Imaging the dynamics and microstructure of fibrin clot polymerization in cardiac surgical patients using spectrally encoded confocal microscopy.

During cardiac surgery with cardiopulmonary bypass (CPB), altered hemostatic balance may disrupt fibrin assembly, predisposing patients to perioperative hemorrhage. We investigated the utility of a novel device termed spectrally-encoded confocal microscopy (SECM) for assessing fibrin clot polymerization following heparin and protamine administration in CPB patients. SECM is a novel, high-speed optical approach to visualize and quantify fibrin clot formation in three dimensions with high spatial resolution (1.0 µm) over a volumetric field-of-view (165 × 4000 × 36 µm). The measurement sensitivity of SECM was first determined using plasma samples from normal subjects spiked with heparin and protamine. Next, SECM was performed in plasma samples from patients on CPB to quantify the extent to which fibrin clot dynamics and microstructure were altered by CPB exposure. In spiked samples, prolonged fibrin time (4.4 ± 1.8 to 49.3 ± 16.8 min, p < 0.001) and diminished fibrin network density (0.079 ± 0.010 to 0.001 ± 0.002 A.U, p < 0.001) with increasing heparin concentration were reported by SECM. Furthermore, fibrin network density was not restored to baseline levels in protamine-treated samples. In CPB patients, SECM reported lower fibrin network density in protaminized samples (0.055 ± 0.01 A.U. [Arbitrary units]) vs baseline values (0.066 ± 0.009 A.U.) (p = 0.03) despite comparable fibrin time (baseline = 6.0 ± 1.3, protamine = 6.4 ± 1.6 min, p = 0.5). In these patients, additional metrics including fibrin heterogeneity, length and straightness were quantified. Note, SECM revealed that following protamine administration with CPB exposure, fibrin clots were more heterogeneous (baseline = 0.11 ± 0.02 A.U, protamine = 0.08 ± 0.01 A.U, p = 0.008) with straighter fibers (baseline = 0.918 ± 0.003A.U, protamine = 0.928 ± 0.0006A.U. p < 0.001). By providing the capability to rapidly visualize and quantify fibrin clot microstructure, SECM could furnish a new approach for assessing clot stability and hemostasis in cardiac surgical patients.

Comprehensive Coagulation Profiling at the Point-of-Care Using a Novel Laser-Based Approach.

Delays in identifying internal bleeding are life-threatening, thus underscoring the need for rapid and comprehensive coagulation profiling at the bedside. The authors review a novel optical coagulation profiler that measures several coagulation metrics including prothrombin time, activated clotting time, clot polymerization rate (α-angle), clot stiffness (maximum amplitude), fibrinolysis (LY), and platelet function, using a single multifunctional instrument. The optical profiler is based on the principles of Laser Speckle Rheology that quantifies tissue viscoelasticity from light scattering patterns called laser speckle. To operate the optical profiler, whole blood (40 µL) is loaded into a disposable cartridge, laser speckle patterns are recorded via a camera, and the viscoelasticity of clotting blood is estimated from speckle intensity fluctuations. By monitoring alterations in viscoelastic moduli over time during clot initiation, thrombin generation, fibrin crosslinking, clot stabilization, and LY, global coagulation parameters are obtained within 10 minutes using a drop of whole blood. Clinical testing in over 500 patients to date has confirmed the accuracy of the optical profiler for comprehensively assessing coagulation status against conventional coagulation tests and thromboelastography. Recent studies have further demonstrated the capability to quantify platelet aggregation induced by adenosine diphosphate in a drop of platelet-rich-plasma in the absence of applied shear stress. Together, these studies demonstrate that global coagulation profiling in addition to platelet function may be accomplished using a single multifunctional device. Thus, by enabling rapid and comprehensive coagulation and platelet function profiling at the bedside, the optical profiler will likely advance the capability to identify and manage patients with an elevated risk for hemorrhage.

Estimation of particle size variations for laser speckle rheology of materials.

