Michael Halter - Academia.edu (original) (raw)
Papers by Michael Halter
BMC Cell Biology, 2009
Background A critical challenge in cell biology is quantifying the interactions of cells with the... more Background A critical challenge in cell biology is quantifying the interactions of cells with their extracellular matrix (ECM) environment and the active remodeling by cells of their ECM. Fluorescence microscopy is a commonly employed technique for examining cell-matrix interactions. A label-free imaging method would provide an alternative that would eliminate the requirement of transfected cells and modified biological molecules, and if collected nondestructively, would allow long term observation and analysis of live cells. Results Using surface plasmon resonance imaging (SPRI), the deposition of protein by vascular smooth muscle cells (vSMC) cultured on fibronectin was quantified as a function of cell density and distance from the cell periphery. We observed that as much as 120 ng/cm2 of protein was deposited by cells in 24 h. Conclusion SPRI is a real-time, low-light-level, label-free imaging technique that allows the simultaneous observation and quantification of protein layers and cellular features. This technique is compatible with live cells such that it is possible to monitor cellular modifications to the extracellular matrix in real-time.
Assay and Drug Development Technologies, 2009
Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are... more Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are important for characterizing decontamination strategies and developing detection technologies for field use. We report here an assay for ricin that provides a response that is relevant to the mechanism of ricin activity and permits a much faster readout than the commonly used assays for cytotoxicity. The assay relies on the response of an engineered reporter cell line that was produced by stably transfecting Vero cells to express green fluorescent protein (GFP) under the control ofa cytomegalovirus (CMV) promoter. The results of the GFP-based assay were compared with the assay results from three commercially available cytotoxicity assays. The GFP assay reports a sensitive response to ricin after 6 h of treatment while the other assays require a 24-h incubation. Unlike the other assays, monitoring cellular GFP on a per-cell basis allows detection of reduced ribosome activity before significant cell death occurs, and the results are not convoluted by the numbers of cells being assayed.
IEEE Transactions on Medical Imaging, 2012
Cell segmentation is a critical step in the analysis pipeline for most imaging cytometry experime... more Cell segmentation is a critical step in the analysis pipeline for most imaging cytometry experiments and evaluating the performance of segmentation algorithms is important for aiding the selection of segmentation algorithms. Four popular algorithms are evaluated based on their cell segmentation performance. Because segmentation involves the classification of pixels belonging to regions within the cell or belonging to background, these algorithms are evaluated based on their total misclassification error. Misclassification error is particularly relevant in the analysis of quantitative descriptors of cell morphology involving pixel counts, such as projected area, aspect ratio and diameter. Since the cumulative distribution function captures completely the stochastic properties of a population of misclassification errors it is used to compare segmentation performance.
Cytometry Part A, 2007
To accurately interpret the data from fluorescent proteins as reporters of gene activation within... more To accurately interpret the data from fluorescent proteins as reporters of gene activation within living cells, it is important to understand the kinetics of the degradation of the reporter proteins. We examined the degradation kinetics over a large number (>1,000) of single, living cells from a clonal population of NIH3T3 fibroblasts that were stably transfected with a destabilized, enhanced green fluorescent protein (eGFP) reporter driven by the tenascin-C promoter. Data collection and quantification of the fluorescence protein within a statistically significant number of individual cells over long times (14 h) by automated microscopy was facilitated by culturing cells on micropatterned arrays that confined their migration and allowed them to be segmented using phase contrast images. To measure GFP degradation rates unambiguously, protein synthesis was inhibited with cycloheximide. Results from automated live cell microscopy and image analysis indicated a wide range of cell-to-cell variability in the GFP fluorescence within individual cells. Degradation for this reporter was analyzed as a first order rate process with a degradation half-life of 2.8 h. We found that GFP degradation rates were independent of the initial intensity of GFP fluorescence within cells. This result indicates that higher GFP abundance in some cells is likely due to higher rates of gene expression, because it is not due to systematically lower rates of protein degradation. The approach described in this study will assist the quantification and understanding of gene activity within live cells using fluorescent protein reporters. Published 2007 Wiley-Liss, Inc.
