Laser Speckle Contrast Research Articles

Multimodal real-time imaging with laser speckle contrast and fluorescent contrast

IntroductionLaser speckle contrast imaging (LSCI) can achieve real-time 2D perfusion maps non-invasively. However, LSCI is still difficult to use in general clinical applications because of movement sensitivity and limitations in blood flow analysis. To overcome this, fluorescence imaging (FI) is combined with LSCI using a light source with a wavelength of 785 nm in near-infrared (NIR) region and validates to visualize real-time blood perfusion. Materials and methodsThe system was performed using Intralipid and indocyanine green (ICG) in a flow phantom that has three tubes and controlled the flow rate in 0–150 μl/min range. First, real-time LSCI was monitored and measured the change in speckle contrast by reperfusion. Then, we visualized blood perfusion of a rabbit ear under the non-invasive condition by intravenous injection using a total of five different ICG concentration solutions from 128 μM to 3.22 mM. ResultsThe combined system achieved the performance of processing laser speckle images at about 37–38 fps, and we simultaneously confirmed the fluorescence of ICG and changes in speckle contrast due to intralipid as a light scatterer. In addition, we obtained real-time contrast variation and fluorescent images occurring in rabbit's blood perfusion. ConclusionsThe aim of this study is to provide a real-time diagnostic imaging system that can be used in general clinical applications. LSCI and FI are combined complementary for observing tissue perfusion using a single NIR light source. The combined system could achieve real-time visualization of blood perfusion non-invasively.

Multimodality imaging reveals angiogenic evolution in vivo during calvarial bone defect healing.

The healing of calvarial bone defects is a pressing clinical problem that involves the dynamic interplay between angiogenesis and osteogenesis within the osteogenic niche. Although structural and functional vascular remodeling (i.e., angiogenic evolution) in the osteogenic niche is a crucial modulator of oxygenation, inflammatory and bone precursor cells, most clinical and pre-clinical investigations have been limited to characterizing structural changes in the vasculature and bone. Therefore, we developed a new multimodality imaging approach that for the first time enabled the longitudinal (i.e., over four weeks) and dynamic characterization of multiple in vivo functional parameters in the remodeled vasculature and its effects on de novo osteogenesis, in a preclinical calvarial defect model. We employed multi-wavelength intrinsic optical signal (IOS) imaging to assess microvascular remodeling, intravascular oxygenation (SO2), and osteogenesis; laser speckle contrast (LSC) imaging to assess concomitant changes in blood flow and vascular maturity; and micro-computed tomography (μCT) to validate volumetric changes in calvarial bone. We found that angiogenic evolution was tightly coupled with calvarial bone regeneration and corresponded to distinct phases of bone healing, such as injury, hematoma formation, revascularization, and remodeling. The first three phases occurred during the initial two weeks of bone healing and were characterized by significant in vivo changes in vascular morphology, blood flow, oxygenation, and maturity. Overall, angiogenic evolution preceded osteogenesis, which only plateaued toward the end of bone healing (i.e., four weeks). Collectively, these data indicate the crucial role of angiogenic evolution in osteogenesis. We believe that such multimodality imaging approaches have the potential to inform the design of more efficacious tissue-engineering calvarial defect treatments.

Non-Newtonian Fluid Flow Modeling in the Inertial Viscometer with a Computer Vision System

Purpose of research. Development of theoretical premises for the new inertial viscometer, as well as the development of an approximate model of the viscosity fluid flow using convolutional neural networks and laser speckle contrast imaging data.Methods. The study consists of two parts. The first is devoted to a theoretical study of viscosity fluid flow in the toroidal channel of еру new inertial viscometer. The mathematical model of the flow includes the dimensionless equations of Navier-Stokes and convective heat conduction, the analysis of which made it possible to estimate the conditions for the uniformity of pressure and temperature fields. The numerical solution of the simplified Navier-Stokes equation was obtained by the control volume method. The computational experiment made it possible to identify additional operating conditions for the viscometer. The second part of the research is aimed at solving the problem of predicting the values of the shear strain rate on the tour surface and the flow rate. The approximate flow model is based on an ensemble of convolutional neural networks trained on data from laser speckle-contrast visualization of a fluid flow in a transparent tube.Results. The recommendations on the operating parameters of the inertial viscometer for the studied types of liquids in a given viscosity range are obtained. An approximate model has been developed in the form of an ensemble of deep neural networks, which makes it possible to determine the volumetric flow rate and the shear strain rate on the flow surface based on fluid flow images.Conclusion. The approximate Navier-Stokes equation obtained as a result of theoretical analysis for the flow of a viscous fluid in a toroidal channel can be used to numerical determination the kinematic viscosity. So, the necessary flow characteristics, such as volumetric flow rate and shear strain rate on the flow surface, can be found using the developed and pretrained convolutional neural network based on laser speck contrast imaging data. The test fluid can be any non-Newtonian fluid capable of reflecting coherent radiation. In particular, it can be physiological fluids, including blood.

