Realtime assessment of vascular occlusion and reperfusion in animal models of intraoperative imaging - a pilot study.

Objectives: Intraoperative monitoring of blood flow (BF) remains vital to guiding surgical decisions. Here, we report the use of SurgeON™ Blood Flow Monitor (BFM), a prototype system that attaches to surgical microscopes and implements laser speckle contrast imaging (LSCI) to noninvasively obtain and present vascular BF information in real-time within the microscope's eyepiece. Methods: The ability of SurgeON BFM to monitor BF status during reversible vascular occlusion procedures was investigated in two large animal models: occlusion of saphenous veins in six NZW rabbit hindlimbs and clipping of middle cerebral artery (MCA) branches in four Dorset sheep brain hemispheres. SurgeON BFM acquired, presented, and stored LSCI-based blood flow velocity index (BFVi) data and performed indocyanine green video angiography (ICG-VA) for corroboration. Results: Stored BFVi data were analyzed for each phase: pre-occlusion (baseline), with the vessel occluded (occlusion), and after reversal of occlusion (re-perfusion). In saphenous veins, BFVi relative to baseline reduced to 5.2±3.7 % during occlusion and returned to 102.9±14.9 % during re-perfusion. Unlike ICG-VA, SurgeON BFM was able to monitor reduced BFVi and characterize re-perfusion robustly during five serial occlusion procedures conducted 2-5 min apart on the same vessel. Across four sheep MCA vessels, BFVi reduced to 18.6±7.7 % and returned to 120.1±27.8 % of baseline during occlusion and re-perfusion phases, respectively. Conclusions: SurgeON BFM can noninvasively monitor vascular occlusion status and provide intuitive visualization of BF information in real-time to an operating surgeon. This technology may find application in vascular, plastic, and neurovascular surgery.

Association of Speckle-Based Blood Flow Measurements and Fluorescein Angiography in Infants with Retinopathy of Prematurity.

Purpose: To determine the correlation between blood flow metrics measured by intravenous fluorescein angiography (IVFA) and the blood flow velocity index (BFVi) obtained by laser speckle contrast imaging (LSCI) in infants with retinopathy of prematurity (ROP). Design: Prospective comparative pilot study. Subjects: Seven eyes from 7 subjects with ROP. Methods: Unilateral LSCI and IVFA data were obtained from each subject in the neonatal intensive care unit. Five LSCI-based metrics and 5 IVFA-based metrics were extracted from images to quantify blood flow patterns in the same region of interest. Correlation between LSCI-based and IVFA-based blood flow metrics was compared between 2 subgroups of ROP severity: moderate ROP (defined as stage ≤ 2 without Plus disease) and severe ROP (defined as stage ≥3 or Plus disease). Main Outcome Measures: Pearson and Kendall rank correlation coefficients between IVFA and LSCI metrics; Student t test P values comparing LSCI metrics between "severe" and "moderate" ROP groups. Results: Pearson correlations between IVFA and LSCI included arterial-venous transit time (AVTT) and peak BFVi (pBFVi; r = -0.917; P = 0.004), AVTT and dip BFVi (dBFVi; r = -0.920; P = 0.003), AVTT and mean BFVi (r = -0.927- P = 0.003), and AVTT and volumetric rise index (r = -0.779; P = 0.039). Kendall rank correlation between AVTT and dBFVi was r = -0.619 (P = 0.051). pBFVi was higher in severe ROP than in moderate ROP (8.4 ± 0.6 and 4.4 ± 1.8, respectively; P = 0.0045 using the 2-sample t test with pooled variance and P = 0.0952 using the Wilcoxon rank-sum test). Conclusions: Correlation was found between blood flow metrics obtained by IVFA and noninvasive LSCI techniques. We demonstrate the feasibility of obtaining quantitative metrics using LSCI in infants with ROP in this pilot study; however, further investigation is needed to evaluate its potential use in clinical assessment of ROP severity. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Dynamic Alterations in Blood Flow in Glaucoma Measured with Laser Speckle Contrast Imaging.

