Ophthalmic Viscosurgical Devices (OVDs) in clinical setting are a major health risk factor for potential endotoxin contamination in the eye, due to their extensive applications in cataract surgery for space creation, stabilization and protection of intraocular tissue and intraocular lens (IOL) during implantation. Endotoxin contamination of OVDs is implicated in toxic anterior syndrome (TASS), a severe complication of cataract surgery that leads to intraocular damage and even blindness. Current standard methods for endotoxin contamination detection utilize rabbit assay or Limulus amoebocyte lysate (LAL) assays. These endotoxin detection strategies are extremely difficult for gel-like type devices such as OVDs. To overcome the endotoxin detection limitations in OVDs, we have developed an alternative optical detection methodology for label-free and real-time sensing of bacterial endotoxin in OVDs, based on fiber-optic Fourier transform infrared (FO-FTIR) transmission spectrometry in the mid-IR spectral range from 2.5 micron to 12 micron. Endotoxin contaminated OVD test samples were prepared by serial dilutions of endotoxins on OVDs. The major results of this study revealed two salient spectral peak shifts (in the regions 2925 to 2890 cm^-1 and 1125 to 1100 cm^-1), which are associated with endotoxin in OVDs. In addition, FO-FTIR experimental results processed using a multivariate analysis confirmed the observed specific peak shifts associated with endotoxin contamination in OVDs. Thus, employing the FO-FTIR sensing methodology integrated with a multivariate analysis could potentially be used as an alternative endotoxin detection technique in OVD.
Biophotonics is an emerging field in modern biomedical technology that has opened up new horizons for transfer of state-of-the-art techniques from the areas of lasers, fiber optics and biomedical optics to the life sciences and medicine. This field continues to vastly expand with advanced developments across the entire spectrum of biomedical applications ranging from fundamental “bench” laboratory studies to clinical patient “bedside” diagnostics and therapeutics. However, in order to translate these technologies to clinical device applications, the scientific and industrial community, and FDA are facing the requirement for a thorough evaluation and review of laser radiation safety and efficacy concerns. In many cases, however, the review process is complicated due the lack of effective means and standard test methods to precisely analyze safety and effectiveness of some of the newly developed biophotonics techniques and devices. There is, therefore, an immediate public health need for new test protocols, guidance documents and standard test methods to precisely evaluate fundamental characteristics, performance quality and safety of these technologies and devices. Here, we will overview our recent developments of novel test methodologies for safety and efficacy evaluation of some emerging biophotonics technologies and medical devices. These methodologies are based on integrating the advanced features of state-of-the-art optical sensor technologies and approaches such as high-resolution fiber-optic sensing, confocal and optical coherence tomography imaging, and infrared spectroscopy. The presentation will also illustrate some methodologies developed and implemented for testing intraocular lens implants, biochemical contaminations of medical devices, ultrahigh-resolution nanoscopy, and femtosecond laser therapeutics.
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.
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.
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.
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.
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