Characterizing the Correlation Between Angiogenesis and Osteogenesis In Vivo Using Multicontrast Functional Imaging in a Calvarial Defect Model

Angiogenesis is a key factor in bone healing that allows the delivery of oxygen, nutrients, inflammatory and bone precursor cells to the defect site. It induces dramatic structural and functional remodeling of the vasculature during the first 4 weeks of bone healing. Although recent advances in optical imaging have elucidated the in vivo relationship between angiogenesis and osteogenesis in a calvarial defect model, these efforts were mostly limited to structural imaging of the vasculature and bone. Therefore, to better characterize changes in vascular function (i.e. blood flow, oxygenation, etc.) during the bone healing cascade we developed a multicontrast optical imaging framework to assess in vivo changes in microvascular architecture using intrinsic optical signal (IOS) imaging; changes in blood flow with laser speckle contrast (LSC) imaging; and bone formation with bright‐field imaging at high spatial (5 µm) and temporal (200 ms) resolutions. With this system we acquired multicontrast images from 10 animals with calvarial defects every 2 days, over 4 weeks (Fig. 1a, d). The bone and vasculature were then digitally segmented from the bright‐field and IOS images for quantitative analysis (Fig. 1b, c). We also euthanized an animal each week for assessing 3D changes in bone volume and vascular architecture using an ex vivo, high‐resolution (10 µm) CT imaging workflow we recently developed called VascuViz (Fig. 2a, b, d). Using this framework, we demonstrated that angiogenic remodeling within the bone defect microenvironment was most robust from post injury day (D) 4 to D10, during which blood vessel length, volume and density all showed significant increase (Fig. 2c). The blood flow exhibited a sudden increase at D6 when angiogenesis started, and the angiogenic vessels were most perfused at D12 (Fig. 1d). These vascular changes peaked by the end of week 2 and plateaued during the next 2 weeks which were correlated with a large increase in bone volume during the first 2 weeks and smaller increase during the last 2 weeks (Fig. 2e). These results indicate that both structural and functional changes of the microvascular system strongly correlate with bone growth during the early stages of bone healing.Next, we plan to include additional indices of vascular function such as vessel maturity and intravascular oxygenation, as well as image‐based hemodynamic models to further characterize the role of the vascular microenvironment during osteogenesis. We believe that this novel imaging framework for characterizing the defect microenvironment can be utilized to inform the design of novel tissue engineering (TE) constructs for craniofacial bone regeneration. Collectively, these advances herald a new era of “image‐based TE”.

Laser speckle reduction via TiO2‐sapphire composite rotating wheel in laser projection

AbstractLaser sources have been considered to be better light sources compared to traditional lamps or light‐emitting diodes used in projectors, which enables the projector to create images with higher color saturation, brightness, and energy efficiency. However, the speckle noise caused by the coherent nature of the laser is a major technology obstacle for laser projection, which severely affects the image quality. To suppress laser speckle, a TiO2‐sapphire composite (TSC) with rough and high reflectivity surface and high thermal conductivity substrate is designed, which reduces the laser speckle by creating spatial diversity. Moreover, combined with a micro motor, the design and fabrication of the TSC rotating wheel are realized, which further reduces the laser speckle by creating time diversity. The experimental results show that the laser speckle contrast can be reduced from 9.0% to 2.2% when placing the TSC rotating wheel in the light path, which is below the speckle perception limit of the human eye (<4%). This new type of laser speckle suppression device is simple to use, low in cost, high in energy utilization, high in thermal conductivity, easy to mass production, and has great application potential in high‐power laser projection.

Transient Hypoperfusion to Ischemic/Anoxic Spreading Depolarization is Related to Autoregulatory Failure in the Rat Cerebral Cortex

