microscopy visualizes structures in vital tissues and could be proposed as an useful tool for the in situ identification of residual tumor mass in patients with a high operative risk for incomplete resection.

Cancer Imaging by Intravital Microscopy: The Dorsal Window Chamber Model.

Intravital microscopy allows a direct visualization of cells' behavior in their environment in a living organism with all its complexity. With appropriated models, longitudinal studies of structural and functional changes can be followed in the same animal on long period. In the field of cancer, the dorsal window chamber model is the model of choice for tumor events such as cells migration, vessels growth, and their permeability or interactions between cells and vessels. Coupled with wide-field, confocal, or multiphoton fluorescence microscopes, high spatial and temporal resolutions of the cellular events can be analyzed in vivo.

Quantification of membrane geometry and protein sorting on cell membrane protrusions using fluorescence microscopy.

Plasma membranes are flexible and can exhibit numerous shapes below the optical diffraction limit. The shape of cell periphery can either induce or be a product of local protein density changes, encoding numerous cellular functions. However, quantifying membrane curvature and the ensuing sorting of proteins in live cells remains technically demanding. Here, we demonstrate the use of simple widefield fluorescence microscopy to study the geometrical properties (i.e., radius, length, and number) of thin membrane protrusions. Importantly, the quantification of protrusion radius establishes a platform for studying the curvature preferences of membrane proteins.

A facile method to determine the molar mass of soft nanoparticles

The determination of the molar mass of soft nanoparticles is essential to estimate their (molar) concentration in dispersions. Measuring this quantity by conventional methods, however, often proves challenging. We describe a facile approach to determine the molar mass of (soft) nanoparticles via counting their number per volume using a widefield fluorescence microscope. The method is exemplified on a microgel dispersion as a model system, while it is applicable to other types of stainable nanoparticles. For this, covalent labeling or modification of the nanoparticles is not required. The dispersion is simply mixed with a Nile Red solution in a defined ratio and measured in an optical fluorescence microscope accessible to most researchers in the field.

A micro-LED array based platform for spatio-temporal optogenetic control of various cardiac models

Optogenetics relies on dynamic spatial and temporal control of light to address emerging fundamental and therapeutic questions in cardiac research. In this work, a compact micro-LED array, consisting of 16 × 16 pixels, is incorporated in a widefield fluorescence microscope for controlled light stimulation. We describe the optical design of the system that allows the micro-LED array to fully cover the field of view regardless of the imaging objective used. Various multicellular cardiac models are used in the experiments such as channelrhodopsin-2 expressing aggregates of cardiomyocytes, termed cardiac bodies, and bioartificial cardiac tissues derived from human induced pluripotent stem cells. The pacing efficiencies of the cardiac bodies and bioartificial cardiac tissues were characterized as a function of illumination time, number of switched-on pixels and frequency of stimulation. To demonstrate dynamic stimulation, steering of calcium waves in HL-1 cell monolayer expressing channelrhodopsin-2 was performed by applying different configurations of patterned light. This work shows that micro-LED arrays are powerful light sources for optogenetic control of contraction and calcium waves in cardiac monolayers, multicellular bodies as well as three-dimensional artificial cardiac tissues.

Single-Molecule Fluorescence Microscopy in Sensory Cilia of Living Caenorhabditis elegans.

Intracellular transport of organelles and biomolecules is vital for several cellular processes. Single-molecule fluorescence microscopy can illuminate molecular aspects of the dynamics of individual biomolecules that remain unresolved in ensemble experiments. For example, studying single-molecule trajectories of moving biomolecules can reveal motility properties such as velocity, diffusivity, location and duration of pauses, etc. We use single-molecule imaging to study the dynamics of microtubule-based motor proteins and their cargo in the primary cilia of living C. elegans. To this end, we employ standard fluorescent proteins, an epi-illuminated, widefield fluorescence microscope, and primarily open-source software. This chapter describes the setup we use, the preparation of samples, a protocol for single-molecule imaging in primary cilia of C. elegans, and data analysis.

