Rapid wide-field Mueller matrix polarimetry imaging based on four photoelastic modulators with no moving parts.

A new polarimetry method is demonstrated to image the entire Mueller matrix of a turbid sample using four photoelastic modulators (PEMs) and a charge coupled device (CCD) camera, with no moving parts. Accurate wide-field imaging is enabled with a field-programmable gate array (FPGA) optical gating technique and an evolutionary algorithm (EA) that optimizes imaging times. This technique accurately and rapidly measured the Mueller matrices of air, polarization elements, and turbid phantoms. The system should prove advantageous for Mueller matrix analysis of turbid samples (e.g., biological tissues) over large fields of view, in less than a second.

Advances in laser speckle imaging: From qualitative to quantitative hemodynamic assessment.

Laser speckle imaging (LSI) techniques have emerged as a promising method for visualizing functional blood vessels and tissue perfusion by analyzing the speckle patterns generated by coherent light interacting with living biological tissue. These patterns carry important biophysical tissue information including blood flow dynamics. The noninvasive, label-free, and wide-field attributes along with relatively simple instrumental schematics make it an appealing imaging modality in preclinical and clinical applications. The review outlines the fundamentals of speckle physics and the three categories of LSI techniques based on their degree of quantification: qualitative, semi-quantitative and quantitative. Qualitative LSI produces microvascular maps by capturing speckle contrast variations between blood vessels containing moving red blood cells and the surrounding static tissue. Semi-quantitative techniques provide a more accurate analysis of blood flow dynamics by accounting for the effect of static scattering on spatiotemporal parameters. Quantitative LSI such as optical speckle image velocimetry provides quantitative flow velocity measurements, which is inspired by the particle image velocimetry in fluid mechanics. Additionally, discussions regarding the prospects of future innovations in LSI techniques for optimizing the vascular flow quantification with associated clinical outlook are presented.

Rapid time-gated polarimetric Stokes imaging using photoelastic modulators.

We report a rapid time-gated full Stokes imaging approach without mechanically moving parts, which is well-suited for biomedical applications, using two photoelastic modulators (PEMs). A charge-coupled device (CCD) with microsecond time-gating capability was used to acquire the images. To synchronize the CCD with the PEMs, thus gaining signal-to-noise ratio advantage, a field programmable gate array was employed. After calibration, an evolutionary algorithm was used to select four time points from which the full Stokes vector can be recovered. Using the images taken by the camera at these four times (in ~80 ms), the images of the full Stokes vectors of different incident polarization states were accurately derived.

Experimental validation of optimum input polarization states for Mueller matrix determination with a dual photoelastic modulator polarimeter.

Dual photoelastic modulator polarimeters can measure light polarization, which is often described as a Stokes vector. By evaluating changes in polarization when light interacts with a sample, the sample Mueller matrix also can be derived, completely describing its interaction with polarized light. The choice of which and how many input Stokes vectors to use for sample investigation is under the experimenter's control. Previous work has predicted that sets of input Stokes vectors forming the vertices of platonic solids on the Poincaré sphere allow for the most robust Mueller matrix determination. Further, when errors specific to the dual photoelastic modulator polarimeter are considered, simulations revealed that one specific shape and orientation of Stokes vectors (cube on the Poincaré sphere with vertices away from principal sphere axes) allows for the most robust Mueller matrix determination. Here we experimentally validate the optimum input Stokes vectors for dual photoelastic modulator Mueller polarimetry, toward developing a robust polarimetric platform of increasing relevance to biophotonics.

Texture analysis of optical coherence tomography speckle for characterizing biological tissues in vivo.

We demonstrate a method for differentiating tissue disease states using the intrinsic texture properties of speckle in optical coherence tomography (OCT) images of normal and tumor tissues obtained in vivo. This approach fits a gamma distribution function to the nonlog-compressed OCT image intensities, thus allowing differentiation of normal and tumor tissues in an ME-180 human cervical cancer mouse xenograft model. Quantitative speckle intensity distribution analysis thus shows promise for identifying tissue pathologies, with potential for early cancer detection in vivo.

Probing multifractality in tissue refractive index: prospects for precancer detection.

Multiresolution analysis on the spatial refractive index inhomogeneities in the epithelium and connective tissue regions of a human cervix reveals a clear signature of multifractality. Importantly, the derived multifractal parameters, namely, the generalized Hurst exponent and the width of the singularity spectrum, derived via multifractal detrended fluctuation analysis, shows interesting differences between tissues having different grades of precancers. The refractive-index fluctuations are found to be more anticorrelated, and the strength of multifractality is observed to be considerably stronger in the higher grades of precancers. These observations on the multifractal nature of tissue refractive-index variations may prove to be valuable for developing light-scattering approaches for noninvasive diagnosis of precancer and early-stage cancer.

