A system is described for performing multicolor fluorescence image cytometry of cell preparations... more A system is described for performing multicolor fluorescence image cytometry of cell preparations. After the setting up stage, the operation is automatic: the microscope fields are found and focused; then images are acquired for each fluorophore, corrected and analyzed, without any operator interaction. Human peripheral blood lymphocytes on microscope slides were used as a test system. In these experiments, three fluorescent antibodies were used to identify lymphocyte sub-populations, and a DNA content probe was used to identify all nucleated cells. The cell subset percentages determined by image cytometry were comparable to percentages obtained when cells from the same preparation were analyzed by flow cytometry. Multicolor fluorescence imaging cytometry can potentially be extended to the analysis of cells in smears, fine needle biopsies, imprints, and tissue sections.
We have investigated the Ca2+ -dependent interactions of villin, a protein of the intestinal micr... more We have investigated the Ca2+ -dependent interactions of villin, a protein of the intestinal microvillar core, with actin by monitoring resonance energy transfer between fluorescently labeled actin subunits. In the presence of elevated free Ca2+ (approximately 20 microM), villin affects both the nucleation and the elongation phases of actin polymerization. Consistent with previous reports, villin stimulates the nucleation process and will form stable nuclei under depolymerization conditions. Compared to the control, the net rate of polymerization is slightly inhibited at low concentrations of villin (villin/actin approximately 1:400) but is stimulated at higher concentrations (villin/actin greater than 1:100). Villin also significantly increases the critical concentration of actin polymerization. Addition of either villin or villin-actin complexes induces depolymerization of preassembled actin filaments. This villin-induced depolymerization is reversible upon removal of free Ca2+ or...
Cell-based target validation, secondary screening, lead optimization, and structure-activity rela... more Cell-based target validation, secondary screening, lead optimization, and structure-activity relationships have been recast with the advent of HCS. Prior to HCS, a computational approach to the characterization of the functions of specific target proteins and other cellular constituents, along with whole-cell functions employing fluorescence cell-based assays and microscopy, required extensive interaction among the researcher, instrumentation, and software tools. Early HCS platforms were instrument-centric and addressed the need to interface fully automated fluorescence microscopy, plate-handling automation, and seamless image analysis. HCS has since evolved into an integrated solution for accelerated drug discovery by encompassing the workflow components of assay and reagent design, robust instrumentation for automated fixed-end-point and live cell kinetic analysis, generalized and specific BioApplication software (Cellomics, Pittsburgh, PA) modules that produce information on drug responses from cell image data, and informatics/bioinformatics solutions that build knowledge from this information while providing a means to globalize HCS throughout an entire organization. This review communicates how these recent advances are incorporated into the drug discovery workflow by presenting a real-world use case.
The formation of lamellipodia in migrating cells involves dynamic processes that occur in a cycli... more The formation of lamellipodia in migrating cells involves dynamic processes that occur in a cyclic manner as the leading edge of a cell slowly advances. We used video-enhanced contrast microscopy (VEC) to monitor the motile behavior of cells to classify protrusions into the temporal stages of initial and established protrusions (Fisher et al.: Cell Motility and the Cytoskeleton 11:235-247, 1988), and to monitor the fixation of cells. Multiple parameter fluorescence imaging methods (DeBiasio et al.: Journal of Cell Biology 105:1613-1622, 1987; Waggoner et al.: Methods in Cell Biology, Vol. 30, Part B, pp. 449-478, 1989) were then used to determine and to map accurately the distributions of actin, myosin and microtubules in specific types of protrusions. Initial protrusions exhibited no substructure as evidenced by VEC and actin was diffusely arranged, while myosin and microtubules were absent. Newly established protrusions contained diffuse actin as well as actin in microspikes. Ther...
