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.

Expanding applications, accuracy, and interpretation of laser speckle contrast imaging of cerebral blood flow.

Laser speckle contrast imaging (LSCI) provides a rapid characterization of cortical flow dynamics for functional monitoring of the microcirculation. The technique stems from interactions of laser light with moving particles. These interactions encode the encountered Doppler phenomena within a random interference pattern imaged in widefield, known as laser speckle. Studies of neurovascular function and coupling with LSCI have benefited from the real-time characterization of functional dynamics in the laboratory setting through quantification of perfusion dynamics. While the technique has largely been relegated to acute small animal imaging, its scalability is being assessed and characterized for both chronic and clinical neurovascular imaging.

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.

Imaging depth and multiple scattering in laser speckle contrast imaging.

Laser speckle contrast imaging (LSCI) is a powerful and simple method for full field imaging of blood flow. However, the depth dependence and the degree of multiple scattering have not been thoroughly investigated. We employ three-dimensional Monte Carlo simulations of photon propagation combined with high resolution vascular anatomy to investigate these two issues. We found that 95% of the detected signal comes from the top 700 µm of tissue. Additionally, we observed that single-intravascular scattering is an accurate description of photon sampling dynamics, but that regions of interest (ROIs) in areas free of obvious surface vessels had fewer intravascular scattering events than ROI over resolved surface vessels. Furthermore, we observed that the local vascular anatomy can strongly affect the depth dependence of LSCI. We performed simulations over a wide range of intravascular and extravascular scattering properties to confirm the applicability of these results to LSCI imaging over a wide range of visible and near-infrared wavelengths.

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.

Low-cost laser speckle contrast imaging of blood flow using a webcam.

Laser speckle contrast imaging has become a widely used tool for dynamic imaging of blood flow, both in animal models and in the clinic. Typically, laser speckle contrast imaging is performed using scientific-grade instrumentation. However, due to recent advances in camera technology, these expensive components may not be necessary to produce accurate images. In this paper, we demonstrate that a consumer-grade webcam can be used to visualize changes in flow, both in a microfluidic flow phantom and in vivo in a mouse model. A two-camera setup was used to simultaneously image with a high performance monochrome CCD camera and the webcam for direct comparison. The webcam was also tested with inexpensive aspheric lenses and a laser pointer for a complete low-cost, compact setup ($90, 5.6 cm length, 25 g). The CCD and webcam showed excellent agreement with the two-camera setup, and the inexpensive setup was used to image dynamic blood flow changes before and after a targeted cerebral occlusion.

Three-dimensional mapping of oxygen tension in cortical arterioles before and after occlusion.

Occlusions in single cortical microvessels lead to a reduction in oxygen supply, but this decrement has not been able to be quantified in three dimensions at the level of individual vessels using a single instrument. We demonstrate a combined optical system using two-photon phosphorescence lifetime and fluorescence microscopy (2PLM) to characterize the partial pressure of oxygen (pO2) in single descending cortical arterioles in the mouse brain before and after generating a targeted photothrombotic occlusion. Integrated real-time Laser Speckle Contrast Imaging (LSCI) provides wide-field perfusion maps that are used to monitor and guide the occlusion process while 2PLM maps changes in intravascular oxygen tension. We present the technique's utility in highlighting the effects of vascular networking on the residual intravascular oxygen tensions measured after occlusion in three dimensions.

Comparison of indocyanine green angiography and laser speckle contrast imaging for the assessment of vasculature perfusion.

BACKGROUND: Assessment of the vasculature is critical for overall success in cranial vascular neurological surgery procedures. Although several methods of monitoring cortical perfusion intraoperatively are available, not all are appropriate or convenient in a surgical environment. Recently, 2 optical methods of care have emerged that are able to obtain high spatial resolution images with easily implemented instrumentation: indocyanine green (ICG) angiography and laser speckle contrast imaging (LSCI). OBJECTIVE: To evaluate the usefulness of ICG and LSCI in measuring vessel perfusion. METHODS: An experimental setup was developed that simultaneously collects measurements of ICG fluorescence and LSCI in a rodent model. A 785-nm laser diode was used for both excitation of the ICG dye and the LSCI illumination. A photothrombotic clot model was used to occlude specific vessels within the field of view to enable comparison of the 2 methods for monitoring vessel perfusion. RESULTS: The induced blood flow change demonstrated that ICG is an excellent method for visualizing the volume and type of vessel at a single point in time; however, it is not always an accurate representation of blood flow. In contrast, LSCI provides a continuous and accurate measurement of blood flow changes without the need of an external contrast agent. CONCLUSION: These 2 methods should be used together to obtain a complete understanding of tissue perfusion.

Dual-wavelength multifrequency photothermal wave imaging combined with optical coherence tomography for macrophage and lipid detection in atherosclerotic plaques using gold nanoparticles.

