Depth resolution in multifocus laser speckle contrast imaging.

Monitoring of partial and full venous outflow obstruction in a porcine flap model using laser speckle contrast imaging

The tissue optical clearing (TOC) technique could significantly improve the biomedical optical imaging depth, but most current investigations are limited to in vitro studies. For in vivo applications, the TOC method must provide a rapid treatment process, sufficient transparency, and safety for animals, which makes it more difficult. Recently developed innovative optical clearing methods for in vivo use show great potential for enhancing the contrast and resolution of laser speckle contrast imaging (LSCI) for blood flow monitoring. This paper gives an overview of recent progress in the use of TOC for vascular visualization with LSCI. First, the principle of TOC-induced improvement of LSCI and a quantitative analysis method for evaluating the improvement are described briefly. Second, the paper introduces transparent windows, including various skin windows and a cranial window, that permit LSCI to monitor dermal or cortical blood flow, respectively, with high resolution and contrast. Third, preliminary investigations of the safety of TOC demonstrate that the transparent skin window is switchable, which enables LSCI to repeatedly image blood flow. However, research on in vivo TOC is currently less advanced than that on in vitro TOC. Future work should focus on developing a highly effective, safe method and extending its applications.

Reactive hyperemia refers to an increase of blood flow in tissue post release of an occlusion in the local vasculature. Measuring the temporal response of reactive hyperemia, post-occlusion in patients has the potential to shed information about microvascular diseases such as systemic sclerosis and diabetes. Laser speckle contrast imaging (LSCI) is an imaging technique capable of sensing superficial blood flow in tissue which can be used to quantitatively assess reactive hyperemia. Here, we employ LSCI using coherent sources in the blue, green and red wavelengths to evaluate reactive hyperemia in healthy human volunteers. Blood flow in the forearms of subjects were measured using LSCI to assess the time-course of reactive hyperemia that was triggered by a pressure cuff applied to the biceps of the subjects. Raw speckle images were acquired and processed to yield blood-flow parameters from a region of interest before, during and after application of occlusion. Reactive hyperemia was quantified via two measures - (1) by calculating the difference between the peak LSCI flow during the hyperemia and baseline flow, and (2) by measuring the amount of time that elapsed between the release of the occlusion and peak flow. These measurements were acquired in three healthy human participants, under the three laser wavelengths employed. The studies shed light on the utility of in vivo LSCI-based flow sensing for non-invasive assessment of reactive hyperemia responses and how they varied with the choice source wavelength influences the measured parameters.

Chronic imaging of cerebral blood flow (CBF) is an important tool for investigating vascular remodeling after injury such as stroke. Although techniques such as Laser Speckle Contrast Imaging (LSCI) have emerged as valuable tools for imaging CBF in acute experiments, their utility for chronic measurements or cross-animal comparisons has been limited. Recently, an extension to LSCI called Multi-Exposure Speckle Imaging (MESI) was introduced that increases the quantitative accuracy of CBF images. In this paper, we show that estimates of chronic blood flow are better with MESI than with traditional LSCI. We evaluate the accuracy of the MESI flow estimates using red blood cell (RBC) photographic tracking as an absolute flow calibration in mice over several days. The flow measures computed using the MESI and LSCI techniques were found to be on average 10% and 24% deviant (n=9 mice), respectively, compared with RBC velocity changes. We also map CBF dynamics after photo-thrombosis of selected cortical microvasculature. Correlations of flow dynamics with RBC tracking were closer with MESI (r=0.88) than with LSCI (r=0.65) up to 2 weeks from baseline. With the increased quantitative accuracy, MESI can provide a platform for studying the efficacy of stroke therapies aimed at flow restoration.

