Showing papers in "Biomedical optics in 2003"
TL;DR: This work focuses on improving the spatial resolution and the quantitative accuracy of Diffuse Optical Tomography by using a priori anatomical information specific to unknown image, which leads to improved spatial resolution, quantitative accuracy and faster convergence than standard least squares approach.
Abstract: Diffuse optical imaging is an emerging modality that uses Near Infrared (NIR) light to reveal structural and functional information of deep biological tissue. It provides contrast mechanisms for molecular, chemical, and anatomical imaging that is not available from other imaging modalities. Diffuse Optical Tomography (DOT) deals with 3D reconstruction of optical properties of tissue given the measurements and a forward model of photon propagation. DOT has inherently low spatial resolution due to diffuse nature of photons. In this work, we focus to improve the spatial resolution and the quantitative accuracy of DOT by using a priori anatomical information specific to unknown image. Such specific a priori information can be obtained from a secondary high-resolution imaging modality such as Magnetic Resonance (MR) or X-ray. Image reconstruction is formulated within a Bayesian framework to determine the spatial distribution of the absorption coefficients of the medium. A spatially varying a priori probability density function is designed based on the segmented anatomical information. Conjugate gradient method is utilized to solve the resulting optimization problem. Proposed method is evaluated using simulation and phantom measurements collected with a novel time-resolved optical imaging system. Results demonstrate that the proposed method leads to improved spatial resolution, quantitative accuracy and faster convergence than standard least squares approach.
123 citations
TL;DR: In this article, an en face coherence gated camera equipped with adaptive optics (AO) was constructed for imaging single cells in the living human retina, where the coherence gate was realized with a free-space Michelson interferometer that employs a scientific-grade 12-bit CCD array for recording 2D retinal interferograms.
Abstract: An en face coherence gated camera equipped with adaptive optics (AO) has been constructed for imaging single cells in the living human retina. The high axial resolution of coherence gating combined with the high transverse resolution of AO provides a powerful imaging tool whose image quality can surpass either methodology performing alone. The AO system relies on a 37-actuator Xinetics mirror and a Shack-Hartmann wavefront sensor that executes up to 22 corrections per second. The coherence gate is realized with a free-space Michelson interferometer that employs a scientific-grade 12-bit CCD array for recording 2-D retinal interferograms. Images were collected of microstructures the size of single cells in the in vivo retina. Early results suggest that a coherence gated adaptive optics camera should substantially improve our ability to detect single cells in the retina over the current state-of-the-art AO retina cameras, including conventional flood illuminated and confocal scanning laser ophthalmoscopes. To our knowledge, this is the first effort to combine coherence gating and adaptive optics.
89 citations
TL;DR: In this article, the authors describe the method of operation of the holographic optical coherence imaging (OCI) on highly scattering specimens of tumor spheroids, which is a coherence-domain imaging technique that uses dynamic holography as the coherence gate.
Abstract: Holographic optical coherence imaging (OCI) has been used to acquire depth resolved images in tumor spheroids. OCI is a coherence-domain imaging technique that uses dynamic holography as the coherence gate. The technique is full-frame (en face) and background free, allowing real-time acquisition to a digital camera without motional reconstruction artifacts. We describe the method of operation of the holographic OCI on highly scattering specimens of tumor spheroids. Because of the sub-resolution structure in the sample, the holograms consist primarily of speckle fields. We present two kinds of volumetric data acquisition. One is uses fly-throughs with a stepping reference delay. Another is static holograms at a fixed reference delay with the coherence gate inside the tumor spheroids. At a fixed reference delay, the holograms consist of time-dependent speckle patterns. The method can be used to study cell motility inside tumor spheroids when metabolic or cross-linking poisons are delivered to the specimens.
83 citations
TL;DR: In this article, a Linnik interferometer illuminated by a tungsten halogen lamp was used for optical coherence tomography (OCT) using white-light interference microscopy.
Abstract: We report on ultrahigh-resolution Optical Coherence Tomography (OCT) using white-light interference microscopy. The experimental setup is based on a Linnik interferometer illuminated by a tungsten halogen lamp. Tomographic images in the en face orientation are calculated by combination of images recorded by a silicon CCD camera. Axial resolution of 0.8 μm is achieved due to the short coherence length of the source and the compensation of dispersion mismatch in the interferometer arms. Transverse resolution of 1.6 μm is obtained by using relatively high numerical aperture microscope objectives. A nearly shot-noise limited detection sensitivity of 90 dB is achieved with 4s acquisition time. Images of the Xenopus Laevis tadpole are presented.
