Annually, about 15 million preterm infants are born in the world. Of these, due to complications resulting from their premature birth, about 1 million would die before the age of five. Since the high incidence of preterm birth (PTB) is partially due to the lack of effective diagnostic modalities, methodologies are needed to determine risk of PTB. We propose a noninvasive tool based on polarized light imaging aimed at measuring the organization of collagen in the cervix. Cervical collagen has been shown to remodel with the approach of parturition. We used a full-field Mueller matrix polarimetric colposcope to assess and compare cervical collagen content and structure in nonpregnant and pregnant women in vivo. Local collagen directional azimuth was used and a total of eight cervices were imaged.
Preterm birth (PTB) presents a serious medical heath concern throughout the world and maintains a high incidence rate in both developed and developing countries ranging between 11-15%, respectively. PTB can be caused by many different morbidities and ultimately results in the disorganization of cervical collagen and the premature alteration of the cervix mechanical properties. Changes in cervical collagen orientation and distribution may prove to be a predictor of PTB. Polarization imaging is an effective means to measure optical anisotropy in birefringent materials such as those rich in collagen. Non-invasive, in-vivo full-field Mueller Matrix polarimetry (MMP) imaging was conducting using a modified colposcope in a clinical study comparing collagen orientation and distribution between non-pregnant and pregnant patients. Six patients threatening PTB were imaged at the Jackson Memorial Hospital Triage Unit and six non-pregnant patients were image at Florida International University STAR center. In pregnant women collagen distributions changed depending on patient age and number of pregnancies in the non-pregnant population age played an important role in collagen organization.
Preterm birth (PTB) presents a serious medical health concern throughout the world. There is a high incidence of PTB in both developed and developing countries ranging from 11% to 15%, respectively. Recent research has shown that cervical collagen orientation and distribution changes during pregnancy may be useful in predicting PTB. Polarization imaging is an effective means to measure optical anisotropy in birefringent materials, such as the cervix’s extracellular matrix. Noninvasive, full-field Mueller matrix polarimetry (MMP) imaging methodologies, and optical coherence tomography (OCT) imaging were used to assess cervical collagen content and structure in nonpregnant porcine cervices. We demonstrate that the highly ordered structure of the nonpregnant porcine cervix can be observed with MMP. Furthermore, when utilized ex vivo, OCT and MMP yield very similar results with a mean error of 3.46% between the two modalities.
Preterm birth (PTB) presents a serious medical heath concern in both economically developed
and developing nations, with incidence rate from 15%-11% respectively. Changes in cervical
collagen bundle orientation and distribution may prove to be a predictor of PTB. Polarization
imaging is an effective means to measure optical anisotropy in birefringent biological tissue such
as those rich in collagen. Non-invasive, full-field Mueller Matrix polarimetry (MMP) imaging
methodologies, optical coherence tomography (OCT), and second harmonic generation (SHG)
microscopy were used to assess cervical collagen content and structure in non-pregnant cervices.
In vivo studies using a Mueller Matrix colposcope are underway. Further studies of cervical
collagen orientation throughout pregnancy are needed to understand if Mueller matrix
polarimetry can effectively identify at-risk conditions for PTB.
Preterm birth (PTB) presents a serious medical heath concern throughout the world. There is a high incidence of
PTB in both developed and developing countries ranging from 11%-15%, respectively. Studies have shown there
may be numerous precursors to PTB including infections, genetic predisposition, nutrition and various other
morbidities which all lead to a premature disorganization in the cervical collagen resulting in the weakening of the
structure designed to keep the fetus in utero. The changes in cervical collagen orientation and distribution may prove
to be a predictor of PTB. Polarization imaging is an effective means to measure optical anisotropy in birefringent
materials such as those rich in collagen as the cervix is. Non-invasive, full-field Mueller Matrix polarimetry (MMP)
imaging methodologies and ex-vivo second harmonic generation (SHG) imaging were used to assess cervical
collagen content and structure in non-pregnant porcine cervices. The SHG microscopy was used to verify the
efficacy of the MMP in assessing changes in collagen orientation.