Correlative Extinction and Single Fluorophore Bleaching Microscopy for Ligand Quantification on Gold Nanoparticles

AbstractNanoparticles (NPs) are promising therapeutic delivery agents, with the number and manner of presentation of cell‐binding ligands on the NP affecting the eventual fate of the therapeutic. Whenever NPs are conjugated with biomolecules, a heterogeneous population of decorated NPs will be produced and these subpopulations of particle‐ligand structures need to be characterized for a reliable interpretation of NP‐based data. An optical microscopy method is reported to quantitatively evaluate the conjugation on a single‐particle basis in samples consisting of gold NPs (GNPs) decorated with holo‐transferrin fluorescently labeled with Alexa647 (Tf). Widefield fluorescence and extinction microscopy are employed on NP‐ligand constructs, alongside a correlative analysis that spatially co‐localizes diffraction‐limited sources of fluorescence with the optical extinction by individual GNPs. A photobleaching step analysis estimates the number of fluorophores contributing to the detected emission rate. The method quantifies the number of fluorescent biomolecules attached per GNP, the numbers of unconjugated GNPs and unbound Tf present within the mixed population, and the size and intraparticle clustering propensity of conjugated GNPs. A high variability is found in the number of Tf ligands per GNP within the GNP population, when analyzed at the single‐particle level, unraveling a non‐trivial statistical distribution not accessible in ensemble‐averaged approaches.

Calcium image analysis in the moving gut.

The neural control of gastrointestinal muscle relies on circuit activity whose underlying motifs remain limited by small-sample calcium imaging recordings confounded by motion artifact, paralytics, and muscle dissections. We present a sequence of resources to register images from moving preparations and identify out-of-focus events in widefield fluorescent microscopy. Our algorithm uses piecewise rigid registration with pathfinding to correct movements associated with smooth muscle contractions. We developed methods to identify loss-of-focus events and to simulate calcium activity to evaluate registration. By combining our methods with principal component analysis, we found populations of neurons exhibit distinct activity patterns in response to distinct stimuli consistent with hypothesized roles. The image analysis pipeline makes deeper insights possible by capturing concurrently calcium dynamics from more neurons in larger fields of view. We provide access to the source code for our algorithms and make experimental and technical recommendations to increase data quality in calcium imaging experiments. These methods make feasible large population, robust calcium imaging recordings and permit more sophisticated network analyses and insights into neural activity patterns in the gut.

Simultaneous spectral differentiation of multiple fluorophores in super-resolution imaging using a glass phase plate.

By engineering the point-spread function (PSF) of single molecules, different fluorophore species can be imaged simultaneously and distinguished by their unique PSF patterns. Here, we insert a silicon-dioxide phase plate at the Fourier plane of the detection path of a wide-field fluorescence microscope to produce distinguishable PSFs (X-PSFs) at different wavelengths. We demonstrate that the resulting PSFs can be localized spatially and spectrally using a maximum-likelihood estimation algorithm and can be utilized for hyper-spectral super-resolution microscopy of biological samples. We produced superresolution images of fixed U2OS cells using X-PSFs for dSTORM imaging with simultaneous illumination of up to three fluorophore species. The species were distinguished only by the PSF pattern. We achieved ∼21-nm lateral localization precision (FWHM) and ∼17-nm axial precision (FWHM) with an average of 1,800 - 3,500 photons per PSF and a background as high as 130 - 400 photons per pixel. The modified PSF distinguished fluorescent probes with ∼80 nm separation between spectral peaks.

Immunocytochemical Visualization of Proteins from Cyanobacterial Cells with High Autofluorescence of Phycoerythrin and Phycourobilin.

Presented is a protocol for visualizing and quantifying a specific protein in cells at the cellular level for the marine cyanobacterium Crocosphaera watsonii, a crucial primary producer and nitrogen fixer in oligotrophic oceans. One of the challenges for marine autotrophic N2 fixers (diazotrophs) is distinguishing probe-derived fluorescence signals from autofluorescence. C. watsonii was selected to represent chlorophyll-, phycoerythrin- and phycourobilin-containing cyanobacteria. The protocol allows for simple and semi-quantitative visualization of proteins in C. watsonii at a single-cell level, enabling investigation of protein production under different environmental conditions to evaluate the metabolic activities of the target cyanobacteria. Furthermore, the fixation and permeabilization methods are optimized to enhance the fluorescence signals from target proteins to distinguish them from autofluorescence, especially from phycoerythrin and phycourobilin. The enhanced signal can be visualized using confocal or widefield fluorescence microscopy. Additionally, fluorescence intensity was semi-quantified using Fiji software. This single-cell analysis workflow allows the evaluation of cell-to-cell variations of specific protein content. The protocol can be performed in any life science laboratory as it requires only standard equipment and can also be easily adapted to other phycoerythrin-containing cyanobacterial cells.

Highly sensitive digital detection of SARS-CoV-2 nucleocapsid protein through single-molecule counting.

