Proceedings Volume 7907

Biomedical Applications of Light Scattering V

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Proceedings Volume 7907

Biomedical Applications of Light Scattering V

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Volume Details

Date Published: 4 February 2011
Contents: 8 Sessions, 26 Papers, 0 Presentations
Conference: SPIE BiOS 2011
Volume Number: 7907

Table of Contents

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Table of Contents

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  • Front Matter: Volume 7907
  • Characterization of Turbid Media
  • Speckle and Dynamic Light Scattering
  • Novel Instruments and Methods
  • Clinical and Pre-Clinical Studies
  • In Vitro Cell Studies
  • Low-coherence Light Scattering
  • Poster Session
Front Matter: Volume 7907
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Front Matter: Volume 7907
This PDF file contains the front matter associated with SPIE Proceedings Volume 7907, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.
Characterization of Turbid Media
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Theoretical and experimental comparison of tissue phantoms using white light spectroscopy
White light spectroscopy has the potential to be used as an optical biopsy method, as backscattered light data can be related to the cellular nucleus size. In this paper, we compare experimental backscattering results against the general Mie theory simulations. Our experimental technique obtains results in terms of intensity vs. wavelength. We also use optical filters, to limit the source spectrum, and when converting the backscattered data into the Fourier domain, we obtain characteristic frequencies which describe the scatterer size. Phantom tissues, comprised of polystyrene spheres in a liquid or gel, are used for the experiments.
Nanoscale nuclear architecture for cancer diagnosis by spatial-domain low-coherence quantitative phase microscopy
Pin Wang, Rajan K. Bista, Walid E. Khalbuss, et al.
Alterations in nuclear architecture are the hallmark diagnostic characteristic of cancer cells. In this work, we show that the nuclear architectural characteristics quantified by spatial-domain low-coherence quantitative phase microscopy (SL-QPM), is more sensitive for the identification of cancer cells than conventional cytopathology. We demonstrated the importance of nuclear architectural characteristics in both an animal model of intestinal carcinogenesis - APC/Min mouse model and human cytology specimens with colorectal cancer by identifying cancer from cytologically noncancerous appearing cells. The determination of nanoscale nuclear architecture using this simple and practical optical instrument is a significant advance towards cancer diagnosis.
Effect of clearing agents on scattering coefficient and anisotropy of scattering of dermis studied by reflectance confocal microscopy
Optical clearing of mouse dermis by glycerol was tested by reflectance-mode confocal microscopy (rCSLM) using 488- nm light. The reflectance signal R(z) was acquired as a function of the depth of the focus (z) within the upper 100 μm of freshly excised mouse dermis. The results specify the scattering coefficient (μs [cm-1]) and the anisotropy of scattering (g [dimensionless]). The absorption is too low to exert an effect. The results, published in Samatham et al., Journal of Innovative Optical Health Sciences 2010, 3(3):183-188, described how the clearing effect of glycerol was to increase g toward nearly 1.0, while having only a modest effect on μs. In other words, glycerol caused light scattering to become very forward-directed, but did not strongly alter the number of scattering events per unit length of photon path. This paper discusses the possible mechanism of action that is responsible for this clearing effect.
Phase function of biological soft tissues for the complete solid angle
R. Michels, S. Rotte, M. T. Heine, et al.
With the help of a solution of the transport equation it is possible to calculate the light propagation in biological tissue quite precisely if the exact phase function of the scattering tissue is known. The phase function of most structured tissues depends on two scattering angles (polar and azimuthal angle) and the direction of the incident light. Even though the use of the complete phase function is crucial for a precise calculation of the light propagation and the only way to understand e.g. the anisotropic light propagation in structured tissue, typically phase functions are used which depend only on one scattering angle. This simplification is most likely due to missing measurement data of more realistic phase functions of biological tissues. In this article we present goniometric measurements of the phase function of porcine skeletal muscle for the whole solid angle and different incident directions.
Monte Carlo simulation of photon migration in turbid random media based on the object-oriented programming paradigm
The advantages of using method Monte Carlo for simulation of radiative transfer in complex turbid random media like biological tissues are well recognized. However, in most practical applications the wave nature of probing optical radiation is ignored, and its propagation is considered in terms of neutral particles, so-called photon packets. Nevertheless, when the interference, polarization or coherent effects of scattering of optical/laser radiation constitute the fundamental principle of a particular optical technique the wave nature of optical radiation must be considered and taken into account. In current report we present the state-of-the art and the development prospects for application of method Monte Carlo to the point of taking into account the wave properties of optical radiation. We also introduce a novel Object-Oriented Programming (OOP) paradigm accelerated by Graphics Processing Unit that provides an opportunity to escalate the performance of standard Monte Carlo simulation up to 100 times.
