Deconvolution techniques for experimental optical imaging in medicine

Optical imaging in biomedicine is essentially determined by the light absorption and scattering interaction of microscopic and macroscopic constituents in the medium. In the near infrared range (NIR) the scattering event, with a coefficient approximately two orders of magnitude greater than absorption, plays a dominant role. The size of the detected scattered light volume is apparently larger than the original one. The purpose of this paper is to quantify these effects for fluorescent light. To this effect, the optical properties of dental composite phantoms were measured and the blurring of a point source – referred to as generalized point spread function (PSF) – was calculated by Monte Carlo simulations. We demonstrate the use of this method for scaling the concentration of fluorophores and for reconstruction of the true shape of fluorescence regions below the scattering layer of the phantom by deconvolution. The combination of these two mathematical techniques – construction of the PSF by Monte Carlo simulation (MCS) and deconvolution of the stray light picture – is of benefit for the interpretation of fluorescence and scattering images used for various medical applications.     

Designing microarray phantoms for hyperspectral imaging validation

The design and fabrication of custom-tailored microarrays for use as phantoms in the characterization of hyperspectral imaging systems is described. Corresponding analysis methods for biologically relevant samples are also discussed. An image-based phantom design was used to program a microarrayer robot to print prescribed mixtures of dyes onto microscope slides. The resulting arrays were imaged by a hyperspectral imaging microscope. The shape of the spots results in significant scattering signals, which can be used to test image analysis algorithms. Separation of the scattering signals allowed elucidation of individual dye spectra. In addition, spectral fitting of the absorbance spectra of complex dye mixtures was performed in order to determine local dye concentrations. Such microarray phantoms provide a robust testing platform for comparisons of hyperspectral imaging acquisition and analysis methods.     

High-speed hyperspectral Raman imaging for label-free compositional microanalysis

We present high-speed hyperspectral Raman imaging with integrated active-illumination for label-free compositional microanalysis. We show that high-quality Raman spectra can be acquired from as many as ~1,000 spots/sec semi-randomly distributed among a ~100x100 μm² area without mechanical scanning. We demonstrate rapid data acquisition from three types of samples: 1) uniform, strong Raman scatterers, e.g., silicon substrates; 2) non-uniform, medium-strength Raman scatterers, e.g., polymer microparticles; and, 3) non-uniform, relatively weak Raman scatterers, e.g., bacterial spores. We compare the system performance to that of point-scan with an electron-multiplied CCD camera, as implemented in some commercial systems. The results suggest that our system not only provides significant imaging speed advantage for various types of samples, but also permits substantially longer integration time per spot, leading to superior signal-to-noise ratio data. Our system enables the rapid collection of high quality Raman spectra for reliable and robust compositional microanalysis that are potentially transformative in applications such as semiconductor material and device, polymer blend and biomedicine.OCIS codes: (180.5655) Raman microscopy, (170.1530) Cell analysis, (170.0110) Imaging systems     

Developing digital tissue phantoms for hyperspectral imaging of ischemic wounds

Hyperspectral imaging has the potential to achieve high spatial resolution and high functional sensitivity for non-invasive assessment of tissue oxygenation. However, clinical acceptance of hyperspectral imaging in ischemic wound assessment is hampered by its poor reproducibility, low accuracy, and misinterpreted biology. These limitations are partially caused by the lack of a traceable calibration standard. We proposed a digital tissue phantom (DTP) platform for quantitative calibration and performance evaluation of spectral wound imaging devices. The technical feasibility of such a DTP platform was demonstrated by both in vitro and in vivo experiments. The in vitro DTPs were developed based on a liquid blood phantom model. The in vivo DTPs were developed based on a porcine ischemic skin flap model. The DTPs were projected by a Hyperspectral Image Projector (HIP) with high fidelity. A wide-gap 2nd derivative oxygenation algorithm was developed to reconstruct tissue functional parameters from hyperspectral measurements. In this study, we have demonstrated not only the technical feasibility of using DTPs for quantitative calibration, evaluation, and optimization of spectral imaging devices but also its potential for ischemic wound assessment in clinical practice.     