BackgroundIn ischemic stroke, cerebral autoregulation and neurovascular coupling may become impaired. The cerebral blood flow (CBF) response to spreading depolarization (SD) is governed by neurovascular coupling. SDs recur in the ischemic penumbra and reduce neuronal viability by the insufficiency of the CBF response. Autoregulatory failure and SD may coexist in acute brain injury. Here, we set out to explore the interplay between the impairment of cerebrovascular autoregulation, SD occurrence, and the evolution of the SD-coupled CBF response.MethodsIncomplete global forebrain ischemia was created by bilateral common carotid artery occlusion in isoflurane-anesthetized rats, which induced ischemic SD (iSD). A subsequent SD was initiated 20–40 min later by transient anoxia SD (aSD), achieved by the withdrawal of oxygen from the anesthetic gas mixture for 4–5 min. SD occurrence was confirmed by the recording of direct current potential together with extracellular K+ concentration by intracortical microelectrodes. Changes in local CBF were acquired with laser Doppler flowmetry. Mean arterial blood pressure (MABP) was continuously measured via a catheter inserted into the left femoral artery. CBF and MABP were used to calculate an index of cerebrovascular autoregulation (rCBFx). In a representative imaging experiment, variation in transmembrane potential was visualized with a voltage-sensitive dye in the exposed parietal cortex, and CBF maps were generated with laser speckle contrast analysis.ResultsIschemia induction and anoxia onset gave rise to iSD and aSD, respectively, albeit aSD occurred at a longer latency, and was superimposed on a gradual elevation of K+ concentration. iSD and aSD were accompanied by a transient drop of CBF (down to 11.9 ± 2.9 and 7.4 ± 3.6%, iSD and aSD), but distinctive features set the hypoperfusion transients apart. During iSD, rCBFx indicated intact autoregulation (rCBFx < 0.3). In contrast, aSD was superimposed on autoregulatory failure (rCBFx > 0.3) because CBF followed the decreasing MABP. CBF dropped 15–20 s after iSD, but the onset of hypoperfusion preceded aSD by almost 3 min. Taken together, the CBF response to iSD displayed typical features of spreading ischemia, whereas the transient CBF reduction with aSD appeared to be a passive decrease of CBF following the anoxia-related hypotension, leading to aSD.ConclusionsWe propose that the dysfunction of cerebrovascular autoregulation that occurs simultaneously with hypotension transients poses a substantial risk of SD occurrence and is not a consequence of SD. Under such circumstances, the evolving SD is not accompanied by any recognizable CBF response, which indicates a severely damaged neurovascular coupling.

A Portable Multicontrast Microscope for Multiscale Imaging of the Microcirculation

Objective To develop a portable microscope capable of interrogating the microcirculation in vivo with multiple contrasts over multiple spatial scales. Methods We used state-of-the-art 3D printing and optics design techniques to fabricate a miniature microscope the size of a US quarter (Fig. 1 a, b)1. It weights 9g and acquires images via three optical contrasts: intrinsic optical signals (IOS)2, laser speckle contrast (LSC)3 and fluorescence (FL). The IOS channel, which relies on light absorption by red blood cell (RBC) bound hemoglobin (HbT), can be used to visualize microvascular morphology, while the LSC channel enables observing blood flow levels by using the orderly flow of RBCs along vessels as a contrast agent. The FL channel can be used for arterio-venous discrimination via the arrival times of an intravenously (iv) injected fluorescent dye (e.g. FITC-Dextran, 70 kDa). The microscope can acquire images at 15 frames per second over a 3x3 mm2 field of view (FoV) at 5 µm resolution. We also designed an adaptor which enhances the resolution tenfold to 0.5 µm. It is operated via USB connections to a laptop, and is portable (Fig. 1c), making it convenient for imaging the microcirculation in a range of preclinical applications. Results We demonstrated the utility of our microscope by using it to interrogate the structure and function of the murine brain and ear microcirculation at different spatial scales. Figs. 1d, e show maps of microvascular morphology and blood flow in a portion of the murine brain under ketamine anesthesia at 5 µm resolution. Fig. 1f shows the map of arrival times of the iv injected fluorescent dye. Dark blue regions corresponding to arrival times less than 0.5 s were used to identify arteries. Figs. 1g, h show plots of changes in HbT (i.e. cerebral blood volume) and cerebral blood flow (CBF) corresponding to an artery and vein (boxes marked “A” and “V” in Fig. 1d) over 3 hours while the animal recovers from anesthesia. Here, the arterial and venous time courses illustrate contrasting flow-volume relationships that indicate the varying systemic effects of anesthesia on components of the microcirculation. Fig 2a shows the microscope with its high-magnification adaptor, while Fig. 2b illustrates the exquisite microvascular details revealed with the adaptor in contrast to images acquired without it. Here, the FL channel was used in conjunction with an iv injected dye to enhance microvascular contrast. One could also “stitch” together these high-resolution images to create a mosaic with wider coverage (Fig. 2c). Since individual RBCs were discernable in these high-resolution images (Fig. 2d), we successfully employed RBC tracking to characterize cellular-scale flow dynamics within the microcirculation (Fig. 2e). Conclusion The multicontrast and multiscale imaging capabilities of our microscope will enable investigators to comprehensively interrogate the structure and function of microcirculation in diverse organ sites and preclinical disease models. Its design and portability enables imaging of unanesthetized animals, convenient usage, and fosters collaborations.