81 citations
TL;DR: Various feature reduction methods are compared and it is found that as few as eight features can provide good classification accuracy for all major subcellular patterns in HeLa cells.
Abstract: The central goal of proteomics is to clarify the mechanism by which each protein in a given cell type carries out its function. Automated protein subcellular location determination by fluorescence microscopy can play an important role in fulfilling this goal. The subcellular location of a protein is critical to understanding its function because each subcellular compartment has a unique biochemical environment. We have previously shown that neural network classifiers using sets of numerical features computed from fluorescence microscope images were able to recognize all major subcellular location patterns with reasonable accuracy. Current classifiers are limited by under-determined classification boundaries due to the limited number of available images compared to the number of features. In this paper, we compare various feature reduction methods that can address this problem. Specifically, principal component analysis, kernel principal component analysis, nonlinear principal component analysis, independent component analysis, classification trees, fractal dimensionality reduction, stepwise discriminant analysis, and genetic algorithms are used to select feature subsets that are evaluated using support vector machine classifiers. The best results were obtained using stepwise discriminant analysis and we found that as few as eight features can provide good classification accuracy for all major subcellular patterns in HeLa cells.
76 citations
TL;DR: This review summarized the basic principles that govern the relationships between thermal exposure and thermal damage, with an emphasis on normal tissue effects, and addressed the relationship of thermal dose-to-effect vs. baseline temperature.
Abstract: In this review we have summarized the basic principles that govern the relationships between thermal exposure (Temperature and time of exposure) and thermal damage, with an emphasis on normal tissue effects. We have also attempted to identify specific thermal dose information (for safety and injury) for a variety of tissues in a variety of species. We address the use, accuracy and difficulty of conversion of an individual time and temperature (thermal doses) to a standardized value (eg equivalent minutes at 43 degrees C) for comparison of thermal treatments. Although, the conversion algorithm appears to work well within a range of moderately elevated temperatures (2-15 deg C) above normal physiologic baseline (37-39 deg C) there is concern that conversion accuracy does not hold up for temperatures which are minimally or significantly above baseline. An extensive review of the literature suggests a comprehensive assessment of the "thermal does-to-tissue effect" has not previously been assembled for most individual tissues and never been viewed in a semi-comprehensive (tissues and species) manner. Finally, we have addressed the relationship of thermal does-to-effect vs. baseline temperature. This issues is important since much of the thermal dose-to-effect information has been accrued in animal models with baseline temperatures 1-2 deg higher than that of humans.
74 citations
TL;DR: In this paper, the Fokker-Planck equation was used to solve the problem of reflection and transmission of a plane wave normally incident on a slab composed of a uniform scattering medium.
Abstract: Biological tissue scatters light mainly in the forward direction where the scattering phase function has a narrow peak. This peak makes it difficult to solve the radiative transport equation. However, it is just for forward peaked scattering that the Fokker-Planck equation provides a good approximation, and it is easier to solve than the transport equation. Furthermore, the modification of the Fokker-Planck equation by Leakeas and Larsen provides an even better approximation and is also easier to solve. We demonstrate the accuracy of these two approximations by solving the problem of reflection and transmission of a plane wave normally incident on a slab composed of a uniform scattering medium.
69 citations
TL;DR: This work describes the application of the numerical features from the previous work to images obtained by random tagging of proteins, and builds subcellular location trees that group the tagged proteins by similarity of location pattern.
Abstract: The overall object of proteomics is to characterize all of the proteins expressed in a given cell type. With the rapid development of random gene tagging technology and high resolution fluorescence microscopy, it has become possible to generate libraries of digital images depicting the location patterns of most proteins in any given cell type. While the subcellular location of a protein is important to its function, no established methods exist for the systematic description, comparison or organization of protein location patterns. We have previously described classification methods that accurately recognize all major subcellular location patterns in both 2D and 3D images, as well as methods for rigorous statistical comparison of such patterns. We describe here the application of the numerical features from the previous work to images obtained by random tagging of proteins. Spinning disk confocal microscopy was used to collect images depicting the location patterns of 46 NIH 3T3 cell clones expressing proteins randomly tagged with a fluorescent protein. A set of 42 numerical features describing both image texture and object morphology were calculated and used to build subcellular location trees that group the tagged proteins by similarity of location pattern.