PURPOSE: To assess the repeatability of blood flow velocity index (BFVi) metrics obtained with a recently Food and Drug Administration-cleared laser speckle contrast imaging device, the XyCAM RI (Vasoptic Medical, Inc), and to characterize differences in these metrics among control, glaucoma suspect, and glaucoma participants. DESIGN: Prospective, observational study. PARTICIPANTS: Forty-six participants: 20 control, 16 glaucoma suspect, and 10 glaucoma participants, 1 eye per participant. METHODS: Key dynamic BFVi metrics-mean, peak, dip, volumetric rise index (VRI), volumetric fall index (VFI), time to rise (TtR), time to fall (TtF), blow-out time (BOT), skew, and acceleration time index-were measured in the optic disc, optic disc vessels, optic disc perfusion region, and macula in 4 imaging sessions on the same day. Intrasession and intersession variability were calculated using the coefficient of variation (CV) for each metric in each region of interest (ROI). Values for each dynamic BFVi variable were compared between glaucoma, glaucoma suspect, and control participants using bivariate and multivariate analysis. Pearson correlation coefficients were used to correlate each variable in each ROI with age, intraocular pressure, cup-to-disc ratio (CDR), mean deviation, pattern standard deviation, retinal nerve fiber layer thickness, and minimum rim width. MAIN OUTCOME MEASURES: Coefficient of variation for the intrasession and intersession variability for each dynamic BFVi metric in each ROI and differences in each metric in each ROI between each diagnostic group. RESULTS: Intersession CV for mean, peak, dip, VRI, VFI, TtR, and TtF ranged from 3.2 ± 2.5% to 11.0 ± 3.8%. Age, CDR, OCT metrics, and visual field metrics showed significant correlations with dynamic BFVi variables. Peak, mean, dip, VRI, and VFI were significantly lower in patients with glaucoma than in control participants in all ROIs except the fovea. These metrics also were significantly lower in glaucoma patients than glaucoma suspect patients in the disc vessels. CONCLUSIONS: Dynamic blood flow metrics measured with the XyCAM RI are reliable, are associated with structural and functional glaucoma metrics, and are significantly different among glaucoma, glaucoma suspect, and control participants. The XyCAM RI may serve as an important tool in glaucoma management in the future.

Current and novel multi-imaging modalities to assess retinal oxygenation and blood flow.

Retinal ischemia characterizes the underlying pathology in a multitude of retinal diseases that can ultimately lead to vision loss. A variety of novel imaging modalities have been developed to characterize retinal ischemia by measuring retinal oxygenation and blood flow in-vivo. These technologies offer valuable insight into the earliest pathophysiologic changes within the retina and provide physicians and researchers with new diagnostic and monitoring capabilities. Future retinal imaging technologies with the capability to provide affordable, noninvasive, and comprehensive data on oxygen saturation, vasculature, and blood flow mechanics are needed. This review will highlight current and future trends in multimodal imaging to assess retinal blood flow and oxygenation.

Use of XyCAM RI for Noninvasive Visualization and Analysis of Retinal Blood Flow Dynamics During Clinical Investigations.

INTRODUCTION: Ocular blood flow plays a critical role in eye health by nourishing the retinal and ocular tissues with oxygen and nutrients and removal of ocular metabolic waste. Imaging of retinal and optic blood flow may provide insights for early and more specific diagnoses of ocular vascular disorder and facilitate eye-based biomarkers applicable to neurological health assessment and research. AREAS COVERED: The ability of the XyCAM RI (Vasoptic Medical Inc., MD, USA) to visualize and to analyze ocular blood flow dynamics XyCAM RI using laser speckle contrast imaging is reviewed and compared with concurrent clinical ophthalmic imaging technologies like optical coherence tomography - angiography (OCT-A), fundus imaging, fluorescein angiography (FA), indocyanine green angiography (ICGA), laser Doppler flowmetry (LDF), and laser speckle flowgraphy (LSFG). EXPERT OPINION: XyCAM RI, with its unprecedented imaging capabilities to assess blood flow dynamics provides a powerful tool to ophthalmic researchers and doctors to obtain greater clinical insights into the physiological status of the posterior segment and treatment approaches for various diseases in a very patient-friendly, noninvasive manner, unlike dye-based angiographic techniques such as FA or ICG. XyCAM RI is well suited as a modality that could close the gap between current screening and comprehensive eye exams.

Portable, non-invasive video imaging of retinal blood flow dynamics.

Retinal blood flow (RBF) information has the potential to offer insight into ophthalmic health and disease that is complementary to traditional anatomical biomarkers as well as to retinal perfusion information provided by fluorescence or optical coherence tomography angiography (OCT-A). The present study was performed to test the functional attributes and performance of the XyCAM RI, a non-invasive imager that obtains and assesses RBF information. The XyCAM RI was installed and used in two different settings to obtain video recordings of the blood flow in the optic nerve head region in eyes of healthy subjects. The mean blood flow velocity index (BFVi) in the optic disc and in each of multiple arterial and venous segments was obtained and shown to reveal a temporal waveform with a peak and trough that correlates with a cardiac cycle as revealed by a reference pulse oximeter (correlation between respective peak-to-peak distances was 0.977). The intra-session repeatability of the XyCAM RI was high with a coefficient of variation (CV) of 1.84 ± 1.13% across both sites. Artery-vein comparisons were made by estimating, in a pair of adjacent arterial and venous segments, various temporal waveform metrics such as pulsatility index, percent time in systole and diastole, and change in vascular blood volume over a cardiac cycle. All arterial metrics were shown to have significant differences with venous metrics (p < 0.001). The XyCAM RI, therefore, by obtaining repeatable blood flow measurements with high temporal resolution, permits the differential assessment of arterial and venous blood flow patterns in the retina that may facilitate research into disease pathophysiology and biomarker development for diagnostics.