Speckle and Dynamic Light Scattering
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The role of cellular environment in dynamic light scattering
Ran An, Kwan Jeong, John Turek, et al.
We have developed motility contrast imaging (MCI) as a coherence-domain volumetric imaging approach that uses subcellular dynamics as an endogenous imaging contrast agent of living tissue. Fluctuation spectroscopy analysis of dynamic light scattering (DLS) from 3-D tissue has identified functional frequency bands related to organelle transport, membrane undulations and cell shape change. In this paper, we track the behavior of dynamic light scattering as we bridge the gap between the two extremes of 2-D cell culture on the one hand, and 3-D tissue spheroids on the other. In a light backscattering geometry, we capture speckle from 2-D cell culture consisting of isolated cells or planar rafts of cells on cell-culture surfaces. DLS from that cell culture shows differences and lower sensitivity to intra-cellular dynamics compared with the 3-D tissue. The motility contrast is weak in this limit. As the cellular density increases to cover the surface, the motility contrast increases. As environmental perturbations or pharmaceuticals are applied, the fluctuation spectral response becomes more dramatic as the dimensionality of the cellular aggregations increases. We show that changing optical thickness of the cellular-to-tissue targets usually causes characteristic frequency shifts in the spectrograms, while changing cellular dimensionality causes characteristic frequencies to be enhanced or suppressed.
Endoscopic laser speckle contrast imaging system using a fibre image guide
There are several challenges when fibre image guides (FIG) are used for endoscopic speckle acquisition: cross talk between fibre cores, FIG fixed pattern noise, the small probe diameter and low sensitivity and resolution due to the decreased number of speckles and their low transmission through the FIG. In this paper, an endoscopic laser speckle contrast analysis system (ELASCA) based on a leached fibre image guide (LFIG) is presented. Different methods of acquiring LASCA images through LFIGs were investigated including the effect of changing the number of speckles per fibre, defocusing the FIG image onto the CCD and processing speckle images with masks and Butterworth filters to deal with the LFIG fixed pattern and noise from the cladding. The experimental results based on a phantom consisting of intralipid suspension pumped at varying speed showed that this system could detect speed changes and that in the case of multiple speckles per fibre the Nyquist frequency criterion need not be applied since the speckle may be transferred through the fibres to some extent. In contrast to the previously reported ELASCA results, this system can both give a map of the observed area and the temporal change in flow. An additional benefit is the small size of the LFIG, which is compatible with current endoscopic instrument channels and may allow additional surgical applications.
Novel Instruments and Methods
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Laser interference fringe tomography: a novel 3D imaging technique for pathology
Farnoud Kazemzadeh, Thomas M. Haylock, Lev M. Chifman, et al.
Laser interference fringe tomography (LIFT) is within the class of optical imaging devices designed for in vivo and ex vivo medical imaging applications. LIFT is a very simple and cost-effective three-dimensional imaging device with performance rivaling some of the leading three-dimensional imaging devices used for histology. Like optical coherence tomography (OCT), it measures the reflectivity as a function of depth within a sample and is capable of producing three-dimensional images from optically scattering media. LIFT has the potential capability to produce high spectral resolution, full-color images. The optical design of LIFT along with the planned iterations for improvements and miniaturization are presented and discussed in addition to the theoretical concepts and preliminary imaging results of the device.
Model-based design of optical diagnostic instrumentation
Chih-Wen Kan, Kort Travis, James Salazar, et al.
