Gregory Fisher - Academia.edu (original) (raw)
Papers by Gregory Fisher
Nature Biotechnology, 2008
In the version of this article initially published online, an author's middle initial was incorre... more In the version of this article initially published online, an author's middle initial was incorrectly given as "A". Brigitte A Schmidt should be Brigitte F Schmidt in the author list, and B.A.S should be B.F.S. in the authors' contribution section. The error has been corrected in the HTML and PDF versions of the article.
Journal of Virology, 2014
The chemokine receptor CCR5 is essential for HIV infection and is thus a potential target for vac... more The chemokine receptor CCR5 is essential for HIV infection and is thus a potential target for vaccine development. However, because CCR5 is a host protein, generation of anti-CCR5 antibodies requires the breaking of immune tolerance and thus carries the risk of autoimmune responses. In this study, performed in mice, we compared 3 different immunogens representing surface domains of murine CCR5, 4 different adjuvants, and 13 different immunization protocols, with the goal of eliciting HIV-blocking activity without inducing autoimmune dysfunction. In all cases the CCR5 sequences were presented as fusions to the Flock House virus (FHV) capsid precursor protein. We found that systemic immunization and mucosal boosting elicited CCR5-specific antibodies and achieved consistent priming in Peyer's patches, where most cells showed a phenotype corresponding to activated B cells and secreted high levels of IgA, representing up to one-third of the total HIV-blocking activity. Histopathologi...
Journal of the American Chemical Society, 2003
Peptide nucleic acid (PNA) is a synthetic analogue of DNA and RNA in which the natural sugar-phos... more Peptide nucleic acid (PNA) is a synthetic analogue of DNA and RNA in which the natural sugar-phosphate backbone has been replaced by achiral N-(2-aminoethyl) glycine units (Scheme 1). 1 PNA can form sequence-specific hybrids with complementary DNA and RNA strands in accordance with the Watson-Crick base pairing rules. The resulting hybrid duplexes exhibit exceptional thermal stability, while the unnatural backbone renders PNA stable to both nucleases and proteases. Since its initial report, PNA has spawned considerable interest in its development as therapeutics, diagnostics, and molecular tools for basic research.
SLAS Discovery, 2010
Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this art... more Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this article, the authors describe a new kind of live-cell biosensor of receptor internalization that takes advantage of fluorogen-activating protein (FAP) technology. Recombinant genes that express the human beta2 adrenergic receptor (β2AR) with FAP domains at their extracellular N-termini were transduced into mammalian cells. Exposure of the cells to membrane-impermeant fluorogens led to a strong fluorescent signal from the cell surface. Agonist-dependent translocation of the receptor from the surface to the cell interior was readily observed and quantified by fluorescence microscopy or flow cytometry in a homogeneous format without wash or separation steps. The approach described here is generalizable to other receptors and cell surface proteins and is adaptable to a variety of fluorescence-based high-throughput screening platforms.
Cytometry Part A, 2010
This study explores the general utility of a new class of biosensor that allows one to selectivel... more This study explores the general utility of a new class of biosensor that allows one to selectively visualize molecules of a chosen membrane protein that are at the cell surface. These biosensors make use of recently described bipartite fluoromodules comprised of a fluorogen-activating protein (FAP) and a small molecule (fluorogen) whose fluorescence increases dramatically when noncovalently bound by the FAP (Szent-Gyorgyi et al., Nat Biotechnol 2008;26:235-240). ' 2010 International Society for Advancement of Cytometry Key terms reporter protein; membrane protein; live cell assay; cell surface labeling; green fluorescent protein, GFP; fluorogen activating protein, FAP; b 2 AR; GLUT4; CFTR CELLS that express single-pass recombinant membrane proteins, each presenting a FAP on the exterior face of the plasma membrane and a standard fluorescent protein (EGFP or mRFP) on the interior face, were generated and examined by fluorescence microscopy. In each case, fluorescent signal was observed exclusively at the cell surface when the FAP domain was imaged using membrane-impermeant fluorogen but was observed in additional intracellular locations when the fluorescent protein domain was imaged. Cells that expressed external N-terminal FAP-fusions to three wellstudied human membrane proteins-the b2 adrenergic receptor (b 2 AR), the insulinregulated glucose transporter (GLUT4), and the cystic fibrosis transmembrane conductance regulator (CFTR)-were also generated and examined; these too showed fluorescent signal exclusively at the cell surface