The Experts below are selected from a list of 9216 Experts worldwide ranked by ideXlab platform
Tianlu Cheng - One of the best experts on this subject based on the ideXlab platform.
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an activity based near infrared glucuronide trapping probe for imaging β glucuronidase expression in deep tissues
Journal of the American Chemical Society, 2012Co-Authors: Tachun Cheng, Kuohsiang Chuang, Chienshu Chen, Ihong Harn, Chihhung Chuang, Yucheng Su, Shey Cherng Tzou, Steve R. Roffler, Tianlu ChengAbstract:β-glucuronidase is an attractive reporter and prodrug-converting enzyme. The development of near-IR (NIR) probes for imaging of β-glucuronidase activity would be ideal to allow estimation of reporter expression and for personalized glucuronide prodrug cancer therapy in preclinical studies. However, NIR glucuronide probes are not yet available. In this work, we developed two fluorescent probes for detection of β-glucuronidase activity, one for the NIR range (containing IR-820 dye) and the other for the visible range [containing fluorescein isothiocyanate (FITC)], by utilizing a difluoromethylphenol–glucuronide moiety (TrapG) to trap the Fluorochromes in the vicinity of the active enzyme. β-glucuronidase-mediated hydrolysis of the glucuronyl bond of TrapG generates a highly reactive alkylating group that facilitates the attachment of the Fluorochrome to nucleophilic moieties located near β-glucuronidase-expressing sites. FITC-TrapG was selectively trapped on purified β-glucuronidase or β-glucuronidase-expre...
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an activity based near infrared glucuronide trapping probe for imaging β glucuronidase expression in deep tissues
Journal of the American Chemical Society, 2012Co-Authors: Tachun Cheng, Kuohsiang Chuang, Chienshu Chen, Ihong Harn, Chihhung Chuang, Shey Cherng Tzou, Steve R. Roffler, Chienhan Kao, Kuanyi Liu, Tianlu ChengAbstract:β-glucuronidase is an attractive reporter and prodrug-converting enzyme. The development of near-IR (NIR) probes for imaging of β-glucuronidase activity would be ideal to allow estimation of reporter expression and for personalized glucuronide prodrug cancer therapy in preclinical studies. However, NIR glucuronide probes are not yet available. In this work, we developed two fluorescent probes for detection of β-glucuronidase activity, one for the NIR range (containing IR-820 dye) and the other for the visible range [containing fluorescein isothiocyanate (FITC)], by utilizing a difluoromethylphenol-glucuronide moiety (TrapG) to trap the Fluorochromes in the vicinity of the active enzyme. β-glucuronidase-mediated hydrolysis of the glucuronyl bond of TrapG generates a highly reactive alkylating group that facilitates the attachment of the Fluorochrome to nucleophilic moieties located near β-glucuronidase-expressing sites. FITC-TrapG was selectively trapped on purified β-glucuronidase or β-glucuronidase-expressing CT26 cells (CT26/mβG) but not on bovine serum albumin or non-β-glucuronidase-expressing CT26 cells used as controls. β-glucuronidase-activated FITC-TrapG did not interfere with β-glucuronidase activity and could label bystander proteins near β-glucuronidase. Both FITC-TrapG and NIR-TrapG specifically imaged subcutaneous CT26/mβG tumors, but only NIR-TrapG could image CT26/mβG tumors transplanted deep in the liver. Thus NIR-TrapG may provide a valuable tool for visualizing β-glucuronidase activity in vivo.
Ralph Weissleder - One of the best experts on this subject based on the ideXlab platform.