67 citations
TL;DR: Treatment of upper tract urinary calculi in patients with uncorrected bleeding diatheses can be safely performed using contemporary small caliber ureteroscopes and holmium laser as the sole modality of lithotripsy.
Abstract: Purpose: To assess the safety and efficacy of ureteroscopy and holmium:YAG (yttrium-aluminum-garnet) laser lithotripsy in the treatment of upper urinary tract calculi in patients with known and uncorrected bleeding diatheses.
Materials and Methods: A retrospective chart review from 2 tertiary stone centers was performed to identify patients with known bleeding diatheses who were treated with holmium:YAG laser lithotripsy for upper urinary tract calculi. Twenty-five patients with 29 upper urinary tract calculi were treated with ureteroscopic holmium laser lithotripsy. Bleeding diatheses identified were coumadin administration for various conditions (17), liver dysfunction (3), thrombocytopenia (4), and von Willebrand's disease (1). Mean international normalized ratio (INR), platelet count and bleeding time were 2.3, 50 x 10 9 /L, and > 16 minutes, for patients receiving coumadin or with liver dysfunction, thrombocytopenia, or von Willebrand's disease, respectively.
Results: Overall, the stone-free rate was 96% (27/28) and 29 of 30 procedures were completed successfully without significant complication. One patient who was treated concomitantly with electrohydraulic lithotripsy (EHL) had a significant retroperitoneal hemorrhage that required blood transfusion.
Conclusions: Treatment of upper tract urinary calculi in patients with uncorrected bleeding diatheses can be safely performed using contemporary small caliber ureteroscopes and holmium laser as the sole modality of lithotripsy. Ureteroscopic holmium laser lithotripsy without preoperative correction of hemostatic parameters limits the risk of thromboembolic complications and costs associated with an extended hospital stay. Avoidance of the use of EHL is crucial in reducing bleeding complications in this cohort of patients.
66 citations
TL;DR: Ureteroscopy and holmium laser lithotripsy can be performed safely in all stages of pregnancy providing definitive management of symptomatic ureteral calculi, and avoids the undesirable features of stents or nephrostomy tubes.
Abstract: Objectives: Symptomatic urolithiasis in pregnancy that does not respond to conservative measures has traditionally been managed with ureteral stent insertion or percutaneous nephrostomy (PCN). Holmium:yttrium-aluminum-garnet (YAG) laser lithotripsy using state-of-the-art ureteroscopes represents an emerging strategy for definitive stone management in pregnancy. The purpose of this study was to review the results of holmium laser lithotripsy in a cohort of patients who presented with symptomatic urolithiasis in pregnancy.
Methods: A retrospective analysis was conducted at 2 tertiary stone centers from January 1996 to August 2001 to identify pregnant patients who were treated with ureteroscopic holmium laser lithotripsy for symptomatic urolithiasis or encrusted stents. Eight patients with a total of 10 symptomatic ureteral calculi and 2 encrusted ureteral stents were treated. Mean gestational age at presentation was 22 weeks. Mean stone size was 8.1 mm. Stones were located in the proximal ureter/ureteropelvic junction (UPJ) (3), mid ureter (1), and distal ureter (6).
Results: Complete stone fragmentation and/or removal of encrusted ureteral stents were achieved in all patients using the holmium:YAG laser. The overall procedural success rate was 91%. The overall stone-free rate was 89%. No obstetrical or urological complications were encountered.
Conclusions: Ureteroscopy and holmium laser lithotripsy can be performed safely in all stages of pregnancy providing definitive management of symptomatic ureteral calculi. The procedure can be done with minimal or no fluoroscopy and avoids the undesirable features of stents or nephrostomy tubes.
60 citations
TL;DR: Application of MCR with nonnegativity and equality constraints reveals several sources of undesired fluorescence that emit in the same wavelength range as the reporter fluorphores that will lead to errors in determining the relative expression of low-expressed genes.