Preterm birth is a worldwide health issue, as the number one cause of infant mortality and neurological disorders.
Although affecting nearly 10% of all births, an accurate, reliable diagnostic method for preterm birth has, yet, to be
developed. The primary constituent of the cervix, collagen, provides the structural support and mechanical strength
to maintain cervical closure, through specific organization, during fetal gestation. As pregnancy progresses, the
disorganization of the cervical collagen occurs to allow eventual cervical pliability so the baby can be birthed
through the cervical opening. This disorganization of collagen affects the mechanical properties of the cervix and,
if the changes occur prematurely, may be a significant factor leading to preterm birth. The organization of collagen
can be analyzed through the use of Mueller Matrix Polarimetric imaging of the characteristic birefringence of
collagen. In this research, we have built a full Mueller Matrix Polarimetry attachment to a standard colposcope to
enable imaging of human cervixes during standard prenatal exams at various stages of fetal gestation. Analysis of
the polarimetric images provides information of quantity and organization of cervical collagen at specific gestational
stages of pregnancy. This quantitative information may provide an indication of risk of preterm birth.
Noncontact optical imaging of curved objects can result in strong artifacts due to the object's shape, leading to curvature biased intensity distributions. This artifact can mask variations due to the object's optical properties, and makes reconstruction of optical/physiological properties difficult. In this work we demonstrate a curvature correction method that removes this artifact and recovers the underlying data, without the necessity of measuring the object's shape. This method is applicable to many optical imaging modalities that suffer from shape-based intensity biases. By separating the spatially varying data (e.g., physiological changes) from the background signal (dc component), we show that the curvature can be extracted by either averaging or fitting the rows and columns of the images. Numerical simulations show that our method is equivalent to directly removing the curvature, when the object's shape is known, and accurately recovers the underlying data. Experiments on phantoms validate the numerical results and show that for a given image with 16.5% error due to curvature, the method reduces that error to 1.2%. Finally, diffuse multispectral images are acquired on forearms in vivo. We demonstrate the enhancement in image quality on intensity images, and consequently on reconstruction results of blood volume and oxygenation distributions.
KEYWORDS: Blood, Principal component analysis, Skin, Data modeling, Chromophores, Multispectral imaging, Associative arrays, Absorption, RGB color model, Biological research
Multispectral images of skin contain information on the spatial distribution of biological chromophores, such as blood and melanin. From this, parameters such as blood volume and blood oxygenation can be retrieved using reconstruction algorithms. Most such approaches use some form of pixelwise or volumetric reconstruction code. We explore the use of principal component analysis (PCA) of multispectral images to access blood volume and blood oxygenation in near real time. We present data from healthy volunteers under arterial occlusion of the forearm, experiencing ischemia and reactive hyperemia. Using a two-layered analytical skin model, we show reconstruction results of blood volume and oxygenation and compare it to the results obtained from our new spectral analysis based on PCA. We demonstrate that PCA applied to multispectral images gives near equivalent results for skin chromophore mapping and quantification with the advantage of being three orders of magnitude faster than the reconstruction algorithm.
HER2 overexpression has been associated with a poor prognosis and resistance to therapy in breast cancer patients.
However, quantitative estimates of this important characteristic have been limited to ex vivo ELISA essays of tissue
biopsies and/or PET. We develop a novel approach in optical imaging, involving specific probes, not interfering
with the binding of the therapeutic agents, thus, excluding competition between therapy and imaging. Affibody-based
molecular probes seem to be ideal for in vivo analysis of HER2 receptors using near-infrared optical imaging.
Fluorescence intensity distributions, originating from specific markers in the tumor area, can reveal the
corresponding fluorophore concentration. We use temporal changes of the signal from a contrast agent, conjugated
with HER2-specific Affibody as a signature to monitor in vivo the receptors status in mice with different HER2
over-expressed tumor models. Kinetic model, incorporating saturation of the bound ligands in the tumor area due to
HER2 receptor concentration, is suggested to analyze relationship between tumor cell characteristics, i.e., HER2
overexpression, obtained by traditional ("golden standard") ex vivo methods (ELISA), and parameters, estimated
from the series of images in vivo. Observed correlation between these parameters and HER2 overexpression
substantiates application of our approach to quantify HER2 concentration in vivo.