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a dual Fluorochrome probe for imaging proteases
Bioconjugate Chemistry, 2004Co-Authors: Moritz F Kircher, Ralph Weissleder, Lee JosephsonAbstract:Near-infrared fluorescence (NIRF) optical probes have been able to provide a noninvasive assessment of enzyme activity for a number of different enzymes and types of pathology. Here we describe a dual Fluorochrome enzyme-activatable probe featuring one NIRF Fluorochrome that is activated by protease activity and a second Fluorochrome that is protease resistant and serves as an internal standard. The probe was prepared by attaching Cy7 directly to an amino-CLIO, an amine functional cross-linked iron oxide (CLIO) nanoparticle carrier, in a protease resistant manner. Cy5.5 was attached to a protease sensitive polyarginine peptide spacer, also attached to amino-CLIO. In vitro and in vivo the ratio of the Cy5.5 to Cy7 fluorescence was increased by protease, reflecting the increase in Cy5.5 fluorescence by protease in the vicinity of the probe. In vitro and in vivo the absolute values of the Cy5.5 and Cy7 fluorescence reflected lesion size and the distance of lesions from the surface, while the ratio of Cy5.5 to Cy7 fluorescence obtained was constant and independent of lesion size and depth. The dual Fluorochrome probe, and related dual wavelength imaging method, represents a novel approach for imaging protease activity in vivo.
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charge coupled device based scanner for tomography of fluorescent near infrared probes in turbid media
Medical Physics, 2002Co-Authors: Vasilis Ntziachristos, Ralph WeisslederAbstract:We present a novel tomographer for three-dimensional reconstructions of Fluorochromes in diffuse media. Photon detection is based on charge-coupled device technology that allows the implementation of a large parallel array of detection channels with high sensitivity. Using this instrument we studied the response and detection limits of near-infrared Fluorochromes in diffuse media as a function of light intensity and for a wide range of biologically relevant concentrations. We further examined the resolution of the scanner and the reconstruction linearity achieved. We demonstrate that the instrument attains better than 3 mm resolution, is linear within more than two orders of magnitude of Fluorochrome concentration, and can detect fluorescent objects at femto-mole quantities in small animal-like geometries. These measurements delineate detection and reconstructioncharacteristics associated with imaging of novel classes of fluorescent probes developed for in vivo molecular and functional probing of tissues.
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novel near infrared cyanine Fluorochromes synthesis properties and bioconjugation
Bioconjugate Chemistry, 2002Co-Authors: Ralph Weissleder, Chinghsuan TungAbstract:Recently, near-infrared (NIR) fluorescence light has been applied to image various biological events in vivo, because it penetrates tissue more efficiently than light in the visible spectrum. Compounds exhibiting fluorescent properties in the NIR range are key elements for this upcoming optical imaging technology. In this paper, we report the synthesis of four new, water-soluble NIR cyanine Fluorochromes which have superior chemical stability and optical properties. Each Fluorochrome was designed with a monoreactive carboxyl group for labeling purposes. When multiple Fluorochromes were attached to a single macromolecule, fluorescence quenching was observed. On the basis of this property, a novel autoquenched enzyme sensitive NIR fluorescence probe was prepared.
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experimental three dimensional fluorescence reconstruction of diffuse media by use of a normalized born approximation
Optics Letters, 2001Co-Authors: Vasilis Ntziachristos, Ralph WeisslederAbstract:We present a normalized Born expansion that facilitates fluorescence reconstructions in turbid, tissuelike media. The algorithm can be particularly useful for tissue investigations of Fluorochrome distributionin vivo, since it does not require absolute photon-field measurements or measurements before contrast-agent administration. This unique advantage can be achieved only in fluorescence mode. We used this algorithm to three-dimensionally image and quantify an indocyanine Fluorochrome phantom, using a novel fluorescence tomographic imager developed for animals.
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preparation of a cathepsin d sensitive near infrared fluorescence probe for imaging
Bioconjugate Chemistry, 1999Co-Authors: Chinghsuan Tung, Umar Mahmood, Sebastian Bredow, Ralph WeisslederAbstract:A variety of proteases are overexpressed or activated during pathogenesis and represent important targets for therapeutic drugs. We have previously shown that optical imaging probes sensitive in the near-infrared fluorescence (NIRF) spectrum can be used for in vivo imaging of enzyme activity. In the current study, we show that these probes can be designed with specificity for specific enzymes, for example, cathepsin D which is known to be overexpressed in many tumors. A NIR cyanine Fluorochrome served as the optical reporter and was attached to the amino terminal of an 11 amino acid peptide sequence with specificity for cathepsin D. The peptides were subsequently attached to a synthetic graft copolymer for efficient tumoral delivery. The close spatial proximity of the multiple Fluorochromes resulted in quenching of fluorescence in the bound state. A 350-fold signal amplification was observed post cleavage during in vitro testing. Cell culture experiments using a rodent tumor cell line stably transfected w...
