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PubMed1999

Telepathology and imaging spectroscopy as a new modality in histopathology.

Telemedicine started in the late 1950's by transmitting data on patients' pulse and heart rates. In the 1980's it expanded to radiology and orthopedics. The technology is now expanding to other specialties that can digitally gather patient data. Telepathology comprises the transmission of microscopic images via telecommunication network. Image compression and multiplexing technologies enabled high-resolution telepathology as well as real-time video consultations over international telephone lines. Organ transplantation has become a viable treatment and offers new life to an increasing number of patients suffering from chronic end stage diseases and from irreversible organ failure. Rejection is still a major problem in kidney, liver, and heart transplantation. To gain further insight into the complex interactions within the components of the immune system, it has become increasingly necessary to develop rapid and simple methods to monitor the status of the immune system in patients. Clinical signs suggest organ rejection and abnormal laboratory test results, but only histological signs on biopsy specimens are adequately specific. The financial cost of organ transplant makes it imperative to develop tools for the early identification and treatment of organ rejection. An increasingly sensitive and accurate way of localizing key structures and abnormalities is through spectroscopy of either H&E stained samples or with a fluorescent tag (fluorophore) or by relying on natural fluorescence. The system is based on a unique Prism and Mirror Imaging Spectroscopy System ("PARISS), spectrometer originally designed and implemented for remote Earth monitoring from space and aircraft and astronomical imaging spectroscopy. Compact and lightweight both the mirror and prism are presently constructed in inexpensive glass but can also be injection molded in plastic. Any number of vendors anywhere in the world can produce all parts of the assembly. This greatly enhances the chances of future commercial viability. The Interactive Histopathology Consultation Network INTERPATH (PL961121) project integrated of remote control imaging microscopy system, imaging spectroscopy, and communication networks called SPECTROMIC. This telepethology unit will be a useful tool in the Regional and International Integrated Telemedicine Network for Medical Assistance in End Stage Diseases and Organ Transplant, RETRANSPLANT HC 4028 (HC) & IN 4028 (HC).

Computer Systems↗
PubMedDate not supplied

A system for real time fluorescence imaging in color for tumor diagnosis.

Early tumors can be diagnosed by the fluorescence of an injected, tumor-specific agent such as dihematoporphyrin ether. However, brightness or intensity contrast is low because of the autofluorescence of tissue. Addition of color (hue) information aids in detection of tumors, and in elimination of false positives. Standard color video cameras are not sensitive enough to image the weak fluorescence. Thus an intensified monochrome video camera has been equipped with a synchronized color filter wheel, and the image displayed by multiplexing alternate lines to a red green blue (RGB) monitor.

Animals↗
PubMed2003

Invariant pattern recognition by use of a spatial code division multiplexing approach.

Invariant pattern recognition can be achieved by use of harmonic decomposition, for example circular harmonics are used for rotation invariant recognition. A common problem with such methods is that often only a single term of the harmonic decomposition is used, and it does not contain a sufficient amount of the reference energy. Thus discrimination capability is limited, especially in the presence of noise or other disturbances. By using several terms of the harmonic decomposition together this problem can be solved; this can be achieved by the use of code division filter multiplexing. Several harmonic terms are encoded onto a single filter, and the signal is simultaneously correlated with all of them, hence producing enhanced discrimination capabilities. Here two methods are suggested for such encoding. The first involves multiplexing the filters in the Fourier plane, while the second involves multiplexing in the image plane.

Journal Article↗
PubMed2005

Theoretical and experimental studies of hologram multiplexing that uses a random wave front generated by photorefractive beam fanning.

A hologram multiplexing technique-that uses random wave fronts generated by photorefractive beam fanning is investigated. A storage photorefractive crystal generates various random wave fronts to be used as reference beams without the external diffusers such as ground glass and multimode optical fiber that are generally employed. We experimentally demonstrate hologram multiplexing with six images and show that the stored holograms can be selectively retrieved. We also simulate photorefractive beam fanning inside a BaTiO3 crystal, in particular regarding the correlation properties of the fanning beams for the first time to our knowledge, and reveal the conditions of incidence of an object beam and a reference beam required for suppressing image degradation, implementing low-cross-talk retrieval, and producing a large number of stored holograms.