Quantitative assessment of skin chromophores in a non-invasive fashion is often desirable. Especially pixel wise
assessment of blood volume and blood oxygenation is beneficial for improved diagnostics. We utilized a multi-spectral
imaging system for acquiring diffuse reflectance images of healthy volunteers' lower forearm. Ischemia and reactive
hyperemia was introduced by occluding the upper arm with a pressure cuff for 5min with 180mmHg. Multi-spectral
images were taken every 30s, before, during and after occlusion. Image reconstruction for blood volume and blood
oxygenation was performed, using a two layered skin model. As the images were taken in a non-contact way, strong
artifacts related to the shape (curvature) of the arms were observed, making reconstruction of optical / physiological
parameters highly inaccurate. We developed a curvature correction method, which is based on extracting the curvature
directly from the intensity images acquired and does not require any additional measures on the object imaged. The
effectiveness of the algorithm was demonstrated, on reconstruction results of blood volume and blood oxygenation for in
vivo data during occlusion of the arm. Pixel wise assessment of blood volume and blood oxygenation was made possible
over the entire image area and comparison of occlusion effects between veins and surrounding skin was performed.
Induced ischemia during occlusion and reactive hyperemia afterwards was observed and quantitatively assessed.
Furthermore, the influence of epidermal thickness on reconstruction results was evaluated and the exact knowledge of
this parameter for fully quantitative assessment was pointed out.
NIR light scattering from ex vivo porcine cardiac tissue was investigated to understand how imaging or point
measurement approaches may assist development of methods for tissue depth assessment. Our results indicate an
increase of average image intensity as thickness increases up to approximately 2 mm. In a dual fiber spectroscopy
configuration, sensitivity up to approximately 3 mm with an increase to 6 mm when spectral ratio between selected
wavelengths was obtained. Preliminary Monte Carlo results provided reasonable fit to the experimental data.
We present a novel method for estimating the intrinsic fluorescence lifetime of deeply embedded localized fluorophores. It is based on scaling relations, characteristic for turbid media. The approach is experimentally substantiated by successfully reconstructing lifetimes for targets at depths up to 14.5 mm. A derived correction factor was determined from the product of the transport-corrected scattering coefficient µ and the index of refraction nr. In addition, data from an array of detectors (2) can be used to estimate µnr. The suggested algorithm is a promising tool for diagnostic fluorescence, since lifetime can be a sensitive indicator of the fluorophore environment.
This research describes a noninvasive, noncontact method used to quantitatively analyze the functional characteristics of tissue. Multispectral images collected at several near-infrared wavelengths are input into a mathematical optical skin model that considers the contributions from different analytes in the epidermis and dermis skin layers. Through a reconstruction algorithm, we can quantify the percent of blood in a given area of tissue and the fraction of that blood that is oxygenated. Imaging normal tissue confirms previously reported values for the percent of blood in tissue and the percent of blood that is oxygenated in tissue and surrounding vasculature, for the normal state and when ischemia is induced. This methodology has been applied to assess vascular Kaposi's sarcoma lesions and the surrounding tissue before and during experimental therapies. The multispectral imaging technique has been combined with laser Doppler imaging to gain additional information. Results indicate that these techniques are able to provide quantitative and functional information about tissue changes during experimental drug therapy and investigate progression of disease before changes are visibly apparent, suggesting a potential for them to be used as complementary imaging techniques to clinical assessment.
Subsurface structural features of biological tissue are visualized using polarized light images. The technique of Pearson correlation coefficient analysis is used to reduce blurring of these features by unpolarized backscattered light and to visualize the regions of high statistical similarities within the noisy tissue images. It is shown that under certain conditions, such correlation coefficient maps are determined by the textural character of tissues and not by the chosen region of interest, providing information on tissue structure. As an example, the subsurface texture of a demineralized tooth sample is enhanced from a noisy polarized light image.
Some recent advances in the optical analysis of anisotropic media, especially those, related with work of our group at National Institutes of Health, are presented. Comparison of theoretical formulas, obtained from random walk theory with available time-resolved (transmission mode) and polarization CW (reflectance mode) experimental data is discussed.