after exposure to membrane-impermeant fluorogen. Further, when endocytosis of tagged b 2 AR was stimulated by agonist treatment in the presence of fluorogen, fluorescent signal was seen to transit from the surface to the cell interior. FAP tagging thus provides a means for selectively visualizing plasma membrane proteins and for monitoring the trafficking of these proteins to and from the cell surface. Plasma membrane proteins play roles in thousands of cellular processes and are the targets of more than half of all therapeutic drugs. Many of these proteins exhibit regulated translocation between the cell surface and the cell interior. For example, most G-protein coupled receptors (GPCRs) and receptor tyrosine kinases are internalized by endocytosis after exposure to agonists (1,2), and numerous ion and metabolite transporters traffic to the membrane in response to particular physiological stimuli (3). Many methods are available to selectively label particular plasma membrane proteins in living cells with fluorescent molecules so as to study membrane protein trafficking. These include immunofluorescence with specific antibodies or antibodies to epitope tags, labeling of tetracysteine (TC) tagged fusion proteins with FlAsH or ReAsH reagents, covalent attachment of fluorophores using SNAP-tag or Halo Tag
Journal of Geophysical Research: Biogeosciences, 2007
The science goals of the Life in the Atacama (LITA) robotic field experiment are to understand ha... more The science goals of the Life in the Atacama (LITA) robotic field experiment are to understand habitat and seek out life in the Atacama Desert, Chile, as an analog to future missions to Mars. To those ends, we present a new data analysis tool, the LITA Data Scoring System (DSS), which (1) integrates rover and orbital data relevant to environmental habitability and life detection, and (2) provides a standard metric, or ''score'' to evaluate (a) the potential habitability, and (b) the strength of evidence for life at all locales along the rover's traverse. Designed and tested during the 2005 field campaign, first results from the DSS indicate that the three selected sites in the Atacama Desert are generally inhospitable. The strength of evidence for life is positively correlated with potential habitability at two of the three sites. Using factor analysis, we find three factors explain 79.9% of the variance in biological observations and five factors explain 96.2% of the variance in potential habitability across all sites. These factors are used to focus a discussion of scoring variable definitions for future robotic missions in the Atacama and of instrument selection and strategy development for future robotic missions on Earth and Mars.
Journal of Geophysical Research, 2007
Journal of biomolecular screening, Jan 6, 2015
A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to tr... more A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluor...
Arthritis & Rheumatology
ABSTRACT
A novel assay using fluorogen activating peptide (FAP) technology for G protein-coupled receptor ... more A novel assay using fluorogen activating peptide (FAP) technology for G protein-coupled receptor (GPCR) activation and internalization was applied to the human β2AR. This technology avoids microscopy and antibody-based detection methods. A major goal for the project was to identify G-protein independent/β2AR ligands or β2AR biased ligands that induce β2AR internalization. Analysis of the most potent hits in the primary project revealed that they interfered with fluorogen activation by the FAP rather than interacting with the receptor itself. These molecules were pursued further because they had the potential to enable improved assay protocols to monitor receptor trafficking and receptor location in real time. A highly potent compound (ML342, CID 2953239) was declared as a Molecular Libraries Probe Center Network (MLPCN) probe molecule.
Cell Differentiation, 1982
Ultrasensitive Biochemical Diagnostics, 1996
ABSTRACT
Journal of biomolecular screening, Jan 12, 2014
G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiologica... more G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology-receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both a...
Cell Structure and Function by Microspectrofluorometry, 1989
BioTechniques, 2002
A self-inactivating CD-tagging retroviral vector was used to introduce epitope and GFP tags into ... more A self-inactivating CD-tagging retroviral vector was used to introduce epitope and GFP tags into genes and proteins in NIH 3T3 cells. Several hundred cell clones, each expressing GFP fluorescence in a distinctive pattern, were isolated. Molecular analysis showed that a wide variety of genes and proteins, some known and some newly discovered, had been tagged. The analysis also revealed that, in the great majority of instances, the abundance and cellular location of the tagged protein mirrored that of its untagged counterpart. This approach provides a systematic means for the functional annotation of mammalian genomes and proteomes in living cells.