Journal Article↗
PubMed2005

Simultaneous real-time imaging of surface and subsurface structures from a single space-frequency multiplexed photodisplacement interferogram.

A new parallel photodisplacement technique has been developed that achieves simultaneous real-time imaging of surface and subsurface structures from a single space-frequency multiplexed interferogram, which greatly simplifies the system and the optical alignment. A linear region of photodisplacement is excited on the sample for subsurface imaging by use of a line-focused intensity-modulated laser beam, and the displacement and surface information on reflectivity and topography are detected by a parallel heterodyne interferometer with a charge-coupled device linear image sensor used as a detector. The frequencies of three control signals for excitation and detection, that is, the heterodyne beat signal, modulation signal, and sensor gate pulse, are optimized such that surface and subsurface information components are space-frequency multiplexed into the sensor signal as orthogonal functions, allowing each to be discretely reproduced from Fourier coefficients. Preliminary experiments demonstrate that this technique is capable of simultaneous imaging of reflectivity, topography, and photodisplacement for the detection of subsurface lattice defects at a remarkable speed of only 0.26 s per 256 x 256 pixel area. This new technique is promising for use in nondestructive hybrid surface and subsurface inspection and other applications.

Journal Article↗
PubMedDate not supplied

Autofluorescence removal, multiplexing, and automated analysis methods for in-vivo fluorescence imaging.

The ability to image and quantitate fluorescently labeled markers in vivo has generally been limited by autofluorescence of the tissue. Skin, in particular, has a strong autofluorescence signal, particularly when excited in the blue or green wavelengths. Fluorescence labels with emission wavelengths in the near-infrared are more amenable to deep-tissue imaging, because both scattering and autofluorescence are reduced as wavelengths are increased, but even in these spectral regions, autofluorescence can still limit sensitivity. Multispectral imaging (MSI), however, can remove the signal degradation caused by autofluorescence while adding enhanced multiplexing capabilities. While the availability of spectral "libraries" makes multispectral analysis routine for well-characterized samples, new software tools have been developed that greatly simplify the application of MSI to novel specimens.

Algorithms↗
PubMed2006

Multicolor FRET silica nanoparticles by single wavelength excitation.

Fluorescent nanoparticles with multiple emission signatures by a single wavelength excitation are needed in multiplex bioanalysis and molecular imaging. We have prepared silica nanoparticles encapsulated with three organic dyes using a modified Stöber synthesis method. By varying the doping ratio of the three tandem dyes, fluorescence resonance energy transfer (FRET)-mediated emission signatures can be tuned to have the nanoparticles exhibit multiple colors under one single wavelength excitation. These nanoparticles are intensely fluorescent, highly photostable, uniform in size, and biocompatible. The acceptor emission of the FRET nanoparticles has generated a large Stokes shift, which implicates broad applications in biological labeling and imaging. Molecular recognition moieties, such as biotin, can be covalently attached to the nanoparticle surface to allow for specific binding to target molecules. These multicolor FRET silica nanoparticles can be used as barcoding tags for multiplexed signaling. By using these NPs, one can envision a dynamic, multicolor, colocalization methodology to follow proteins, nucleic acids, molecular machines, and assemblies within living systems.

Journal Article↗
PubMed1983

Three-dimensional holographic display of images of otological specimens.

Three-dimensional displays of anatomical structures and clinical findings are very persuasive and instructive. Using multiplex holograms, we designed a display of three-dimensional images of otological specimens. Multiplex holograms, reported by Cross of the United States in 1975, enable reconstruction of three-dimensional moving images and are used for artistic display as well as for teaching in medicine and general education. Multiplex holograms were recorded in a two-step process. The first step is to make a series of original cine-pictures of an object from different horizontal directions, rotating it on a turntable. In the second step, one frame of the original film is recorded on a narrow strip hologram. All frames of the original film are recorded one after another and a complete multiplex hologram can be synthesized. In the reconstruction stage, the multiplex hologram is formed into a cylinder and illuminated from below by a small white light source. Reconstructions of the three-dimensional bright images of the object inside the cylindrical holographic screen are shown.

Ear↗
PubMed1994

A four-channel time domain multiplexer: a cost-effective alternative to multiple receivers.

