Individual Capillaries Research Articles

Robotic microfluidic imaging of blood stimulation- towards high-throughput portable measurement of haemostasis

Measuringblood and platelet function is vital for the development and use of drugs thatcombat cardiovascular disease, such as anti-platelet drugs and other medicinesthat reduce the risk of thrombosis. We propose combining mass-produced microfluidicdevices with open-source robotic instrumentation to enable development of affordableand portable, yet high-throughput and high-performance haematological testing.A time- and distance-resolved fluid flow analysis by Raspberry Pi imagingintegrated with controlled sample addition and illumination, enablessimultaneous tracking of capillary rise in 120 individual capillaries within 5minutes. We showed that time-resolved microcapillary rise imaging permits bloodfunction measurement by measuring thrombin-triggered activation of globalhaemostasis. Thrombin stimulation slowed vertical fluid velocity, consistentwith a dynamic increase in viscosity. Microfluidic systems expandhaematological testing towards high-efficiency, multi-parameter blood analysisnecessary for understanding and improving cardiovascular health.

Visual Acuity-Related Outer Retinal Structural Parameters on Swept Source Optical Coherence Tomography and Angiography in XLRS Patients and Carriers.

To assess the quantitative differences in vessel density and retinal thickness of X-linked retinoschisis (XLRS) patients and RS1 mutation carriers, and the correlation with best-corrected visual acuity (BCVA) with swept source optical coherence tomography (SS-OCT) and OCT angiography (OCTA). We analyzed the correlation between the BCVA of XLRS patients and the SS-OCT and OCTA findings including the detailed structural characteristics of XLRS patients. Besides the schitic changes in various retinal layers, the structural disturbance of outer retina was universally found. In 29 eyes included in the quantitative analysis, XLRS patients showed lower vessel density of the superficial capillary plexus, deep capillary plexus and lower thickness of the outer nuclear layer. BCVA was correlated with the thickness of the outer plexiform layer and outer nuclear layer and the thickness from the outer limiting membrane to the retinal pigment epithelium. Carriers showed higher thickness of outer plexiform layer and smaller foveal avascular zone area. SS-OCT and OCTA could identify the pathological alterations of the individual retinal layers and capillaries, which could pinpoint the exact location of the damages related to visual impairment. In the carriers, the subtle alterations that can be detected with SS-OCT, despite their normal visual acuity, may be caused by the lyonization. Swept source optical coherence tomography can be used as an efficient technique to expose the retinal damage related to visual impairment for prognosis and follow-up.

Making Nanofiber Membrane Stand on End to Construct Vertically Interfacial Evaporators for Efficient Solar Evaporation, Omnidirectional Solar Absorption, and Ultrahigh-Salinity Brine Desalination.

Solar-driven interfacial desalination is widely considered to be a promising technology to address the global water crisis. This study proposes a novel electrospun nanofiber-based all-in-one vertically interfacial solar evaporator endowed with a high steam generation rate, steady omnidirectional evaporation, and enduring ultrahigh-salinity brine desalination. In particular, the electrospun nanofiber is collected into the tubular structure, followed by spraying with a dense crosslinked poly(vinyl alcohol) film, which renders them sufficiently strong for the preparation of a vertically array evaporator. The integrated evaporator made an individual capillary as a unit to form multiple thermal localization interfaces and steam dissipation channels, realizing zone heating of water. Thus a high steam generation rate exceeding 4.0kgm-2h-1 in pure water is demonstrated even under omnidirectional sunlight, and outperforms existing evaporators. Moreover, salt ions in the photothermal layer can be effectively transported to the water in capillaries and subsequently exchanged with the bulk water due to the strong action of capillary force, which ensures an ultrahigh desalination rate (≈12.5kgm-2h-1 under 3 sun) in 25wt% concentration brine over 300min. As such, this work provides a meaningful roadmap for the development of state-of-the-art solar-driven interfacial desalination.