The objective of this study was to assess the ability of combined photothermal wave (PTW) imaging and optical coherence tomography (OCT) to detect, and further characterize the distribution of macrophages (having taken up plasmonic gold nanorose as a contrast agent) and lipid deposits in atherosclerotic plaques. Aortas with atherosclerotic plaques were harvested from nine male New Zealand white rabbits divided into nanorose- and saline-injected groups and were imaged by dual-wavelength (800 and 1210 nm) multifrequency (0.1, 1 and 4 Hz) PTW imaging in combination with OCT. Amplitude PTW images suggest that lateral and depth distribution of nanorose-loaded macrophages (confirmed by two-photon luminescence microscopy and RAM-11 macrophage stain) and lipid deposits can be identified at selected modulation frequencies. Radiometric temperature increase and modulation amplitude of superficial nanoroses in response to 4 Hz laser irradiation (800 nm) were significantly higher than native plaque (P<0.001). Amplitude PTW images (4 Hz) were merged into a coregistered OCT image, suggesting that superficial nanorose-loaded macrophages are distributed at shoulders on the upstream side of atherosclerotic plaques (P<0.001) at edges of lipid deposits. Results suggest that combined PTW-OCT imaging can simultaneously reveal plaque structure and composition, permitting characterization of nanorose-loaded macrophages and lipid deposits in atherosclerotic plaques.

Narrow band imaging of squamous cell carcinoma tumors using topically delivered anti-EGFR antibody conjugated gold nanorods.

BACKGROUND: Nanoparticles have recently gained interest as exogenous contrast agents in a variety of biomedical applications related to cancer detection and treatment. The objective of this study was to determine the potential of topically administered antibody conjugated gold nanorods (GNRs) for imaging squamous cell carcinomas (SCCs) of the skin using near-infrared narrowband imaging (NBI). Near-infrared (NIR) NBI images narrow wavelength bands to enhance contrast from plasmonic particles in a wide field portable and noncontact device that is clinically compatible for real-time tumor imaging and tumor margin demarcation. STUDY DESIGN: We conjugated GNRs to Cetuximab, a clinically approved humanized antibody that targets the epidermal growth factor receptor (EGFR), which is overexpressed on the surface of many tumor cells, especially SCCs. We excised subcutaneous xenografts of SCCs (A431) from Swiss nu/nu mice and divided the tumors into two groups: (1) the targeted group (Cetuximab conjugated GNRs) and (2) the control group (polyethylene glycol-conjugated GNRs). After topical application of particles and incubation for 30 minutes, the tumors were washed and imaged using NBI. In addition, we performed two-photon imaging to quantify the binding of EGFR targeted GNRs in tumors and their depth profile. RESULTS: The NBI images showed a visual increase in contrast from tumors after topical administration of targeted GNR. Targeted GNR tumors showed increased contrast compared to tumors administered with the control GNR. There was a statistically significant increase in mean pixel intensity (∼2.5×) from targeted GNR tumors (n = 6). Two-photon microscopy images of targeted GNRs confirmed their binding affinity to the EGF receptors over expressed in the A431 tumors. CONCLUSION: We have demonstrated that a topical application of gold nanorods targeted specifically to tumor growth factor receptors results in a significantly higher image contrast compared to nontargeted gold nanorods. These results demonstrate the feasibility of near-infrared NBI to image and demarcate tumor margins during surgical resection using topical administration of targeted GNR.

Combined two-photon luminescence microscopy and OCT for macrophage detection in the hypercholesterolemic rabbit aorta using plasmonic gold nanorose.

BACKGROUND AND OBJECTIVES: The macrophage is an important early cellular marker related to risk of future rupture of atherosclerotic plaques. Two-channel two-photon luminescence (TPL) microscopy combined with optical coherence tomography (OCT) was used to detect, and further characterize the distribution of aorta-based macrophages using plasmonic gold nanorose as an imaging contrast agent. STUDY DESIGN/MATERIALS AND METHODS: Nanorose uptake by macrophages was identified by TPL microscopy in macrophage cell culture. Ex vivo aorta segments (8 × 8 × 2 mm(3) ) rich in macrophages from a rabbit model of aorta inflammation were imaged by TPL microscopy in combination with OCT. Aorta histological sections (5 µm in thickness) were also imaged by TPL microscopy. RESULTS: Merged two-channel TPL images showed the lateral and depth distribution of nanorose-loaded macrophages (confirmed by RAM-11 stain) and other aorta components (e.g., elastin fiber and lipid droplet), suggesting that nanorose-loaded macrophages are diffusively distributed and mostly detected superficially within 20 µm from the luminal surface of the aorta. Moreover, OCT images depicted detailed surface structure of the diseased aorta. CONCLUSIONS: Results suggest that TPL microscopy combined with OCT can simultaneously reveal macrophage distribution with respect to aorta surface structure, which has the potential to detect vulnerable plaques and monitor plaque-based macrophages overtime during cardiovascular interventions.

Quantitative imaging of ischemic stroke through thinned skull in mice with Multi Exposure Speckle Imaging.

Laser Speckle Contrast Imaging (LSCI) has become a widely used technique to image cerebral blood flow in vivo. However, the quantitative accuracy of blood flow changes measured through the thin skull has not been investigated thoroughly. We recently developed a new Multi Exposure Speckle Imaging (MESI) technique to image blood flow while accounting for the effect of scattering from static tissue elements. In this paper we present the first in vivo demonstration of the MESI technique. The MESI technique was used to image the blood flow changes in a mouse cortex following photothrombotic occlusion of the middle cerebral artery. The Multi Exposure Speckle Imaging technique was found to accurately estimate flow changes due to ischemia in mice brains in vivo. These estimates of these flow changes were found to be unaffected by scattering from thinned skull.