Biophotonics methods are attractive since they allow for the non-invasive diagnosis of cancer. Experiments were carried out to investigate the feasibility of detecting early pre-cancer using optical spectroscopy. However, optimization of instrumentation design parameters remains challenging because of the lack of metrics to evaluate the performance of certain design parameters. For example, although using angled-collection geometry has been shown to collect depth sensitive spatial origins, the performance of devices with angled-collection geometries are not well characterized or quantified. In this study, we use a polarization-sensitive Monte Carlo simulation (Pol-MC) to aid in the design of instrumentation for the early detection of epithelial cancer. The tissue is modeled in layers: (0) air outside the tissue, (1) epithelial layer, (2) thin pre-cancer layer of cells, (3) thin basement membrane, implemented as a thin transparent layer, and (4) the stroma, implemented as a thick layer of scattering material. We propose a new metric, Target Signal Ratio (TSR), to evaluate the proportion of signal that is scattered from a target layer, which is the basal/pre-cancer layer. This study is a proof-of-concept for the application of computational techniques to facilitate instrument design.
A dark-field scanning spectroscopy platform for localized scatter and fluorescence imaging of tissue
Tissue ultra-structure and molecular composition provide native contrast mechanisms for discriminating across pathologically distinct tissue-types. Multi-modality optical probe designs combined with spatially confined sampling techniques have been shown to be sensitive to this type of contrast but their extension to imaging has only been realized recently. A modular scanning spectroscopy platform has been developed to allow imaging localized morphology and molecular contrast measures in breast cancer surgical specimens. A custom designed dark-field telecentric scanning spectroscopy system forms the core of this imaging platform. The system allows imaging localized elastic scatter and fluorescence measures over fields of up to 15 mm x 15 mm at 100 microns resolution in tissue. Results from intralipid and blood phantom measurements demonstrate the ability of the system to quantify localized scatter parameters despite significant changes in local absorption. A co-registered fluorescence spectroscopy mode is also demonstrated in a protophorphyrin-IX phantom.
Clinical and Pre-Clinical Studies
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High-speed simulation of skin spectral reflectance based on an optical path-length matrix method and its application
Izumi Fujiwara, Satoshi Yamamoto, Midori Yamauchi, et al.
In this paper, we propose optical path-length matrix method for high-speed simulation of photon migration in human skin. The optical path-length matrix is defined as the probability density distribution of optical pathlength in the skin. Generally, Monte Carlo simulation is used to simulate a skin reflectance, since it can simulate the reflectance accurately. However, it requires a huge computation time, thus this is not easily applicable in practical imaging system with large number of pixels. On the other hand, the proposed optical path-length matrix method achieves the simulation in shorter time. The skin model was assumed to be two-layered media of the epidermal and dermal layers. For obtaining the path-length matrix, photon migration in the model without any absorption was simulated only once by Monte Carlo simulation for each wavelength, and the probabilistic density histograms of the optical path-length at each layer were acquired and stored in the optical path-length matrix. Skin spectral reflectance for arbitrary absorption can be calculated easily by accumulating all combination of an element in the above pre-recomputed path-length matrix and absorption coefficient based on the Beer-Lambert law. Our proposed method was compared with the conventional Monte Carlo simulation. Computational time of the proposed method was approximately two minutes; while that of the conventional method was 15 hours. In addition, error margin of the proposed method was approximately less than 1.6%. This method would applied to skin spectral image analysis for skin chromophore quantification.
Retention of indocyanine green as a potential marker for optical detection of blood brain barrier disruption
A. Ergin, S. Joshi, M. Wang, et al.
Preliminary studies have shown that there is great variability in the degree of disruption of blood-brain barrier (BBBD) after the intraarterial injection of mannitol in rabbit models. The disruption of blood-brain barrier (BBB) is affected by a number of factors, and the variations could have a profound impact on regional delivery of chemotherapeutics. Optically measured brain tissue concentrations of indocyanine green (ICG) and Evan's blue (EB) enable the quantification of BBBD after intraarterial administration of mannitol. Using the optical pharmacokinetics technique, a variation of diffuse reflectance spectroscopy, we are able to track in vivo brain tissue concentrations of ICG and EB in rabbits, before and after barrier disruption. This study shows the feasibility of optical monitoring of BBBD, a method that can help improve intraarterial delivery of chemotherapeutic drugs.
Near-infrared scattering imaging of depolarization waves in a rat hypoxic brain model and its application to assessment of brain tissue reversibility
Satoko Kawauchi, Shunichi Sato, Yoichi Uozumi, et al.