Purpose: Laser speckle contrast imaging (LSCI) provides an innovative approach to the non-invasive evaluation of systemic microvascular endothelial function. In the present work, we assessed skin microvascular reactivity in individuals with early onset coronary artery disease (EOCAD, diagnosis at less than 45 years of age) using LSCI coupled to pharmacological and physiological stimuli. Methods: Skin microvascular blood flow was continuously monitored in the forearm using LSCI. Acetylcholine (ACh) dose-response curves were performed using an iontophoresis micropharmacology system and post-occlusive reactive hyperemia (PORH) was analyzed after a 3-minute occlusion of blood flow to the forearm. Measurements of microvascular skin blood flow in arbitrary perfusion units (APU) were divided by mean arterial pressure to give cutaneous vascular conductance (CVC) in APU/mm Hg. The results were presented as mean ± SEM and were analyzed using two-tailed unpaired Student's t tests. Results: Fifty-eight patients with EOCAD aged 45±0.4 years (34 men) and 25 healthy age- and sex-matched individuals were included in the study. ACh- and PORH-mediated vasodilation was significantly reduced in EOCAD individuals. Area under the curve of ACh-induced vasodilation was of 6445±842 and 3975±540 APU/sec in controls vs. EOCAD, respectively; p=0.0087; figure 1). Increase in CVC resulting from PORH-induced vasodilation was of 0.46±0.03 and 0.38±0.02 APU/mmHg in controls vs. EOCAD, respectively; p=0.0182; figure 1). ![Figure][1] Conclusions: LSCI identifies systemic microvascular endothelial-dysfunction in individuals presenting with EOCAD and thus could be used as an early peripheral marker of the atherothrombotic disease. [1]: pending:yes

A comparative application of major dynamic light scattering (DLS)‐based image methodologies applied to transcranial cerebral blood flow imaging is presented. In particular, the study delves into assessing capability of Laser Doppler Flowmetry (LDF), Laser Speckle Contrast Imaging (LSCI), and Diffuse Correlation Spectroscopy (DCS) in enhancing the spatial and temporal resolution of transcranial blood flow imaging. An integral part of the study is focused on the modulation of blood flow through the administration of the vasodilator drug, Sodium Nitroprusside (SNP). This pharmacological intervention facilitated a direct observation of cerebral vasculature's responsiveness to external stimuli, illuminating the physiological adaptations within the brain's microvascular architecture. Advanced LSCI processing techniques are incorporated, notably entropy and principal component analysis (PCA). Entropy is providing a quantifiable measure of the randomness and complexity within the speckle patterns of transcranial blood flow images, revealing remarkably similar outcomes with DSC approach in terms of blood flow dynamics and its quantitative evaluation. The application of PCA approach is provided a more nuanced understanding of blood flow dynamics, facilitating the identification of subtle changes induced by drug administration. This method proved instrumental in enhancing the visualization and detection of nuanced blood flow dynamics, thereby allowing for a more detailed examination of cerebral circulation alterations induced by SNP administration. The study seeks to offer a wider‐ranging insight into comprehending the translating further the concept of DLS into transcrainial blood flow vizualization and explore its practical applications, considering hardware, advanced quantitative image processing, and data acquisition.

Monitoring cerebral blood flow (CBF) during neurosurgery can provide important physiological information for a variety of surgical procedures. Although multiple intraoperative vascular monitoring technologies are currently available, a quantitative method that allows for continuous monitoring is still needed. Laser speckle contrast imaging (LSCI) is an optical imaging method with high spatial and temporal resolution that has been widely used to image CBF in animal models in vivo. In this pilot clinical study, we adapted a Zeiss OPMI Pentero neurosurgical microscope to obtain LSCI images by attaching a camera and a laser diode. This LSCI adapted instrument has been used to acquire full field flow images from 10 patients during tumor resection procedures. The patient's ECG was recorded during acquisition and image registration was performed in post-processing to account for pulsatile motion artifacts. Digital photographs confirmed alignment of vasculature and flow images in four cases, and a relative change in blood flow was observed in two patients after bipolar cautery. The LSCI adapted instrument has the capability to produce real-time, full field CBF image maps with excellent spatial resolution and minimal intervention to the surgical procedure. Results from this study demonstrate the feasibility of using LSCI to monitor blood flow during neurosurgery.

The visualization and localization of blood vessels in Laser Speckle Contrast Imaging is an important task in biomedical applications such as dermatology, neurosciences, and ophthalmology, as it allows to determine the presence of blood vessels and also to estimate properties such as blood flow. This work establishes a review of visualization methods for contrast calculation and improvement in Laser Speckle Contrast Imaging. In addition, it is presented the localization of blood vessels in an automatic way through clustering. The results suggest that the localization of blood vessels depends largely on the calculation and improvement of contrast. If the blood vessel and biological tissue regions are well separated from each other, and the noise level is low, K-means clustering is a powerful tool for locating blood vessels in Laser Speckle Contrast Imaging.