Intraoperative Laser Speckle Contrast Imaging For Real-Time Visualization of Cerebral Blood Flow in Cerebrovascular Surgery: Results From Pre-Clinical Studies.

Cerebrovascular surgery can benefit from an intraoperative system that conducts continuous monitoring of cerebral blood flow (CBF). Such a system must be handy, non-invasive, and directly integrated into the surgical workflow. None of the currently available techniques, considered alone, meets all these criteria. Here, we introduce the SurgeON™ system: a newly developed non-invasive modular tool which transmits high-resolution Laser Speckle Contrast Imaging (LSCI) directly onto the eyepiece of the surgical microscope. In preclinical rodent and rabbit models, we show that this system enabled the detection of acute perfusion changes as well as the recording of temporal response patterns and degrees of flow changes in various microvascular settings, such as middle cerebral artery occlusion, femoral artery clipping, and complete or incomplete cortical vessel cautery. During these procedures, a real-time visualization of vasculature and CBF was available in high spatial resolution through the eyepiece as a direct overlay on the live morphological view of the surgical field. Upon comparison with indocyanine green angiography videoangiography (ICG-VA) imaging, also operable via SurgeON, we found that direct-LSCI can produce greater information than ICG-VA and that continuous display of data is advantageous for performing immediate LSCI-guided adjustments in real time.

Noninvasive Assessment of Retinal Blood Flow Using a Novel Handheld Laser Speckle Contrast Imager.

PURPOSE: We assessed the image quality and reproducibility of blood flow measurements from a novel handheld laser speckle imager in handheld and stabilized use cases. METHODS: Eleven dilated human subjects were imaged with the XyCAM Handheld Retinal Imager investigational device (XyCAM HRI) in the handheld and stabilized use case in nine consecutive imaging sessions. Subjects then underwent standard color fundus photography using a Topcon TRC 50DX. The vessel-to-background contrast of the XyCAM HRI red-free photo was compared to the fundus photograph, while the coefficient of variation of blood flow measurements in specific arteries and veins also was determined. RESULTS: Vessel-to-background contrast was statistically greater in the handheld use case when compared to the standard color fundus photographs (P = 0.01). Estimates of mean blood flow velocity (BFV) were highly correlated between the stabilized and handheld use case (r 2 = 0.96). Peak velocity estimates in arteries were significantly higher than those in veins (P < 0.05). CONCLUSIONS: The XyCAM HRI prototype can acquire fundus photographs with the same or better level of clarity as color fundus photographs, and reproducibly acquire functional blood flow information in the handheld use case. TRANSLATIONAL RELEVANCE: To our knowledge, this is the first human study of a handheld laser speckle retinal imaging device. Determination of retinal blood flow has applications to ophthalmic and systemic disease and a portable handheld retinal imager that determines blood flow may be widely adopted at the point of care.

Laser Speckle Contrast Imaging: theory, instrumentation and applications.

Laser Speckle Contrast Imaging (LSCI) is a wide field of view, non scanning optical technique for observing blood flow. Speckles are produced when coherent light scattered back from biological tissue is diffracted through the limiting aperture of focusing optics. Mobile scatterers cause the speckle pattern to blur; a model can be constructed by inversely relating the degree of blur, termed speckle contrast to the scatterer speed. In tissue, red blood cells are the main source of moving scatterers. Therefore, blood flow acts as a virtual contrast agent, outlining blood vessels. The spatial resolution (~10 µm) and temporal resolution (10 ms to 10 s) of LSCI can be tailored to the application. Restricted by the penetration depth of light, LSCI can only visualize superficial blood flow. Additionally, due to its non scanning nature, LSCI is unable to provide depth resolved images. The simple setup and non-dependence on exogenous contrast agents have made LSCI a popular tool for studying vascular structure and blood flow dynamics. We discuss the theory and practice of LSCI and critically analyze its merit in major areas of application such as retinal imaging, imaging of skin perfusion as well as imaging of neurophysiology.

Longitudinal in vivo monitoring of rodent glioma models through thinned skull using laser speckle contrast imaging.