Sexual Dimorphisms in Hypertension‐Associated Cerebrovascular Damage

Hypertension is a major risk factor for cerebral small vessel disease (CVSD), which increases stroke and dementia risk. Hypertension alters artery structure, impairing blood flow regulation and preventing nutrient delivery to the brain parenchyma. We previously showed angiotensin II (AngII)-hypertension in male mice reduces overall pial blood flow measured by scanning laser Doppler and causes inward hypotrophic remodeling in cerebral parenchymal arterioles (PAs). Epidemiological evidence suggests sex differences exist in CVSD, with large vessel damage occurring more in males and small vessel damage occurring more in females. We hypothesized hypertensive male and female mice would have reductions in pial blood flow measured by laser speckle contrast and inward hypotrophic remodeling in PAs. AngII-filled osmotic minipumps (800ng/kg/min, 4 weeks) were implanted in 16-18-week-old male C56BL/6 mice. Age-matched female mice received either an AngII dose that matches the dose used in male mice (800ng/kg/min) or a higher dose (1200ng/kg/day) that produces an elevation in blood pressure similar to male mice. Sham mice served as control. Blood pressure was measured by tail-cuff plethysmography. Pial artery blood flow was measured in the frontal, parietal, and temporal areas of both brain hemispheres. After euthanasia, PAs were isolated and mounted on a pressure myograph for structure assessment by increasing intraluminal pressure from 3 to 120mmHg in 20mmHg increments. Data are presented as means ± SEM (n=6-9). PA data are reported at an intraluminal pressure of 40mmHg. Systolic blood pressure was elevated in AngII-treated male mice (sham: 133 ± 10, AngII: 181 ± 11mmHg; p<0.05) and in 1200ng AngII-treated female mice (sham: 146 ± 8, 800ng AngII: 161 ± 13, 1200ng AngII: 179* ± 7mmHg; p<0.05) vs sham. There was a sex difference in blood flow alterations during hypertension. Male AngII treated mice had reduced flow in the parietal region (sham: 361 ± 19, AngII: 301 ± 9.3A.U.; p<0.05) but not frontal (sham: 359 ± 19, AngII: 327 ± 11A.U.; p=0.19) or temporal regions (sham: 292 ± 13, AngII: 284 ± 15A.U.; p=0.68) vs sham. AngII treated female mice had reductions in pial flow in the parietal (sham: 403 ± 9, 800ng AngII: 336* ± 19, 1200ng AngII: 314* ± 33 A.U.; p<0.05), frontal (sham: 378 ± 7, 800ng AngII: 314* ± 13, 1200ng AngII: 279* ± 24 A.U.; p<0.05), and temporal (sham: 338 ± 8.4, 800ng AngII: 273* ± 11, 1200ng AngII: 278* ± 25 A.U.; p<0.05) regions vs sham. PAs from male AngII mice had reduced outer diameter (sham: 56 ± 3, AngII: 46 ± 2μm; p<0.05), lumen diameter (sham: 47 ± 1, AngII: 40 ± 2μm; p=0.05), and wall area (sham: 719 ± 115, AngII: 405 ± 24μm2; p<0.05) vs sham, reproducing previous findings. PAs from AngII treated female mice did not exhibit significant reductions in outer diameter (sham: 57 ± 3, 800ng AngII: 50 ± 5, 1200ng AngII: 47 ± 4μm; p=0.17) or lumen diameter (sham: 49 ± 2, 800ng AngII: 43 ± 5, 1200ng AngII: 41 ± 4μm; p=0.22, but showed a modest reduction in wall area (sham: 647 ± 64, 800ng AngII: 534 ± 78, 1200ng AngII: 439 ± 58μm2; p=0.09) vs sham. These data suggest the mechanism of hypertension-induced vascular damage differs between males and females. Understanding sex differences in CVSD is critical for dementia and stroke prevention.

Intensity-dependent speckle contrast of second harmonic light in a nonlinear disordered medium.

Coherent light propagation and scattering in diffuse media realize an interference effect that manifests as a random spatial distribution of bright and dark spots called speckle. Laser speckle contrast is a crucial parameter that quantifies light behavior in diffuse media, often of help in biomedical imaging applications. With several ultra-intense lasers coming onto the scene in recent years, the probability of nonlinearly generating harmonic light has considerably increased. To our knowledge, there are no studies on the simultaneous measurement of local speckle contrast (LSC) at fundamental and harmonic wavelengths. In this work, we experimentally characterize the intensity-dependent global and local speckle contrast at λ=1064nm (IR) and λ=532nm [second-harmonic generation (SHG)] in a diffuse sample made of potassium dihydrogen phosphate. Under pulsed excitation with IR light, second-harmonic light is generated and undergoes scattering, leading to speckle at both wavelengths. The radiation pressure on the sample induces displacement of the particles of the medium, which leads to the temporal averaging and hence reduction in speckle contrast of IR. The decrease in speckle contrast with input power is consistent with the theoretically predicted behavior. Next, the probability distribution of LSC for IR and SHG are observed to follow log-normal distributions with distinctive parameters, measured for co-polarized and counter-polarized channels. Parameter evolution with box-size is fit to the established global model for speckle contrast and also another alternative model. The fit parameters, illustrated as a function of input power, indicate possibilities of widening the global model to second-harmonic light.