ABSTRACT Stress fibers in serum-deprived fibroblasts provide an excellent system for studying the... more ABSTRACT Stress fibers in serum-deprived fibroblasts provide an excellent system for studying the assembly dynamics of the actin-based cytoskeleton. Previous studies suggested that fibers containing actin and myosin II are restructured via two alternative pathways: coupled solation-contraction and disassembly without contraction. The former process appears to be regulated by Ca++, but regulation of the latter process has not been extensively explored. To assess the possible role of protein phosphorylation, we examined the dynamic behavior of stress fibers in cells treated with inhibitors of protein kinases. Swiss 3T3 fibroblasts were microinjected with fluorescent analogs of actin and myosin II and fiber dynamics monitored using light-microscope imaging. Staurosporine and KT5926 caused reversible dispersal of stress fibers without contraction, along with dephosphorylation of the regulatory light chain of myosin II, LC20. In order to make direct comparisons between the dose responses of these biochemical and morphological effects, fiber disruption was quantitated using two independent measures: total edge strength in fluorescent images of actin and myosin II, and fiber length determined by automated object-identification. Loss of stress fibers was shown to parallel LC20 dephosphorylation. Quantitation of cytoskeletal organization provides a framework for testing relationships between structural events and potential biochemical regulatory signals.
Optical Diagnostics of Living Cells and Biofluids, 1996
ABSTRACT The Automated Interactive Microscope is a robotic light microscope workstation that comb... more ABSTRACT The Automated Interactive Microscope is a robotic light microscope workstation that combines high performance light microscopy and computing to explore the chemical and molecular dynamics of cells and tissues.
A system is described for performing multicolor fluorescence image cytometry of cell preparations... more A system is described for performing multicolor fluorescence image cytometry of cell preparations. After the setting up stage, the operation is automatic: the microscope fields are found and focused; then images are acquired for each fluorophore, corrected and analyzed, without any operator interaction. Human peripheral blood lymphocytes on microscope slides were used as a test system. In these experiments, three fluorescent antibodies were used to identify lymphocyte sub-populations, and a DNA content probe was used to identify all nucleated cells. The cell subset percentages determined by image cytometry were comparable to percentages obtained when cells from the same preparation were analyzed by flow cytometry. Multicolor fluorescence imaging cytometry can potentially be extended to the analysis of cells in smears, fine needle biopsies, imprints, and tissue sections.
A system is described for performing multicolor fluorescence image cytometry of cell preparations... more A system is described for performing multicolor fluorescence image cytometry of cell preparations. After the setting up stage, the operation is automatic: the microscope fields are found and focused; then images are acquired for each fluorophore, corrected and analyzed, without any operator interaction. Human peripheral blood lymphocytes on microscope slides were used as a test system. In these experiments, three fluorescent antibodies were used to identify lymphocyte sub-populations, and a DNA content probe was used to identify all nucleated cells. The cell subset percentages determined by image cytometry were comparable to percentages obtained when cells from the same preparation were analyzed by flow cytometry. Multicolor fluorescence imaging cytometry can potentially be extended to the analysis of cells in smears, fine needle biopsies, imprints, and tissue sections.
We have investigated the Ca2+ -dependent interactions of villin, a protein of the intestinal micr... more We have investigated the Ca2+ -dependent interactions of villin, a protein of the intestinal microvillar core, with actin by monitoring resonance energy transfer between fluorescently labeled actin subunits. In the presence of elevated free Ca2+ (approximately 20 microM), villin affects both the nucleation and the elongation phases of actin polymerization. Consistent with previous reports, villin stimulates the nucleation process and will form stable nuclei under depolymerization conditions. Compared to the control, the net rate of polymerization is slightly inhibited at low concentrations of villin (villin/actin approximately 1:400) but is stimulated at higher concentrations (villin/actin greater than 1:100). Villin also significantly increases the critical concentration of actin polymerization. Addition of either villin or villin-actin complexes induces depolymerization of preassembled actin filaments. This villin-induced depolymerization is reversible upon removal of free Ca2+ or...