Laser speckle contrast imaging (LSCI) is a contrast agent free imaging technique suited for longitudinal assessment of vascular remodeling that accompanies brain tumor growth. We report the use of LSCI to monitor vascular changes in a rodent glioma model. Ten rats are inoculated with 9L gliosarcoma cells, and the angiogenic response is monitored five times over two weeks through a thinned skull imaging window. We are able to visualize neovascularization and measure the number of vessels per unit area to assess quantitatively the microvessel density (MVD). Spatial spread of MVD reveals regions of high MVD that may correspond to tumor location. Whole-field average MVD values increase with time in the tumor group but are fairly stable in the control groups. Statistical analysis shows significant differences in MVD values between the tumor group and both saline-receiving and unperturbed control groups over the two-week period (p<0.05). In conclusion, LSCI is suitable for investigation of tumor angiogenesis in rodent models. In addition, the statistical difference (p<0.02) between MVD values of the tumor (24.40 ± 1.41) and control groups (15.40 ± 1.60) on the 14th day after inoculation suggests a potential use of LSCI in the clinic in distinguishing tumor environments from normal vasculature.

Anisotropic processing of laser speckle images improves spatiotemporal resolution.

Laser speckle contrast imaging (LSCI) is a full field optical imaging technique, capable of imaging blood flow without the introduction of any exogenous dyes. Spatial and temporal resolution in LSCI images depend on how pixels are chosen from the raw image stack for contrast processing. However, all processing schemes are based on isotropic treatment of the spatial neighborhood about each pixel, restricting further improvement in spatiotemporal resolution and image quality. We present a novel spatiotemporal processing scheme for LSCI where the spatial neighborhood is anisotropic, that is, restricted along a specific direction that matches direction of blood flow. The technique allows for a significant increase in temporal resolution, from conventionally used 40 or 80 frames to just three frames; while simultaneously achieving 23% and 47% higher signal-to-noise ratios over concurrent spatiotemporal schemes, when imaging rapid and slow functional changes in blood flow, respectively. We present the concept, justification, and performance evaluation of the novel scheme and demonstrate its suitability for imaging rapid changes in blood flow. Anisotropic LSCI was able to monitor the heart rate associated fluctuations in intravascular blood flow and showed them to be as high as 28% of the mean.

In vivo laser speckle imaging reveals microvascular remodeling and hemodynamic changes during wound healing angiogenesis.

Laser speckle contrast imaging (LSCI) is a high-resolution and high contrast optical imaging technique often used to characterize hemodynamic changes in short-term physiological experiments. In this study, we demonstrate the utility of LSCI for characterizing microvascular remodeling and hemodynamic changes during wound healing angiogenesis in vivo. A 2 mm diameter hole was made in the mouse ear and the periphery of the wound imaged in vivo using LSCI over 12 days. We were able to visualize and quantify the vascular and perfusion changes that accompanied wound healing in the microenvironment proximal to the wound, and validated these changes with histology. We found that consistent with the stages of wound healing, microvessel density increased during the initial inflammatory phase (i.e., day 0-3), stayed elevated through the tissue formation phase (i.e., until day 7) and returned to baseline during the tissue remodeling phase (i.e., by day 12). Concomitant "wide area mapping" of blood flow revealed that tissue perfusion in the wound periphery initially decreased, gradually increased from day 3-7, and subsided as healing completed. Interestingly, some regions exhibited a reestablishment of tissue perfusion approximately 6 days earlier than the ~18 days usually reported for the long term remodeling phase. The results from this study demonstrate that LSCI is an ideal platform for elucidating in vivo changes in microvascular hemodynamics and angiogenesis, and has the potential to offer invaluable insights in a range of disease models involving abnormal hemodynamics, such as diabetes and tumors.

Multiexposure laser speckle contrast imaging of the angiogenic microenvironment.

We report the novel use of laser speckle contrast imaging (LSCI) at multiple exposure times (meLSCI) for enhanced in vivo imaging of the microvascular changes that accompany angiogenesis. LSCI is an optical imaging technique that can monitor blood vessels and the flow therein at a high spatial resolution without requiring the administration of an exogenous contrast agent. LSCI images are obtained under red (632 nm) laser illumination at seven exposure times (1-7 ms) and combined using a curve-fitting approach to obtain high-resolution meLSCI images of the rat brain vasculature. To evaluate enhancement in in vivo imaging performance, meLSCI images are statistically compared to individual LSCI images obtained at a single exposure time. We find that meLSCI reduced the observed variability in the LSCI-based blood-flow estimates by 30% and improved the contrast-to-noise ratio in regions with high microvessel density by 41%. The ability to better distinguish microvessels, makes meLSCI uniquely suited to longitudinal imaging of changes in the vascular microenvironment induced by pathological angiogenesis. We demonstrate this utility of meLSCI by sequentially monitoring, over days, the microvascular changes that accompany wound healing in a mouse ear model.