Laser speckle rheology (LSR) is an optical technique for assessing the viscoelastic properties of materials with several industrial, biological, and medical applications. In LSR, the viscoelastic modulus, G*(ω), of a material is quantified by analyzing the temporal fluctuations of speckle patterns. However, the size of scattering particles within the material also influences the rate of speckle fluctuations, independent of sample mechanical properties, and complicates the accurate estimation of G*(ω). Here, we demonstrate that the average particle size may be retrieved from the azimuth-angle dependence of time-averaged speckle intensities, permitting the accurate quantification of the viscoelastic moduli of materials with unknown particle size distribution using LSR.

Correction of optical absorption and scattering variations in Laser Speckle Rheology measurements.

Laser Speckle Rheology (LSR) is an optical technique to evaluate the viscoelastic properties by analyzing the temporal fluctuations of backscattered speckle patterns. Variations of optical absorption and reduced scattering coefficients further modulate speckle fluctuations, posing a critical challenge for quantitative evaluation of viscoelasticity. We compare and contrast two different approaches applicable for correcting and isolating the collective influence of absorption and scattering, to accurately measure mechanical properties. Our results indicate that the numerical approach of Monte-Carlo ray tracing (MCRT) reliably compensates for any arbitrary optical variations. When scattering dominates absorption, yet absorption is non-negligible, diffusing wave spectroscopy (DWS) formalisms perform similar to MCRT, superseding other analytical compensation approaches such as Telegrapher equation. The computational convenience of DWS greatly simplifies the extraction of viscoelastic properties from LSR measurements in a number of chemical, industrial, and biomedical applications.

The influence of optical fiber bundle parameters on the transmission of laser speckle patterns.

Laser speckle imaging (LSI) techniques provide important functional information about tissue perfusion and mechanical properties. To perform LSI in vivo, laser speckle patterns are transmitted via optical fiber bundles incorporated within small-diameter endoscopes. Inter-fiber crosstalk due to mode coupling in fiber bundles can result in erroneous speckle statistics and therefore reduces the accuracy of LSI analysis. In this paper, we investigate the influence of multiple parameters that influence crosstalk between neighboring cores within optical fiber bundles and govern the modulation of transmitted laser speckle patterns. Our results show that in addition to large core-to-core separation, large refractive index contrast between core and cladding material, reduced number of propagating modes and variability in core size are essential parameters for accurate speckle pattern transmission to conduct endoscopic LSI.

Laser speckle rheological microscopy reveals wideband viscoelastic spectra of biological tissues.

Viscoelastic transformation of tissue drives aberrant cellular functions and is an early biomarker of disease pathogenesis. Tissues scale a range of viscoelastic moduli, from biofluids to bone. Moreover, viscoelastic behavior is governed by the frequency at which tissue is probed, yielding distinct viscous and elastic responses modulated over a wide frequency band. Existing tools do not quantify wideband viscoelastic spectra in tissues, leaving a vast knowledge gap. We present wideband laser speckle rheological microscopy (WB-SHEAR) that reveals elastic and viscous response over sub-megahertz frequencies previously not investigated in tissue. WB-SHEAR uses an optical, noncontact approach to quantify wideband viscoelastic spectra in specimens spanning a range of moduli from low-viscosity fibrin to highly elastic bone. Via laser scanning, micromechanical imaging is enabled to access wideband viscoelastic spectra in heterogeneous tumor specimens with high spatial resolution (25 micrometers). The ability to interrogate the viscoelastic landscape of diverse biospecimens could transform our understanding of mechanobiological processes in various diseases.

Spectral binning for mitigation of polarization mode dispersion artifacts in catheter-based optical frequency domain imaging.

Polarization mode dispersion (PMD) has been recognized as a significant barrier to sensitive and reproducible birefringence measurements with fiber-based, polarization-sensitive optical coherence tomography systems. Here, we present a signal processing strategy that reconstructs the local retardation robustly in the presence of system PMD. The algorithm uses a spectral binning approach to limit the detrimental impact of system PMD and benefits from the final averaging of the PMD-corrected retardation vectors of the spectral bins. The algorithm was validated with numerical simulations and experimental measurements of a rubber phantom. When applied to the imaging of human cadaveric coronary arteries, the algorithm was found to yield a substantial improvement in the reconstructed birefringence maps.

Speckle rheological spectroscopy reveals wideband viscoelastic spectra of biological tissues.