Joost D De Bruijn - One of the best experts on this subject based on the ideXlab platform.
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use of Fluorochrome labels in in vivo bone tissue engineering research
Tissue Engineering Part B-reviews, 2010Co-Authors: Steven M Van Gaalen, Moyo C Kruyt, Ruth E Geuze, Joost D De Bruijn, Jacqueline Alblas, Wouter J A DhertAbstract:The use of Fluorochromes in bone research is a widely accepted technique that dates back to the 1950s. Several pioneers, such as Harold Frost, have thoroughly investigated the potential of Fluorochrome use for the study on bone formation and bone remodeling dynamics. Since the development of bone tissue engineering, a renewed interest in the benefits of Fluorochrome use was perceived. Fluorochrome use in animal models makes it possible to determine the onset time and location of osteogenesis, which are the fundamental parameters in bone tissue engineering studies. There is, however, a lack of standardized procedures for using this technique. In addition, many types of Fluorochromes exist and one could be confused upon selecting the appropriate type, the appropriate concentration, the route of administration, and methods of visualization. All these variables can potentially affect the outcome during fluorescence microscopy. This work aims at providing the bone tissue engineering researcher with an overview of the history, working mechanism, and the potential pitfalls in the use of Fluorochromes in animal studies. Experiments using some of the more frequently used Fluorochromes are explained and illustrated.
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analysis of ectopic and orthotopic bone formation in cell based tissue engineered constructs in goats
Biomaterials, 2007Co-Authors: Moyo C Kruyt, Wouter J A Dhert, Cumhur F Oner, Clemens Van Blitterswijk, A J Verbout, Joost D De BruijnAbstract:Despite decades of extensive research, the application of cell-based bone tissue engineering in clinically relevant models remains challenging. To improve effectiveness, a better understanding of how the technique should work is crucial. In the current study, we investigated the onset time, rate, location and direction of bone formation in ectopically and orthotopically implanted clinically sized tissue-engineered constructs to gain insight the mechanism behind it. Bone marrow stromal cells (BMSCs) were obtained from 10 goats, culture expanded and cryopreserved. Porous biphasic calcium phosphate (BCP) disks of 17 mm×6 mm were per-operatively seeded with BMSCs or left empty. Both conditions were implanted intramuscularly and in bilateral critical-sized iliac wing defects. Fluorochromes were administered at 3, 5 and 7 weeks and samples were retrieved after 9 weeks. Histology showed abundant and homogeneous bone formation throughout the intramuscular BMSC samples and little bone in the controls. Histomorphometry and measurements of the Fluorochrome labels of the ectopical BMSC samples indicated that osteogenesis started at the periphery and subsequent osteoconduction filled the whole scaffold within 7 weeks. In the orthotopically implanted disks, there was good integration with the surrounding bone, but minimal bone in the center of the implants, in both conditions. Bone was only derived from the interface with the surrounding bone, there was no early bone at the surfaces in contact to soft tissue as was seen in the ectopical samples. Apparently cell survival was minimal and insufficient for relevant additional bone formation. However, the speed of integration with surrounding bone and subsequent bone apposition on the BMSC-seeded orthotopic scaffolds were found to be significantly enhanced, which may be relevant especially in challenging environments.
Elzbieta Bedner - One of the best experts on this subject based on the ideXlab platform.