Blood Flow Visualization by Means of Laser Speckle-Contrast Measurements under the Conditions of Nonergodicity

The influence of stationary structural inclusions in inhomogeneous strongly scattering media such as biotissues on the results of speckle-contrast measurements using temporal and spatial methods of image processing by means of laser speckle-contrast imaging is investigated. The limits of applicability of the method of laser speckle-contrast imaging are established in detail for the case in which ergodicity conditions are not fulfilled. Based on model experiments, it is demonstrated that an increase in the number of stationary scatterers relative to that of the dynamic ones in the sampling volume introduces substantial error into the results of spatial and temporal processing of speckle images for given exposure time of the detector. At the same time, analysis of spatial and temporal speckle contrasts, the values of the coefficient of speckle dynamics, along with the results of Monte-Carlo simulation of the sampling volume, revealed that the presence of a relatively thin, up to 30% of entire volume, static layer does not introduce considerable changes into the results of measurements by the method of laser speckle-contrast imaging. The exposure time of the camera, along with the number of frames used for image processing, can be varied and chosen individually for each experiment. The developed algorithms of spatial and temporal processing of images obtained by the method of laser speckle-contrast imaging were tested in the experiments on transcranial visualization of the cerebral blood flow of a mouse.

Female Mice Are Resistant to Inward Remodeling of Parenchymal Arterioles Observed in Male Mice During Angiotensin II‐Induced Hypertension

Hypertension increases risk of vascular cognitive impairment and dementia (VCID) by impairing the structure and function of cerebral arteries. These arterial changes result in chronic hypoperfusion of the brain parenchyma and, eventually, cognitive impairment. Epidemiological studies suggest a sex difference in development of VCID, with women being protected from its development until late in age. We have previously shown that angiotensin II (AngII)‐hypertension led to inward hypotrophic remodeling and decreased wall stress in the parenchymal arterioles (PAs) of male mice. This PA remodeling in AngII‐hypertension was associated with reduced cerebral perfusion, increased myogenic tone, and cognitive dysfunction. Female mice, however, were protected from memory impairments despite a similar increase in blood pressure and decrease in cerebral perfusion measured by scanning laser Doppler. We hypothesized that AngII‐hypertension in female mice will cause a decrease in cerebral perfusion measured by laser speckle contrast, as well as a decrease in outer diameter, lumen diameter, and wall thickness in the PAs. Sixteen to eighteen‐week‐old female C56BL/6 mice were treated with AngII (1200 ng/kg/min via osmotic mini‐pump) for four weeks. This dose was selected to produce a similar increase in blood pressure to the previously studied male mice. Sham mice served as control. Blood pressure was measured by tail‐cuff plethysmography and pial blood flow was measured. After euthanasia, brains were collected and PAs were isolated and mounted on a pressure myograph for assessment of spontaneous myogenic tone generation and structure. PA structure was assessed by increasing intralumenal pressure from 3mmHg to 120mmHg in 20mmHg increments. Results are presented as means ± SEM and PA data are reported at an intralumenal pressure of 40mmHg (n=5–12). Systolic blood pressure was increased (Sham: 141 ± 7, AngII: 179 ± 7mmHg; p&lt;0.05) and cerebral perfusion was reduced in AngII‐hypertension (Sham: 328 ± 6, AngII: 288 ± 11 perfusion units; p&lt;0.05). AngII infusion slightly decreased lumen (Sham: 49 ± 3, AngII: 42 ± 4μm; p=0.17) and outer (Sham: 57 ± 4, AngII: 48 ± 4μm; p=0.11) diameters, although not significantly. AngII‐hypertension reduced wall thickness (Sham: 4 ± 0.3, AngII: 3 ± 0.2μm; p&lt;0.05) and wall area (Sham: 691 ± 83, AngII: 441 ± 52μm2; p&lt;0.05). PAs from female mice did not show reductions in wall stress (Sham: 241.8 ± 19, AngII: 268 ± 20dynes/cm2; p&gt;0.05) or generate more myogenic tone in response to hypertension (Sham: 27 ± 4, AngII: 22 ± 2%; p&gt;0.05). While AngII‐hypertension resulted in reductions in lumen diameter, outer diameter, wall thickness, and wall stress in the PAs of male mice, PAs of female mice had only reduced wall thickness with modest reductions in lumen and outer diameters. Preliminary data suggest that females may be resistant to inward PA remodeling during AngII‐hypertension, despite similar reductions in cerebral blood flow as male mice. This potential protection of the cerebrovascular structure and myogenic tone generation could explain the absence of cognitive dysfunction in response to hypertension in female mice.Support or Funding Information5T32GM092715‐14

HemoSYS: An Image‐based Toolkit for Quantifying Hemodynamics in the Microcirculation