Algorithm for mapping cutaneous tissue oxygen concentration using hyperspectral imaging

The measurement of tissue oxygenation plays an important role in the diagnosis and therapeutic assessment of a large variety of diseases. Many different methods have been developed and are currently applied in clinical practice for the measurement of tissue oxygenation. Unfortunately, each of these methods has its own limitations. In this paper we proposed the use of hyperspectral imaging as new method for the assessment of the tissue oxygenation level. To extract this information from hyperspectral images a new algorithm for mapping cutaneous tissue oxygen concentration was developed. This algorithm takes into account and solves some problems related to setting and calculation of some parameters derived from hyperspectral images. The algorithm was tested with good results on synthetic images and then validated on the fingers of a hand with different blood irrigation states. The results obtained have proved the ability of hyperspectral imaging together with the developed algorithm to map the oxy- and deoxyhemoglobin distribution on the analyzed fingers. These are only preliminary results and other studies should be done before this approach to be used in the clinical setting for the diagnosis and monitoring of various diseases.OCIS codes: (000.2170) Equipment and techniques, (110.4234) Multispectral and hyperspectral imaging, (170.6935) Tissue characterization, (300.6550) Spectroscopy, visible     

Spectral-profile-based algorithm for hemoglobin oxygen saturation determination from diffuse reflectance spectra

Variations of hemoglobin (Hb) oxygenation in tissue provide important indications concerning the physiological conditions of tissue, and the data related to these variations are of intense interest in medical research as well as in clinical care. In this study, we derived a new algorithm to estimate Hb oxygenation from diffuse reflectance spectra. The algorithm was developed based on the unique spectral profile differences between the extinction coefficient spectra of oxy-Hb and deoxy-Hb within the visible wavelength region. Using differential wavelet transformation, these differences were quantified using the locations of certain spectral features, and, then, they were related to the oxygenation saturation level of Hb. The applicability of the algorithm was evaluated using a set of diffuse reflectance spectra produced by a Monte Carlo simulation model of photon migration and by tissue phantoms experimentally. The algorithm was further applied to the diffuse reflectance spectra acquired from in vivo experiments to demonstrate its clinical utility. The validation and evaluation results concluded that the algorithm is applicable to various tissue types (i.e., scattering properties) and can be easily used in conjunction with a diverse range of probe geometries for real-time monitoring of Hb oxygenation.     

An advanced method for mineral mapping applicable to hyperspectral images: the composite MSAM

We developed a new method for mineral mapping based on the continuum-removal Modified Spectral Angle Mapper (MSAM) method to distinguish minerals, such as calcite and chlorite, which have diagnostic spectral features in the visible near infrared (VNIR) and shortwave infrared (SWIR) regions. This method was applied to the reflectance data obtained by HyMap, a hyperspectral airborne sensor with 126 spectral bands (0.44–2.48 μm) in Cuprite, Nevada, USA. Reflectance spectra in the VNIR and SWIR regions are characterized by a convex background (continuum). Continuum-removal processing results in emphasis of the shape and location of absorption peaks in reflectance spectra, and thus in recognition of the chemical species which are causing the absorption spectrum. On the other hand, the broad features in continuum spectra contain essential information for mineral classification as well. Therefore, we produced mineral index maps from both the continuum and continuum-removed images based on the MSAM method and then combined them to generate a mineral distribution map. This composite MSAM method was applied to remotely sensed hyperspectral data for the first time. It was confirmed that our mineral index maps were consistent with the existing mineral maps. We can conclude that the composite MSAM method has a great advantage over our previously developed method in discrimination between calcite and chlorite, in particular.     

On-line automatic slice positioning for brain MR imaging

In clinical brain MR imaging protocols, the technician collects a quick localizer and manually positions the subsequent scans using the localizer as a guide. We present a method for automatic slice positioning using a rapidly acquired 3D localizer. The localizer is automatically aligned to a statistical atlas representing 40 healthy subjects. The atlas contains the probability of a given tissue type occurring at a given location in atlas space and the conditional probability distribution of the multi-spectral MRI intensity values for a given tissue class. Accurate rigid alignment of each subject to an atlas ensures that all patients' scans are acquired in a consistent manner. A further benefit is that slices are positioned consistently over time, so that scans of patients returning for follow-up imaging can be compared side-by-side to accurately monitor the progression of illness. The procedure also helps ensure that left/right asymmetries reflect true anatomy rather than being the result of oblique slice positioning relative to the underlying anatomy. The use of an atlas-based procedure eliminates the need to refer to a database of previously scanned images of the same patient and ensures corresponding alignment across scanners and sites, without requiring fiducial markers. Since the registration method is probabilistic, the registration error tends to increase smoothly in the presence of increasing noise and unusual anatomy or pathology rather than failing catastrophically. Translations and rotations relative to the atlas can be set so that planning can be done in anatomical space, rather than scanner coordinates, and stored as part of the protocol allowing standardization of slice orientations.     