The skin of athymic nude mice is irradiated with a single dose of x-ray irradiation that initiated fibrosis. Digital photographs of the irradiated mice are taken by illuminating the mouse skin with linearly polarized probe light of 650 nm. The specific pattern of the surface distribution of the degree of polarization enables the detection of initial skin fibrosis structures that were not visually apparent. Data processing of the raw spatial distributions of the degree of polarization based on Fourier filtering of the high-frequency noise improves subjective perception of the revealed structure in the images. In addition, Pearson correlation analysis provides information about skin structural size and directionality.
Potentials of two modalities of skin diagnostic with focused and expanded linearly polarized probe light are studied. For the focused beam (wavelength 650 nm) the photometric patterns of light backscattered from skin and collagenous tissue phantoms were recorded using digital camera. It is shown that equiintensity contours are well fitted with ellipses that appeared to follow the orientation of collagen fibers. In the peripheral zone from the entry point of the probe beam the ratio of the ellipses semi-axes is correlated with the ratio ofreduced scattering coefficients obtained from intensity profiles. In the vicinity of the entry point it depends on the mutual orientation of polarization vector and collagen fibers. For the expanded probe beam the digital mapping of the residual polarization degree of backscattered linearly polarized light allowed visualization of the hidden structure of earlier fibrosis of the mouse skin arisen from X-ray treatment. The structure of the skin fibrosis was enhanced using Fourier transform filtering of polarization degree pattern. The pattern scanning with Pearson correlation coefficient was developed to determine the orientation and characteristic size of hidden structure. Both modalities may be potentially used for diagnostic ofskin abnormalities, such as fibrosis.
Anisotropy of mouse and human skin is investigated in vivo using polarized videoreflectometry. An incident beam (linearly polarized, wavelength 650 nm) is focused at the sample surface. Two types of tissuelike media are used as controls to verify the technique: isotropic delrin and highly anisotropic demineralized bone with a priori knowledge of preferential orientation of collagen fibers. Equi-intensity profiles of light, backscattered from the sample, are fitted with ellipses that appear to follow the orientation of the collagen fibers. The ratio of the ellipse semiaxes is well correlated with the ratio of reduced scattering coefficients obtained from radial intensity distributions. Variation of equi-intensity profiles with distance from the incident beam is analyzed for different initial polarization states of the light and the relative orientation of polarization filters for incident and backscattered light. For the anisotropic media (demineralized bone and human and mouse skin), a qualitative difference between intensity distributions for cross- and co-polarized orientations of the polarization analyzer is observed up to a distance of 1.5 to 2.5 mm from the entry point. The polarized videoreflectometry of the skin may be a useful tool to assess skin fibrosis resulting from radiation treatment.
Increasing evidence suggests that inflammation may contribute to the process of carcinogenesis. This is the basis of several clinical trials evaluating potential chemopreventive drugs. These trials require quantitative assessments of inflammation, which, for the oral epithelium, are traditionally provided by histopathological evaluation. To reduce patient discomfort and morbidity of tissue biopsy procedures, we develop a noninvasive alternative using diffuse reflectance spectroscopy to measure epithelial thickness as an index of tissue inflammation. Although any optical system has the potential for probing near-surface structures, traditional methods of accounting for scattering of photons are generally invalid for typical epithelial thicknesses. We develop a single-scattering theory that is valid for typical epithelial thicknesses. The theory accurately predicts a distinctive feature that can be used to quantify epithelial thickness given intensity measurements with sources at two different angles relative to the tissue surface. This differential measure approach has acute sensitivity to small, layer-related changes in scattering coefficients. To assess the capability of our method to quantify epithelial thickness, detailed Monte Carlo simulations and measurements on phantom models of a two-layered structure are performed. The results show that the intensity ratio maximum feature can be used to quantify epithelial thickness with an error less than 30% despite fourfold changes in scattering coefficients and 10-fold changes in absorption coefficients. An initial study using a simple two-source, four-detector probe on patients shows that the technique has promise. We believe that this new method will perform well on patients with diverse tissue optical characteristics and therefore be of practical clinical value for quantifying epithelial thickness in vivo.