Mechanical transformation of tissue is not merely a symptom but a decisive driver in pathological processes. Comprising intricate network of cells, fibrillar proteins, and interstitial fluid, tissues exhibit distinct solid-(elastic) and liquid-like (viscous) behaviours that span a wide band of frequencies. Yet, characterization of wideband viscoelastic behaviour in whole tissue has not been investigated, leaving a vast knowledge gap in the higher frequency range that is linked to fundamental intracellular processes and microstructural dynamics. Here, we present wideband Speckle rHEologicAl spectRoScopy (SHEARS) to address this need. We demonstrate, for the first time, analysis of frequency-dependent elastic and viscous moduli up to the sub-MHz regime in biomimetic scaffolds and tissue specimens of blood clots, breast tumours, and bone. By capturing previously inaccessible viscoelastic behaviour across the wide frequency spectrum, our approach provides distinct and comprehensive mechanical signatures of tissues that may provide new mechanobiological insights and inform novel disease prognostication.

Regeneration of collecting lymphatic vessels following injury.

Secondary lymphedema is a debilitating condition driven by impaired regeneration of lymphatic vasculature following lymphatic injury, surgical removal of lymph nodes in cancer patients or infection. However, the extent to which collecting lymphatic vessels regenerate following injury remains unclear. Here, we employed a novel mouse model of lymphatic injury in combination with state-of-the-art lymphatic imaging to demonstrate that the implantation of an optimized fibrin gel following lymphatic vessel injury leads to the growth and reconnection of the injured lymphatic vessel network, resulting in the restoration of lymph flow to the draining node. Intriguingly, we found that fibrin implantation elevates the tissue levels of CCL5, a potent macrophage-recruiting chemokine. Notably, CCL5-KO mice displayed a reduced ability to reconnect injured vessels following fibrin gel implantation. These novel findings shed light on the mechanisms underlying lymphatic regeneration and suggest that enhancing CCL5 signaling may be a promising therapeutic strategy for enhancing lymphatic regeneration.

Nanosecond SRS fiber amplifier for label-free near-infrared photoacoustic microscopy of lipids.

Near-infrared photoacoustics receives increasing interest as an intravital modality to sense key biomolecules. One of the most central types of biomolecules of interest are lipids as they constitute essential bio-hallmarks of cardiovascular and metabolic diseases and their in-vivo detection holds insightful information about disease progression and treatment monitoring. However, the full potential of near-infrared photoacoustic for high-resolution and high-sensitivity biomedical studies of lipids has so far not been exploited due a lack of appropriate excitation sources delivering short-pulses at high-repetition-rate, high-pulse-energy, and wavelength around 1200 nm. Here, we demonstrate a custom-built SRS fiber amplifier that provides optical excitations at 1192.8 nm, repetition rates of 200 kHz, pulse durations below 2 ns, and pulse energies beyond 5 µJ. We capitalize on the performance of our excitation source and show near-infrared photoacoustics resolving intrinsic lipid contrast in biomedically relevant specimens ranging from single cells to lipid-rich tissue with subcellular resolution.

Technological perspectives on laser speckle micro-rheology for cancer mechanobiology research.

SIGNIFICANCE: The ability to measure the micro-mechanical properties of biological tissues and biomaterials is crucial for numerous fields of cancer research, including tumor mechanobiology, tumor-targeting drug delivery, and therapeutic development. AIM: Our goal is to provide a renewed perspective on the mainstream techniques used for micro-mechanical evaluation of biological tissues and biomimetic scaffoldings. We specifically focus on portraying the outlook of laser speckle micro-rheology (LSM), a technology that quantifies the mechanical properties of biomaterials and tissues in a rapid, non-contact manner. APPROACH: First, we briefly explain the motivation and significance of evaluating the tissue micro-mechanics in various fields of basic and translational cancer research and introduce the key concepts and quantitative metrics used to explain the mechanical properties of tissue. This is followed by reviewing the general active and passive themes of measuring micro-mechanics. Next, we focus on LSM and elaborate on the theoretical grounds and working principles of this technique. Then, the perspective for measuring the micro-mechanical properties via LSM is outlined. Finally, we draw an overview picture of LSM in cancer mechanobiology research. RESULTS: With the continued emergence of new approaches for measuring the mechanical attributes of biological tissues, the field of micro-mechanical imaging is at its boom. As one of these competent innovations, LSM presents a tremendous potential for both technical maturation and prospective applications in cancer biomechanics and mechanobiology research. CONCLUSION: By elaborating the current viewpoint of LSM, we expect to accelerate the expansion of this approach to new territories in both technological domains and applied fields. This renewed perspective on LSM may also serve as a road map for other micro-mechanical measurement concepts to be applied for answering mechanobiological questions.