Abstract: Multivariate curve resolution (MCR) using constrained alternating least squares algorithms represents a powerful analysis capability for a quantitative analysis of hyperspectral image data. We will demonstrate the application of MCR using data from a new hyperspectral fluorescence imaging microarray scanner for monitoring gene expression in cells from thousands of genes on the array. The new scanner collects the entire fluorescence spectrum from each pixel of the scanned microarray. Application of MCR with nonnegativity and equality constraints reveals several sources of undesired fluorescence that emit in the same wavelength range as the reporter fluorphores. MCR analysis of the hyperspectral images confirms that one of the sources of fluorescence is due to contaminant fluorescence under the printed DNA spots that is spot localized. Thus, traditional background subtraction methods used with data collected from the current commercial microarray scanners will lead to errors in determining the relative expression of low-expressed genes. With the new scanner and MCR analysis, we generate relative concentration maps of the background, impurity, and fluroescent labels over the entire image. Since the concentration maps of the fluorescent labels are relativly uaffected by the presence of background and impurity emissions, the accuracy and useful dynamic range of the gene expression data are both greatly improved over those obtained by commercial microarray scanners.
TL;DR: In this paper, the authors demonstrate that the light scattering of tissue can be effectively reduced by topical applications of the biocompatible chemical agents, such as propylene glycol and glucose solutions.
Abstract: The highly scattering nature of human tissue limits light penetration depth in the near infrared range, which prevents the deeper microstructures from imaging. In order to enhance the imaging depth for the current high resolution optical imaging techniques, the light scattering in tissue must be reduced. This paper demonstrates that the light scattering of tissue can be effectively reduced by the topical applications of the biocompatible chemical agents. In this study the propylene glycol and glucose solutions were chosen for the demonstrations through topical applications and intra-dermis injection, respectively. The experiments were performed in vitro and in vivo by the use of the optical coherence tomography system. The results clearly show that the OCT imaging depth and contrast are dramatically improved after the topical applications of propylene glycol solution. Such improvement was discussed on the basis of refractive index matching environment created by the chemical agents, which effectively reduces the light scattering of tissue. Rayleigh-Gans approximation of light scattering was also used to show theoretically how the increase of refractive index of background medium would have effect on the reduced scattering coefficient of tissue. The theoretical and experimental results were qualitatively consistent.
TL;DR: In this article, the spectral information of diffuse reflectance spectroscopy with the spatial information of 2D imaging is combined to produce a quantitative distribution map of chromophores contributing to the color appearance of the skin.
Abstract: Hyperspectral imaging of skin combines the spectral information of diffuse reflectance spectroscopy with the spatial information of 2D imaging. Skin chromophore maps can be reconstructed in which features such as pigmented lesions, diffuse and localized erythema, areas of increased blood stasis, etc. could be identified and the relative
parameters quantified. Hyperspectral imaging is the only reliable method to produce a quantitative distribution map of chromophores contributing to the color appearance of the skin.
TL;DR: In this paper, a classical medical ultrasound system was combined with a pulsed laser source to allow laser-induced ultrasound imaging(optoacoustics), which is based on reection and scattering of an incident acoustic pulse at internal tissuestructures.
Abstract: A classical medical ultrasound system was combined with a pulsed laser source to allow laser-induced ultrasound imaging(optoacoustics). Classical ultrasound is based on re ection and scattering of an incident acoustic pulse at internal tissuestructures. Laser-induced ultrasound is generated in situ by heating optical absorbing structures, such as blood vessels,witha5nslaser pulse (few degrees or fraction of degree), which generates pressure transients. Laser-induced ultrasoundprobes optical properties and therefore provides much higher contrast and complementary information compared to clas-sical ultrasound. An ultrasound array transducer in combination with a commercial medical imaging system was used torecord acoustic transients of both methods. Veins and arteries in a human forearm were identi ed in vivo using classi-cal color doppler and oxygenation dependent optical absorption at 660 nm and 1064 nm laser wavelength. Safety limitsof both methods were explored. Laser-induced ultrasound seems well suited to improve classical ultrasound imaging ofsubcutaneous regions.Keywords: photoacoustics, optoacoustics, ultrasound, imaging
TL;DR: The feasibility of highly focused transducers with several array structures as well as the combination of these technologies with MRI and Ultrasonic imaging systems will be illustrated through various examples.
Abstract: The development of therapeutic ultrasound requires specially adapted transducers for the generation of high intensity focused beams.