MuscleJ2: a rebuilding of MuscleJ with new features for high-content analysis of skeletal muscle immunofluorescence slides

Histological analysis of skeletal muscle is of major interest for understanding its behavior in different pathophysiological conditions, such as the response to different environments or myopathies. In this context, many software programs have been developed to perform automated high-content analysis. We created MuscleJ, a macro that runs in ImageJ/Fiji on batches of images. MuscleJ is a multianalysis tool that initially allows the analysis of muscle fibers, capillaries, and satellite cells. Since its creation, it has been used in many studies, and we have further developed the software and added new features, which are presented in this article. We converted the macro into a Java-language plugin with an improved user interface. MuscleJ2 provides quantitative analysis of fibrosis, vascularization, and cell phenotype in whole muscle sections. It also performs analysis of the peri-myonuclei, the individual capillaries, and any staining in the muscle fibers, providing accurate quantification within regional sublocalizations of the fiber. A multicartography option allows users to visualize multiple results simultaneously. The plugin is freely available to the muscle science community.

Distinctive effects of NMDA receptor modulators on cerebral microcirculation in a schizophrenia mouse model

Substantial evidence demonstrates that schizophrenia patients have altered cerebral microcirculation. However, little is known regarding how cerebral microcirculatory blood flow (microCBF) changes in schizophrenia. Here, using time-lapse two-photon imaging of individual capillaries, we demonstrated a substantial decrease in cerebral microcirculation in a mouse model of schizophrenia. The involvement of NMDA receptor (NMDAR) functions was investigated to understand further the mechanism of microcirculation reduction in this animal model. Administration of D-serine, a selective full agonist at the glycine site of NMDAR, significantly increased the microCBF in the schizophrenia mouse. Interestingly, administration of GNE-8324, a GluN2A-selective positive allosteric modulator that selectively enhances NMDAR-mediated synaptic responses in inhibitory but not excitatory neurons, had no effect on the microCBF of the schizophrenia mice. Together, these data indicated that NMDAR participated in the regulation of microcirculation in schizophrenia using a mechanism dependent on the tonic NMDAR signaling and the selective modulation of inhibitory neuron activity. Further studies are warranted to establish NMDAR's role in modulating microcirculation in schizophrenia.

Abstract 11913: In vivo Optical Coherence Tomography Angiography of the Coronary Microcirculation

Introduction: Imaging the coronary microcirculation in the beating heart is challenging due to motion artifact from cardiac contraction and respiration, which limits the application of high-resolution microscopy techniques. In the brain and other tissues with less motion artifact, optical coherence tomography angiography (OCTA) has been successfully used in rodents to create label-free maps of blood flow down to the capillary level. We hypothesized that advanced cardiac and respiratory gating together with high-speed optical coherence tomography can be used to implement OCTA of the capillary microcirculation in vivo in the mouse heart. Methods: C57BL/6 mice (n=30) were imaged through a sternotomy while anesthetized and ventilated. OCTA was performed using a custom tissue stabilizer and a Thorlabs TEL 321 Spectral Domain OCT system, which was synchronized to physiologic signals using custom hardware and software and cardiac pacing protocols. The system had an optical center wavelength of 1300 nm and produced axial (depth) and lateral resolutions of 4.2 μm and 13 μm, respectively. Angiograms were created from the difference between two consecutive images acquired during diastole, and a volumetric three-dimensional scan was acquired over multiple cardiac cycles. Images were analyzed in MATLAB and ImageJ to produce projection angiograms of the coronary microcirculation. Results: OCTA with cardiac and respiratory gating can produce high-resolution angiograms of the coronary microcirculation without exogenous contrast agents. OCTA has improved imaging depth and field of view compared to conventional confocal microscopy, and can produce widefield coronary angiograms (Fig. 1A) while maintaining the ability to visualize individual capillaries (Fig. 1B). Conclusion: In vivo OCTA can image the coronary microcirculation in the heart and will provide a powerful new tool for investigating coronary microvascular dysfunction in mouse models of heart disease.

Investigation of heterocellular features of the mouse retinal neurovascular unit by 3D electron microscopy.

