Multi-exposure Speckle Imaging Research Articles

Multiexposure laser speckle contrast analysis system calibration limited by perfusion-dependent scattering on the skin.

Application of multiexposure speckle contrast imaging (MESI) methods for perfusion measurements can correct for the contribution of static scattering of the skin, at the expense of reduced temporal resolution as compared to classical single-exposure methods. Persistence of tissue scattering properties during the measurements could allow for an initial calibration and enhancement of the temporal resolution of the measurements. We aim to study the influence of the perfusion on the light scattering of the forearm skin and to use the obtained data for the enhancement of the temporal resolution. A wide range of skin perfusion states was induced while monitoring the changes in the dynamic range of the exposure-dependent contrast. Different measurement and evaluation methods were tested based on an initial MESI calibration followed by image recording with reduced number of exposure time values. The changes in the skin perfusion can alter not only the contribution of the static scattering to the speckle images but also the short-exposure time contrast limit. The perfusion-dependent scattering of the skin can invalidate the precalibrations (e.g., calibration) characterizing the combination of the given tissue and the measurement system.

Quasi-analytic solution for real-time multi-exposure speckle imaging of tissue perfusion.

Laser speckle contrast imaging (LSCI) is a widefield imaging technique that enables high spatiotemporal resolution measurement of blood flow. Laser coherence, optical aberrations, and static scattering effects restrict LSCI to relative and qualitative measurements. Multi-exposure speckle imaging (MESI) is a quantitative extension of LSCI that accounts for these factors but has been limited to post-acquisition analysis due to long data processing times. Here we propose and test a real-time quasi-analytic solution to fitting MESI data, using both simulated and real-world data from a mouse model of photothrombotic stroke. This rapid estimation of multi-exposure imaging (REMI) enables processing of full-frame MESI images at up to 8 Hz with negligible errors relative to time-intensive least-squares methods. REMI opens the door to real-time, quantitative measures of perfusion change using simple optical systems.

Using pressure-driven flow systems to evaluate laser speckle contrast imaging.

Microfluidic flow phantom studies are commonly used for characterizing the performance of laser speckle contrast imaging (LSCI) instruments. The selection of the flow control system is critical for the reliable generation of flow during testing. The majority of recent LSCI studies using microfluidics used syringe pumps for flow control. We quantified the uncertainty in flow generation for a syringe pump and a pressure-regulated flow system. We then assessed the performance of both LSCI and multi-exposure speckle imaging (MESI) using the pressure-regulated flow system across a range of flow speeds. The syringe pump and pressure-regulated flow systems were evaluated during stepped flow profile experiments in a microfluidic device using an inline flow sensor. The uncertainty associated with each flow system was calculated and used to determine the reliability for instrument testing. The pressure-regulated flow system was then used to characterize the relative performance of LSCI and MESI during stepped flow profile experiments while using the inline flow sensor as reference. The pressure-regulated flow system produced much more stable and reproducible flow outputs compared to the syringe pump. The expanded uncertainty for the syringe pump was 8 to higher than that of the pressure-regulated flow system across the tested flow speeds. Using the pressure-regulated flow system, MESI outperformed single-exposure LSCI at all flow speeds and closely mirrored the flow sensor measurements, with average errors of and , respectively. Pressure-regulated flow systems should be used instead of syringe pumps when assessing the performance of flow measurement techniques with microfluidic studies. MESI offers more accurate relative flow measurements than traditional LSCI across a wide range of flow speeds.

All fiber-based illumination system for multi-exposure speckle imaging.

Monitoring blood flow is critical to treatment efficacy in many surgical settings. Laser speckle contrast imaging (LSCI) is a simple, real-time, label-free optical technique for monitoring blood flow that has emerged as a promising technique but lacks the ability to make repeatable quantitative measurements. Multi-exposure speckle imaging (MESI) is an extension of LSCI that requires increased complexity of instrumentation, which has limited its adoption. In this paper, we design and fabricate a compact, fiber-coupled MESI illumination system (FCMESI) that is substantially smaller and less complex than previous systems. Using microfluidics flow phantoms, we demonstrate that the FCMESI system measures flow with an accuracy and repeatability equivalent to traditional free space MESI illumination systems. With an in vivo stroke model, we also demonstrate the ability of FCMESI to monitor cerebral blood flow changes.

Multi-exposure Speckle Imaging for Quantitative Evaluation of Cortical Blood Flow.