New piezocomposite technologies have been developed to take into account the high power requirements and constraints like the high degree of focussing, the capability to withstand thermal shocks, the requirement for safe and reliable transducers, the compatibility with MRI and Ultrasonic imaging systems. This approach enables a wide variety of shapes and the design of array transducers for electronic focusing, scanning and steering of the therapeutic beam. Recent results will be presented on power and efficiency aspects. The feasibility of highly focused transducers with several array structures as well as the combination of these technologies with MRI and Ultrasonic imaging systems will be illustrated through various examples.
TL;DR: In this paper, a micro-optical scanner at the tip of an ultrathin flexible endoscope with an overall diameter of 1 mm is presented, using a small diameter piezoelectric tube actuator to drive a cantilevered optical fiber in mechanical resonance to scan a beam of light in a space-filling, spiral scan pattern.
Abstract: Our goal is to produce a micro-optical scanner at the tip of an ultrathin flexible endoscope with an overall diameter of 1 mm. Using a small diameter piezoelectric tube actuator, a cantilevered optical fiber can be driven in mechanical resonance to scan a beam of light in a space-filling, spiral scan pattern. By knowing and/or controlling the fiber position and acquiring backscattered intensity with a photodetector, an image is acquired. A microfabrication process of computer-controlled acid etching is used to reduce the mass along the fiber scanner shaft to allow for high scan amplitude and frequency. A microlens ( 50 degrees full angle), up to video scan rates (>10 KHz), while maintaining a scanner diameter of 1 mm. A comparison can be made to bi-axial mirror scanners being fabricated as a MEMS device (micro-electro-mechanical system). Based on the opto-mechanical performance of these microlensed fiber scanners, flexible catheter scopes are possible for new microendoscopies that combine imaging with laser diagnoses.
TL;DR: In this article, a multi-dimensional histogram was used to record the photon density versus the time within the fluorescence decay function, the x-y coordinates of the scanning area and the detector channel number.
Abstract: Time-correlated single photon counting (TCSPC) fluorescence lifetime imaging in laser scanning microscopes can be combined with a multi-detector technique that allows to record time-resolved images in several wavelength channels simultaneously. The technique is based on a multi-dimensional histogramming process that records the photon density versus the time within the fluorescence decay function, the x-y coordinates of the scanning area and the detector channel number. It avoids any time gating or wavelength switching and therefore yields a near-ideal counting efficiency. We show an instrument that records dual wavelength lifetime images with up to 512 x 512 pixels, and single wavelength lifetime images with up to 1024 × 1024 pixels. It resolves the components of doubleexponential decay functions down to 30 ps, and works at the full scanning speed of a two-photon laser scanning microscope. The performance of the instrument is demonstrated for simultaneous lifetime imaging of the donor and acceptor fluorescence in CFP / YFP FRET systems and for tissue samples stained with several fluorophores.
TL;DR: A new SLP system, GDx VCC (Laser Diagnostic Technologies, Inc., San Diego, CA), with a variable corneal compensator (VCC) to achieve individualized cornea compensation is presented.
Abstract: One of the earliest signs of glaucoma presence is defects in the retinal nerve fiber layer (RNFL). Scanning laser polarimetry (SLP) provides objective assessment of RNFL, a birefringent tissue, by measuring the total retardation in the reflected light. SLP provides a potential tool for early detection of glaucoma and its progression. The birefringence of the anterior segment of the eye, mainly the cornea, is a confounding variable to SLP's clinical application, if compensation cannot be achieved properly. This paper presents a new SLP system, GDx VCC (Laser Diagnostic Technologies, Inc., San Diego, CA), with a variable corneal compensator (VCC) to achieve individualized corneal compensation. Clinical application of this device in glaucoma detection is also demonstrated.
TL;DR: A stepwise rotation of the γ-subunit is detected under conditions for ATP synthesis (i.e. energization of the proteoliposomes by an acid-base-transition).