Multispectral near-IR reflectance and transillumination imaging of teeth

NIR imaging methods do not require ionizing radiation and have great potential for detecting caries lesions (tooth decay) on high-risk proximal and occlusal tooth surfaces and at the earliest stages of development. Previous in vitro and in vivo studies at 1300-nm demonstrated that high contrast reflectance and transillumination images could be acquired of caries lesions on tooth proximal and occlusal surfaces where most new decay is found. Water absorption varies markedly between 1200 and 1600-nm and the scattering properties of enamel and the underlying dentin have not been characterized in this region. Hyperspectral reflectance studies show lower reflectivity from sound enamel and dentin at NIR wavelengths with higher water absorption. The purpose of this imaging study was to determine which NIR wavelengths between 1200 and 1600-nm provide the highest contrast of demineralization or caries lesions for each of the different modes of NIR imaging, including transillumination of proximal and occlusal surfaces along with cross polarization reflectance measurements. A tungsten halogen lamp with several spectral filters and a Ge-enhanced CMOS focal plane array (FPA) sensitive from 400 to 1600-nm were used to acquire the images of caries lesions on extracted teeth. Artificial interproximal lesions were created on twelve tooth sections of 5 & 6-mm thickness that were used for transillumination imaging. Fifty-four extracted teeth with suspected occlusal lesions were also examined in both occlusal transillumination and reflectance imaging modes. Cavity preparations were also cut into whole teeth and filled with composite and used to compare the contrast between composite and enamel at NIR wavelengths. NIR wavelengths longer than 1400-nm are likely to have better performance for the transillumination of occlusal caries lesions while 1300-nm appears best for the transillumination of proximal surfaces. Loss of mobile water in enamel markedly reduced the transparency of the enamel at all NIR wavelengths. Significantly higher contrast was attained for reflectance measurements at wavelengths that have higher water absorption, namely 1460-nm. Wavelengths with higher water absorption also provided higher contrast of composite restorations.     

Combination of computer-aided detection algorithms for automatic lung nodule identification

Purpose

The aim of this work is to evaluate the potential of combining different computer-aided detection (CADe) methods to increase the actual support for radiologists of automated systems in the identification of pulmonary nodules in CT scans.

Methods

The outputs of three different CADe systems developed by researchers of the Italian MAGIC-5 collaboration were combined. The systems are: the CAMCADe (based on a Channeler-Ant-Model which segments vessel tree and nodule candidates and a neural classifier), the RGVPCADe (a Region-Growing- Volume-Plateau algorithm detects nodule candidates and a neural network reduces false positives); the VBNACADe (two dedicated procedures, based respectively on a 3D dot-enhancement algorithm and on intersections of pleura surface normals, identifies internal and juxtapleural nodules, and a Voxel-Based-Neural-Approach reduces false positives. A dedicated OsiriX plugin implemented with the Cocoa environments of MacOSX allows annotating nodules and visualizing singles and combined CADe findings.

Results

The combined CADe has been tested on thin slice (lower than 2?mm) CTs of the LIDC public research database and the results have been compared with those obtained by the single systems. The FROC (Free Receiver Operating Characteristic) curves show better results than the best of the single approaches.

Conclusions

Has been demonstrated that the combination of different approaches offers better results than each single CADe system. A clinical validation of the combined CADe as second reader is being addressed by means of the dedicated OsiriX plugin.     

Comparison of the reflectance method (Reflotron reflectance photometer) with the absorbance method (automatic analysers) for the determination of cholesterol