Mapping Mechanical Properties of the Tumor Microenvironment by Laser Speckle Rheological Microscopy.

Altered mechanical properties of the tumor matrix have emerged as both the cause and consequence of breast carcinogenesis. Increased tumor stiffness has traditionally provided a viable metric to screen for malignancies via palpation or imaging. Previous studies have demonstrated that the microscale mechanical properties of the cell substrate influence tumor proliferation and invasive migration in vitro. Nevertheless, the association of the mechanical microenvironment with clinical hallmarks of aggressiveness in human breast tumors, including histopathological subtype, grade, receptor expression status, and lymph node involvement is poorly understood. This is largely due to the lack of tools for mapping tumor viscoelastic properties in clinical specimens with high spatial resolution over a large field of view (FoV). Here we introduce laser Speckle rHEologicAl micRoscopy (SHEAR) that for the first time enables mapping the magnitude viscoelastic or shear modulus, |G*(x,y,ω)|, over a range of frequencies (ω = 1-250 rad/second) in excised tumors within minutes with a spatial resolution of approximately 50 µm, over multiple cm2 FoV. Application of SHEAR in a cohort of 251 breast cancer specimens from 148 patients demonstrated that |G*(x,y,ω)| (ω = 2π rad/second) closely corresponds with histological features of the tumor, and that the spatial gradient of the shear modulus, |∇|G*(x,y,ω)||, is elevated at the tumor invasive front. Multivariate analyses established that the metrics, (|G* |) and (|∇|G* ||), measured by SHEAR are associated with prognosis. These findings implicate the viscoelastic properties of the tumor microenvironment in breast cancer prognosis and likely pave the path for identifying new modifiable targets for treatment. SIGNIFICANCE: Laser speckle rheological microscopy establishes the links between microscale heterogeneities of viscoelasticity and histopathological subtype, tumor grade, receptor expression, as well as lymph node status in breast carcinoma.

In-vivo mechanical characterization of coronary atherosclerotic plaques in living swine using intravascular laser speckle imaging.

The ability to evaluate the viscoelastic properties of coronary arteries is crucial for identifying mechanically unstable atherosclerotic plaques. Here, we demonstrate for the first time in living swine, the capability of intravascular laser speckle imaging (ILSI) to measure an index of coronary plaque viscoelasticity, τ, using a human coronary to swine xenograft model. Cardiac motion effects are evaluated by comparing the EKG-non-gated τ ¯ N G , and EKG-gated τ ¯ G among different plaque types. Results show that both τ ¯ N G and τ ¯ G are significantly lower in necrotic-core plaques compared with stable lesions. Discrete-point pullback measurements demonstrate the capability of ILSI for rapid mechanical characterization of coronary segments under physiological conditions, in-vivo.

Tutorial on laser speckle rheology: technology, applications, and opportunities.

SIGNIFICANCE: The onset of several diseases is frequently marked with anomalous mechanical alteration of the affected tissue at the intersection of cells and their microenvironment. Therefore, mapping the micromechanical attributes of the tissues could enhance our understanding of the etiology of human disease, improve the diagnosis, and help stratify therapies that target these mechanical aberrations. AIM: We review the tremendous opportunities offered through using optics for imaging the micromechanical properties, at length scales inaccessible to other modalities, in both basic research and clinical medicine. We specifically focus on laser speckle rheology (LSR), a technology that quantifies the mechanical properties of tissues in a rapid, noncontact manner. APPROACH: In LSR, the shear viscoelastic modulus is measured from the time-variant speckle intensity fluctuations reflected off the tissue. The LSR technology is engineered and configured into several embodiments, including bench-top optical systems, endoscopes for minimally invasive procedures, portable point-of-care devices, and microscopes. RESULTS: These technological nuances have primed the LSR for widespread applications in diagnosis and therapeutic monitoring, as demonstrated here, in cardiovascular disease, coagulation disorders, and tumor malignancies. CONCLUSION: The fast-paced technological advancements, elaborated here, position the LSR as a competent candidate for many more exciting opportunities in basic research and medicine.