Light scattering signal, which is sensitive to cellular/subcellular structural integrity, is a potential indicator of tissue viability in brain, because metabolic energy is used in part to maintain the structure of the cells. We performed near-infrared scattering imaging of rat brain during hypoxia followed by reoxygenation to examine spatiotemporal scattering change due to anoxic depolarization and its correlation with tissue reversibility. For imaging change in light scattering of rat brain, NIR light was transmitted from a halogen lamp with a bandpass filter (800 ± 70 nm) and it was incident onto the entire cortex through the intact skull at an oblique angle; diffusely reflected light from the brain was imaged with a charge-coupled device. About 2 min after starting hypoxia, scattering waves were generated focally in the bilateral outermost regions in the cortex and spread toward the midline at a rate of ~6 mm/min. When reoxygenation was started before the leading edges of scattering waves reached the midline of the brain, the scattering waves disappeared gradually and the tissue was saved. Reoxygenation when scattering wave reached the midline did not save the brain. These results suggest that the coverage of the scattering waves determine the brain tissue reversibility after hypoxia.
Optical assessment of pathology in surgically resected tissues
Ashley Laughney, Venkataramanan Krishnaswamy, Wendy A. Wells, et al.
Multi-spectral spatially modulated light is used to guide localized spectroscopy of surgically resected tissues for cancer involvement. Modulated imaging rapidly quantifies near-infrared optical parameters with sub-millimeter resolution over the entire field for identification of residual disease in resected tissues. Suspicious lesions are further evaluated using a spectroscopy platform designed to image thick tissue samples at a spatial resolution sensitive to the diagnostic gold standard, pathology. MI employs a spatial frequency domain sampling and model-based analysis of the spatial modulation transfer function to interpret a tissue's absorption and scattering parameters at depth. The spectroscopy platform employs a scanning-beam, telecentric dark-field illumination and confocal detection to image fields up to 1cm2 with a broadband source (480:750nm). The sampling spot size (100μm lateral resolution) confines the volume of tissue probed to within a few transport pathlengths so that multiple-scattering effects are minimized and simple empirical models may be used to analyze spectra. Localized spectroscopy of Intralipid and hemoglobin phantoms demonstrate insensitivity of recovered scattering parameters to changes in absorption, but a non-linear dependence of scattering power on Intralipid concentration is observed due to the phase sensitivity of the measurement system. Both systems were validated independently in phantom and murine studies. Ongoing work focuses on assessing the combined utility of these systems to identify cancer involvement in vitro, particularly in the margins of resected breast tumors.
Variations in the optical scattering properties of skin in murine animal models
Katherine Calabro, Allison Curtis, Jean-Rene Galarneau, et al.
In the work presented here, the optical scattering properties of mouse skin are investigated in depth with the use of Elastic Scattering Spectroscopy (ESS). In particular, sources of variation that lead to experimental error are identified and examined. The thickness of the dermal layer of the skin is determined to be the primary source of variation due to its high collagen content. Specifically, gender differences in skin thickness are found to cause increases in the reflectance and scattering coefficient value by a factor of two in males as opposed to females. Changes in the hair growth cycle are found to influence scattering strength not only due to changes in skin thickness, but also from melanin collection in hair follicles. Because direct and/or indirect measurement of mouse skin is common in the development of novel biomedical optics techniques (optical biopsy, molecular imaging, in vivo monitoring of glucose/blood oxygenation, etc.), the purpose of this work is to identify sources of experimental variation that may arise in these studies such that care can be taken to avoid or compensate for their affects.
In Vitro Cell Studies
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Rapid analysis of white blood cells with diffraction imaging flow cytometry
Xin-Hua Hu, Jun Q. Lu, Yuanming Feng
From a foundation of cell optics research program we have recently developed a diffraction imaging flow cytometer method which can be used to rapidly acquire and analyze diffraction image data from single flowing cells. Modeling and experimental investigation of light scattering by white blood cells (WBC) have been conducted which suggest a strong correlation between the fringe patterns related to the texture of the diffraction images and 3D morphological features of the cells. In particular, we present results of calculated and measured diffraction images obtained with three cell lines derived from leukemia patients to demonstrate that texture features of the diffraction images extracted with a gray-scale co-occurrence-matrix algorithm can be used for rapid cell classification.
Time-lapsed integrated Raman and angular-scattering microscopy of single cells
Integrated Raman- and Angular-scatteringMicroscopy (IRAM) combines two light scattering techniques to make chemical and morphological measurements of intact, single cells without the use of external labeling. IRAM has previously demonstrated its ability to differentiate between activated and non-activated CD8+ T cells based on both chemical and morphological differences. Activated cells showed an increase in protein and lipid content as well as an increase in the size and number of 0.5-1.0 μm diameter scatterers (likely lysosomes). Recent improvements to the IRAM system enable studies over an extended period of time. The applications of IRAM to chemical and structural changes of single cells during biological processes and treatments will be discussed.