Laser speckle contrast imaging (LSCI) is a label-free optical imaging technique that can quantify flow dynamics across an entire image. Multi-exposure speckle imaging (MESI) is an extension of LSCI that allows for reproducible and quantifiable measurements of flow. MESI has the potential to provide quantitative cerebral blood flow information in both preclinical and clinical applications; in fact, MESI can be extended to resolve the flow dynamics in any exposed tissue. A MESI system can be divided into three primary components: (i) the illumination optics, consisting of the optical source and a method of modulating and gating the illumination intensity; (ii) the collection optics, consisting of a high-speed camera that can be triggered and gated to match the pulsed illumination; and finally (iii) post-processing hardware and software to extract the flow information from the recorded raw intensity images. In the following protocol, we offer a guide to design, operate, and test a MESI system.

Dynamics of isoflurane-induced vasodilation and blood flow of cerebral vasculature revealed by multi-exposure speckle imaging

BackgroundAnesthetized animal models are used extensively during neurophysiological and behavioral studies despite systemic effects from anesthesia that undermine both accurate interpretation and translation to awake human physiology. The majority of work examining the impact of anesthesia on cerebral blood flow (CBF) has been restricted to before and after measurements with limited spatial resolution. New methodWe used multi-exposure speckle imaging (MESI), an advanced form of laser speckle contrast imaging (LSCI), to characterize the dynamics of isoflurane anesthesia induction on cerebral vasculature and blood flow in the mouse brain. ResultsThe large anatomical changes caused by isoflurane are depicted with wide-field imagery and video highlighting the induction of general anesthesia. Within minutes of exposure, both vessel diameter and blood flow increased drastically compared to the awake state and remained elevated for the duration of imaging. An examination of the dynamics of anesthesia induction reveals that blood flow increased faster in arteries than in veins or parenchyma regions. Comparison with existing methodsMESI offers robust hemodynamic measurements across large fields-of-view and high temporal resolutions sufficient for continuous visualization of cerebrovascular events featuring major changes in blood flow. ConclusionThe large alterations caused by isoflurane anesthesia to the cortical vasculature and CBF are readily characterized using MESI. These changes are unrepresentative of normal physiology and provide further evidence that neuroscience experiments would benefit from transitioning to un-anesthetized awake animal models.

Laparoscopic multi-exposure speckle imaging for quantitative flow measurement

Multiple studies have demonstrated that laser speckle contrast imaging (LSCI) can be a valuable blood flow monitoring technique during laparoscopic operations. However, the quantitative accuracy of traditional single-exposure LSCI is limited. In this paper, an extension to LSCI called multi-exposure speckle imaging (MESI) can overcome this limitation and is evaluated in a commercially available laparoscopic setting. Our laparoscopic MESI system records multiple exposure times spanning 3 ms–20 ms. We demonstrate that the laparoscopic MESI estimates provide the sensitivity for sampling the flow magnitude in a phantom with different channel sizes. Conservation of flow analysis on the rabbit vascular branch is also used to validate the accuracy of flow estimates from MESI. Our results demonstrate that laparoscopic MESI can be performed with the ability of quantitative blood flow measurements and shows the potential to be clinically useful for monitoring blood flow.

Obesity in Midlife Hampers Resting and Sensory-Evoked Cerebral Blood Flow in Mice.

This study aimed to investigate the effects of a high-fat diet (HFD) and aging on resting and activity-dependent cerebral blood flow (CBF). To run a comparison between obese and age-matched control animals, 6-week-old mice were fed either with regular chow or an HFD for 3 months or 8 months. Glucose tolerance and insulin sensitivity were assessed for metabolic phenotyping. Resting and odor-evoked CBF at the microvascular scale in the olfactory bulb (OB) was investigated by multiexposure speckle imaging. Immunolabeling-enabled imaging of solvent-cleared organs was used to analyze vascular density. The ejection fraction was studied by using cardioechography. Olfactory sensitivity was tested by using a buried-food test. Glucose intolerance and compromised odor-evoked CBF were observed in obese mice in the younger group. Prolonged HFD feeding triggered insulin resistance and stronger impairment in activity-dependent CBF. Aging had a specific negative impact on resting CBF. There was no decrease in vascular density in the OB of obese mice, although cardiac function was impaired at both ages. In addition, decreased olfactory sensitivity was observed only in the older, middle-aged obese mice. OB microvasculature in obese mice showed a specific functional feature characterized by impaired sensory-evoked CBF and a specific deleterious effect of aging on resting CBF.

A Window of Vascular Plasticity Coupled to Behavioral Recovery after Stroke.