Imaging microvascular flow characteristics using laser speckle contrast imaging.

Laser speckle contrast imaging (LSCI) has classically been used to image regional blood flow changes in animal models. In this paper, we demonstrate the use of LSCI for elucidating blood flow characteristics in individual microvessels with diameters as small as 24µm. We extracted profiles of speckle contrast values within individual vessels, both along their diameters and along their lengths and inferred that they could be attributed to the flow within the vessel. Profiles along the diameter of vessels revealed maxima at the center of vessels, consistent with fluid dynamics. These observed profiles could be fitted with parabolic curves with a mean coefficient of determination of 0.92. Similarly, analysis of speckle contrast values in the axial direction revealed profiles that progressively decreased in discreet quanta at branch points indicating blood flow bifurcations. Flow estimates obtained from speckle contrast values within branches of vessels obeyed the law of mass conservation with a mean error of only 3.5%. This allowed us to elucidate the percentage distribution of blood flow into each of the downstream branches. This ability of LSCI to resolve blood flow distribution in branching microvessel trees in a minimally invasive and dye free environment over a wide field of view promises to find application in both the neuroscience laboratory as well as intraoperative neurosurgery.

High resolution cerebral blood flow imaging by registered laser speckle contrast analysis.

Laser speckle imaging (LSI) has been widely used for in vivo detecting cerebral blood flow (CBF) under various physiological and pathological conditions. So far, nearly all literature on in vivo LSI does not consider the influence of disturbances due to respiration and/or heart beating of animals. In this paper, we analyze how such physiologic motions affect the spatial resolution of the conventional laser speckle contrast analysis (LASCA). We propose a registered laser speckle contrast analysis (rLASCA) method which first registers raw speckle images with a 3 x 3 convolution kernel, normalized correlation metric and cubic B-spline interpolator, and then constructs the contrast image for CBF. rLASCA not only significantly improves the distinguishability of small vessels, but also efficiently suppresses the noises induced by respiration and/or heart beating. In an application of imaging the angiogenesis of rat's brain tumor, rLASCA outperformed the conventional LASCA in providing a much higher resolution for new small vessels. As a processing method for LSI, rLASCA can be directly applied to other LSI experiments where the disturbances from different sources (like respiration, heart beating) exist.

Model based reconstruction for simultaneously imaging cerebral blood flow and De-oxygen hemoglobin distribution.

Laser speckle imaging (LSI) and optical intrinsic signal (OIS) imaging are shown to produce high resolution information of cerebral blood flow(CBF) and de-oxygen hemoglobin(dHb) respectively. However, the cortex curvature and non-uniform illumination always result in the inhomo-geneous impact and thus distort the CBF and dHb results. In this paper, we propose to extract both CBF and dHb images from raw speckle images. A parametric model for such inhomogeneity is estimated by adaptive window median-mean filtering and curve fitting technique to reconstruct the CBF and dHb image.

High spatiotemporal resolution imaging of the neurovascular response to electrical stimulation of rat peripheral trigeminal nerve as revealed by in vivo temporal laser speckle contrast.

Previous studies have implicated the abnormal activation of the trigeminal system to be a factor in the pathogenesis of migraine. The relationship between vascular changes and migraine, however, is under considerable debate. In this study, temporal laser speckle contrast imaging is combined with ridge tracking based vessel detection to obtain high resolution (6.7 microm x 6.7 microm), high contrast images of cerebral vascular structure. For the first time, the vasomotor and blood flow responses to electrical stimulation in rat peripheral trigeminal system were obtained simultaneously. The system is capable of picking up individual vessels with diameters down to 30 microm. The spatial spread of the blood velocity response relative to the point of stimulation was studied. Analysis of branching vessels showed a 50+/-5% vs. 30+/-5% change in mean peak magnitude and a 54% per second vs. 17% per second change in mean rate of increase for vessels proximal vs. distal to the stimulation site. The penetration depth of the laser used was proven to be sufficient to image dural as well as cortical vessels through a thinned skull preparation. Different responses were observed from cortical and dural vessels. While the diameter of cortical vessels did not change in response to the stimulation the blood velocity went up by 65+/-5% per second. Dural vessels enlarged by 40+/-8% and the blood velocity increased by 50+/-5%. The method described here could be very useful in understanding and studying disorders in the neurovascular system.