Intravascular Polarimetry: Clinical Translation and Future Applications of Catheter-Based Polarization Sensitive Optical Frequency Domain Imaging.

Optical coherence tomography (OCT) and optical frequency domain imaging (OFDI) visualize the coronary artery wall and plaque morphology in great detail. The advent of these high-resolution intracoronary imaging modalities has propelled our understanding of coronary atherosclerosis and provided enhanced guidance for percutaneous coronary intervention. Yet, the lack of contrast between distinct tissue types and plaque compositions impedes further elucidation of the complex mechanisms that contribute to acute coronary syndrome (ACS) and hinders the prospective identification of plaques susceptible to rupture. Intravascular polarimetry with polarization-sensitive OFDI measures polarization properties of the coronary arterial wall using conventional intravascular imaging catheters. The quantitative polarization metrics display notable image contrast between several relevant coronary plaque microstructures that are difficult to identify with conventional OCT and OFDI. Tissues rich in collagen and smooth muscle cells exhibit birefringence, while lipid and macrophages cause depolarization. In this review, we describe the basic principles of intravascular polarimetry, discuss the interpretation of the polarization signatures, and outline promising avenues for future research and clinical implications.

Comprehensive Blood Coagulation Profiling in Patients Using iCoagLab: Comparison Against Thromboelastography.

Delayed identification of coagulopathy and bleeding increases the risk of organ failure and death in hospitalized patients. Timely and accurate identification of impaired coagulation at the point-of-care can proactively identify bleeding risk and guide resuscitation, resulting in improved outcomes for patients. We test the accuracy of a novel optical coagulation sensing approach, termed iCoagLab, for comprehensive whole blood coagulation profiling and investigate its diagnostic accuracy in identifying patients at elevated bleeding risk. Whole blood samples from patients (N = 270) undergoing conventional coagulation testing were measured using the iCoagLab device. Recalcified and kaolin-activated blood samples were loaded in disposable cartridges and time-varying intensity fluctuation of laser speckle patterns were measured to quantify the clot viscoelastic modulus during coagulation. Coagulation parameters including the reaction time (R), clot progression time (K), clot progression rate (α), and maximum clot strength (MA) were derived from clot viscoelasticity traces and compared with mechanical thromboelastography (TEG). In all patients, a good correlation between iCoagLab- and TEG-derived parameters was observed (p < 0.001). Multivariate analysis showed that iCoagLab-derived parameters identified bleeding risk with sensitivity (94%) identical to, and diagnostic accuracy (89%) higher than TEG (87%). The diagnostic specificity of iCoagLab (77%) was significantly higher than TEG (69%). By rapidly and comprehensively permitting blood coagulation profiling the iCoagLab innovation is likely to advance the capability to identify patients with elevated risk for bleeding, with the ultimate goal of preventing life-threatening hemorrhage.

Biomechanical Stress Profiling of Coronary Atherosclerosis: Identifying a Multifactorial Metric to Evaluate Plaque Rupture Risk.

OBJECTIVES: The purpose of this study was to derive a biomechanical stress metric that was based on the multifactorial assessment of coronary plaque morphology, likely related to the propensity of plaque rupture in patients. BACKGROUND: Plaque rupture, the most frequent cause of coronary thrombosis, occurs at locations of elevated tensile stress in necrotic core fibroatheromas (NCFAs). Finite element modeling (FEM), typically used to calculate tensile stress, is computationally intensive and impractical as a clinical tool for locating rupture-prone plaques. This study derived a multifactorial stress equation (MSE) that accurately computes peak stress in NCFAs by combining the influence of several morphological parameters. METHODS: Intravascular ultrasound and optical frequency domain imaging were conducted in 30 patients, and plaque morphological parameters were defined in 61 NCFAs. Multivariate regression analysis was applied to derive the MSE and compute a peak stress metric (PSM) that was based on the analysis of plaque morphological parameters. The accuracy of the MSE was determined by comparing PSM with FEM-derived peak stress values. The ability of the PSM in locating plaque rupture sites was tested in 3 additional patients. RESULTS: The following parameters were found to be independently associated with peak stress: fibrous cap thickness (p < 0.0001), necrotic core angle (p = 0.024), necrotic core thickness (p < 0.0001), lumen area (p < 0.0001), necrotic core including calcium areas (p = 0.017), and plaque area (p = 0.003). The PSM showed excellent correlation (R = 0.85; p < 0.0001) with FEM-derived peak stress, thus confirming the accuracy of the MSE. In only 56% (n = 34) of plaques, the thinnest fibrous cap thickness was a determining parameter in identifying the cross section with highest PSM. In coronary segments with plaque ruptures, the MSE precisely located the rupture site. CONCLUSIONS: The MSE shows potential to calculate the PSM in coronary lesions rapidly. However, further studies are warranted to investigate the use of biomechanical stress profiling for the prognostic evaluation of patients with atherosclerosis.