ObjectiveTo develop an easy‐to‐use image‐based toolkit for quantifying spatiotemporal hemodynamic changes in the healthy and pathologic microcirculation.MethodsWe combined the capabilities of two well‐known image processing platforms: Matlab® (MathWorks, MA) and ImageJ into a single framework, and created an interactive graphical user interface (GUI) to manage all aspects of user interactions. The resulting toolkit for quantifying hemodynamic changes, HemoSYS, can be utilized without any prior programming knowledge, and consists of five analysis modules: propagation, clustering, coupling, perturbation, and Fourier. We demonstrate the utility of HemoSYS by using it to quantify hemodynamic changes within the tumor microenvironment (TME) of a murine breast cancer model. To acquire multi‐variable microcirculatory dynamics of the TME, we used a multi‐contrast optical imaging system and obtained wide field (i.e. 5 × 7 mm2) images of tumor extent via fluorescence (FL) imaging of GFP tagged tumor cells, and concurrent image‐sequences of tumor microvasculature or blood volume (BV) via hemoglobin‐based intrinsic optical signal (IOS) imaging, intravascular oxygen saturation (HbSat) via IOS imaging, and blood flow (BF) via laser speckle contrast (LSC) imaging every 30 seconds, for 1 hour.ResultsFig. 1 shows a schematic of HemoSYS illustrating its input data and capabilities. Using HemoSYS on a cohort of 5 animals, we identified the tumor extent and interrogated: (i) the propagation characteristics of an acute hypoxic episode; (ii) the formation and cessation of clusters with distinct microcirculatory dynamics; (iii) the dysregulation of blood flow; (iv) the multi‐variable hemodynamic response to carbogen gas inhalation;and (v) the heterogeneity of microcirculatory dynamics in the frequency domain.ConclusionHemoSYS (freely available upon request) enables characterization of hemodynamic changes in microvascular beds using a systems biology approach. It can be used by anyone interested in quantifying hemodynamic changes from imaging data because it does not require any programming experience. While we demonstrated its utility using wide‐field optical imaging data, HemoSYS could be readily modified to accept input data from additional imaging modalities such as MR, PET, and Photoacoustic imaging. We believe that HemoSYS will become a tool‐of‐choice for image‐based characterization of the microcirculation in any preclinical disease model or tissue bed.Support or Funding InformationThis work was supported by NCI 1R01CA196701, 5R01CA138264 and a Kavli Neuroscience Distinguished Fellowship (JS).Schematic of HemoSYS illustrating its inputs and capabilities.HemoSYS accepts a broad range of inputs, from static images indicating an area of interest (e.g. the tumor extent) to multiple image‐sequences showing concurrent spatiotemporal changes in multiple hemodynamic variables (e.g. BV, HbSat and BF). One can then interactively use HemoSYS’s five modules (i.e. propagation, clustering, coupling, perturbation and Fourier) to quantify and characterize the underlying microcirculation.Figure 1

Assessing Renal Microvascular Reactivity by Laser Speckle-Contrast Imaging in Angiotensin-II-Treated Mice.

IntroductionThe kidney is one of the main organs affected by microvascular damage wrought by hypertension. We developed an approach to investigate renal microcirculatory disturbance in live mice by measuring post-occlusive reactive hyperemia (PORH), a reactivity test exploring endothelial and neuro-microvascular functioning. Laser speckle-contrast analysis (LASCA) assesses microvascular blood flow; it provides real-time images of spatial and temporal blood flow dynamics. We compared basal blood flow and PORH test between control and angiotensin-II-treated mice (Ang-II) to validate the model.ObjectiveThe study objective was to develop an approach to investigate renal microcirculation, and then to compare microvascular reactivity assessed on LASCA in control versus Ang-II mice.MethodsThirty 7-week-old wild-type C57BL/6J mice were allocated into two groups. One received angiotensin-II via osmotic minipumps (Ang-II; n=15); the other served as control (n=15). Basal blood flow was measured on LASCA. The PORH test was then performed in the two groups.ResultsControl mice had significantly lower basal renal microcirculatory flow, expressed in perfusion units (PU), than Ang-II-treated mice (1448 ± 96 vs 1703 ± 185 PU, respectively; P < 0.05). Peak flow was lower in controls than in Ang-II mice (1617±104 vs.1724 ± 205 PU, respectively; P=0.21). Control mice had significantly higher kidney PORH than Ang-II mice (8±3 vs 1±4%, respectively; P < 0.05).ConclusionWe developed an innovative technique to study renal microcirculation in mice. Ang-II-treated mice showed significantly higher basal blood flow than controls, while PORH was significantly higher in controls than in Ang-II mice.

Imaging the Landmarks of Vascular Recovery.