Laser speckle contrast imaging (LSCI) has been widely used in in vivo blood flow imaging. However, the effect of local scattering property (scattering coefficient µs) on blood flow speed estimation has not been well investigated. In this study, such an effect was quantified and involved in relation between speckle autocorrelation time τc and flow speed v based on simulation flow experiments. For in vivo blood flow imaging, an improved estimation strategy was developed to eliminate the estimation bias due to the inhomogeneous distribution of the scattering property. Compared to traditional LSCI, a new estimation method significantly suppressed the imaging noise and improves the imaging contrast of vasculatures. Furthermore, the new method successfully captured the blood flow changes and vascular constriction patterns in rats’ cerebral cortex from normothermia to mild and moderate hypothermia.

Introduction The ability of laser speckle contrast imaging (LSCI) to provide real-time images of blood flow makes this modality appealing in the assessment of burn wounds, particularly for clinicians making treatment decisions based on burn wound depth and presumed progression. Here we present 2 preclinical studies that used LSCI to assess wound progress, both immediately and months after injury. Methods LSCI images were taken 10-40cm away from the wound and captured with a 1388x1038-pixel CCD camera. In the first study, LSCI images were captured prior to and immediately following creation of superficial partial-thickness (SPTB, 10s), deep partial-thickness (DPTB, 15s), and full-thickness burns (FTB, 20s), and on post-burn day (PBD) 1, 2, and 3. In the second study, LSCI images were obtained before and after DPTB creation and on PBD 7, 14, 21, 28, 60, 90, and 120. Results 92 wounds from 9 swine were included. Speckle data was normalized to control sites and converted to percentages ([speckle wound/speckle control] x 100), producing speckle percentage of control (SPOC) which quantifies the relative decrease or increase in speckle output (vascularity). SPOC was significantly decreased for all burn times on PBD 0, 1, and 2. By PBD 3, only DPTB and FTB remained diminished (p=0.028 and p=0.005, respectively), and FTB SPOC was significantly less than the SPTB (p=0.015). In the second study, SPOC showed an increase post-debridement on PBD 7, noted as post-debridement day (PDD) 0. SPOC continued to increase significantly to a peak at PDD 7 (p< 0.0001) and remained elevated until PDD 28. By PDD 60, SPOC was no longer significantly increased. Conclusions LSCI is a reliable method for analyzing burn depth and wound progression in the preclinical setting. LSCI data shows an immediate decrease in vascularity at all burn depths immediately following burn creation, followed by a peak in vascularity on PDD 7, with a trend back to normal by PDD 60. Applicability of Research to Practice The correlation of wound bed vascularity based on LSCI to known data on burn depth and progression suggests that LSCI could be a useful measurement tool in the clinical setting for the provider determining wound viability.

Real-time intraoperative perfusion assessment may reduce anastomotic leaks. Laser speckle contrast imaging (LSCI) provides dye-free visualization of perfusion by capturing coherent laser light scatter from red blood cells and displays perfusion as a colormap. Herein, we report a novel method to precisely quantify intestinal perfusion using LSCI. ActivSight™ is an FDA-cleared multi-modal visualization system that can detect and display perfusion via both indocyanine green imaging (ICG) and LSCI in minimally invasive surgery. An experimental prototype LSCI perfusion quantification algorithm was evaluated in porcine models. Porcine small bowel was selectively devascularized to create regions of perfused/watershed/ischemic bowel, and progressive aortic inflow/portal vein outflow clamping was performed to study arterial vs. venous ischemia. Continuous arterial pressure was monitored via femoral line. LSCI perfusion colormaps and quantification distinguished between perfused, watershed, and ischemic bowel in all vascular control settings: no vascular occlusion (p < 0.001), aortic occlusion (p < 0.001), and portal venous occlusion (p < 0.001). LSCI quantification demonstrated similar levels of ischemia induced both by states of arterial inflow and venous outflow occlusion. LSCI-quantified perfusion values correlated positively with higher mean arterial pressure and with increasing distance from ischemic bowel. LSCI relative perfusion quantification may provide more objective real-time assessment of intestinal perfusion compared to conventional naked eye assessment by quantifying currently subjective gradients of bowel ischemia and identifying both arterial/venous etiologies of ischemia.