Stroke causes remodeling of vasculature surrounding the infarct, but whether and how vascular remodeling contributes to recovery are unclear. We established an approach to monitor and compare changes in vascular structure and blood flow with high spatiotemporal precision after photothrombotic infarcts in motor cortex using longitudinal 2-photon and multiexposure speckle imaging in mice of both sexes. A spatially graded pattern of vascular structural remodeling in peri-infarct cortex unfolded over the first 2 weeks after stroke, characterized by vessel loss and formation, and selective stabilization of a subset of new vessels. This vascular structural plasticity was coincident with transient activation of transcriptional programs relevant for vascular remodeling, reestablishment of peri-infarct blood flow, and large improvements in motor performance. Local vascular plasticity was strongly predictive of restoration of blood flow, which was in turn predictive of behavioral recovery. These findings reveal the spatiotemporal evolution of vascular remodeling after stroke and demonstrate that a window of heightened vascular plasticity is coupled to the reestablishment of blood flow and behavioral recovery. Our findings support that neovascularization contributes to behavioral recovery after stroke by restoring blood flow to peri-infarct regions. These findings may inform strategies for enhancing recovery from stroke and other types of brain injury.SIGNIFICANCE STATEMENT An improved understanding of neural repair could inform strategies for enhancing recovery from stroke and other types of brain injury. Stroke causes remodeling of vasculature surrounding the lesion, but whether and how the process of vascular remodeling contributes to recovery of behavioral function have been unclear. Here we used longitudinal in vivo imaging to track vascular structure and blood flow in residual peri-infarct cortex after ischemic stroke in mice. We found that stroke created a restricted period of heightened vascular plasticity that was associated with restoration of blood flow, which was in turn predictive of recovery of motor function. Therefore, our findings support that vascular remodeling facilitates behavioral recovery after stroke by restoring blood flow to peri-infarct cortex.

On the equivalence of speckle contrast-based and diffuse correlation spectroscopy methods in measuring in vivo blood flow.

We establish the equivalence between laser speckle contrast-based and diffuse correlation spectroscopy methods inin vivo imaging of blood flow using the Volterra integral equation theory. We further substantiate the need of regularized fitting while employing the multiexposure speckle contrast imaging to recover autocorrelation function.

Artery targeted photothrombosis widens the vascular penumbra, instigates peri-infarct neovascularization and models forelimb impairments

The photothrombotic stroke model generates localized and reproducible ischemic infarcts that are useful for studying recovery mechanisms, but its failure to produce a substantial ischemic penumbra weakens its resemblance to human stroke. We examined whether a modification of this approach, confining photodamage to arteries on the cortical surface (artery-targeted photothrombosis), could better reproduce aspects of the penumbra. Following artery-targeted or traditional photothrombosis to the motor cortex of mice, post-ischemic cerebral blood flow was measured using multi-exposure speckle imaging at 6, 48, and 120 h post-occlusion. Artery-targeted photothrombosis produced a more graded penumbra at 48 and 120 h. The density of isolectin B4+ vessels in peri-infarct cortex was similarly increased after both types of infarcts compared to sham at 2 weeks. These results indicate that both models instigated post-ischemic vascular structural changes. Finally, we determined whether the strength of the traditional photothrombotic approach for modeling upper-extremity motor impairments extends to the artery-targeted approach. In adult mice that were proficient in a skilled reaching task, small motor-cortical infarcts impaired skilled-reaching performance for up to 10 days. These results support that artery-targeted photothrombosis widens the penumbra while maintaining the ability to create localized infarcts useful for modeling post-stroke impairments.

Abstract WP149: Post-stroke Vascular Remodeling and the Role of Neural Precursor Cells

The time course, mode, regulation, and relevance to functional recovery of post-stroke vascular remodeling are poorly defined. We tracked peri-infarct vascular remodeling in mice with repeated in vivo two-photon imaging of vascular structure (labeled with transgenic expression of green fluorescent protein) and multi-exposure speckle imaging of cortical blood flow over months after photothrombotic cortical lesions. Vascular structure in the intact brain is remarkably stable. In contrast, we found that peri-infarct vasculature (micro- and macrovessels) underwent long-term structural changes, including formation and elimination of vessel segments, beginning within days of infarction. Remodeling continued after peri-infarct blood flow was restored and was most pronounced in superficial vessels. Histological methods indicated that vascular density was rapidly (within days of ischemia) and persistently (at least one month) increased surrounding the infarct. Past findings suggest that neural precursor cells (NPCs) originating in the subventricular zone associate closely with remodeling blood vessels in peri-infarct cortex. To evaluate the role of NPCs in vascular remodeling, we used a pharmacogenetic method to ablate NPCs. NPC ablation prior to infarct altered the pattern of vascular remodeling: the neovascularization response was exaggerated and the orientation of peri-infarct vessels was disordered compared to the normal pattern of vessels oriented towards the lesion. As well, NPC ablation worsened recovery of behavioral function after ischemia. Collectively, these results suggest that vascular remodeling is a chronic process instigated by stroke and that proper vascular patterning is directed by NPCs. NPCs likely contribute to behavioral recovery after brain injury at least in part through their influence on neovascularization.