Background: Peripheral arterial disease (PAD) is a major worldwide health concern. Since the late 1990s therapeutic angiogenesis has been investigated as an alternative to traditional PAD treatments. Although positive preclinical results abound in the literature, the outcomes of human clinical trials have been discouraging. Among the challenges the field has faced has been a lack of standardization of the timings and measures used to validate new treatment approaches.Methods: In order to study the spatiotemporal dynamics of both perfusion and neovascularization in mice subjected to surgically-induced hindlimb ischemia (n= 30), we employed three label-free imaging modalities (a novel high-sensitivity ultrasonic Power Doppler methodology, laser speckle contrast, and photoacoustic imaging), as well as a tandem of radio-labeled molecular probes, 99mTc-NC100692 and 99mTc-BRU-5921 respectively, designed to detect two key modulators of angiogenic activity, αVβ3 and HIF-1α , via scintigraphic imaging.Results: The multimodal imaging strategy reveals a set of “landmarks”—key physiological and molecular events in the healing process—that can serve as a standardized framework for describing the impact of emerging PAD treatments. These landmarks span the entire process of neovascularization, beginning with the rapid decreases in perfusion and oxygenation associated with ligation surgery, extending through pro-angiogenic changes in gene expression driven by the master regulator HIF-1α , and ultimately leading to complete functional revascularization of the affected tissues.Conclusions: This study represents an important step in the development of multimodal non-invasive imaging strategies for vascular research; the combined results offer more insight than can be gleaned through any of the individual imaging methods alone. Researchers adopting similar imaging strategies and will be better able to describe changes in the onset, duration, and strength of each of the landmarks of vascular recovery, yielding greater biological insight, and enabling more comprehensive cross-study comparisons. Perhaps most important, this study paves the road for more efficient translation of PAD research; emerging experimental treatments can be more effectively assessed and refined at the preclinical stage, ultimately leading to better next-generation therapies.

A miniature multi-contrast microscope for functional imaging in freely behaving animals

Neurovascular coupling, cerebrovascular remodeling and hemodynamic changes are critical to brain function, and dysregulated in neuropathologies such as brain tumors. Interrogating these phenomena in freely behaving animals requires a portable microscope with multiple optical contrast mechanisms. Therefore, we developed a miniaturized microscope with: a fluorescence (FL) channel for imaging neural activity (e.g., GCaMP) or fluorescent cancer cells (e.g., 9L-GFP); an intrinsic optical signal (IOS) channel for imaging hemoglobin absorption (i.e., cerebral blood volume); and a laser speckle contrast (LSC) channel for imaging perfusion (i.e., cerebral blood flow). Following extensive validation, we demonstrate the microscope’s capabilities via experiments in unanesthetized murine brains that include: (i) multi-contrast imaging of neurovascular changes following auditory stimulation; (ii) wide-area tonotopic mapping; (iii) EEG-synchronized imaging during anesthesia recovery; and (iv) microvascular connectivity mapping over the life-cycle of a brain tumor. This affordable, flexible, plug-and-play microscope heralds a new era in functional imaging of freely behaving animals.

Comparison of speckleplethysmographic (SPG) and photoplethysmographic (PPG) imaging by Monte Carlo simulations and in vivo measurements.

Noncontact photoplethysmography (PPG) is limited by a poor signal-to-noise ratio (SNR). A solution to this limitation is the use of alternate sources of optical contrast to generate a complementary pulsatile waveform. One such source is laser speckle contrast, which is modulated in biological tissues by the flow rate of red blood cells. Averaging a region of interest from a speckle contrast image over time allows for the calculation of a speckleplethysmogram (SPG). Similar to PPG, SPG enables monitoring of heart rate and respiratory rate. A gap in the knowledge base exists as to the precise spatiotemporal relationship between PPG and SPG signals. We have developed an eight-layer tissue model to simulate both PPG and SPG signals in a reflectance geometry via Monte Carlo methods. We modeled PPG by compression of the upper and lower blood nets due to expansion of the larger arterial layer below. The in silico PPG peak-to-peak amplitude percent was greater at 532 nm than at 860 nm (5.6% vs. 3.0%, respectively), which matches trends from the literature. We modeled SPG by changing flow speeds of red blood cells in both the capillaries and arterioles over the cardiac cycle. The in silico SPG peak-to-peak amplitude percent was 24% at 532 nm and 40% at 860 nm. In silico results are similar to in vivo results measured with a two-camera set up for simultaneous imaging of PPG and SPG. Both in silico and in vivo data suggest SPG has a much larger SNR than PPG, which may prove beneficial for noncontact, wide-field optical monitoring of cardiovascular health.

Phenotyping the Microvasculature in Critical-Sized Calvarial Defects via Multimodal Optical Imaging.