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analysis of apoptotic cells by flow and laser scanning cytometry
Methods in Enzymology, 2000Co-Authors: Zbigniew Darzynkiewicz, Elzbieta BednerAbstract:A large number of flow cytometric methods to identify apoptotic cells and analyze morphological, biochemical, and molecular changes that occur during apoptosis have been developed. These methods are also applicable to the laser scanning cytometer (LSC), a microscope-based cytofluorometer that combines advantages of flow and image cytometry and that, by offering a possibility of assessment of cell morphology, is of particular utility in analysis of apoptosis. Apoptosis-related changes in cell morphology associated with cell shrinkage and condensation of cytoplasm and chromatin are detected by measurements of the intensity of light scatter of the laser beam in the forward and 90 degrees angle directions. Changes in plasma membrane composition and function are analyzed by its altered permeability to certain dyes and by the appearance of phosphatidylserine, which reacts with annexin V-Fluorochrome conjugates on the external surface of the membrane. Decrease in mitochondrial transmembrane potential is measured with several Fluorochromes of the rhodamine or carbocyanine family. DNA fragmentation is detected either by measurement of cellular DNA content after elution of the degraded DNA from the cell before or during the staining procedure or by in situ labeling DNA strand breaks. Apoptotic cells are then recognized either on the basis of their reduced DNA-associated fluorescence as the cells with fractional DNA content ("sub-G1 cells"), or as the cells with an extensive number of DNA breaks, respectively. Advantages and limitations of the preceding methods are discussed and their adaptation to LSC is presented.
Wouter J A Dhert - One of the best experts on this subject based on the ideXlab platform.
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use of Fluorochrome labels in in vivo bone tissue engineering research
Tissue Engineering Part B-reviews, 2010Co-Authors: Steven M Van Gaalen, Moyo C Kruyt, Ruth E Geuze, Joost D De Bruijn, Jacqueline Alblas, Wouter J A DhertAbstract:The use of Fluorochromes in bone research is a widely accepted technique that dates back to the 1950s. Several pioneers, such as Harold Frost, have thoroughly investigated the potential of Fluorochrome use for the study on bone formation and bone remodeling dynamics. Since the development of bone tissue engineering, a renewed interest in the benefits of Fluorochrome use was perceived. Fluorochrome use in animal models makes it possible to determine the onset time and location of osteogenesis, which are the fundamental parameters in bone tissue engineering studies. There is, however, a lack of standardized procedures for using this technique. In addition, many types of Fluorochromes exist and one could be confused upon selecting the appropriate type, the appropriate concentration, the route of administration, and methods of visualization. All these variables can potentially affect the outcome during fluorescence microscopy. This work aims at providing the bone tissue engineering researcher with an overview of the history, working mechanism, and the potential pitfalls in the use of Fluorochromes in animal studies. Experiments using some of the more frequently used Fluorochromes are explained and illustrated.
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analysis of ectopic and orthotopic bone formation in cell based tissue engineered constructs in goats
Biomaterials, 2007Co-Authors: Moyo C Kruyt, Wouter J A Dhert, Cumhur F Oner, Clemens Van Blitterswijk, A J Verbout, Joost D De BruijnAbstract:Despite decades of extensive research, the application of cell-based bone tissue engineering in clinically relevant models remains challenging. To improve effectiveness, a better understanding of how the technique should work is crucial. In the current study, we investigated the onset time, rate, location and direction of bone formation in ectopically and orthotopically implanted clinically sized tissue-engineered constructs to gain insight the mechanism behind it. Bone marrow stromal cells (BMSCs) were obtained from 10 goats, culture expanded and cryopreserved. Porous biphasic calcium phosphate (BCP) disks of 17 mm×6 mm were per-operatively seeded with BMSCs or left empty. Both conditions were implanted intramuscularly and in bilateral critical-sized iliac wing defects. Fluorochromes were administered at 3, 5 and 7 weeks and samples were retrieved after 9 weeks. Histology showed abundant and homogeneous bone formation throughout the intramuscular BMSC samples and little bone in the controls. Histomorphometry and measurements of the Fluorochrome labels of the ectopical BMSC samples indicated that osteogenesis started at the periphery and subsequent osteoconduction filled the whole scaffold within 7 weeks. In the orthotopically implanted disks, there was good integration with the surrounding bone, but minimal bone in the center of the implants, in both conditions. Bone was only derived from the interface with the surrounding bone, there was no early bone at the surfaces in contact to soft tissue as was seen in the ectopical samples. Apparently cell survival was minimal and insufficient for relevant additional bone formation. However, the speed of integration with surrounding bone and subsequent bone apposition on the BMSC-seeded orthotopic scaffolds were found to be significantly enhanced, which may be relevant especially in challenging environments.