Abstract: F o F 1 -ATP synthases couple proton translocation with the synthesis of ATP using two rotary motors within the enzyme. To monitor inter-subunit movements during catalysis, we selectively attached two fluorophores to the F 1 part, sulforhodamine B at one of three β-subunits and Cy5 at the γ-subunit. Reassembly with F o parts embedded in liposomes yielded functional holoenzymes. Fluorescence resonance energy transfer (FRET) was investigated in photon bursts of freely diffusing liposomes with reconstituted ATP synthases using a confocal set-up for single-molecule detection. Incubation with AMPPNP resulted in stable intensity ratios within a burst and three different FRET efficiencies. Upon ATP addition, a repeating sequence of three distinct FRET efficiencies was observed, indicating the stepwise movement of the γ-subunit during ATP hydrolysis. With this single-molecule FRET approach we detected a stepwise rotation of the γ-subunit under conditions for ATP synthesis (i.e. energization of the proteoliposomes by an acid-base-transition). The direction of rotation is opposite to the direction observed during ATP hydrolysis.
TL;DR: The tissue effects of and response to cryothermic and heat injury appear to be different and the heat-treated tissues lacked a prominent wound healing response and appeared to resist breakdown/repair by the body.
Abstract: Minimally invasive cryothermic and hyperthermic therapies are being increasingly used to destroy dysfunctional and neoplastic tissues in several organ systems. This report morphologically compares the acute tissue response that follow cryothermic and microwave therapy in porcine kidneys. Three cryothermic and hyperthermic groups of treated kidneys were pooled from other studies for evaluation: 1) in vitro treated non-perfused, 2) in situ treated with 2-hour post in vivo perfusion, and 3) in situ treated with 3-day or 7-day post in vivo perfusion. The cryolesions showed uniform central coagulative-type necrosis and interstitial hemorrhage. The hyperthermic lesions showed central thermal fixation and a rim of coagulative necrosis. The cryothermic and hyperthermic lesions both had a similar narrow transition zone of partial cell injury. The cryothermic lesions developed a wound healing response that advanced into the central lesion. In contrast, the heat-treated tissues lacked a prominent wound healing response and appeared to resist breakdown/repair by the body. Thus, the tissue effects of and response to cryothermic and heat injury appear to be different.
TL;DR: In this paper, a spectral imaging technique applied to in vivo bioluminescent imaging is presented that provides an estimate of the depth of bioluminous reporters inside living animals.
Abstract: A spectral imaging technique applied to in vivo bioluminescent imaging is presented that provides an estimate of the depth of bioluminescent reporters inside living animals. The model, based on the standard diffusion approximation of light propagating in a slab sample, is described in this paper. Validation experiments performed on phantom and tissue models, as well as preliminary in vivo mouse images, demonstrate the ability of spectral imaging to provide a correct estimate of depth based upon a single view imaging system.
TL;DR: In this paper, a low coherence interferometric system with axial resolution better than coherence length providing simultaneous measurement of the geometrical thickness and refractive index of transparent layers by sharp focusing of light on the measured object is considered.
Abstract: The idea of a new low coherence interferometric system with axial resolution better than coherence length providing simultaneous measurement of the geometrical thickness and refractive index of transparent layers by sharp focusing of light on the measured object is considered. The presented interferometric system consists of two parts -- the so-called Wave Front Matching Interferometer (WFMI) and a low coherence Michelson interferometer (LCMI) as a light source for the first. The WFMI provides high separation of interference signal peaks from demarcations in layer object at high numerical aperture focusing of light on the object. The tandem optical scheme of these interferometers allows to make this system very compact and mobile.
TL;DR: First results from a rigorous study of image formation in SHG microscopy of collagen are presented, which shows promise as a technique for characterizing collagen.
Abstract: Collagen is known to be a very effective generator of the second harmonic of incident light from 700 to 1100nm, and second harmonic generation (SHG) microscopy is coming into use as a tool for studying the distribution of collagen in tissue. It also shows promise as a technique for characterizing collagen - both in distinguishing different collagen types and their packing and in identifying degradation of collagen in pathologic conditions. However many aspects of image formation in SHG microscopy of collagen remain imperfectly understood, and we have commenced a rigorous study of these factors. The present paper presents the first results from this program.
TL;DR: The SherlockTM system, a laser-scanning confocal imaging system that automatically scans all 96 bundles of an array matrix at variable resolution down to 0.8 micron, has been developed to meet the needs of large-scale genomic analysis.