Spatial helicity response metric to quantify particle size and turbidity of heterogeneous media through circular polarization imaging.

Backscattered circularly polarized light from turbid media consists of helicity-flipped and helicity-preserved photon sub-populations (i.e., photons of perpendicular and parallel circular handedness). Their intensities and spatial distributions are found to be acutely sensitive to average scatterer size and modestly sensitive to the scattering coefficient (medium turbidity) through an interplay of single and multiple scattering effects. Using a highly sensitive intensified-CCD camera, helicity-based images of backscattered light are captured, which, with the aid of corroborating Monte Carlo simulation images and statistics, enable (1) investigation of subsurface photonic pathways and (2) development of the novel 'spatial helicity response' metric to quantify average scatterer size and turbidity of tissue-like samples. An exciting potential application of this work is noninvasive early cancer detection since malignant tissues exhibit alterations in scatterer size (larger nuclei) and turbidity (increased cell density).

Elastin overexpression by cell-based gene therapy preserves matrix and prevents cardiac dilation.

After a myocardial infarction, thinning and expansion of the fibrotic scar contribute to progressive heart failure. The loss of elastin is a major contributor to adverse extracellular matrix remodelling of the infarcted heart, and restoration of the elastic properties of the infarct region can prevent ventricular dysfunction. We implanted cells genetically modified to overexpress elastin to re-establish the elastic properties of the infarcted myocardium and prevent cardiac failure. A full-length human elastin cDNA was cloned, subcloned into an adenoviral vector and then transduced into rat bone marrow stromal cells (BMSCs). In vitro studies showed that BMSCs expressed the elastin protein, which was deposited into the extracellular matrix. Transduced BMSCs were injected into the infarcted myocardium of adult rats. Control groups received either BMSCs transduced with the green fluorescent protein gene or medium alone. Elastin deposition in the infarcted myocardium was associated with preservation of myocardial tissue structural integrity (by birefringence of polarized light; P < 0.05 versus controls). As a result, infarct scar thickness and diastolic compliance were maintained and infarct expansion was prevented (P < 0.05 versus controls). Over a 9-week period, rats implanted with BMSCs demonstrated better cardiac function than medium controls; however, rats receiving BMSCs overexpressing elastin showed the greatest functional improvement (P < 0.01). Overexpression of elastin in the infarcted heart preserved the elastic structure of the extracellular matrix, which, in turn, preserved diastolic function, prevented ventricular dilation and preserved cardiac function. This cell-based gene therapy provides a new approach to cardiac regeneration.

Quantifying tissue microvasculature with speckle variance optical coherence tomography.

In this Letter, we demonstrate high resolution, three-dimensional optical imaging of in vivo blood vessel networks using speckle variance optical coherence tomography, and the quantification of these images through the development of biologically relevant metrics using image processing and segmentation techniques. Extracted three-dimensional metrics include vascular density, vessel tortuosity, vascular network fractal dimension, and tissue vascularity. We demonstrate the ability of this quantitative imaging approach to characterize normal and tumor vascular networks in a preclinical animal model and the potential for quantitative, longitudinal vascular treatment response monitoring.

Bridging the macro to micro resolution gap with angiographic optical coherence tomography and dynamic contrast enhanced MRI.

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) is emerging as a valuable tool for non-invasive volumetric monitoring of the tumor vascular status and its therapeutic response. However, clinical utility of DCE-MRI is challenged by uncertainty in its ability to quantify the tumor microvasculature ([Formula: see text] scale) given its relatively poor spatial resolution (mm scale at best). To address this challenge, we directly compared DCE-MRI parameter maps with co-registered micron-scale-resolution speckle variance optical coherence tomography (svOCT) microvascular images in a window chamber tumor mouse model. Both semi and fully quantitative (Toft's model) DCE-MRI metrics were tested for correlation with microvascular svOCT biomarkers. svOCT's derived vascular volume fraction (VVF) and the mean distance to nearest vessel ([Formula: see text]) metrics were correlated with DCE-MRI vascular biomarkers such as time to peak contrast enhancement ([Formula: see text] and [Formula: see text] respectively, [Formula: see text] for both), the area under the gadolinium-time concentration curve ([Formula: see text] and [Formula: see text] respectively, [Formula: see text] for both) and [Formula: see text] ([Formula: see text] and [Formula: see text] respectively, [Formula: see text] for both). Several other correlated micro-macro vascular metric pairs were also noted. The microvascular insights afforded by svOCT may help improve the clinical utility of DCE-MRI for tissue functional status assessment and therapeutic response monitoring applications.

Binary dose level classification of tumour microvascular response to radiotherapy using artificial intelligence analysis of optical coherence tomography images.