Diseased tissue may be specifically marked by an exogenous fluorescent marker and then, following laser activation of the marker, optically and non-invasively detected through fluorescence imaging. Interaction of a fluorophore, conjugated to an appropriate antibody, with the antigen expressed by the diseased tissue, can indicate the presence of a specific disease. Using an optical detection system and a reconstruction algorithm, we were able to determine the fluorophore’s position in the tissue. We present 3D reconstructions of the location of a fluorescent marker, FITC, in the tongues of mice. One group of BALB/c mice was injected with squamous cell carcinoma (SqCC) cell line to the tongue, while another group served as the control. After tumor development, the mice’s tongues were injected with FITC conjugated to anti-CD3 and anti-CD 19 antibodies. An Argon laser excited the marker at 488 nm while a high precision fluorescent camera collected the emitted fluorescence. Measurements were performed with the fluorescent marker embedded at various simulated depths. The simulation was performed using agarose-based gel slabs applied to the tongue as tissue-like phantoms. A biopsy was taken from every mouse after the procedure and the excised tissue was histologically evaluated. We reconstruct the fluorescent marker’s location in 3D using an algorithm based on the random walk theory.
For individuals with cancer risk factors, reducing tissue inflammation may reduce the risk of developing cancer. This is the basis of several clinical trials evaluating potential chemoprevention drugs. These trials require quantitative assessments of inflammation which, for the oral epithelium, are traditionally provided by punch biopsies. To reduce patient discomfort and morbidity, we have developed a non-invasive alternative using diffuse reflectance spectroscopy. Though any optical system has the potential for probing near-surface structures, traditional methods of accounting for scattering of photons are generally invalid for typical epithelial thicknesses. We have previously developed a theory that is valid in this regime and validated it with Monte Carlo simulations. We use a differential measure with acute sensitivity to small changes in layer scattering coefficients. To assess the capability of the approach to quantify epithelial
thickness, detailed Monte Carlo simulations and measurements on phantom models of a two layered structure have been performed. Preliminary results from this work show that our key feature varies less than 20 percent despite four-fold changes in scattering coefficients and ten-fold changes in absorption coefficients. This indicates that the method will be of practical clinical value for quantifying epithelial thickness in vivo.
Recent studies suggest that inflammatory cell products may contribute to the evolution of particular cancers leading to new chemoprevention trials exploring the benefit of anti-inflammatory drugs such as aspirin and related products. As part of a prospective trial evaluating this anti-inflammatory strategy for oral cancer, we evaluated a non-invasive optical system to determine if we could use an indirect measure of oral inflammation, mucosal thickness, as a monitoring parameter to evaluate the effectiveness of anti-inflammatory drug therapy. Diffuse reflectance spectroscopy has the potential for probing near-surface structures, however, traditional methods for accounting for scattering of photons are generally invalid for typical epithelial thicknesses. Monte Carlo simulations have shown that, with proper scaling, a simple photon model may be used to predict photon behavior under these conditions. A differential measure, which is very sensitive to small changes, has been shown to have the potential to quantify epithelial thickness. A simple prototype device has been brought from desk, to bench and bedside in a rapid manner to fill a need for a non-invasive measure of oral inflammation. From the theory, a simple feature has been identified that corresponds to patient oral inflammation. Preliminary results from this work are presented and indicate that further development of the approach to enable quantification of epithelial thickness in vivo is warranted.
Intrinsic and exogenous fluorescent molecules may be used as specific markers of disease processes, or metabolic status. A variety of fluorescent markers have been successfully used for transparent tissue, in-vitro studies, and in cases where the markers are located close to the tissue surface. For example, given fluorescence lifetime measurements of a fluorophore such as bis(carboxylic acid) dye, the known relationship of pH on its lifetime may be used to determine the pH of tissue at the fluorophore's location. For fluorophore depths greater than approximately one millimeter in normal tissue, such as might be encountered in in vivo studies, multiple scattering makes it impossible to make direct measurements of characteristics such as fluorophore lifetime. In a multiple scattering environment, the collected intensity depends heavily on the scattering and absorption coefficients of the tissue at both the excitation and emission frequencies. Thus, to obtain values for specific fluorophore characteristics such as the lifetime, a theoretical description of the complex photon paths is required. We have applied Random-walk theory to successfully model photon migration in turbid medias such as tissue. We show how time-resolve intensity measurements may be used to determine fluorophore location and lifetime even when the fluorophore site is located many mean photon scattering lengths from the emitter and detector.