Intrinsic optical signal imaging of glucose-stimulated physiological responses in the insulin secreting INS-1 β-cell line
Yi-Chao Li, Wan-Xing Cui, Xu-Jing Wang, et al.
Intrinsic optical signal (IOS) imaging has been established for noninvasive monitoring of stimulus-evoked physiological responses in the retina and other neural tissues. Recently, we extended the IOS imaging technology for functional evaluation of insulin secreting INS-1 cells. INS-1 cells provide a popular model for investigating β-cell dysfunction and diabetes. Our experiments indicate that IOS imaging allows simultaneous monitoring of glucose-stimulated physiological responses in multiple cells with high spatial (sub-cellular) and temporal (sub-second) resolution. Rapid image sequences reveal transient optical responses that have time courses comparable to glucose-evoked β-cell electrical activities.
Low-coherence Light Scattering
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Optical coherence tomography speckle decorrelation for detecting cell death
Golnaz Farhat, Adrian Mariampillai, Victor X. D. Yang, et al.
We present a dynamic light scattering technique applied to optical coherence tomography (OCT) for detecting changes in intracellular motion caused by cellular reorganization during apoptosis. We have validated our method by measuring Brownian motion in microsphere suspensions and comparing the measured values to those derived based on particle diffusion calculated using the Einstein-Stokes equation. Autocorrelations of OCT signal intensities acquired from acute myeloid leukemia cells as a function of treatment time demonstrated a significant drop in the decorrelation time after 24 hours of cisplatin treatment. This corresponded with nuclear fragmentation and irregular cell shape observed in histological sections. A similar analysis conducted with multicellular tumor spheroids indicated a shorter decorrelation time in the spheroid core relative to its edges. The spheroid core corresponded to a region exhibiting signs of cell death in histological sections and increased backscatter intensity in OCT images.
Determining size, shape, and orientation of non-spherical scatterers using the fiber optic interferometric two-dimensional scattering (FITS) system
Michael Giacomelli, Yizheng Zhu, John Lee, et al.
Angle-resolved scattering measurements have shown promise as a method of detecting neoplasia and analyzing cellular structure. Recently we have developed new systems for interferometric measurement of two-dimensional, depth resolved scattered fields with excellent depth resolution and polarization sensitivity. We present inverse analysis of oriented ensembles of micro-spheroidal phantoms and cells showing sub-wavelength accuracy in size and shape determination, and additionally precise estimates of scatterer orientation. Finally we show that inverse fits provided are essentially free of multiple solutions over a wide range of possible scatterer sizes and shapes.
Fourier-domain low-coherence interferometry for detection of early colorectal cancer development in the AOM rat model
Francisco E. Robles, Yizheng Zhu, Jin Lee, et al.
We present Fourier domain low coherence interferometry (fLCI) applied to the detection of preneoplastic changes in the colon using the ex-vivo azoxymethane (AOM) rat carcinogenesis model. fLCI measures depth resolved spectral oscillations, also known as local oscillations, resulting from coherent fields induced by the scattering of cell nuclei. The depth resolution of fLCI permits nuclear morphology measurements within thick tissues, making the technique sensitive to the earliest stages of precancerous development. To achieve depth resolved spectroscopic analysis, we use the dual window method, which obtains simultaneously high spectral and depth resolution and yields access to the local oscillations. The results show highly statistically significant differences between the AOM-treated and control group samples. Further, the results suggest that fLCI may be used to detect the field effect of carcinogenesis, in addition to identifying specific areas where more advanced neoplastic development has occurred.
Cell death monitoring using quantitative optical coherence tomography methods
Golnaz Farhat, Victor X. D. Yang, Michael C. Kolios, et al.