The dominant consequence of irradiating biological systems is cellular damage, yet microvascular damage begins to assume an increasingly important role as the radiation dose levels increase. This is currently becoming more relevant in radiation medicine with its pivot towards higher-dose-per-fraction/fewer fractions treatment paradigm (e.g., stereotactic body radiotherapy (SBRT)). We have thus developed a 3D preclinical imaging platform based on speckle-variance optical coherence tomography (svOCT) for longitudinal monitoring of tumour microvascular radiation responses in vivo. Here we present an artificial intelligence (AI) approach to analyze the resultant microvascular data. In this initial study, we show that AI can successfully classify SBRT-relevant clinical radiation dose levels at multiple timepoints (t = 2-4 weeks) following irradiation (10 Gy and 30 Gy cohorts) based on induced changes in the detected microvascular networks. Practicality of the obtained results, challenges associated with modest number of animals, their successful mitigation via augmented data approaches, and advantages of using 3D deep learning methodologies, are discussed. Extension of this encouraging initial study to longitudinal AI-based time-series analysis for treatment outcome predictions at finer dose level gradations is envisioned.

Discriminating turbid media by scatterer size and scattering coefficient using backscattered linearly and circularly polarized light.

The effects of scatterer size and scattering coefficient on backscattered linearly and circularly polarized light are investigated through Stokes polarimetry. High-SNR polarization modulation/synchronous detection measurements are corroborated by polarization-sensitive Monte Carlo simulations. Circular degree of polarization (DOP) is found to be sensitive to scatterer size, but is equivocal at times due to helicity flipping effects; linear DOP appears to be mostly dependent on the medium scattering coefficient. We exploit these trends to generate a DOPC - DOPL response surface which clusters turbid samples based on these medium properties. This work may prove useful in biomedicine, for example in noninvasive assessment of epithelial precancer progression.

Polarimetry-based method to extract geometry-independent metrics of tissue anisotropy.

Recently, we have used polarimetry as a method for assessing the linear retardance of infarcted myocardium. While linear retardance reflects tissue anisotropy, experimental geometry has a confounding effect due to dependence of the linear retardance on the orientation of the sample with respect to the probing beam. Here, polarimetry imaging of an 8mm diameter birefringent polystyrene sphere of known anisotropy axis was used to test a dual-projection method by which the anisotropy axis and its true magnitude can be reconstructed, thus eliminating the confounding effect of anisotropy axis orientation. Feasibility is demonstrated in ex-vivo tissue imaging.

Depolarization of light in turbid media: a scattering event resolved Monte Carlo study.

Details of light depolarization in turbid media were investigated using polarization-sensitive Monte Carlo simulations. The surviving linear and circular polarization fractions of photons undergoing a particular number of scattering events were studied for different optical properties of the turbid media. It was found that the threshold number of photon scattering interactions that fully randomize the incident polarization (defined here as <1% surviving polarization fraction) is not a constant, but varies with the photon detection angle. Larger detection angles, close to backscattering direction, show lower full depolarization threshold number for a given set of sample's optical properties. The Monte Carlo simulations also confirm that depolarization is not only controlled by the number of scattering events and detection geometry, but is also strongly influenced by other factors such as anisotropy g, medium linear birefringence, and the polarization state of the incident light.

Dual-Agent Photodynamic Therapy with Optical Clearing Eradicates Pigmented Melanoma in Preclinical Tumor Models.

Treatment using light-activated photosensitizers (photodynamic therapy, PDT) has shown limited efficacy in pigmented melanoma, mainly due to the poor penetration of light in this tissue. Here, an optical clearing agent (OCA) was applied topically to a cutaneous melanoma model in mice shortly before PDT to increase the effective treatment depth by reducing the light scattering. This was used together with cellular and vascular-PDT, or a combination of both. The effect on tumor growth was measured by longitudinal ultrasound/photoacoustic imaging in vivo and by immunohistology after sacrifice. In a separate dorsal window chamber tumor model, angiographic optical coherence tomography (OCT) generated 3D tissue microvascular images, enabling direct in vivo assessment of treatment response. The optical clearing had minimal therapeutic effect on the in control, non-pigmented cutaneous melanomas but a statistically significant effect (p < 0.05) in pigmented lesions for both single- and dual-photosensitizer treatment regimes. The latter enabled full-depth eradication of tumor tissue, demonstrated by the absence of S100 and Ki67 immunostaining. These studies are the first to demonstrate complete melanoma response to PDT in an immunocompromised model in vivo, with quantitative assessment of tumor volume and thickness, confirmed by (immuno) histological analyses, and with non-pigmented melanomas used as controls to clarify the critical role of melanin in the PDT response. The results indicate the potential of OCA-enhanced PDT for the treatment of pigmented lesions, including melanoma.

High-power wavelength-swept laser in Littman telescope-less polygon filter and dual-amplifier configuration for multichannel optical coherence tomography.