The effect of lateral boundaries on time-resolved measurements of light transmitted through slabs of finite thickness is considered in the framework of a random walk model of photon transport in tissue. A model for a single lateral boundary is derived from the result obtained previously for an infinite slab by employing a standard technique known as the method of images. The predictions of the model are compared with time of flight data from Monte Carlo simulations (University of Florence) and experiments (University College London) using a homogenous phantom having tissue-like optical properties. Agreements in both cases are very good, indicating that the simple formalism of the random walk model is quite adequate to describe the influence of the side boundaries of the tissue slab on the observed characteristics of the transmitted light. The same methodology is applied to assess the influence of side boundaries on the time-dependent contrast functions observed in time-resolved transillumination experiments when an abnormally scattering and absorbing target is embedded in the slab. The potential use of suggested lateral boundary corrections in optical tomography is briefly discussed.
We have developed a random walk model that uses time-dependent contrast functions to quantify the crosssection and the diffusion and absorption coefficients of an optically abnormal target from time-of-flight (TOF) data obtained in time-resolved transillumination experiments1. To substantiate our methodology we have used two different sets of data. The first set of data, provided by colleagues at University College London, are TOF measurements obtained using a solid phantom whose thickness (55mm), optical properties (absorption, ?a=0.006mm-1, transport corrected scattering, ?sc'=0.7mm-1), and characteristics of its abnormal target (size=5mm, optical properties twice those of the background) are close to those of a human breast. The second set of data, provided by colleagues at Politecnico di Milan, are TOF measurements on a 50mm thick phantom (?a=0.01mm-1, ?sc'= 1mm-1) in which two 10mm abnormal targets (one abnormally scattering, ?sc'=2mm-1; one abnormally absorbing and scattering, ?a =0.04mm-1, ?sc'=2mm-1), are embedded. None of these data includes very short path photons whose measurements are clinically impractical. Using our time-dependent contrast functions, we were able to estimate the size and optical properties of the targets with an error margin of 3-25%.
Fluorescence lifetime imaging is a useful tool for quantifying site-dependent environmental conditions in tissue. Fluorophores exist with known lifetime dependencies on factors such as concentrations of O2 and other specific molecules, as well as on temperature and pH. Extracting fluorophore lifetime for deeply embedded sites in turbid media such as tissue is made difficult by the multiple scattering of photons traveling through tissue. This scattering introduces photon arrival delays that have similar characteristics to the delays resulting from the excitation and subsequent emission of photons by fluorophores. Random walk theory (RWT) provides a framework in which the two sources of diffusion-like delays can be separated so that the part due to fluorescent lifetime can be quantified. We derive a closed-form solution that predicts time-resolved photon arrivals from a deeply embedded fluorophore site. The solution requires that an average absorption coefficient be used. However, it is shown that this assumption introduces only a small error. This RWT-derived solution is also shown to be valid for a range of geometries in which the fluorophore site is embedded at least 10 mean scattering lengths and in which the fluorophore lifetime is less than 1 ns.
The success of time-resolved imaging of an abnormal site embedded in thick tissue may rely on one's ability to quantify the absorption coefficient of the target as a specific spectroscopic signature. This task is particularly complicated when the scattering properties of the target differ from those of the surrounding tissue. Using data obtained from time- resolved transillumination experiments of abnormally absorbing and differentially scattering objects embedded in a tissue- like phantom, we show how a new deconvolution algorithm enables us to quantify the optical properties of the target. The algorithm is based on a photon random walk theory that expresses different time-dependent point spread functions to calculate the diffusive and absorptive contrasts obtained in time-of-flight measurements.
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