The retina has a complex structure with a diverse collection of component cells that work together to facilitate vision. The retinal capillaries supplying the nutritional requirements to the inner retina have an intricate system of neural, glial and vascular elements that interconnect to form the neurovascular unit (NVU). The retina has no autonomic nervous system and so relies on the NVU as an interdependent, physical and functional unit to alter blood flow appropriately to changes in the physiological environment. The importance of this is demonstrated by alterations in NVU function being apparent in the blinding disease diabetic retinopathy and other diseases of the retina. It is, therefore, imperative to understand the anatomy of the components of the NVU that underlie its functioning and in particular the nanoscale arrangements of its heterocellular components. However, information on this in three spatial dimensions is limited. In the present study, we utilised the technique of serial block-face scanning electron microscopy (SBF-SEM), and computational image reconstruction, to enable the first three-dimensional ultrastructural analysis of the NVU in mouse retinal capillaries. Mouse isolated retina was prepared for SBF-SEM and up to 150 serial scanning electron microscopy images (covering z-axes distances of 12-8 mm) of individual capillaries in the superficial plexus and NVU cellular components digitally aligned. Examination of the data in the x-, y- and z-planes was performed with the use of semi-automated computational image analysis tools including segmentation, 3D image reconstruction and quantitation of cell proximities. A prominent feature of the capillary arrangements in 3D was the extensive sheath-like coverage by singular pericytes. They appeared in close register to the basement membrane with which they interwove in a complex mesh-like appearance. Breaks in the basement membrane appeared to facilitate pericyte interactions with other NVU cell types. There were frequent, close (<10nm) pericyte-endothelial interactions with direct contact points and peg-and-socket-like morphology. Macroglia typically intervened between neurons and capillary structures; however, regions were identified where neurons came into closer contact with the basement membrane. A software-generated analysis to assess the morphology of the different cellular components of the NVU, including quantifications of convexity, sphericity and cell-to-cell closeness, has enabled preliminary semi-quantitative characterisation of cell arrangements with neighbouring structures. This study presents new data on the nanoscale spatial characteristics of components of the murine retinal NVU in 3D that has implications for our understanding of structural integrity (e.g. pericyte-endothelial cell anchoring) and function (e.g. possible paracrine communication between macroglia and pericytes). It also serves as a platform to inform future studies examining changes in NVU characteristics with different biological and disease circumstances. All raw and processed image data have been deposited for public viewing.

Acoustic Molecular Imaging Beyond the Diffraction Limit In Vivo.

Ultrasound molecular imaging (USMI) is a technique used to noninvasively estimate the distribution of molecular markers in vivo by imaging microbubble contrast agents (MCAs) that have been modified to target receptors of interest on the vascular endothelium. USMI is especially relevant for preclinical and clinical cancer research and has been used to predict tumor malignancy and response to treatment. In the last decade, methods that improve the resolution of contrast-enhanced ultrasound by an order of magnitude and allow researchers to noninvasively image individual capillaries have emerged. However, these approaches do not translate directly to molecular imaging. In this work, we demonstrate super-resolution visualization of biomarker expression in vivo using superharmonic ultrasound imaging (SpHI) with dual-frequency transducers, targeted contrast agents, and localization microscopy processing. We validate and optimize the proposed method in vitro using concurrent optical and ultrasound microscopy and a microvessel phantom. With the same technique, we perform a proof-of-concept experiment in vivo in a rat fibrosarcoma model and create maps of biomarker expression co-registered with images of microvasculature. From these images, we measure a resolution of 23 μm, a nearly fivefold improvement in resolution compared to previous diffraction-limited molecular imaging studies.

Measuring Temporal and Spatial Variability of Red Blood Cell Velocity in Human Retinal Vessels.

PurposeThe retinal circulation regulates blood flow through various internal and external factors; however, it is unclear how locally these factors act within the retinal microcirculation. We measured the temporal and spatial variability of blood velocity in small retinal vessels using a dual-beam adaptive optics scanning laser ophthalmoscope.MethodsIn young healthy subjects (n = 3), temporal blood velocity variability was measured in a local vascular region consisting of an arteriole, capillary, and venule repeatedly over 2 days. Data consisted of 10 imaging periods separated into two sessions: (1) five 6-minute image acquisition periods with 30-minute breaks, and (2) five 6-minute image acquisition periods with 10-minute breaks. In another group of young healthy subjects (n = 5), spatial distribution of velocity variability was measured by imaging three capillary segments during three 2-minute conditions: (1) baseline imaging condition (no flicker), (2) full-field flicker, and (3) no flicker condition again.ResultsBlood velocities were measurable in all subjects with a reliability of about 2%. The coefficient of variation (CV) was used as an estimate of the physiological variability of each vessel. Over 2 days, the average CV in arterioles was 7% (±2%); in capillaries, it was 19% (±6%); and, in venules, it was 8% (±2%). During flicker stimulation, the average capillary CV was 16% during baseline, 15% during flicker stimulation, and 18% after flicker stimulation.ConclusionsCapillaries in the human retina exhibit spatial and temporal variations in blood velocity. This inherent variation in blood velocity places limits on studying the vascular regulation of individual capillaries, and the study presented here serves as a foundation for future endeavors.