The concept of simultaneous image acquisition as originally conceived by Hyde has been implemented by Roemer et al. using a multiple receiver system. This article describes an alternative technique that uses time domain multiplexing (TDM) to acquire simultaneous images using a single receiver channel. This method requires few modifications to the existing receiver and has been shown to be equivalent to a true four channel receiver in most applications. The multiplexing technique was implemented and tested on a standard commercial scanner that has also been equipped with a four channel receiver. Signal-to-noise results demonstrate that four independent images can be multiplexed through a single receiver channel with no degradation in image quality. Additionally, spine images that were obtained from a normal volunteer with both a multiplexed single channel receiver and a true four channel receiver system are presented.

Cost-Benefit Analysis↗
PubMed2005

Normalization of multicolor fluorescence in situ hybridization (M-FISH) images for improving color karyotyping.

BACKGROUND: Multiplex or multicolor fluorescence in situ hybridization (M-FISH) is a recently developed cytogenetic technique for cancer diagnosis and research on genetic disorders. By simultaneously viewing the multiply labeled specimens in different color channels, M-FISH facilitates the detection of subtle chromosomal aberrations. The success of this technique largely depends on the accuracy of pixel classification (color karyotyping). Improvements in classifier performance would allow the elucidation of more complex and more subtle chromosomal rearrangements. Normalization of M-FISH images has a significant effect on the accuracy of classification. In particular, misalignment or misregistration across multiple channels seriously affects classification accuracy. Image normalization, including automated registration, must be done before pixel classification. METHODS AND RESULTS: We studied several image normalization approaches that affect image classification. In particular, we developed an automated registration technique to correct misalignment across the different fluor images (caused by chromatic aberration and other factors). This new registration algorithm is based on wavelets and spline approximations that have computational advantages and improved accuracy. To evaluate the performance improvement brought about by these data normalization approaches, we used the downstream pixel classification accuracy as a measurement. A Bayesian classifier assumed that each of 24 chromosome classes had a normal probability distribution. The effects that this registration and other normalization steps have on subsequent classification accuracy were evaluated on a comprehensive M-FISH database established by Advanced Digital Imaging Research (http://www.adires.com/05/Project/MFISH_DB/MFISH_DB.shtml). CONCLUSIONS: Pixel misclassification errors result from different factors. These include uneven hybridization, spectral overlap among fluors, and image misregistration. Effective preprocessing of M-FISH images can decrease the effects of those factors and thereby increase pixel classification accuracy. The data normalization steps described in this report, such as image registration and background flattening, can significantly improve subsequent classification accuracy. An improved classifier in turn would allow subtle DNA rearrangements to be identified in genetic diagnosis and cancer research.

Algorithms↗
PubMed2025

Population-Specific Immunogenomic Alterations in Gallbladder Cancer and Prognostic Significance.

Gallbladder carcinoma is a deadly disease with a poor prognosis, and recent clinical data suggest only a modest benefit of PD1/PDL1 inhibitors in this disease. Optimizing immunotherapeutic approaches will require a detailed understanding of the immunogenomic landscape of this disease worldwide. We combined targeted next-generation sequencing and immunohistochemistry to create detailed immunogenomic landscapes from 2 cohorts of gallbladder cancer cases from the United States (n = 60) and Chile (n = 62). Mutations in TP53, SMAD4, KRAS, PIK3CA, ARID2, ARID1A, ATM, FBXW7, ERBB2, and NF1 were found in both the US and Chilean primary cohorts, as well as amplifications in ERBB2, CCNE1, MDM2/CDK4, and CCND1. Despite similar mutation profiles, the immune profiles were distinct, with the Latin American cohort having higher densities of biomarkers associated with CD4+ T cells and PD-1 but lower densities of CD68+ macrophages compared with the North American cohort. Clustering and correlation analyses suggest novel immune subgroups and clinical associations independently of any specific mutations. Additionally, supported by multiplexed single-cell imaging technology, we identified low CD4 and high V-domain Ig suppressor of T cell activation as a candidate biomarker pair of poor outcomes. In summary, our findings highlight the importance of sensitivity to geographic location when considering therapeutic developments and pave a path for further immune investigations of this understudied disease.

Humans↗
PubMed2005

Imaging orientational order and lipid density in multilamellar vesicles with multiplex CARS microscopy.

