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.

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.

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.

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.

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.

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.

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.

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.

Coronary Plaque Microstructure and Composition Modify Optical Polarization: A New Endogenous Contrast Mechanism for Optical Frequency Domain Imaging.

OBJECTIVES: This study aimed to evaluate whether polarimetry, performed using a modified optical frequency domain imaging (OFDI) system, can improve the assessment of histological features relevant to characterizing human coronary atherosclerosis. BACKGROUND: The microscopic structure and organization of the arterial wall influence the polarization of the infrared light used by OFDI. Modification of the OFDI apparatus, along with recently developed image reconstruction methods, permits polarimetric measurements simultaneously with conventional OFDI cross-sectional imaging through standard intravascular imaging catheters. METHODS: The main coronary arteries of 5 cadaveric human hearts were imaged with an OFDI system capable of providing polarimetric assessment. Cross-sectional views of tissue birefringence, measured in refractive index units, and depolarization, expressed as the ratio of depolarized signal to total intensity, were reconstructed, together with conventional OFDI images. Following imaging, the vessels underwent histological evaluation to enable interpretation of the observed polarization features of individual tissue components. RESULTS: Birefringence in fibrous tissue was significantly higher than in intimal tissue with minimal abnormality (0.44 × 10-3 vs. 0.33 × 10-3; p < 0.0001). Birefringence was highest in the tunica media (p < 0.0001), consistent with its high smooth muscle cell content, cells known to associate with birefringence. In fibrous areas, birefringence showed fine spatial features and close correspondence with the histological appearance of collagen. In contrast, necrotic cores and regions rich in lipid elicited significant depolarization (p < 0.0001). Depolarization was also evident in locations of cholesterol crystals and macrophages. CONCLUSIONS: Intravascular measurements of birefringence and depolarization can be obtained using conventional OFDI catheters in conjunction with a modified console and signal processing algorithms. Polarimetric measurements enhance conventional OFDI by providing additional information related to the tissue composition and offer quantitative metrics enabling characterization of plaque features.

Evaluating platelet aggregation dynamics from laser speckle fluctuations.

Platelets are key to maintaining hemostasis and impaired platelet aggregation could lead to hemorrhage or thrombosis. We report a new approach that exploits laser speckle intensity fluctuations, emanated from a drop of platelet-rich-plasma (PRP), to profile aggregation. Speckle fluctuation rate is quantified by the speckle intensity autocorrelation, g2(t), from which the aggregate size is deduced. We first apply this approach to evaluate polystyrene bead aggregation, triggered by salt. Next, we assess dose-dependent platelet aggregation and inhibition in human PRP spiked with adenosine diphosphate and clopidogrel. Additional spatio-temporal speckle analyses yield 2-dimensional maps of particle displacements to visualize platelet aggregate foci within minutes and quantify aggregation dynamics. These findings demonstrate the unique opportunity for assessing platelet health within minutes for diagnosing bleeding disorders and monitoring anti-platelet therapies.

Evaluating the viscoelastic properties of tissue from laser speckle fluctuations.

Most pathological conditions such as atherosclerosis, cancer, neurodegenerative, and orthopedic disorders are accompanied with alterations in tissue viscoelasticity. Laser Speckle Rheology (LSR) is a novel optical technology that provides the invaluable potential for mechanical assessment of tissue in situ. In LSR, the specimen is illuminated with coherent light and the time constant of speckle fluctuations, τ, is measured using a high speed camera. Prior work indicates that τ is closely correlated with tissue microstructure and composition. Here, we investigate the relationship between LSR measurements of τ and sample mechanical properties defined by the viscoelastic modulus, G*. Phantoms and tissue samples over a broad range of viscoelastic properties are evaluated using LSR and conventional mechanical testing. Results demonstrate a strong correlation between τ and |G*| for both phantom (r = 0.79, p <0.0001) and tissue (r = 0.88, p<0.0001) specimens, establishing the unique capability of LSR in characterizing tissue viscoelasticity.

Intravascular optical imaging technology for investigating the coronary artery.

There is an ever-increasing demand for new imaging methods that can provide additional information about the coronary wall to better characterize and stratify high-risk plaques, and to guide interventional and pharmacologic management of patients with coronary artery disease. While there are a number of imaging modalities that facilitate the assessment of coronary artery pathology, this review paper focuses on intravascular optical imaging modalities that provide information on the microstructural, compositional, biochemical, biomechanical, and molecular features of coronary lesions and stents. The optical imaging modalities discussed include angioscopy, optical coherence tomography, polarization sensitive-optical coherence tomography, laser speckle imaging, near-infrared spectroscopy, time-resolved laser induced fluorescence spectroscopy, Raman spectroscopy, and near-infrared fluorescence molecular imaging. Given the wealth of information that these techniques can provide, optical imaging modalities are poised to play an increasingly significant role in the evaluation of the coronary artery in the future.

Diagnostic accuracy of optical coherence tomography and integrated backscatter intravascular ultrasound images for tissue characterization of human coronary plaques.

OBJECTIVES: The purpose of the present study was to validate the diagnostic accuracy of optical coherence tomography (OCT), integrated backscatter intravascular ultrasound (IB-IVUS), and conventional intravascular ultrasound (C-IVUS) for tissue characterization of coronary plaques and to evaluate the advantages and limitations of each of these modalities. BACKGROUND: The diagnostic accuracy of OCT for characterizing tissue types is well established. However, comparisons among OCT, C-IVUS, and IB-IVUS have not been done. METHODS: We examined 128 coronary arterial sites (42 coronary arteries) from 17 cadavers; IVUS and OCT images were acquired on the same slice as histology. Ultrasound signals were obtained using an IVUS system with a 40-MHz catheter and digitized at 1 GHz with 8-bit resolution. The IB values of the ultrasound signals were calculated with a fast Fourier transform. RESULTS: Using histological images as a gold standard, the sensitivity of OCT for characterizing calcification, fibrosis, and lipid pool was 100%, 98%, and 95%, respectively. The specificity of OCT was 100%, 94%, and 98%, respectively (Cohen's kappa = 0.92). The sensitivity of IB-IVUS was 100%, 94%, and 84%, respectively. The specificity of IB-IVUS was 99%, 84%, and 97%, respectively (Cohen's kappa = 0.80). The sensitivity of C-IVUS was 100%, 93%, and 67%, respectively. The specificity of C-IVUS was 99%, 61%, and 95%, respectively (Cohen's kappa = 0.59). CONCLUSIONS: Within the penetration depth of OCT, OCT has a best potential for tissue characterization of coronary plaques. Integrated backscatter IVUS has a better potential for characterizing fibrous lesions and lipid pools than C-IVUS.