Direct measurement of pulse wave propagation in capillaries of the human retina.

With each contraction of the heart's left ventricle, a pulse pressure wave surges into the aorta and propagates throughout the vascular tree. The pulse wave drives blood flow forward. Its passage is complex, but it passes more quickly through non-compliant, or stiff, vessels, providing an important signpost of cardiovascular disease. The transparent media of the eye allow direct and non-invasive measurement of this phenomenon within the microvasculature of neural tissue. However, previous estimates differ over three orders of magnitude. Here, we used high spatiotemporal resolution adaptive optics imaging to directly track the pulse wave within individual retinal capillaries in three human subjects. Across 74 unique capillary segments, pulse wave velocity averaged 6.4±0.5mm/sec (mean±SEM). There was large variation between vessels; the slowest pulse wave was at most 0.8 mm/sec and the fastest at least 17.6 mm/sec. In 44% of vessels, the pulse wave traveled upstream, in the opposite direction to flow, suggesting wave reflection from downstream collecting junctions.

Role of endothelium-pericyte signaling in capillary blood flow response to neuronal activity

Local blood flow in the brain is tightly coupled to metabolic demands, a phenomenon termed functional hyperemia. Both capillaries and arterioles contribute to the hyperemic response to neuronal activity via different mechanisms and timescales. The nature and specific signaling involved in the hyperemic response of capillaries versus arterioles, and their temporal relationship are not fully defined. We determined the time-dependent changes in capillary flux and diameter versus arteriolar velocity and flow following whisker stimulation using optical microangiography (OMAG) and two-photon microscopy. We further characterized depth-resolved responses of individual capillaries versus capillary networks. We hypothesized that capillaries respond first to neuronal activation, and that they exhibit a coordinated response mediated via endothelial-derived epoxyeicosatrienoates (EETs) acting on pericytes. To visualize peri-capillary pericytes, we used Tie2-GFP/NG2-DsRed mice, and to determine the role of endothelial-derived EETs, we compared cerebrovascular responses to whisker stimulation between wild-type mice and mice with lower endothelial EETs (Tie2-hsEH). We found that capillaries respond immediately to neuronal activation in an orchestrated network-level manner, a response attenuated in Tie2-hsEH and inhibited by blocking EETs action on pericytes. These results demonstrate that capillaries are first responders during functional hyperemia, and that they exhibit a network-level response mediated via endothelial-derived EETs’ action on peri-capillary pericytes.

Do skeletal muscle motor units and microvascular units align to help match blood flow to metabolic demand?

We explore the motor unit recruitment and control of perfusion of microvascular units in skeletal muscle to determine whether they coordinate to match blood flow to metabolic demand. The PubMed database was searched for historical, current and relevant literature. A microvascular, or capillary unit consists of 2-20 individual capillaries. Individual capillaries within a capillary unit cannot increase perfusion independently of other capillaries within the unit. Capillary units perfuse a short segment of approx. 12 muscle fibres located beside each other. Motor units consist of muscle fibres that can be dispersed widely within the muscle volume. During a contraction, where not all motor units are recruited, muscle fibre contraction will result in increased perfusion of associated capillaries as well as all capillaries within that capillary unit. Perfusion of the entire capillary unit will result in an increased blood flow delivery to muscle fibres associated with active motor unit plus approximately 11 other inactive muscle fibres within the same region. This will result in an overperfusion of the muscle resulting in blood flow in excess of the muscle fibre needs. Given the architecture of the capillary units and the dispersed nature of muscle fibres within a motor unit, during submaximal contractions, where not all motor units are recruited, there will be a greater perfusion to the muscle than that predicted by the number of active muscle fibres. Such overperfusion brings into question if blood flow and metabolic demand are as tightly matched as previously assumed.

Assessing haemorrhage-critical values of cerebral blood flow by modelling biomechanical stresses on capillaries in the immature brain

The development of intraventricular haemorrhages (IVH) in preterm newborns is triggered by a disruption of the vessels responsible for cerebral microcirculation. Analysis of the stresses exerted on vessel walls enables the identification of the critical values of cerebral blood flow (CBF) associated with the development of IVH in preterm infants. The purpose of the present study is the estimation of these critical CBF values using the biomechanical stresses obtained by the finite element modelling of immature brain capillaries. The properties of the endothelial cells and basement membranes employed were selected on the basis of published nanoindentation measurements using atomic force microscopes. The forces acting on individual capillaries were derived with a mathematical model that accounts for the peculiarities of microvascularity in the immature brain. Calculations were based on clinical measurements obtained from 254 preterm infants with the gestational age ranging from 23 to 30 weeks, with and without diagnosis of IVH. No distinction between the affected and control groups with the gestational age of 23 to 26 weeks was possible. For infants with the gestational age of 27 to 30 weeks, the CBF value of 17.03 ml/100 g/min was determined as the critical upper value, above which the likelihood of IVH increases.