Recombinant human erythropoietin (rHuEPO) has been used as a performance‐enhancing agent by athletes in a variety of sports. The resulting increase in hematocrit levels leads to increased blood viscosity and can affect blood flow, potentially increasing the athlete's risk of developing health complications. However, the actual effects of using rHuEPO on microvascular blood flow and post‐occlusive reactive hyperemia are currently unknown. We therefore evaluated the effect of rHuEPO on the cutaneous microcirculation in well‐trained cyclists using laser speckle contrast imaging (LSCI). This study was part of a randomized, double‐blind, placebo‐controlled, parallel trial designed to investigate the effects of rHuEPO in 47 well‐trained adult cyclists (age 18–50 years). Subjects received a weekly dose of either rHuEPO or placebo for 8 weeks, and LSCI was performed at baseline, after a maximal exercise test in week 6, and before maximal exercise in week 8. Endpoints included basal blood flux, maximum post‐occlusion reperfusion, and time to return to baseline. Despite an increase in hematocrit levels in the rHuEPO‐treated group, we found no statistically significant difference in microvascular function measured between the rHuEPO‐treated group and the placebo group. Our results suggest that the increased hematocrit levels in rHuEPO‐treated well‐trained cyclists are not associated with changes in microvascular blood flow or post‐occlusive reactive hyperemia measured using LSCI.

Laser speckle contrast imaging is a robust and versatile blood flow imaging tool in basic and clinical research for its relatively simple construction and ease of customization. One of its key features is the scalability of the imaged field of view. With minimal changes to the system or analysis, laser speckle contrast imaging allows for high-resolution blood flow imaging through cranial windows or low-resolution perfusion visualization of perfusion over large areas, e.g. in human skin. We further utilize this feature and introduce a multi-scale laser speckle contrast imaging system, which we apply to study vasoreactivity in renal microcirculation. We combine high resolution (small field of view) to segment blood flow in individual vessels with low resolution (large field of view) to monitor global blood flow changes across the renal surface. Furthermore, we compare their performance when analyzing blood flow dynamics potentially associated with a single nephron and show that the previously published approaches, based on low-zoom imaging alone, provide inaccurate results in such applications.

In the last decade, Laser Speckle Contrast Imaging (LSCI) has been proposed and validated for imaging cerebral blood flow at the rodent brain surface in vivo. The technique relies on the calculation of the spatial speckle contrast, which is related to the velocity of scatterers (red blood cells). The implementation of the technique requires a partial craniotomy so that the brain tissues of interest can be illuminated with a laser diode. However, the studies of changes in the microcirculation during disease progression or treatment require longitudinal studies (i.e. imaging is done repeatedly over weeks or even months). Practically, the less invasive way to obtain such data is to image through the thinned skull without a craniotomy. However the presence of static scatterers (skull) will affect the speckle calculation and produce a bias in the estimation of the microcirculation changes. An extension to LSCI, termed Multi-Exposure Speckle Imaging (MESI) was proposed and validated a few years ago that address these limitations. It relies on a model of the speckle contrast as a function of the exposure time and the proportion of static scatterers. Here, we used MESI with the aim of repeatedly imaging the olfactory bulb of mice models of obesity. First, we have developed a MESI set up which was characterized on microfluidic flow phantoms with different flow-rates and channel diameters to simulate blood flow in animal model characteristics. Second, we show that MESI can discriminate flows in the presence of static scatterers and it can measure flow changes consistently. Finally we provide an in vivo validation of the technique in mice with and without a craniotomy.

The liver controls blood homeostasis and depends critically on adequate blood supply. While the global regulation of liver blood flow via the hepatic arterial buffer response is well established, the mechanisms governing hepatic sinusoidal hemodynamics remain elusive. We use laser speckle contrast imaging to investigate the hepatic microvascular blood flow in anesthetized rats. Laser speckle contrast imaging offers a spatial resolution of a few micrometers, enabling visualization of individual microvessels, and a temporal resolution sufficient to track flow dynamics. This allowed us to resolve individual sinusoids and venules on the liver surface and to detect a reduction of the blood flow following local Angiotensin II injections. We show that the microvascular blood flow oscillates with frequencies within the range of 0.05-0.4 Hz, which may be linked to rhythmic contraction of upstream blood vessels. Our findings provide insights into vessel-specific liver microcirculation in vivo, offering new opportunities to explore vascular dysfunction mechanisms in metabolic liver diseases.