Molecular medicine (Cambridge, Mass.), 2002
Two prominent biological features of the advanced stages of human melanoma are their high degree ... more Two prominent biological features of the advanced stages of human melanoma are their high degree of vascularity and high-level expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1). Given these characteristics, human melanoma serves as an ideal model to address an important question regarding the efficacy of angiogenesis-based cancer therapy. To induce tumor growth arrest and regression, does it suffice to block expression of bFGF and/or FGFR-1 in only the melanoma cells, or is it essential to inhibit expression of bFGF and/or FGFR-1 in both the melanoma cells and the melanoma cell-interspersing vasculature? Primary and metastatic human melanomas, grown as subcutaneous tumors in nude mice, were injected twice a week with vector constructs containing the human tyrosinase promoter and antisense- oriented human bFGF or FGFR-1 cDNA. On alternating days, the bFGF and FGFR-1 antisense-targeted tumors received injections of cyanine fluorochrom...
The Journal of cell biology, 1987
Fluorescence ratio imaging microscopy (Tanasugarn, L., P. McNeil, G. Reynolds, and D. L. Taylor, ... more Fluorescence ratio imaging microscopy (Tanasugarn, L., P. McNeil, G. Reynolds, and D. L. Taylor, 1984, J. Cell Biol., 98:717-724) has been used to measure the spatial variations in cytoplasmic pH of individual quiescent and nonquiescent Swiss 3T3 cells. Fundamental issues of ratio imaging that permit precise and accurate temporal and spatial measurements have been addressed including: excitation light levels, lamp operation, intracellular probe concentrations, methods of threshold selection, photobleaching, and spatial signal-to-noise ratio measurements. Subcellular measurements can be measured accurately (less than 3% coefficient of variation) in an area of 3.65 microns 2 with the present imaging system. Quiescent Swiss 3T3 cells have a measured cytoplasmic pH of 7.09 (0.01 SEM), whereas nonquiescent cells have a pH of 7.35 (0.01 SEM) in the presence of bicarbonate buffer. A unimodal distribution of mean cytoplasmic pH in both quiescent and nonquiescent cells was identified from po...
Lecture Notes in Computer Science, 1997
Light is a most versatile tool for investigating biological systems and phenomena; the range, non... more Light is a most versatile tool for investigating biological systems and phenomena; the range, non-destructiveness, spatial discrimination and speed of optical imaging are all important for investigating biological structure and function at the cellular, tissue or even whole organism level. In live biological imaging, where the technological requirements are heightened by the challenges posed, other features of light, such as coherence and wavelength, are used to generate the additional contrast and resolution needed. We report here the recent improvements in our ability to image biological specimens optically, focusing on (a) spectral imaging and the related image processing issues, and (b) tomographic threedimensional fluorescence imaging in vivo.
Ultrasensitive Instrumentation for DNA Sequencing and Biochemical Diagnostics, 1995
ABSTRACT There is a clear trend today towards non-invasive, dynamic, digital approaches to biomed... more ABSTRACT There is a clear trend today towards non-invasive, dynamic, digital approaches to biomedical imaging, and a need for even higher resolution. Light is particularly well suited for such investigations, as its temporal, spatial and intensity range are unparalleled. A convergence of new capabilities from fields as diverse as electronics, optics, molecular biology, computer science and dye chemistry have transformed light microscopy from a traditional, static, 2D tool into a highly useful, dynamic, 3D research capability for biology and medicine. We believe that the understanding of certain fundamental biological functions by dynamic mapping of events in living systems is within reach, based on novel, interdisciplinary methods. For imaging molecular events with high resolution (live cells, in vitro), light microscopy has continued to improve in performance, and we survey here some of our recent progress. The same dynamic mapping can be extended to organs, whole animals and humans, by monitoring molecules labeled with the long-wavelength dyes that proved useful in microscopy. We report here results obtained by in vivo imaging of fluorescently labeled monoclonal antibodies, indicative of tumor location and evolution in nude mice.