Abstract: Illumina is developing a BeadArray TM technology that supports SNP genotyping, mRNA expression analysis and protein expression analysis on the same platform. We use fiber-optic bundles with a density of approximately 40,000 fibers/mm 2 . At hte end of each fiber, a derivatized silica bead forms an array element for reading out a genotyping or expression assay data point. Each bead contains oligonucleotide probes that hybridize with high specificity to complementary sequences in a complex nucleic acid mixture. We derivatize the beads in bulk, pool them to form a quality-controlled source of microarray elements, and allow them to assemble spontaneously into pits etched into the end of each optical fiber bundle. We load our fiber bundles, containing 49,777 fibers, with up to 1520 different bead types. The presence of many beads of each type greatly improves the accuracy of each assay. As the final step in our manufacturing process, we decode the identity of each bead by a series of rapid hybridizations with fluroescent oligos. Decoding accuracy and the number of beads of each type is recorded for each array. Decoding also serves as a quality control procedure for the performance of each element in the array. To facilitate high-throughput analysis of many samples, the fiber bundles are arranged in an array matrix (Sentrix TM arrays). Using a 96-bundle array matrix, up to 1520 assays can be performed on each of 96 samples simultaneously for a total of 145,920 assays. Using a 384-bundle array matrix, up to 583,680 assays can be performed simultaneously.
The BeadArray platform is the highest density microarray in commercial use, requiring development of a high-performance array scanner. To meet this need, we developed the Sherlock TM system, a laser-scanning confocal imaging system that automatically scans all 96 bundles of an array matrix at variable resolution down to 0.8 micron. The system scans with both 532 and 635 nm lasers simultaneously, collecting two fluorescence images. The optical train is designed around a telecentric, flat field, macro scan lens with a field of view of 2 mm.
Our BeadArray platform is adaptable to many different assays. In our genotyping services lab, we automated the development and production of highly multiplexed SNP genotyping assays. Each SNP call is made automatically and assigned a quality score based on objective measures of allele clustering across multiple samples. The quality score correlates directly with genotyping accuracy. With a small number of robots and thermal cyclers, and a team of 5 people, we have the capacity to perform over 1 million genotypes per day. The system is modular so that scale-up is limited only by demand. The system has the capacity, versatility, and cost structure to meet the needs of large-scale genomic analysis.
TL;DR: In this article, a two-element GRIN lens system was used for color-encoded optical sectioning with a wavelength encoding range of 200 nm and a sectioning range of 40 μm from the tip of the probe into the tissue.
Abstract: Placing a spatial light modulator, such as the Texas Instruments Digital Micromirror Device (DMD), in the light path of a microscope enables a variety of novel applications. One application enables reflectance in vivo confocal imaging of cells and tissue structure through a fiber-optic image guide. While multi-wavelength reflectance confocal microendoscopy with optical sectioning is a requirement for a clinically useful device, some form of axial scanning is also necessary. This is readily achieved using a multi-element lens system with some form of mechanical translation, however, this generally results in large probes and high cost. These limitations can be overcome using a two-element GRIN lens system in which the traditionally undesirable chromatic aberration of such a system can be exploited to allow for color-encoded optical sectioning. In our system a wavelength encoding range of 200 nm permits a sectioning range of 40 μm from the tip of the probe into the tissue.
TL;DR: In this paper, a photoacoustic probe was used for quantifying epidermal melanin content using a Q-switched, frequency doubled Nd:YAG laser operating at 532nm to generate acoustic pulses in skin.
Abstract: Most dermatologic laser procedures must consider epidermal melanin, as it is a broadband optical absorber which affects subsurface fluence, effectively limiting the amount of light reaching the dermis and targeted chromophores. An accurate method for quantifying epidermal melanin content would aid clinicians in determining proper light dosage for therapeutic laser procedures. While epidermal melanin content has been quantified non-invasively using optical methods, there is currently no way to determine the melanin distribution in the epidermis. We have developed a photoacoustic probe that uses a Q-switched, frequency doubled Nd:YAG laser operating at 532nm to generate acoustic pulses in skin in vivo . The probe contained a piezoelectric element that detected photoacoustic waves which were then analyzed for epidermal melanin content, using a photoacoustic melanin index (PAMI). We tested 15 human subjects with skin types I--VI using the photoacoustic probe. We also present photoacoustic data for a human subject with vitiligo. Photoacoustic measurement showed melanin in the vitiligo subject was almost completely absent.