Nucleocapsid protein (NP) is one of the structural proteins of SARS-CoV-2 which is stable, well-conserved, highly immunogenic, and abundantly expressed due to the host's adaptive immune response, making it a promising antigenic biomarker for the early and rapid identification and diagnosis of SARS-CoV-2. Traditional antigen analytical methods with NP as the detection marker often have insufficient sensitivity. To achieve rapid and highly sensitive detection of NP, we constructed a novel single-molecule (digital) fluorescence-linked immunosorbent assay (FLISA) based on streptavidin-modified transparent 96-well microplates. Streptavidin was immobilized on the microplate under optimized conditions with a 15mM carbonate buffer solution (pH 9.6) as the coating solution, biotinylated antibodies conjugated with streptavidin as capture probes, and carboxylated fluorescent microsphere-conjugated monoclonal antibodies (FMs-mAbs) as fluorescent probes. Individual sandwich immunolabeled complexes of the SARS-CoV-2 diagnostic marker NP were detected and counted though wide-field inverted fluorescence microscopy (1.1 × 1.4 mm2). FLISA had a linear detection range of 0.2pg/mL to 200ng/mL and a limit of detection (LOD) of 0.73fg/mL and 8fg/mL for NP in phosphate buffer saline and spiked nasal swab samples, respectively. The sensitivity was much higher than commercial antigen detection kits, providing wide detection prospects in future clinical diagnosis, environmental monitoring, and other fields.

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues.

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in autoimmune diseases and sterile inflammation. They are web-like structures composed of double-stranded DNA filaments, histones, and antimicrobial proteins. Once released, NETs can trap and kill extracellular pathogens in blood and tissue. Furthermore, NETs participate in homeostatic regulation by stimulating platelet adhesion and coagulation. However, the dysregulated production of NETs has also been associated with various diseases, including sepsis or autoimmune disorders, which makes them a promising target for therapeutic intervention. Apart from electron microscopy, visualizing NETs using immunofluorescence imaging is currently one of the only known methods to demonstrate NET interactions in tissue. Therefore, various staining methods to visualize NETs have been utilized. In the literature, different staining protocols are described, and we identified four key components showing high variability between protocols: (1) the types of antibodies used, (2) the usage of autofluorescence-reducing agents, (3) antigen retrieval methods, and (4) permeabilization. Therefore, in vitro immunofluorescence staining protocols were systemically adapted and improved in this work to make them applicable for different species (mouse, human) and tissues (skin, intestine, lung, liver, heart, spinal disc). After fixation and paraffin-embedding, 3 µm thick sections were mounted onto slides. These samples were stained with primary antibodies for myeloperoxidase (MPO), citrullinated histone H3 (H3cit), and neutrophil elastase (NE) according to a modified staining protocol. The slides were stained with secondary antibodies and examined using a widefield fluorescence microscope. The results were analyzed according to an evaluation sheet, and differences were recorded semi-quantitatively. Here, we present an optimized NET staining protocol suitable for different tissues. We used a novel primary antibody to stain for H3cit and reduced non-specific staining with an autofluorescence-reducing agent. Furthermore, we demonstrated that NET staining requires a constant high temperature and careful handling of samples.

Surfactant-Mediated Structural Modulations to Planar, Amphiphilic Multilamellar Stacks.

The hydrophobic effect, a ubiquitous process in biology, is a primary thermodynamic driver of amphiphilic self-assembly. It leads to the formation of unique morphologies including two highly important classes of lamellar and micellar mesophases. The interactions between these two types of structures and their involved components have garnered significant interest because of their importance in key biochemical technologies related to the isolation, purification, and reconstitution of membrane proteins. This work investigates the structural organization of mixtures of the lamellar-forming phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and two zwitterionic micelle-forming surfactants, being n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent 3-12 or DDAPS) and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (O-Lyso-PC), when assembled by water vapor hydration with X-ray diffraction measurements, brightfield optical microscopy, wide-field fluorescence microscopy, and atomic force microscopy. The results reveal that multilamellar mesophases of these mixtures can be assembled across a wide range of POPC to surfactant (POPC:surfactant) concentration ratios, including ratios far surpassing the classical detergent-saturation limit of POPC bilayers without significant morphological disruptions to the lamellar motif. The mixed mesophases generally decreased in lamellar spacing (D) and headgroup-to-headgroup distance (Dhh) with a higher concentration of the doped surfactant, but trends in water layer thickness (Dw) between each bilayer in the stack are highly variable. Further structural characteristics including mesophase topography, bilayer thickness, and lamellar rupture force were revealed by atomic force microscopy (AFM), exhibiting homogeneous multilamellar stacks with no significant physical differences with changes in the surfactant concentration within the mesophases. Taken together, the outcomes present the assembly of unanticipated and highly unique mixed mesophases with varied structural trends from the involved surfactant and lipidic components. Modulations in their structural properties can be attributed to the surfactant's chemical specificity in relation to POPC, such as the headgroup hydration and the hydrophobic chain tail mismatch. Taken together, our results illustrate how specific chemical complexities of surfactant-lipid interactions can alter the morphologies of mixed mesophases and thereby alter the kinetic pathways by which surfactants dissolve lipid mesophases in bulk aqueous solutions.