Cell-based target validation, secondary screening, lead optimization, and structure-activity rela... more Cell-based target validation, secondary screening, lead optimization, and structure-activity relationships have been recast with the advent of HCS. Prior to HCS, a computational approach to the characterization of the functions of specific target proteins and other cellular constituents, along with whole-cell functions employing fluorescence cell-based assays and microscopy, required extensive interaction among the researcher, instrumentation, and software tools. Early HCS platforms were instrument-centric and addressed the need to interface fully automated fluorescence microscopy, plate-handling automation, and seamless image analysis. HCS has since evolved into an integrated solution for accelerated drug discovery by encompassing the workflow components of assay and reagent design, robust instrumentation for automated fixed-end-point and live cell kinetic analysis, generalized and specific BioApplication software (Cellomics, Pittsburgh, PA) modules that produce information on drug responses from cell image data, and informatics/bioinformatics solutions that build knowledge from this information while providing a means to globalize HCS throughout an entire organization. This review communicates how these recent advances are incorporated into the drug discovery workflow by presenting a real-world use case.
The formation of lamellipodia in migrating cells involves dynamic processes that occur in a cycli... more The formation of lamellipodia in migrating cells involves dynamic processes that occur in a cyclic manner as the leading edge of a cell slowly advances. We used video-enhanced contrast microscopy (VEC) to monitor the motile behavior of cells to classify protrusions into the temporal stages of initial and established protrusions (Fisher et al.: Cell Motility and the Cytoskeleton 11:235-247, 1988), and to monitor the fixation of cells. Multiple parameter fluorescence imaging methods (DeBiasio et al.: Journal of Cell Biology 105:1613-1622, 1987; Waggoner et al.: Methods in Cell Biology, Vol. 30, Part B, pp. 449-478, 1989) were then used to determine and to map accurately the distributions of actin, myosin and microtubules in specific types of protrusions. Initial protrusions exhibited no substructure as evidenced by VEC and actin was diffusely arranged, while myosin and microtubules were absent. Newly established protrusions contained diffuse actin as well as actin in microspikes. Ther...
ABSTRACT Stress fibers in serum-deprived fibroblasts provide an excellent system for studying the... more ABSTRACT Stress fibers in serum-deprived fibroblasts provide an excellent system for studying the assembly dynamics of the actin-based cytoskeleton. Previous studies suggested that fibers containing actin and myosin II are restructured via two alternative pathways: coupled solation-contraction and disassembly without contraction. The former process appears to be regulated by Ca++, but regulation of the latter process has not been extensively explored. To assess the possible role of protein phosphorylation, we examined the dynamic behavior of stress fibers in cells treated with inhibitors of protein kinases. Swiss 3T3 fibroblasts were microinjected with fluorescent analogs of actin and myosin II and fiber dynamics monitored using light-microscope imaging. Staurosporine and KT5926 caused reversible dispersal of stress fibers without contraction, along with dephosphorylation of the regulatory light chain of myosin II, LC20. In order to make direct comparisons between the dose responses of these biochemical and morphological effects, fiber disruption was quantitated using two independent measures: total edge strength in fluorescent images of actin and myosin II, and fiber length determined by automated object-identification. Loss of stress fibers was shown to parallel LC20 dephosphorylation. Quantitation of cytoskeletal organization provides a framework for testing relationships between structural events and potential biochemical regulatory signals.
Optical Diagnostics of Living Cells and Biofluids, 1996
ABSTRACT The Automated Interactive Microscope is a robotic light microscope workstation that comb... more ABSTRACT The Automated Interactive Microscope is a robotic light microscope workstation that combines high performance light microscopy and computing to explore the chemical and molecular dynamics of cells and tissues.
A system is described for performing multicolor fluorescence image cytometry of cell preparations... more A system is described for performing multicolor fluorescence image cytometry of cell preparations. After the setting up stage, the operation is automatic: the microscope fields are found and focused; then images are acquired for each fluorophore, corrected and analyzed, without any operator interaction. Human peripheral blood lymphocytes on microscope slides were used as a test system. In these experiments, three fluorescent antibodies were used to identify lymphocyte sub-populations, and a DNA content probe was used to identify all nucleated cells. The cell subset percentages determined by image cytometry were comparable to percentages obtained when cells from the same preparation were analyzed by flow cytometry. Multicolor fluorescence imaging cytometry can potentially be extended to the analysis of cells in smears, fine needle biopsies, imprints, and tissue sections.
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Papers by D. Taylor