Nature Biotechnology, 2008
In the version of this article initially published online, an author's middle initial was incorre... more In the version of this article initially published online, an author's middle initial was incorrectly given as "A". Brigitte A Schmidt should be Brigitte F Schmidt in the author list, and B.A.S should be B.F.S. in the authors' contribution section. The error has been corrected in the HTML and PDF versions of the article.
Journal of Virology, 2014
The chemokine receptor CCR5 is essential for HIV infection and is thus a potential target for vac... more The chemokine receptor CCR5 is essential for HIV infection and is thus a potential target for vaccine development. However, because CCR5 is a host protein, generation of anti-CCR5 antibodies requires the breaking of immune tolerance and thus carries the risk of autoimmune responses. In this study, performed in mice, we compared 3 different immunogens representing surface domains of murine CCR5, 4 different adjuvants, and 13 different immunization protocols, with the goal of eliciting HIV-blocking activity without inducing autoimmune dysfunction. In all cases the CCR5 sequences were presented as fusions to the Flock House virus (FHV) capsid precursor protein. We found that systemic immunization and mucosal boosting elicited CCR5-specific antibodies and achieved consistent priming in Peyer's patches, where most cells showed a phenotype corresponding to activated B cells and secreted high levels of IgA, representing up to one-third of the total HIV-blocking activity. Histopathologi...
Journal of the American Chemical Society, 2003
Peptide nucleic acid (PNA) is a synthetic analogue of DNA and RNA in which the natural sugar-phos... more Peptide nucleic acid (PNA) is a synthetic analogue of DNA and RNA in which the natural sugar-phosphate backbone has been replaced by achiral N-(2-aminoethyl) glycine units (Scheme 1). 1 PNA can form sequence-specific hybrids with complementary DNA and RNA strands in accordance with the Watson-Crick base pairing rules. The resulting hybrid duplexes exhibit exceptional thermal stability, while the unnatural backbone renders PNA stable to both nucleases and proteases. Since its initial report, PNA has spawned considerable interest in its development as therapeutics, diagnostics, and molecular tools for basic research.
SLAS Discovery, 2010
Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this art... more Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this article, the authors describe a new kind of live-cell biosensor of receptor internalization that takes advantage of fluorogen-activating protein (FAP) technology. Recombinant genes that express the human beta2 adrenergic receptor (β2AR) with FAP domains at their extracellular N-termini were transduced into mammalian cells. Exposure of the cells to membrane-impermeant fluorogens led to a strong fluorescent signal from the cell surface. Agonist-dependent translocation of the receptor from the surface to the cell interior was readily observed and quantified by fluorescence microscopy or flow cytometry in a homogeneous format without wash or separation steps. The approach described here is generalizable to other receptors and cell surface proteins and is adaptable to a variety of fluorescence-based high-throughput screening platforms.
Cytometry Part A, 2010
This study explores the general utility of a new class of biosensor that allows one to selectivel... more This study explores the general utility of a new class of biosensor that allows one to selectively visualize molecules of a chosen membrane protein that are at the cell surface. These biosensors make use of recently described bipartite fluoromodules comprised of a fluorogen-activating protein (FAP) and a small molecule (fluorogen) whose fluorescence increases dramatically when noncovalently bound by the FAP (Szent-Gyorgyi et al., Nat Biotechnol 2008;26:235-240). ' 2010 International Society for Advancement of Cytometry Key terms reporter protein; membrane protein; live cell assay; cell surface labeling; green fluorescent protein, GFP; fluorogen activating protein, FAP; b 2 AR; GLUT4; CFTR CELLS that express single-pass recombinant membrane proteins, each presenting a FAP on the exterior face of the plasma membrane and a standard fluorescent protein (EGFP or mRFP) on the interior face, were generated and examined by fluorescence microscopy. In each case, fluorescent signal was observed exclusively at the cell surface when the FAP domain was imaged using membrane-impermeant fluorogen but was observed in additional intracellular locations when the fluorescent protein domain was imaged. Cells that expressed external N-terminal FAP-fusions to three wellstudied human membrane proteins-the b2 adrenergic receptor (b 2 AR), the insulinregulated glucose transporter (GLUT4), and the cystic fibrosis transmembrane conductance regulator (CFTR)-were also generated and examined; these too showed fluorescent signal exclusively at the cell surface after exposure to membrane-impermeant