Quantitative model of diffuse speckle contrast analysis for flow measurement

Diffuse speckle contrast analysis (DSCA) is a noninvasive optical technique capable of monitoring deep tissue blood flow. However, a detailed study of the speckle contrast model for DSCA has yet to be presented. We deduced the theoretical relationship between speckle contrast and exposure time and further simplified it to a linear approximation model. The feasibility of this linear model was validated by the liquid phantoms which demonstrated that the slope of this linear approximation was able to rapidly determine the Brownian diffusion coefficient of the turbid media at multiple distances using multiexposure speckle imaging. Furthermore, we have theoretically quantified the influence of optical property on the measurements of the Brownian diffusion coefficient which was a consequence of the fact that the slope of this linear approximation was demonstrated to be equal to the inverse of correlation time of the speckle.

Intraoperative multi-exposure speckle imaging of cerebral blood flow

Multiple studies have demonstrated that laser speckle contrast imaging (LSCI) has high potential to be a valuable cerebral blood flow monitoring technique during neurosurgery. However, the quantitative accuracy and sensitivity of LSCI is limited, and highly dependent on the exposure time. An extension to LSCI called multi-exposure speckle imaging (MESI) overcomes these limitations, and was evaluated intraoperatively in patients undergoing brain tumor resection. This clinical study (n = 8) recorded multiple exposure times from the same cortical tissue area spanning 0.5–20 ms, and evaluated images individually as single-exposure LSCI and jointly using the MESI model. This study demonstrated that the MESI estimates provided the broadest flow sensitivity for sampling the flow magnitude in the human brain, closely followed by the shorter exposure times. Conservation of flow analysis on vascular bifurcations was used to validate physiological accuracy, with highly conserved flow estimates (<10%) from both MESI and 1 ms LSCI (n = 14 branches). The MESI model had high goodness-of-fit with proper image calibration and acquisition, and was used to monitor blood flow changes after tissue cautery. Results from this study demonstrate that intraoperative MESI can be performed with high quantitative accuracy and sensitivity for cerebral blood flow monitoring.

Evaluating multi-exposure speckle imaging estimates of absolute autocorrelation times.

Multi-exposure speckle imaging (MESI) is a camera-based flow-imaging technique for quantitative blood-flow monitoring by mapping the speckle-contrast dependence on camera exposure duration. The ability of laser speckle contrast imaging to measure the temporal dynamics of backscattered and interfering coherent fields, in terms of the accuracy of autocorrelation measurements, is a major unresolved issue in quantitative speckle flowmetry. MESI fits for a number of parameters including an estimate of the electric field autocorrelation decay time from the imaged speckles. We compare the MESI-determined correlation times in vitro and in vivo with accepted true values from direct temporal measurements acquired with a photon-counting photon-multiplier tube and an autocorrelator board. The correlation times estimated by MESI in vivo remain on average within 14±11% of those obtained from direct temporal autocorrelation measurements, demonstrating that MESI yields highly comparable statistics of the time-varying fields that can be useful for applications seeking not only quantitative blood flow dynamics but also absolute perfusion.

Flux or speed? Examining speckle contrast imaging of vascular flows.

Speckle contrast imaging enables rapid mapping of relative blood flow distributions using camera detection of back-scattered laser light. However, speckle derived flow measures deviate from direct measurements of erythrocyte speeds by 47 ± 15% (n = 13 mice) in vessels of various calibers. Alternatively, deviations with estimates of volumetric flux are on average 91 ± 43%. We highlight and attempt to alleviate this discrepancy by accounting for the effects of multiple dynamic scattering with speckle imaging of microfluidic channels of varying sizes and then with red blood cell (RBC) tracking correlated speckle imaging of vascular flows in the cerebral cortex. By revisiting the governing dynamic light scattering models, we test the ability to predict the degree of multiple dynamic scattering across vessels in order to correct for the observed discrepancies between relative RBC speeds and multi-exposure speckle imaging estimates of inverse correlation times. The analysis reveals that traditional speckle contrast imagery of vascular flows is neither a measure of volumetric flux nor particle speed, but rather the product of speed and vessel diameter. The corrected speckle estimates of the relative RBC speeds have an average 10 ± 3% deviation in vivo with those obtained from RBC tracking.