Stages of NETosis Development upon Stimulation of Neutrophils with Activators of Different Types.

Before NETs are released, the neutrophil undergoes structural changes. First, it flattens, accompanied by a change in cell shape and rearrangement of the cytoskeleton. Then, nuclear swelling begins, which ends with the ejection of NETs into the extracellular space. We used widefield and confocal fluorescence microscopy to register morphological and structural changes in neutrophils during activation and NETosis. Different types of activators were used, such as NOX-dependent PMA and calcium ionophore A23187. The measurements were performed in a series of sequential stages. In the first stage (30 s after addition of activators and immediately after stimulation of neutrophils), the response of neutrophils to A23187 and PMA exposure was studied. Subsequently, the characteristics of neutrophils in different phases of activation were examined over a longer period of time (30, 60, 120, 180, and 240 min). The specific features of NETosis development were analyzed separately. During the first 30 s, neutrophils appeared to be heterogeneous in shape and structure of the actin cytoskeleton. Characteristic cell shapes included 30″ type 1 cells, similar in shape to the control, with F-actin concentrated in the center of the cytoplasm, and 30″ type 2 cells, which had flattened (spread) shapes with increased frontal dimensions and F-actin distributed throughout the cell. Later, the development of nuclear swelling, the corresponding changes in neutrophil membranes, and NET release into the extracellular space were evaluated. The conditions determining the initiation of chromatin ejection and two characteristic types of decondensed chromatin ejection were revealed. The results obtained contribute to a better understanding of the biophysical mechanisms of neutrophil activation and NETosis development.

Immunofluorescence Microscopy.

Visualizing fluorescence-tagged molecules is a powerful strategy that can reveal the complex dynamics of the cell. One robust and broadly applicable method is immunofluorescence microscopy, in which a fluorescence-labeled antibody binds the molecule of interest and then the location of the antibody is determined by fluorescence microscopy. The effective application of this technique includes several considerations, such as the nature of the antigen, specificity of the antibody, permeabilization and fixation of the specimen, and fluorescence imaging of the cell. Although each protocol will require fine-tuning depending on the cell type, antibody, and antigen, there are steps common to nearly all applications. This article provides protocols for staining the cytoskeleton and organelles in two very different kinds of cells: flat, adherent fibroblasts and thick, free-swimming Tetrahymena cells. Additional protocols enable visualization with widefield, laser scanning confocal, and eSRRF super-resolution fluorescence microscopy. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescence staining of adherent cells such as fibroblasts Basic Protocol 2: Immunofluorescence of suspension cells such as Tetrahymena Basic Protocol 3: Visualizing samples with a widefield fluorescence microscope Alternate Protocol 1: Staining suspension cells adhered to poly-l-lysine-coated coverslips Alternate Protocol 2: Visualizing samples with a laser scanning confocal microscope Alternate Protocol 3: Generating super-resolution images with SRRF microscopy.

Engineering and characterization of carbohydrate-binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts.

Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM-based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type-A CBMs from families 3a and 64 with crystalline cellulose-I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose-I using equilibrium binding assays. To compute the adsorption (nkon ) and desorption (koff ) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide-field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.

Gold Nanoparticles Inhibit Extravasation of Canine Osteosarcoma Cells in the Ex Ovo Chicken Embryo Chorioallantoic Membrane Model.