Cell death is characterized by a series of predictable morphological changes, which modify the light scattering properties of cells. We present a multi-parametric approach to detecting changes in subcellular morphology related to cell death using optical coherence tomography (OCT). Optical coherence tomography data were acquired from acute myeloid leukemia (AML) cells undergoing apoptosis over a period of 48 hours. Integrated backscatter (IB) and spectral slope (SS) were computed from OCT backscatter spectra and statistical parameters were extracted from a generalized gamma (GG) distribution fit to OCT signal intensity histograms. The IB increased by 2-fold over 48 hours with significant increases observed as early as 4 hours. The SS increased in steepness by 2.5-fold with significant changes at 12 hours, while the GG parameters were sensitive to apoptotic changes at 24 to 48 hours. Histology slides indicated nuclear condensation and fragmentation at 24 hours, suggesting the late scattering changes could be related to nuclear structure. A second series of measurements from AML cells treated with cisplatin, colchicine or ionizing radiation suggested that the GG parameters could potentially differentiate between modes of cell death. Distinct cellular morphology was observed in histology slides obtained from cells treated under each condition.
Lidar-like equation model for optical coherence tomography signal solution
Marcello M. Amaral, Marcus P. Raele, Eduardo Landulfo, et al.
The objective of this work was to develop a LIDAR-like equation model to analyze the measured Optical coherence tomography (OCT) signal and determine the total extinction coefficient of a scattering sample. OCT is an interferometric technique that explore sample backscattering feature to acquire in depth cross-section images using a low coherence light source. Although, almost of the OCT applications are intended to generate images for diagnostic, similar to histological images, but the backscattering signal carries much more information. The backscattering problem is similar to those found on LIDAR (Light Detection And Ranging) problem, this similar situation indicate a path that should be followed to solve the OCT problem. To determine the total extinction coefficient three inversion methods was used: the slope, boundary point and optical depth methods solutions. These algorithms were used to analyze the OCT signal of a single and double layer dentist resin polymer. The total extinction coefficient variations along the optical path were obtained in order to evaluate the potential of this technique to differentiate structures with different optical properties. The sample optical characteristics extracted from OCT signal can be use as an additional quantitative method to help clinical diagnoses when applied on biological tissues among others.
Poster Session
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Images of depolarization power and retardance to study stages of dysplasia in human cervical tissues
Meghdoot Mozumder, Jaidip Jagtap, Prashant Shukla, et al.
Optical techniques, especially the ones related to light scattering, has been seen to capture morphological changes, such as increase in size and density of nuclei in cells. Mueller imaging of the epithelium and basal layer based on polarized elastic scattering in human cervical tissue sections has been used to identify the dysplastic conditions of the human cervix. The effect of dysplasia strongly manifests in the depolarization power and retardance, which differs significantly in normal and dysplastic tissues sections. Principal Component Analysis (PCA) of the depolarization power images derived from polar decomposition of Mueller matrices of 36 patients clearly identifies the tissue region responsible for clear discrimination between the diseased and normal tissues. Significantly, the principal components are found to be sensitive in discriminating normal cervical tissue and the two different stages of dysplastic conditions grade I (GD1) and grade II (GD2) and provide cut-off depolarization values for each of these stages.Though the depolarization values of GD1 are quite random as compared to normal and GD2 states, PCA is able to effectively separate it out by capturing subtle changes in the depolarization values.It is worth noting that in the GD2 stage concentration of cells is high in the epithelial region near the basal layer than the epithelium layer near the surface though this difference between these two regions is not as significant as in GD1. Interestingly, this phenomenon is well reflected in the depolarization values, which PCA uses effectively to segment GD1 and GD2 into different clusters. Retardance values show little variation along the stroma. However, covariance matrix images of dysplastic and normal are able to capture depletion of retardance below the basal layer due to progressive disruption of collagen network in dysplasia.
Image reconstruction using measurements in volume speckle fields formed by different wavelengths
Nikolay V. Petrov, Mikhail V. Volkov, Andrei A. Gorodetsky, et al.
An iterative wavefront retrieval method based on intensity measurements formed by several wavelengths is investigated in the present contribution. This multiwavelength technique is extended to use the intensity distributions recorded in various planes of the volume speckle field. The ability to retrieve the wavefront using speckle patterns is demonstrated in experiment. Two different experimental techniques have been used. The first proposed method allows one to record three different intensity distributions corresponding to the three CCD RGB channels at single exposure. This gives the advantage in the analysis of fast processes, e.g. phase microscopy of moving biological cell-like objects investigation. The second technique involves using a large number of wavelengths of supercontinuum radiation formed by photonic-crystal fiber. This approach provides faster and more accurate convergence of the proposed method, has simple and rugged recording scheme with fiber optic elements.