We report a high-power wavelength-swept laser source for multichannel optical coherence tomography (OCT) imaging. Wavelength tuning is performed by a compact telescope-less polygon-based filter in Littman arrangement. High output power is achieved by incorporating two serial semiconductor optical amplifiers in the laser cavity in Fourier domain mode-locked configuration. The measured wavelength tuning range of the laser is 111 nm centered at 1329 nm, coherence length of 5.5 mm, and total average output power of 131 mW at 43 kHz sweeping rate. Multichannel simultaneous OCT imaging at an equivalent A-scan rate of 258 kHz is demonstrated.

Proof-of-principle demonstration of a Mueller matrix decomposition method for polarized light tissue characterization in vivo.

We demonstrate the first in vivo use of a Mueller matrix decomposition method for polarization-based characterization of tissue. Collagenase is injected into a region of dermal tissue in a dorsal skin window chamber in a nude mouse to alter the structure of the extracellular matrix. Mueller matrices for polarized light transmitted through the window chamber in the collagenase-treated region, as well as a distal control region, are measured. From the measured matrices, the individual constituent polarization properties of the tissue are extracted through polar matrix decomposition. Large decreases in birefringence and depolarization are seen in the collagenase-treated region due to the destruction of collagen, showing the potential for this method to monitor the organization and structural anisotropy of tissue. This study represents the first in vivo demonstration of a Mueller matrix decomposition method for polarimetric tissue characterization.

Analysis of low-scattering regions in optical coherence tomography: applications to neurography and lymphangiography.

Analysis of semi-transparent low scattering biological structures in optical coherence tomography (OCT) has been actively pursued in the context of lymphatic imaging, with most approaches relying on the relative absence of signal as a means of detection. Here we present an alternate methodology based on spatial speckle statistics, utilizing the similarity of a distribution of given voxel intensities to the power distribution function of pure noise, to visualize the low-scattering biological structures of interest. In a human tumor xenograft murine model, we show that these correspond to lymphatic vessels and nerves; extensive histopathologic validation studies are reported to unequivocally establish this correspondence. The emerging possibility of OCT lymphangiography and neurography is novel and potentially impactful (especially the latter), although further methodology refinement is needed to distinguish between the visualized lymphatics and nerves.

High power wavelength linearly swept mode locked fiber laser for OCT imaging.

We report a long coherence length, high power, and wide tuning range wavelength linearly swept fiber mode-locked laser based on polygon scanning filters. An output power of 52.6 mW with 112 nm wavelength tuning range at 62.6 kHz sweeping rate has been achieved. The coherence length is long enough to enable imaging over 8.1 mm depth when the sensitivity decreases by 8.7 dB (1/e(2)). The Fourier components are still distinguishable when the ranging depth exceeds 15 mm, which corresponds to 30 mm optical path difference in air. The parameters that are critical to OCT imaging quality such as polygon filter linewidth, the laser coherence length, output power, axial resolution and the Fourier sensitivity have been investigated theoretically and experimentally. Since the wavelength is swept linearly with time, an analytical approach has been developed for transforming the interference signal from equidistant spacing in wavelength to equidistant spacing in frequency. Axial resolution of 7.9 microm in air has been achieved experimentally that approaches the theoretical limit.

Mueller matrix decomposition for extraction of individual polarization parameters from complex turbid media exhibiting multiple scattering, optical activity, and linear birefringence.

Linear birefringence and optical activity are two common optical polarization effects present in biological tissue, and determination of these properties has useful biomedical applications. However, measurement and unique interpretation of these parameters in tissue is hindered by strong multiple scattering effects and by the fact that these and other polarization effects are often present simultaneously. We have investigated the efficacy of a Mueller matrix decomposition methodology to extract the individual intrinsic polarimetry characteristics (linear retardance delta and optical rotation psi, in particular) from a multiply scattering medium exhibiting simultaneous linear birefringence and optical activity. In the experimental studies, a photoelastic modulation polarimeter was used to record Mueller matrices from polyacrylamide phantoms having strain-induced birefringence, sucrose-induced optical activity, and polystyrene microspheres-induced scattering. Decomposition of the Mueller matrices recorded in the forward detection geometry from these phantoms with controlled polarization properties yielded reasonable estimates for delta and psi parameters. The confounding effects of scattering, the propagation path of multiple scattered photons, and detection geometry on the estimated values for delta and psi were further investigated using polarization-sensitive Monte Carlo simulations. The results show that in the forward detection geometry, the effects of scattering induced linear retardance and diattenuation are weak, and the decomposition of the Mueller matrix can retrieve the intrinsic values for delta and psi with reasonable accuracy. The ability of this approach to extract the individual intrinsic polarimetry characteristics should prove valuable in diagnostic photomedicine, for example, in quantifying the small optical rotations due to the presence of glucose in tissue and for monitoring changes in tissue birefringence as a signature of tissue abnormality.