TL;DR: In this article, the authors discuss the requirements for successful production of defined fiber core compositions and the knowledge of the incorporation mechanism of the rare earths into the high silica matrix.
Abstract: Rare earth doped silica based fiber lasers and amplifiers have been shown to be suitable for a variety of applications in industry, science and medicine. They can yield very high power output both in the pulsed and cw regime with high efficiency, reliability and beam quality. Great progress was possible by new design concepts as non-symmetric double clads and large mode area guiding structures and by carefully tailoring the material properties. Extreme power load and complicated fiber structures make high demands on the preparation technology. Special aspects of material and technology development will be discussed in the following. Basic requirement for a successful production of defined fiber core compositions is the knowledge of the incorporation mechanism of the rare earths into the high silica matrix. Diffusion processes during the preparation steps determine refractive index distribution, geometry and numerical aperture. Strong concentration dependences of codopants and the interaction between codopants and active components must be taken into account. The optical properties of rare earths ions can be tailored by defined codopant relations according to the intended application. Some unusual basic loss contributions of certain rare earths have to be considered and controlled in order to get high laser performance. Loss effects by UV radiation play a role if UV cured coatings are used or if the fibers are provided with Bragg gratings as laser mirrors.
TL;DR: The mechanisms underlying material ejection in pulsed laser ablation of biological tissues are reviewed, with special emphasis on the thermodynamics and kinetics of phase transitions and their modifications arising from the presence of a tissue matrix.
Abstract: We review the mechanisms underlying material ejection in pulsed laser ablation of biological tissues, with special emphasis on the thermodynamics and kinetics of phase transitions and their modifications arising from the presence of a tissue matrix.
TL;DR: Two methods of acquiring spectral images are described, along with their application to multicolor immunohistochemistry and transmission in-situ hybridization (TRISH): 1) liquid crystal tunable filters; and 2) a novel, spectrally agile light source.
Abstract: Molecular medicine now requires molecular pathology. While fluorescence has traditionally been used for high-resolution multiplexed molecular imaging, clinical practitioners prefer non-fluorescent multicolor methods. However, in brightfield, typically, only one color is used at a time, which precludes assessment of co-expression on a cell-by-cell basis. Similar constraints apply to brightfield in-situ hybridization techniques. Double- and triple-staining procedures are rarely performed in non-research settings not only because the wet chemistry can be difficult, but also because it can be challenging or impossible to determine visually where and to what extent different chromogens may physically overlap. Spectral imaging can be useful in this context. Two methods of acquiring spectral images are described, along with their application to multicolor immunohistochemistry and transmission in-situ hybridization (TRISH): 1) liquid crystal tunable filters; and 2) a novel, spectrally agile light source. This source emits white light of any desired color temperature, or single 10-nm wavelength bands in the range 420 to 700 nm, or any combination of wavelengths with individual intensity control. Both methods are allied with a grayscale camera and appropriate algorithms to analyze multicolor samples of clinical significance. Spectrally unmixed images clearly separate signals linked to different chromogens, even with spectral and spatial overlap. Intriguing challenges in matching mathematical algorithms to these specific problems remain: how many bands are enough? What are the optimal unmixing procedures? What automated tools can be applied to speed and simplify the procedures?
TL;DR: The weld strength as a function of laser wavelength demonstrated a correlation with the absorption spectrum of native water suggests that absorption of light by water in the tissue plays a significant role in laser tissue welding.
Abstract: Ex vivo specimens of human and porcine aorta and skin were welded using either Cr4+:YAG or Erbium fiber lasers tuned to the water absorption band at 1440-1460 nm. Welding was performed without the use of protein solders or glues. Welding efficacy was monitored by measuring the tensile strength of the welded tissue and the extent of collateral tissue damage. Full thickness tissue bonding with no collateral damage was observed with porcine aorta samples. The optimum tensile strength for porcine and human aorta was 1.33 ± 0.15 kg/cm2 and 1.13 ± 0.27 kg/cm2 respectively for welding at 1460 nm, while that for porcine and human skin was 0.94 ± 0.15 kg/cm2 and 1.05 ± 0.19 kg/cm2 respectively achieved with welding at 1455 nm. The weld strength as a function of laser wavelength demonstrated a correlation with the absorption spectrum of native water suggests that absorption of light by water in the tissue plays a significant role in laser tissue welding.