The European Atherosclerosis Society (1) and the Expert Panel of the US National Cholesterol Education Program (2) have issued detailed guide values for recognition and management of hyperlipidaemia in adults. In these guidelines, the diagnosis of dyslipidaemia based on the measurements of total cholesterol, triacylglycerols, HDL and LDL cholesterol plays an important role. A prerequisite for the desired success of interventive measures is the reliability of the analytical data. The aim of this study was to investigate the precision and accuracy of Reflotron Cholesterol, a method based on the dry chemistry principle. Accuracy was assessed by establishing the correlation with the standardized automated methods used in routine lipid diagnosis. In addition, it was also examined whether the Reflotron Cholesterol results in plasma and blood are comparable. The Reflotron cholesterol (sample: blood) showed a good correlation with the CHOD/PAP method on a Hitachi 737 instrument (sample: plasma). The median value of the differences of the test results was -0.4%. Similarly, the method comparison of Reflotron Cholesterol (sample: blood) versus CHOD/PAP method on a SMAC instrument (sample: plasma) showed that Reflotron produces slightly (1.8%) higher results. The Reflotron Cholesterol values obtained from blood samples were slightly lower than those from plasma samples (median value of the differences: -2.2%). The results suggest that for routine purposes Reflotron Cholesterol provides results which are in good agreement with those obtained by standardized wet chemistry methods.     

Near-IR fluorescence and reflectance confocal microscopy for imaging of quantum dots in mammalian skin

Understanding the skin penetration of nanoparticles (NPs) is an important concern due to the increasing presence of NPs in consumer products, including cosmetics. Technical challenges have slowed progress in evaluating skin barrier and NP factors that contribute to skin penetration risk. To limit sampling error and other problems associated with histological processing, many researchers are implementing whole tissue confocal or multiphoton microscopies. This work introduces a fluorescence and reflectance confocal microscopy system that utilizes near-IR excitation and emission to detect near-IR lead sulfide quantum dots (QDs) through ex vivo human epidermis. We provide a detailed prediction and experimental analysis of QD detection sensitivity and demonstrate detection of QD skin penetration in a barrier disrupted model. The unique properties of near-IR lead-based QDs will enable future studies that examine the impact of further barrier-disrupting agents on skin penetration of QDs and elucidate mechanistic insight into QD tissue interactions at the cellular level.     

A new method for automatic identification of coronary arteries in standard biplane angiograms

A new computerized method for analyzing biplane coronary arteriograms was proposed. The method extracted the vessels in an image, detected branching points, and identified vessel segments according to their anatomical names. The method was applied to a pair of orthogonal projection images acquired in the right anterior oblique and left anterior oblique views. It recognized the corresponding vessel segments in the images and labeled them correctly by their anatomical names. For diameter measurement, a combined first and second derivative filter process was applied to the images along the skeleton lines of the extracted vessel segments. We also designed a method of representation in which vessel diameters measured in the two projection images could be displayed in a single image.     

Fluorescence and reflectance spectral imaging system for a murine mammary window chamber model

A spectral imaging system was developed to study the development of breast cancer xenografts in a murine mammary window chamber model. The instrument is configured to work with either a laser to excite fluorescence or a broadband light source for diffuse reflectance imaging. Two applications were demonstrated. First, spectral imaging of fluorescence signals was demonstrated with a GFP-breast cancer tumor and fluorescein injection. Second, based on the principles of broadband reflectance spectroscopy, the instrument was used to monitor dynamic changes of tissue absorbance to yield tissue oxygenation maps at different time points during tumor progression.OCIS codes: (110.4234) Multispectral and hyperspectral imaging, (170.0110) Imaging systems, (170.1470) Blood or tissue constituent monitoring, (170.6280) Spectroscopy, fluorescence and luminescence, (170.2655) Functional monitoring and imaging     

Label-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging

In vivo two-photon imaging combined with targeted fluorescent indicators is currently extensively used for attaining critical insights into brain functionality and structural plasticity. Additional information might be gained from back-scattered photons from the near-infrared (NIR) laser without introducing any exogenous labelling. Here, we describe a complimentary and versatile approach that, by collecting the reflected NIR light, provides structural details on axons and blood vessels in the brain, both in fixed samples and in live animals under a cranial window. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from a Thy1-GFPm mouse, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Interestingly, NIR reflectance microscopy allowed the label-free detection of axonal elongations over the superficial layers of mouse cortex under a cranial window in vivo. Finally, blood flow can be measured in live preparations, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated.OCIS codes: (170.0110) Imaging systems, (170.3880) Medical and biological imaging     

An average enumeration method of hyperspectral imaging data for quantitative evaluation of medical device surface contamination