Chronic monitoring of vascular progression after ischemic stroke using multiexposure speckle imaging and two-photon fluorescence microscopy.

Monitoring the progression of the vascular structure and cerebral blood flow (CBF) after brain injury is vital to understand the neurovascular recovery process. Multiexposure speckle imaging (MESI) provides a quantitatively accurate technique for chronically measuring the postocclusion CBF perfusion of the infarct and peri-infarct regions in rodent stroke models, while multiphoton microscopy offers direct visualization of the microvascular structure. In this paper, we present imaging outcomes extending 35 days after photo-thrombotic occlusion, tracking the progression of the vasculature throughout this period. We compare MESI flow estimates within the unresolvable parenchyma with subsurface microvascular volume fractions taken with two-photon microscopy in the same regions to assess how the vascular density influences the surface-integrated MESI flow values. The MESI flow measurements and volume fractions are shown to have high correlations (r=0.90) within areas of recovering vasculature in the peri-infarct region. We also observe vascular reorientation occurring within the microvascular structure throughout the 35-day postocclusion period. With the combination of a chronic mouse model and relatively noninvasive optical imaging techniques, we present an imaging protocol for monitoring long-term vascular progression after photo-thrombotic occlusion with the potential to test the efficacy of rehabilitation and pharmacological therapies.

Quantitatively assessing flow velocity by the slope of the inverse square of the contrast values versus camera exposure time.

The slope of the inverse square of the contrast values versus camera exposure time at multi-exposure speckle imaging (MESI) can be a new indicator of flow velocity. The slope is linear as the diffuse coefficient in Brownian motion diffusion model and the mean velocity in ballistic motion model. Combining diffuse speckle contrast analysis (DSCA) and MESI, we demonstrate theoretically and experimentally that the flow velocity can be obtained from this slope. The calculation results processes of the slop don't need tedious Newtonian iterative method and are computationally inexpensive. The new indicator can play an important role in quantitatively assessing tissue blood flow velocity.

Optimization of camera exposure durations for multi-exposure speckle imaging of the microcirculation.

Improved Laser Speckle Contrast Imaging (LSCI) blood flow analyses that incorporate inverse models of the underlying laser-tissue interaction have been used to develop more quantitative implementations of speckle flowmetry such as Multi-Exposure Speckle Imaging (MESI). In this paper, we determine the optimal camera exposure durations required for obtaining flow information with comparable accuracy with the prevailing MESI implementation utilized in recent in vivo rodent studies. A looping leave-one-out (LOO) algorithm was used to identify exposure subsets which were analyzed for accuracy against flows obtained from analysis with the original full exposure set over 9 animals comprising n = 314 regional flow measurements. From the 15 original exposures, 6 exposures were found using the LOO process to provide comparable accuracy, defined as being no more than 10% deviant, with the original flow measurements. The optimal subset of exposures provides a basis set of camera durations for speckle flowmetry studies of the microcirculation and confers a two-fold faster acquisition rate and a 28% reduction in processing time without sacrificing accuracy. Additionally, the optimization process can be used to identify further reductions in the exposure subsets for tailoring imaging over less expansive flow distributions to enable even faster imaging.

Quantitative, depth-resolved determination of particle motion using multi-exposure, spatial frequency domain laser speckle imaging

Laser Speckle Imaging (LSI) is a simple, noninvasive technique for rapid imaging of particle motion in scattering media such as biological tissue. LSI is generally used to derive a qualitative index of relative blood flow due to unknown impact from several variables that affect speckle contrast. These variables may include optical absorption and scattering coefficients, multi-layer dynamics including static, non-ergodic regions, and systematic effects such as laser coherence length. In order to account for these effects and move toward quantitative, depth-resolved LSI, we have developed a method that combines Monte Carlo modeling, multi-exposure speckle imaging (MESI), spatial frequency domain imaging (SFDI), and careful instrument calibration. Monte Carlo models were used to generate total and layer-specific fractional momentum transfer distributions. This information was used to predict speckle contrast as a function of exposure time, spatial frequency, layer thickness, and layer dynamics. To verify with experimental data, controlled phantom experiments with characteristic tissue optical properties were performed using a structured light speckle imaging system. Three main geometries were explored: 1) diffusive dynamic layer beneath a static layer, 2) static layer beneath a diffuse dynamic layer, and 3) directed flow (tube) submerged in a dynamic scattering layer. Data fits were performed using the Monte Carlo model, which accurately reconstructed the type of particle flow (diffusive or directed) in each layer, the layer thickness, and absolute flow speeds to within 15% or better.