fluorogen. Further, when endocytosis of tagged b 2 AR was stimulated by agonist treatment in the presence of fluorogen, fluorescent signal was seen to transit from the surface to the cell interior. FAP tagging thus provides a means for selectively visualizing plasma membrane proteins and for monitoring the trafficking of these proteins to and from the cell surface. Plasma membrane proteins play roles in thousands of cellular processes and are the targets of more than half of all therapeutic drugs. Many of these proteins exhibit regulated translocation between the cell surface and the cell interior. For example, most G-protein coupled receptors (GPCRs) and receptor tyrosine kinases are internalized by endocytosis after exposure to agonists (1,2), and numerous ion and metabolite transporters traffic to the membrane in response to particular physiological stimuli (3). Many methods are available to selectively label particular plasma membrane proteins in living cells with fluorescent molecules so as to study membrane protein trafficking. These include immunofluorescence with specific antibodies or antibodies to epitope tags, labeling of tetracysteine (TC) tagged fusion proteins with FlAsH or ReAsH reagents, covalent attachment of fluorophores using SNAP-tag or Halo Tag
Journal of Geophysical Research: Biogeosciences, 2007
The science goals of the Life in the Atacama (LITA) robotic field experiment are to understand ha... more The science goals of the Life in the Atacama (LITA) robotic field experiment are to understand habitat and seek out life in the Atacama Desert, Chile, as an analog to future missions to Mars. To those ends, we present a new data analysis tool, the LITA Data Scoring System (DSS), which (1) integrates rover and orbital data relevant to environmental habitability and life detection, and (2) provides a standard metric, or ''score'' to evaluate (a) the potential habitability, and (b) the strength of evidence for life at all locales along the rover's traverse. Designed and tested during the 2005 field campaign, first results from the DSS indicate that the three selected sites in the Atacama Desert are generally inhospitable. The strength of evidence for life is positively correlated with potential habitability at two of the three sites. Using factor analysis, we find three factors explain 79.9% of the variance in biological observations and five factors explain 96.2% of the variance in potential habitability across all sites. These factors are used to focus a discussion of scoring variable definitions for future robotic missions in the Atacama and of instrument selection and strategy development for future robotic missions on Earth and Mars.
Journal of Geophysical Research, 2007
Journal of biomolecular screening, Jan 6, 2015
A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to tr... more A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluor...
Arthritis & Rheumatology
ABSTRACT
A novel assay using fluorogen activating peptide (FAP) technology for G protein-coupled receptor ... more A novel assay using fluorogen activating peptide (FAP) technology for G protein-coupled receptor (GPCR) activation and internalization was applied to the human β2AR. This technology avoids microscopy and antibody-based detection methods. A major goal for the project was to identify G-protein independent/β2AR ligands or β2AR biased ligands that induce β2AR internalization. Analysis of the most potent hits in the primary project revealed that they interfered with fluorogen activation by the FAP rather than interacting with the receptor itself. These molecules were pursued further because they had the potential to enable improved assay protocols to monitor receptor trafficking and receptor location in real time. A highly potent compound (ML342, CID 2953239) was declared as a Molecular Libraries Probe Center Network (MLPCN) probe molecule.
Cell Differentiation, 1982
Ultrasensitive Biochemical Diagnostics, 1996
ABSTRACT
Journal of biomolecular screening, Jan 12, 2014
G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiologica... more G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology-receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both a...
Cell Structure and Function by Microspectrofluorometry, 1989
BioTechniques, 2002
A self-inactivating CD-tagging retroviral vector was used to introduce epitope and GFP tags into ... more A self-inactivating CD-tagging retroviral vector was used to introduce epitope and GFP tags into genes and proteins in NIH 3T3 cells. Several hundred cell clones, each expressing GFP fluorescence in a distinctive pattern, were isolated. Molecular analysis showed that a wide variety of genes and proteins, some known and some newly discovered, had been tagged. The analysis also revealed that, in the great majority of instances, the abundance and cellular location of the tagged protein mirrored that of its untagged counterpart. This approach provides a systematic means for the functional annotation of mammalian genomes and proteomes in living cells.