In Vivo Confocal Imaging of Fluorescently Labeled Microbubbles: Implications for Ultrasound Localization Microscopy.

We report the time kinetics of fluorescently labeled microbubbles (MBs) in capillary-level microvasculature as measured via confocal microscopy and compare these results to ultrasound localization microscopy (ULM). The observed 19.4 ± 4.2 MBs per confocal field-of-view ( [Formula: see text]) are in excellent agreement with the expected count of 19.1 MBs per frame. The estimated time to fully perfuse this capillary network was 193 s, which corroborates the values reported in the literature. We then modeled the capillary network as an empirically determined discrete-time Markov chain with adjustable MB transition probabilities though individual capillaries. The Monte Carlo random walk simulations found perfusion times ranging from 24.5 s for unbiased Markov chains up to 182 s for heterogeneous flow distributions. This pilot study confirms a probability-derived explanation for the long acquisition times required for super-resolution ULM.

Non-invasive real-time characterization of hollow-core photonic crystal fibers using whispering gallery mode spectroscopy.

Single-ring hollow-core photonic crystal fibers, consisting of a ring of one or two thin-walled glass capillaries surrounding a central hollow core, hold great promise for use in optical communications and beam delivery, and are already being successfully exploited for extreme pulse compression and efficient wavelength conversion in gases. However, achieving low loss over long (km) lengths requires highly accurate maintenance of the microstructure-a major fabrication challenge. In certain applications, for example adiabatic mode transformers, it is advantageous to taper the fibers, but no technique exists for measuring the delicate and complex microstructure without first cleaving the taper at several positions along its length. In this Letter, we present a simple non-destructive optical method for measuring the diameter of individual capillaries. Based on recording the spectrum scattered from whispering gallery modes excited in the capillary walls, the technique is highly robust, allowing real-time measurement of fiber structure during the draw with sub-micron accuracy.

One-step deposition and in-situ reduction of graphene oxide in photonic crystal fiber for all-fiber laser mode locking

Films of graphene oxide (GO) were produced on the inner walls of the glass microcapillaries of photonic crystal fibers via insertion of a GO water suspension followed by quick drying with a hot finger (no previous surface functionalization required). Individual capillaries from an array could also be selectively GO coated. Raman hyperspectral images revealed the films to be continuous along tens of centimeters. Furthermore, the films were thermally reduced and, through an annealing process, the concentration of defects in the resulting reduced graphene oxide (rGO) could be decreased. In particular, the two larger microcapillaries of a polarization maintaining photonic crystal fibers were covered with an rGO film, leading to the demonstration of fibers with polarization dependent loss, which were used as mode lockers for pulsed fiber lasers. Pulses as short as 350 fs, with a spectral bandwidth of 8.1 nm, were generated, being shorter than those previously obtained with GO- or rGO-coated non-polarization maintaining photonic crystal fibers.

PECULIARITIES OF MYOCARDIUM STRUCTURE OF RATS WITH INSULIN RESISTANCE AND IODINE DEFICIENCY

Insulin is a key hormone that functions as a regulator of cellular metabolism in many tissues of the body. Pathogenetically, with the development of IR (oxidative stress, inflammation), endothelial dysfunction is associated with the development of vasoconstrictors and vasodilator synthesis, as well as the functional properties of vascular smooth muscle cells, endothelial cell precursors, fibroblasts and macrophages, which ultimately leads to development cardiovascular pathologies. The studies were carried out on 60 white, nonbreeding, sexually mature male rats weighing 150-180 g, which were divided into two groups: animals with IP (1st experimental group, n = 20) and animals with IP, burdened with YD (2nd experimental group , n = 20). For the purpose of simulating IR in the drinking water of animals for eight weeks, a 10% solution of fructose was added. The structural features of the myocardium of rats with insulin resistance (IR), that were on standard or iodine deficient diets, were considered. It was established that in animals with an IR, a part of myocytes had diminished in diameter, some capillaries in the lumen contained the accumulation of red blood cells and leukocytes. In arterioles and small arteries, individual capillaries endothelial hypercellularity was marked. The wall of a part of such vessels is unevenly homogeneous, with areas of lumens. More significant changes in the structure of the myocardium were detected in rats with iodine deficiency. It was found that in some cases, loose connective tissue between cardiomyocytes was enlarged due to edema (parenchyma-interstitial index was lower by 83 %, p&lt;0.001 for control). In most cardiomyocytes there is a decrease in the content of glycogen, and in individual cells – not observed at all. Parenchymatous cells of the myocardium with the undefined borders, the cytoplasm is lightened and granular, difficult to verified transverse strain.