This paper presents preliminary results on the use of transferrin protein nanospheres (TfpNS) for... more This paper presents preliminary results on the use of transferrin protein nanospheres (TfpNS) for targeting cancer cells in vitro. Protein nanospheres represent an easily prepared and modifiable nanoplatform for receptor-specific targeting, molecular imaging and gene delivery. Rhodamine B isothiocyanate conjugated TfpNS (RBITC-TfpNS) show significantly enhanced uptake in vitro in SK-MEL-28 human malignant melanoma cells known to overexpress transferrin receptors compared to controls. RBITCTfpNS labeling of the cancer cells is due to transferrin receptor-mediated uptake, as demonstrated by competitive inhibition with native transferrin. Initial fluorescence microscopy studies indicate GFP plasmid can be transfected into melanoma cells via GFP plasmid encapsulated by TfpNS.
Analytical Chemistry, 2009
Oxygen tension in mammalian cell culture can profoundly affect cellular differentiation, viabilit... more Oxygen tension in mammalian cell culture can profoundly affect cellular differentiation, viability, and proliferation. However, precise measurement of dissolved oxygen in real time remains difficult. We report a new noninvasive sensor that can accurately measure oxygen concentration during cell culture while being compatible with live-cell imaging techniques such as fluorescence and phase contrast microscopy. The sensor is prepared by integrating the porphyrin dye, Pt(II) meso-tetrakis(pentafluorophenyl)porphine (PtTFPP) into polydimethylsiloxane (PDMS) thin films. Response of the sensor in the presence of oxygen can be characterized by the linear Stern-Volmer relationship with high sensitivity (K(SV) = 584 +/- 71 atm(-1)). A multilayer sensor design, created by sandwiching the PtTFPP-PDMS with a layer of Teflon AF followed by a second PDMS layer, effectively mitigates against dye cytotoxicity while providing a substrate for cell attachment. Using this sensor, changes in oxygen tension could be monitored in real-time as attached cells proliferated. The oxygen tension was found to decrease due to oxygen consumption by the cells, and the data could be analyzed using Fick's law to obtain the per-cell oxygen consumption rate. This sensor is likely to enable new studies on the effects of dissolved oxygen on cellular behavior.
Biointerphases, 2008
While it is well-appreciated that the extracellular matrix plays a critical role in influencing c... more While it is well-appreciated that the extracellular matrix plays a critical role in influencing cell responses, well-defined and reproducible presentation of extracellular matrix proteins poses a challenge for in vitro experiments. Films of type 1 collagen fibrils assembled on alkanethiolate monolayers formed at gold-coated surfaces have been shown to elicit a cellular response comparable to collagen gels, but with the advantages of excellent optical properties, and high reproducibility and robustness. To make this collagen matrix more accessible to laboratories that do not have access to gold film deposition the authors have examined the use of untreated polystyrene as a substrate for forming fibrillar collagen films. Direct comparison of films of fibrillar collagen fibrils formed at polystyrene with those formed at alkanethiolate monolayers indicates that films of collagen formed on these two surfaces compare very favorably to one another, both in their supramolecular structural characteristics as well as in the cell response that they elicit. Both substrates exhibit a dense covering of fibrils approximately 200 nm in diameter. The spreading of fibroblasts and activation of the tenascin-C gene promoter are statistically equivalent as determined by a metric derived from the D-statistic normally used in the Kolmogorov-Smirnov statistical test. The results of this study suggest that biologically relevant, robust thin films of collagen fibrils can be formed in any laboratory in untreated polystyrene dishes and multi-well polystyrene plates.
Biophysical Journal, 2010
Assembly of a high numerical objective platform for surface plasmon resonance imaging (SPRI) was ... more Assembly of a high numerical objective platform for surface plasmon resonance imaging (SPRI) was used to investigate a high resolution diffraction limited refractive index image of cell-substrate contacts. SPRI is a highly sensitive biochemical surface sensor measurement technique that has only recently been applied to the field of cell-biology. The nature of the refractive index measurement made at the cell-surface interface is investigated. We present SPRI measurements of several different cell types as well as multimode image comparisons with fluorescence and transmission mode microscopies to scrutinize the signal composition of the SPRI refractive index signal. While the primary correlation of the SPRI signal appears to be the cellular membrane distance to substrate, depending on the cell type, cell focal contacts, and cellular organelles can also contribute to subcellular refractive index changes at the cell-substrate interface.