Molecular medicine (Cambridge, Mass.), 2002
Two prominent biological features of the advanced stages of human melanoma are their high degree ... more Two prominent biological features of the advanced stages of human melanoma are their high degree of vascularity and high-level expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1). Given these characteristics, human melanoma serves as an ideal model to address an important question regarding the efficacy of angiogenesis-based cancer therapy. To induce tumor growth arrest and regression, does it suffice to block expression of bFGF and/or FGFR-1 in only the melanoma cells, or is it essential to inhibit expression of bFGF and/or FGFR-1 in both the melanoma cells and the melanoma cell-interspersing vasculature? Primary and metastatic human melanomas, grown as subcutaneous tumors in nude mice, were injected twice a week with vector constructs containing the human tyrosinase promoter and antisense- oriented human bFGF or FGFR-1 cDNA. On alternating days, the bFGF and FGFR-1 antisense-targeted tumors received injections of cyanine fluorochrom...
The Journal of cell biology, 1987
Fluorescence ratio imaging microscopy (Tanasugarn, L., P. McNeil, G. Reynolds, and D. L. Taylor, ... more Fluorescence ratio imaging microscopy (Tanasugarn, L., P. McNeil, G. Reynolds, and D. L. Taylor, 1984, J. Cell Biol., 98:717-724) has been used to measure the spatial variations in cytoplasmic pH of individual quiescent and nonquiescent Swiss 3T3 cells. Fundamental issues of ratio imaging that permit precise and accurate temporal and spatial measurements have been addressed including: excitation light levels, lamp operation, intracellular probe concentrations, methods of threshold selection, photobleaching, and spatial signal-to-noise ratio measurements. Subcellular measurements can be measured accurately (less than 3% coefficient of variation) in an area of 3.65 microns 2 with the present imaging system. Quiescent Swiss 3T3 cells have a measured cytoplasmic pH of 7.09 (0.01 SEM), whereas nonquiescent cells have a pH of 7.35 (0.01 SEM) in the presence of bicarbonate buffer. A unimodal distribution of mean cytoplasmic pH in both quiescent and nonquiescent cells was identified from po...
Lecture Notes in Computer Science, 1997
Light is a most versatile tool for investigating biological systems and phenomena; the range, non... more Light is a most versatile tool for investigating biological systems and phenomena; the range, non-destructiveness, spatial discrimination and speed of optical imaging are all important for investigating biological structure and function at the cellular, tissue or even whole organism level. In live biological imaging, where the technological requirements are heightened by the challenges posed, other features of light, such as coherence and wavelength, are used to generate the additional contrast and resolution needed. We report here the recent improvements in our ability to image biological specimens optically, focusing on (a) spectral imaging and the related image processing issues, and (b) tomographic threedimensional fluorescence imaging in vivo.
Ultrasensitive Instrumentation for DNA Sequencing and Biochemical Diagnostics, 1995
ABSTRACT There is a clear trend today towards non-invasive, dynamic, digital approaches to biomed... more ABSTRACT There is a clear trend today towards non-invasive, dynamic, digital approaches to biomedical imaging, and a need for even higher resolution. Light is particularly well suited for such investigations, as its temporal, spatial and intensity range are unparalleled. A convergence of new capabilities from fields as diverse as electronics, optics, molecular biology, computer science and dye chemistry have transformed light microscopy from a traditional, static, 2D tool into a highly useful, dynamic, 3D research capability for biology and medicine. We believe that the understanding of certain fundamental biological functions by dynamic mapping of events in living systems is within reach, based on novel, interdisciplinary methods. For imaging molecular events with high resolution (live cells, in vitro), light microscopy has continued to improve in performance, and we survey here some of our recent progress. The same dynamic mapping can be extended to organs, whole animals and humans, by monitoring molecules labeled with the long-wavelength dyes that proved useful in microscopy. We report here results obtained by in vivo imaging of fluorescently labeled monoclonal antibodies, indicative of tumor location and evolution in nude mice.