Visualizing choriocapillaris using swept-source optical coherence tomography angiography with various probe beam sizes.

Imaging choriocapillaris (CC) is a long-term challenge for commercial OCT angiography (OCTA) systems due to limited transverse resolution. Effects of transverse resolution on the visualization of a CC microvascular network are explored and demonstrated in this paper. We use three probe beams with sizes of ~1.12 mm, ~2.51 mm and ~3.50 mm at the pupil plane, which deliver an estimated transverse resolution at the retina of 17.5 µm, 8.8 µm and 7.0 µm, respectively, to investigate the ability of OCTA to resolve the CC capillary vessels. The complex optical microangiography algorithm is applied to extract blood flow in the CC slab. Mean retinal pigment epithelium (RPE) to CC (RPE-CC) distance, mean CC inter-vascular spacing and the magnitude in the radially-averaged power spectrum are quantified. We demonstrate that a clearer CC lobular capillary network is resolved in the angiograms provided by a larger beam size. The image contrast of the CC angiogram with a large beam size of 3.50 mm is 114% higher than that with a small beam size of 1.12 mm. While the measurements of the mean RPE-CC distance and CC inter-vascular spacing are almost consistent regardless of the beam sizes, they are more reliable and stable with the larger beam size of 3.50 mm. We conclude that the beam size is a key parameter for CC angiography if the purpose of the investigation is to visualize the individual CC capillaries.

Mathematical synthesis of the cortical circulation for the whole mouse brain-part I. theory and image integration

Microcirculation plays a significant role in cerebral metabolism and blood flow control, yet explaining and predicting functional mechanisms remains elusive because it is difficult to make physiologically accurate mathematical models of the vascular network. As a precursor to the human brain, this paper presents a computational framework for synthesizing anatomically accurate network models for the cortical blood supply in mouse. It addresses two critical deficiencies in cerebrovascular modeling. At the microscopic length scale of individual capillaries, we present a novel synthesis method for building anatomically consistent capillary networks with loops and anastomoses (=microcirculatory closure). This overcomes shortcomings in existing algorithms which are unable to create closed circulatory networks. A second critical innovation allows the incorporation of detailed anatomical features from image data into vascular growth. Specifically, computed tomography and two photon laser scanning microscopy data are input into the novel synthesis algorithm to build the cortical circulation for the entire mouse brain in silico. Computer predictions of blood flow and oxygen exchange executed on synthetic large-scale network models are expected to elucidate poorly understood functional mechanisms of the cerebral circulation.

Intravital Imaging of Blood Flow and HSPC Homing in Bone Marrow Microvessels.

Two-photon intravital microscopy (2P-IVM) is an advanced imaging technique that allows the visualization of dynamic cellular behavior deeply inside tissues and organs of living animals. Due to the deep tissue penetration, imaging of highly light-scattering tissue as the bone becomes feasible at subcellular resolution.To better understand the influence of blood flow on hematopoietic stem and progenitor cell (HSPC) homing to the bone marrow (BM) microvasculature of the calvarial bone, we analyzed blood flow dynamics and the influence of flow on the early homing behavior of HSPCs during their passage through BM microvessels. Here, we describe a 2P-IVM approach for direct measurements of red blood cell (RBC) velocities in the BM microvasculature using repetitive centerline scans at the level of individual arterial vessels and sinusoidal capillaries to obtain a detailed flow profile map. Furthermore, we explain the isolation and enrichment of HSPCs from long bones and the transplantation of these cells to study the early homing behavior of HSPCs in BM sinusoids at cellular resolution. This is achieved by high-resolution spatiotemporal imaging through a chronic cranial window using transgenic reporter mice.