Intravascular Polarimetry in Patients With Coronary Artery Disease.

OBJECTIVES: The aims of this first-in-human pilot study of intravascular polarimetry were to investigate polarization properties of coronary plaques in patients and to examine the relationship of these features with established structural characteristics available to conventional optical frequency domain imaging (OFDI) and with clinical presentation. BACKGROUND: Polarization-sensitive OFDI measures birefringence and depolarization of tissue together with conventional cross-sectional optical frequency domain images of subsurface microstructure. METHODS: Thirty patients undergoing polarization-sensitive OFDI (acute coronary syndrome, n = 12; stable angina pectoris, n = 18) participated in this study. Three hundred forty-two cross-sectional images evenly distributed along all imaged coronary arteries were classified into 1 of 7 plaque categories according to conventional OFDI. Polarization features averaged over the entire intimal area of each cross section were compared among plaque types and with structural parameters. Furthermore, the polarization properties in cross sections (n = 244) of the fibrous caps of acute coronary syndrome and stable angina pectoris culprit lesions were assessed and compared with structural features using a generalized linear model. RESULTS: The median birefringence and depolarization showed statistically significant differences among plaque types (p < 0.001 for both, one-way analysis of variance). Depolarization differed significantly among individual plaque types (p < 0.05), except between normal arteries and fibrous plaques and between fibrofatty and fibrocalcified plaques. Caps of acute coronary syndrome lesions and ruptured caps exhibited lower birefringence than caps of stable angina pectoris lesions (p < 0.01). In addition to clinical presentation, cap birefringence was also associated with macrophage accumulation as assessed using normalized SD. CONCLUSIONS: Intravascular polarimetry provides quantitative metrics that help characterize coronary arterial tissues and may offer refined insight into coronary arterial atherosclerotic lesions in patients.

Evaluation of collagen in atherosclerotic plaques: the use of two coherent laser-based imaging methods.

Acute coronary events such as myocardial infarction are frequently caused by the rupture of unstable atherosclerotic plaque. Collagen plays a key role in determining plaque stability. Methods to measure plaque collagen content are invaluable in detecting unstable atherosclerotic plaques. Recently, novel coherent laser-based imaging techniques, such as polarization-sensitive optical coherence tomography (PSOCT) and laser speckle imaging (LSI) have been investigated, and they provide a wealth of information related to collagen content and plaque stability. Additionally, given their potential for intravascular use, these technologies will be invaluable for improving our understanding of the natural history of plaque development and rupture and, hence, enable the detection of unstable plaques. In this article we review recent developments in these techniques and potential challenges in translating these methods into intra-arterial use in patients.

Intravascular Polarimetry for Tissue Characterization of Coronary Atherosclerosis.

The microscopic tissue structure and organization influence the polarization of light. Intravascular polarimetry leverages this compelling intrinsic contrast mechanism by using polarization-sensitive optical frequency domain imaging to measure the polarization properties of the coronary arterial wall. Tissues rich in collagen and smooth muscle cells appear birefringent, while the presence of lipid causes depolarization, offering quantitative metrics related to the presence of important components of coronary atherosclerosis. Here, we review the basic principle, the interpretation of polarization signatures, and first clinical investigations of intravascular polarimetry and discuss how this extension of contemporary intravascular imaging may advance our knowledge and improve clinical practice in the future.