Multiplex coherent anti-Stokes Raman scattering (CARS) microscopy is used to measure the width of the orientational order distribution of lipid acyl chains within a three-dimensionally confined microscopic probing volume. A theoretical model is developed to describe and simulate the polarization-dependent measurements. We observe that the orientational order in phosphatidylcholine multilammellar vesicles increases significantly upon addition of small amounts (<or=15 mol%) of cholesterol and is significantly reduced for unsaturated lipids. Based on these measurements and using the quantitative nature of multiplex CARS microscopy the exact local concentration of lipid molecules within the vesicles can be measured in terms of the number of lipid bilayers present in the microscopic probing volume.

Lipids↗
PubMed1994

Making global telemedicine practical and affordable: demonstrations from the Middle East.

OBJECTIVE: The purpose of this study was to demonstrate the first use of voice-grade telephone lines for the international transmission of both high-resolution digital images (radiology and pathology) and video in near real-time. MATERIALS AND METHODS: Eight live demonstrations were performed from the United Arab Emirates and the Kingdom of Saudi Arabia at the invitation of the respective ministries of health. Thirty radiologic studies (CT, MR, and radiographs) were digitized, compressed, and transmitted to Cambridge, MA, where they were interpreted on diagnostic workstations (1792 x 2252 display matrix) by a team of subspecialist radiologists. Near real-time image transmission was achieved by combining wavelet-based image compression (average compression ratio of 23:1) and multiplexing technology that used four phone lines simultaneously. During each demonstration, one pathology image was transmitted from Cambridge to the demonstration site, where it was interpreted by a visiting pathologist. Video-conferencing was implemented with a 64-kilobits-per-sec leased line from the United Arab Emirates and with four multiplexed telephone lines from Saudi Arabia. RESULTS: For teleradiology and telepathology, transmission times ranged from 2-5 min per image. Image fidelity was judged to be of diagnostic quality in all transmitted cases. The video link to the United Arab Emirates was highly reliable. Bandwidth for videoconferencing from Saudi Arabia was marginal on four voice-grade telephone lines, resulting in some downtime (10-20%). Live consultations provided by subspecialists in Cambridge assisted in the management of patients at both venues. The system was well received by both the referring physicians in the Middle East and the participants in the United States. CONCLUSION: Image compression and multiplexing technologies enabled high-resolution teleradiology and telepathology as well as real-time video consultations over international telephone lines. While telecommunications systems are advancing rapidly in many parts of the world, those areas most in need of telemedicine services are likely to be the last to upgrade their telecommunications infrastructures. This "proof of concept" article outlines a practical and affordable approach that makes telemedicine more accessible to underserved areas worldwide.

Humans↗
PubMed2005

Multiplexed G-protein-coupled receptor Ca2+ flux assays for high-throughput screening.

An early drug discovery approach focusing on gene families can benefit from strategies that exploit common signaling mechanisms to more effectively identify and characterize novel chemical lead structures. Multiplexing, defined as the screening of multiple targets within the same experiment, is an example of this strategy. Here, the authors describe a technique that allows multiplexing of a common assay type used to study G-protein-coupled receptors: changes in intracellular Ca2+ levels as measured by Molecular Device's fluorometric imaging plate reader (FLIPR). The multiplexed FLIPR assays showed the expected pharmacological properties of single assays, with good reproducibility and Z* factors. The authors used them to screen large compound libraries in 2 multiplexed assay designs. The 1st used a single-cell line expressing 2 different receptors and the 2nd a mixture of 2 cell lines of the same type each expressing distinct receptors. Screening using these multiplexed assays produced significant savings in reagents, time, and human resources and allowed the authors to quickly identify specific and selective hits.

Biological Assay↗
PubMed2003

High-content proteomics: fluorescence multiplexing using an integrated, high-sensitivity, multiwavelength charge-coupled device imaging system.