Canine osteosarcoma (OS) is an aggressive bone tumor with high metastatic potential and poor prognosis, mainly due to metastatic disease. Nanomedicine-based agents can be used to improve both primary and metastatic tumor treatment. Recently, gold nanoparticles were shown to inhibit different stages of the metastatic cascade in various human cancers. Here, we assessed the potential inhibitory effect of the glutathione-stabilized gold nanoparticles (Au-GSH NPs) on canine OS cells extravasation, utilizing the ex ovo chick embryo chorioallantoic membrane (CAM) model. The calculation of cells extravasation rates was performed using wide-field fluorescent microscopy. Transmission electron microscopy and Microwave Plasma Atomic Emission Spectroscopy revealed Au-GSH NPs absorption by OS cells. We demonstrated that Au-GSH NPs are non-toxic and significantly inhibit canine OS cells extravasation rates, regardless of their aggressiveness phenotype. The results indicate that Au-GSH NPs can act as a possible anti metastatic agent for OS treatment. Furthermore, the implemented CAM model may be used as a valuable preclinical platform in veterinary medicine, such as testing anti-metastatic agents.

Deep ultraviolet fluorescence microscopy of three-dimensional structures in the mouse brain

Three-dimensional (3D) imaging at cellular resolution improves our understanding of the brain architecture and is crucial for structural and functional integration as well as for the understanding of normal and pathological conditions in the brain. We developed a wide-field fluorescent microscope for 3D imaging of the brain structures using deep ultraviolet (DUV) light. This microscope allowed fluorescence imaging with optical sectioning due to the large absorption at the surface of the tissue and hence low tissue penetration of DUV light. Multiple channels of fluorophore signals were detected using single or a combination of dyes emitting fluorescence in the visible range of spectrum upon DUV excitation. Combination of this DUV microscope with microcontroller-based motorized stage enabled wide-field imaging of a coronal section of the cerebral hemisphere in mouse for deciphering cytoarchitecture of each substructure in detail. We extended this by integrating vibrating microtome which allowed serial block-face imaging of the brain structure such as the habenula in mouse. Acquired images were with resolution high enough for quantification of the cell numbers and density in the mouse habenula. Upon block-face imaging of the tissues covering entire extent of the cerebral hemisphere of the mouse brain, acquired data were registered and segmented for quantification of cell number in each brain regions. Results in the current analysis indicated that this novel microscope could be a convenient tool for large-scale 3D analysis of the brain in mice.

Enhancing Total Optical Throughput of Microscopy with Deep Learning for Intravital Observation.

The significance of performing large-depth dynamic microscopic imaging in vivo for life science research cannot be overstated. However, the optical throughput of the microscope limits the available information per unit of time, i.e., it is difficult to obtain both high spatial and temporal resolution at once. Here, a method is proposed to construct a kind of intravital microscopy with high optical throughput, by making near-infrared-II (NIR-II, 900-1880nm) wide-field fluorescence microscopy learn from two-photon fluorescence microscopy based on a scale-recurrent network. Using this upgraded NIR-II fluorescence microscope, vessels in the opaque brain of a rodent are reconstructed three-dimensionally. Five-fold axial and thirteen-fold lateral resolution improvements are achieved without sacrificing temporal resolution and light utilization. Also, tiny cerebral vessel dilatations in early acute respiratory failure mice are observed, with this high optical throughput NIR-II microscope at an imaging speed of 30fps.

Measurement of hippocampal arteriolar diameter and blood velocity in awake head-fixed mice

Cerebral hypoperfusion is associated with neurodegeneration. However, the influence of disease on the microcirculation of specific brain regions is poorly understood. In the current study, we validated a technical approach that allows us to simultaneously image arteriolar diameter and blood velocity in the hippocampi of awake, head-fixed mice. The approach involved implanting a gradient-Index (GRIN) lens into the hippocampus and cementing it in place together with a steel head-fixation bar. Imaging was performed after 7-14 days recovery. On the day of imaging, we intravenously administered Alexa Fluor 633 hydrazide (1.25 mg/kg) to label arterioles, and after 90 minutes, secured the mice on a custom-built stage of a widefield fluorescence microscope. We measured blood velocity in parallel with vessel diameter by imaging fluorescent latex beads infused by tail vein cannulation. Vessel diameter and blood velocity were assessed in 14 vessels from 6 mice. Hypercapnia induces cerebral vasodilation and increases cerebral blood flow. In our experiments, inhalation of 10% CO 2 caused a 12.1 ± 6% (mean ± SEM) increase in vessel diameter and a significant (P<0.01, student’s t-test) increase in blood velocity from 1.09 ± 0.1 to 1.73 ± 0.2 cm/s (mean ± SEM). These data demonstrate the feasibility of this approach to assess microvascular function and perfusion in highly localized brain regions. NIH grant (HL142906) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.