Tissue-engineered scaffolds are a powerful means of healing craniofacial bone defects arising from trauma or disease. Murine models of critical-sized bone defects are especially useful in understanding the role of microenvironmental factors such as vascularization on bone regeneration. Here, we demonstrate the capability of a novel multimodality imaging platform capable of acquiring in vivo images of microvascular architecture, microvascular blood flow, and tracer/cell tracking via intrinsic optical signaling (IOS), laser speckle contrast (LSC), and fluorescence (FL) imaging, respectively, in a critical-sized calvarial defect model. Defects that were 4 mm in diameter were made in the calvarial regions of mice followed by the implantation of osteoconductive scaffolds loaded with human adipose-derived stem cells embedded in fibrin gel. Using IOS imaging, we were able to visualize microvascular angiogenesis at the graft site and extracted morphological information such as vessel radius, length, and tortuosity two weeks after scaffold implantation. FL imaging allowed us to assess functional characteristics of the angiogenic vessel bed, such as time-to-peak of a fluorescent tracer, and also allowed us to track the distribution of fluorescently tagged human umbilical vein endothelial cells. Finally, we used LSC to characterize the in vivo hemodynamic response and maturity of the remodeled microvessels in the scaffold microenvironment. In this study, we provide a methodical framework for imaging tissue-engineered scaffolds, processing the images to extract key microenvironmental parameters, and visualizing these data in a manner that enables the characterization of the vascular phenotype and its effect on bone regeneration. Such multimodality imaging platforms can inform optimization and design of tissue-engineered scaffolds and elucidate the factors that promote enhanced vascularization and bone formation.

Laser Speckle Contrast Imaging for Measuring Cerebral Blood Flow Changes Caused by Electrical Sensory Stimulation

Recently laser speckle contrast (LSC) imaging has become a widely used optical method for in vivo assessment of blood flow in the animal brain. LSC imaging is useful for monitoring brain hemodynamics with relatively high spatio-temporal resolution. A speckle contrast imaging system has been implemented with electrical sensory stimulation apparatus. LSC imaging is combined with optical intrinsic signal imaging in order to measure changes in cerebral blood flow as well as neural activity in response to electrical sensory stimulation applied to the hindlimb region of the mouse brain. We found that blood flow and oxygen consumption are correlated and both sides of hindlimb activation regions are symmetrically located. This apparatus could be used to monitor spatial or temporal responses of cerebral blood flow in animal disease models such as ischemic stroke or cortical spreading depression.

Early Recruitment of Cerebral Microcirculation by Neuronal Nitric Oxide Synthase Inhibition in a Juvenile Ischemic Rat Model

Background: The development of collateral circulation is proposed as an inherent compensatory mechanism to restore impaired blood perfusion after ischemia, at least in the penumbra. We have studied the dynamic macro- and microcirculation after ischemia-reperfusion in the juvenile rat brain and evaluated the impact of neuronal nitric oxide synthase (nNOS) inhibition on the collateral flow. Methods: Fourteen-day-old (P14) rats were subjected to ischemia-reperfusion and treated with either PBS or 7-nitroindazole (7-NI, an nNOS inhibitor, 25 mg/kg). Arterial blood flow (BF) was measured using 2D-color-coded pulsed ultrasound imaging. Laser speckle contrast (LSC) imaging and sidestream dark-field videomicroscopy were used to measure cortical and microvascular BF, respectively. Results: In basal conditions, 7-NI reduced BF in the internal carotids (by ∼25%) and cortical (by ∼30%) BF, as compared to PBS. During ischemia, the increased mean BF velocity in the basilar trunk after both PBS and 7-NI demonstrated the establishment of collateral support and patency. Upon re-flow, BF immediately recovered to basal values in the internal carotid arteries under both conditions. The 7-NI group showed increased collateral flow in the penumbral tissue during early re-flow compared to PBS, as shown with both LSC imaging and side-stream dark-field videomicroscopy. The proportion of perfused capillaries was significantly increased under 7-NI as compared to PBS when given before ischemia (67.0 ± 3.9 vs. 46.8 ± 8.8, p < 0.01). Perfused capillaries (63.1 ± 17.7 vs. 81.1 ± 20.7, p < 0.001) and the BF index (2.4 ± 0.6 vs. 1.3 ± 0.1, p < 0.001) significantly increased under 7-NI given at the re-flow onset. Conclusions: Collateral support in the penumbra is initiated during ischemia, and may be increased during early re-flow by neuronal NOS inhibition (given in pre- and post-treatment), which may preserve brain tissue in juvenile rats.

Continuous monitoring of arthritis in animal models using optical imaging modalities.

Given the several difficulties associated with histology, including difficulty in continuous monitoring, this study aimed to investigate the feasibility of optical imaging modalities—cross-polarization color (CPC) imaging, erythema index (EI) imaging, and laser speckle contrast (LSC) imaging—for continuous evaluation and monitoring of arthritis in animal models. C57BL/6 mice, used for the evaluation of arthritis, were divided into three groups: arthritic mice group (AMG), positive control mice group (PCMG), and negative control mice group (NCMG). Complete Freund’s adjuvant, mineral oil, and saline were injected into the footpad for AMG, PCMG, and NCMG, respectively. LSC and CPC images were acquired from 0 through 144 h after injection for all groups. EI images were calculated from CPC images. Variations in feet area, EI, and speckle index for each mice group over time were calculated for quantitative evaluation of arthritis. Histological examinations were performed, and the results were found to be consistent with those from optical imaging analysis. Thus, optical imaging modalities may be successfully applied for continuous evaluation and monitoring of arthritis in animal models.