The detection of proteins in 2-D gels and their subsequent identification by MS is still the "gold standard" in proteomics. Fluorescent detection has increasingly replaced colorimetric and radiometric detection on gels and blots. The reasons for this are multiple and varied and include higher sensitivity, better quantitation, increased dynamic range, speed, safety and ease of use. Unlike other methods, fluorescent protein detection is also typically very consistent in response from protein to protein and in many cases is compatible with MS methods for protein identification. The superior sensitivity and benefits achieved by fluorescent techniques have spurred the development of instrumentation capable of delivering precise, sensitive, high-resolution image acquisition over a wide variety of excitation and emission wavelengths. This report focuses on applications using the highly sensitive, charge-coupled device based ProXPRESS multilabel imager, readily configurable for image acquisition over a wide variety of wavelengths (380-700 nm and ultraviolet (UV)) using xenon lamp or UV excitation. The ability to simultaneously detect enzyme activities or protein modifications with different color fluorescent probes in addition to total protein amounts (multiplexing) allows the further mining of proteomic data content from a single set of protein samples. To this end, the development of instrumentation that enables a multiplexing strategy will become central to in-depth proteomic studies. The ProXPRESS maximizes the efficiency of experimental strategies that require flexibility and multicolor fluorescence detection.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

Spectral imaging perspective on cytomics.

BACKGROUND: Cytomics involves the analysis of cellular morphology and molecular phenotypes, with reference to tissue architecture and to additional metadata. To this end, a variety of imaging and nonimaging technologies need to be integrated. Spectral imaging is proposed as a tool that can simplify and enrich the extraction of morphological and molecular information. Simple-to-use instrumentation is available that mounts on standard microscopes and can generate spectral image datasets with excellent spatial and spectral resolution; these can be exploited by sophisticated analysis tools. METHODS: This report focuses on brightfield microscopy-based approaches. Cytological and histological samples were stained using nonspecific standard stains (Giemsa; hematoxylin and eosin (H&E)) or immunohistochemical (IHC) techniques employing three chromogens plus a hematoxylin counterstain. The samples were imaged using the Nuance system, a commercially available, liquid-crystal tunable-filter-based multispectral imaging platform. The resulting data sets were analyzed using spectral unmixing algorithms and/or learn-by-example classification tools. RESULTS: Spectral unmixing of Giemsa-stained guinea-pig blood films readily classified the major blood elements. Machine-learning classifiers were also successful at the same task, as well in distinguishing normal from malignant regions in a colon-cancer example, and in delineating regions of inflammation in an H&E-stained kidney sample. In an example of a multiplexed ICH sample, brown, red, and blue chromogens were isolated into separate images without crosstalk or interference from the (also blue) hematoxylin counterstain. CONCLUSION: Cytomics requires both accurate architectural segmentation as well as multiplexed molecular imaging to associate molecular phenotypes with relevant cellular and tissue compartments. Multispectral imaging can assist in both these tasks, and conveys new utility to brightfield-based microscopy approaches.

Animals↗
PubMed2026

Menopausal timing and senescent-immune coupling in age-related lobular involution of the human breast: a longitudinal cohort study.

BACKGROUND: Incomplete postmenopausal breast involution leaves persistent epithelial-rich lobules and elevated breast density in about 40% of women and is associated with higher breast cancer risk, but why remodelling stalls remains unclear. METHODS: We studied a longitudinal cohort of 81 women with paired benign breast biopsies (baseline age 45-55 years; follow-up 2-10 years), all with baseline NanoString transcriptomics and two-timepoint digital morphometry, and with multiplex immunofluorescence in spatial-imaging subsets (baseline n = 14-16 depending on panel; follow-up n = 14). A separate postmenopausal endpoint cohort (12 women: eight noninvoluted, four completely involuted), profiled by genome-wide expression array and multiplex immunofluorescence, defined the persistent-lobule phenotype. FINDINGS: Noninvoluted postmenopausal tissue retained a proliferation-competent, tumour-associated epithelial state and showed immune accumulation at lobular boundaries with reduced access to p16+ (senescence-associated) epithelial foci. The same SASP and innate immune programmes that predicted slower involution across the menopausal transition predicted faster involution after menopause. Follow-up boundary CD45&#x2192;p16 engagement was directionally consistent with this reversal in Pre&#x2192;Post and Post&#x2192;Post women. Spatial imaging resolved this reversal into a perimenopausal stall architecture and a postmenopausal clearance-associated architecture marked by direct CD16+ innate-effector engagement of p16+ epithelium; macrophage targeting provided convergent support (two-sided exact permutation interaction p = 0.0077). INTERPRETATION: Menopausal timing conditions whether senescent-immune programmes couple to productive clearance or to spatially uncoupled surveillance and persistent risk-associated tissue. Biomarker interpretation should therefore be anchored to menopausal timing. FUNDING: Casey DeSantis Cancer Fund and US National Cancer Institute.

Humans↗