We propose a quantification method called Mapped Average Principal component analysis Score (MAPS) to enumerate the contamination coverage on common medical device surfaces. The method was adapted from conventional Principal Component Analysis (PCA) on non-overlapped regions of a full frame hyperspectral image to resolve the percentage of contamination from the substrate. The concept was proven by using a controlled contamination sample with artificial test soil and color simulating organic mixture, and was further validated using a bacterial system including biofilm on stainless steel surface. We also validate the results of MAPS with other statistical spectral analysis including Spectral Angle Mapper (SAM). The proposed method provides an alternative quantification method for hyperspectral imaging data, which can be easily implemented by basic PCA analysis.OCIS codes: (110.4234) Multispectral and hyperspectral imaging, (170.3880) Medical and biological imaging     

An automatic identification and removal method for eye-blink artifacts in event-related magnetoencephalographic measurements

While measuring event-related magnetoencephalographic (MEG) signals using visual stimuli, eye blinks were inevitable and generated large magnetic artifacts. Since trials containing eye blinks were excluded from the analyses, the signal-to-noise ratio of the event-related signals was decreased. In this study, we propose a method to identify the eye blink magnetic artifacts and remove them automatically using independent component analysis preprocessed by principal component analysis. The method evaluates the spatiotemporal similarity between independent components and both MEG and electro-oculogram data based on a newly devised cost function. Testing of the method on event-related MEG signals measured by a 306-channel whole-head system in a visual perception task led to the successful identification and removal of eye-blink artifacts in all trials containing eye blinks from all the seven subjects.     

In vivo hyperspectral imaging of microvessel response to trastuzumab treatment in breast cancer xenografts

HER2-amplified (HER2 + ) breast cancers are treated with the anti-HER2 monoclonal antibody trastuzumab. Although trastuzumab reduces production of the angiogenic factor VEGF in HER2 + tumors, the acute and sustained effects of trastuzumab on the tumor vasculature are not understood fully, particularly in trastuzumab-resistant tumors. We used mouse models of trastuzumab sensitive and trastuzumab-resistant HER2 + breast cancers to measure dynamic changes in tumor microvessel density and hemoglobin oxygenation (sO₂) in vivo using quantitative hyperspectral imaging at 2, 5, 9, and 14 days after antibody treatment. Further analysis quantified the distribution of microvessels into low and high oxygenation groups, and monitored changes in these distributions with trastuzumab treatment. Gold standard immunohistochemistry was performed to validate complementary markers of tumor cell and vascular response to treatment. Trastuzumab treatment in both responsive and resistant tumors resulted in decreased sO₂ 5 days after initial treatment when compared to IgG-treated controls (p<0.05). Importantly, responsive tumors showed significantly higher vessel density and significantly lower sO₂ than all other groups at 5 days post-treatment (p<0.05). Distribution analysis of vessel sO₂ showed a significant (p<0.05) shift of highly oxygenated vessels towards lower oxygenation over the time-course in both trastuzumab-treated responsive and resistant tumors. This study suggests that longitudinal hyperspectral imaging of microvessel sO₂ and density could distinguish trastuzumab-responsive from trastuzumab-resistant tumors, a finding that could be exploited in the post-neoadjuvant setting to guide post-surgical treatment decisions.OCIS codes: (110.4234) Multispectral and hyperspectral imaging, (170.3880) Medical and biological imaging, (170.1470) Blood or tissue constituent monitoring     

脑功能区自动提取法对99mTc-ECD脑显像63个脑区的灰度分析

目的：验证脑解剖功能区的自动提取法在99mTc-ECD脑显像中提取位置准确性，并初步探讨63个功能区药物相对摄取分布。方法：采用图像配准和图像形变方法，将99mTc-ECD脑SPECT图像标化到标准的Talairach坐标空间，按各脑部功能区对全脑中坐标点分类，计算每个脑区摄取信息即平均灰度，并计算其与全脑平均灰度比值，比较左右半球及不同脑区间摄取比值差异。结果：该方法成功地将目标图像与标准坐标空间配准，提取的脑功能区通过不同透视显示与实际目测的功能区吻合，通过对63个脑解剖区分析，人的大脑左右对99mTc-ECD摄取是不对称的，各个脑区间的分布悬殊也很大，枕叶楔前叶、舌回以及顶下小叶等在脑内99mTc-ECD摄取最高，而眶回、胼胝体以及尾状核尾部摄取最低。结论：脑功能区自动提取法对99mTc-ECD脑显像对特定脑区分析显示快速，准确，重现性好的特点，不但适合多样本统计分析，也适宜于单个样本的处理，为研究特定脑感兴趣区提供了一个客观、细致的分析方法。