Cytometry Part A, 2010
Spatially resolved details of the interactions of cells with a fibronectin modified surface were ... more Spatially resolved details of the interactions of cells with a fibronectin modified surface were examined using surface plasmon resonance imaging (SPRI). SPRI is a label-free technique that is based on the spatial measurement of interfacial refractive index. SPRI is sensitive to short range interactions between cells and their substratum. The high contrast in SPR signal between cell edges and substratum facilitates identification of cell edges and segmentation of cell areas. With this novel technique, we demonstrate visualization of cell-substratum interactions, and how cell-substratum interactions change over time as cells spread, migrate, and undergo membrane ruffling. Published 2010 Wiley-Liss, Inc.
Journal of Theoretical Biology, 2009
A population of cells in culture displays a range of phenotypic responses even when those cells a... more A population of cells in culture displays a range of phenotypic responses even when those cells are derived from a single cell and are exposed to a homogeneous environment. Phenotypic variability can have a number of sources including the variable rates at which individual cells within the population grow and divide. We have examined how such variations contribute to population responses by measuring cell volumes within genetically identical populations of cells where individual members of the population are continuously growing and dividing, and we have derived a function describing the stationary distribution of cell volumes that arises from these dynamics. The model includes stochastic parameters for the variability in cell cycle times and growth rates for individual cells in a proliferating cell line. We used the model to analyze the volume distributions obtained for two different cell lines and one cell line in the absence and presence of aphidicolin, a DNA polymerase inhibitor. The derivation and application of the model allows one to relate the stationary population distribution of cell volumes to extrinsic biological noise present in growing and dividing cell cultures.
Assay and Drug Development Technologies, 2009
Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are... more Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are important for characterizing decontamination strategies and developing detection technologies for field use. We report here an assay for ricin that provides a response that is relevant to the mechanism of ricin activity and permits a much faster readout than the commonly used assays for cytotoxicity. The assay relies on the response of an engineered reporter cell line that was produced by stably transfecting Vero cells to express green fluorescent protein (GFP) under the control ofa cytomegalovirus (CMV) promoter. The results of the GFP-based assay were compared with the assay results from three commercially available cytotoxicity assays. The GFP assay reports a sensitive response to ricin after 6 h of treatment while the other assays require a 24-h incubation. Unlike the other assays, monitoring cellular GFP on a per-cell basis allows detection of reduced ribosome activity before significant cell death occurs, and the results are not convoluted by the numbers of cells being assayed.
BMC Cell Biology, 2009
Background A critical challenge in cell biology is quantifying the interactions of cells with the... more Background A critical challenge in cell biology is quantifying the interactions of cells with their extracellular matrix (ECM) environment and the active remodeling by cells of their ECM. Fluorescence microscopy is a commonly employed technique for examining cell-matrix interactions. A label-free imaging method would provide an alternative that would eliminate the requirement of transfected cells and modified biological molecules, and if collected nondestructively, would allow long term observation and analysis of live cells. Results Using surface plasmon resonance imaging (SPRI), the deposition of protein by vascular smooth muscle cells (vSMC) cultured on fibronectin was quantified as a function of cell density and distance from the cell periphery. We observed that as much as 120 ng/cm2 of protein was deposited by cells in 24 h. Conclusion SPRI is a real-time, low-light-level, label-free imaging technique that allows the simultaneous observation and quantification of protein layers and cellular features. This technique is compatible with live cells such that it is possible to monitor cellular modifications to the extracellular matrix in real-time.
Assay and Drug Development Technologies, 2009
Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are... more Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are important for characterizing decontamination strategies and developing detection technologies for field use. We report here an assay for ricin that provides a response that is relevant to the mechanism of ricin activity and permits a much faster readout than the commonly used assays for cytotoxicity. The assay relies on the response of an engineered reporter cell line that was produced by stably transfecting Vero cells to express green fluorescent protein (GFP) under the control ofa cytomegalovirus (CMV) promoter. The results of the GFP-based assay were compared with the assay results from three commercially available cytotoxicity assays. The GFP assay reports a sensitive response to ricin after 6 h of treatment while the other assays require a 24-h incubation. Unlike the other assays, monitoring cellular GFP on a per-cell basis allows detection of reduced ribosome activity before significant cell death occurs, and the results are not convoluted by the numbers of cells being assayed.
IEEE Transactions on Medical Imaging, 2012
Cell segmentation is a critical step in the analysis pipeline for most imaging cytometry experime... more Cell segmentation is a critical step in the analysis pipeline for most imaging cytometry experiments and evaluating the performance of segmentation algorithms is important for aiding the selection of segmentation algorithms. Four popular algorithms are evaluated based on their cell segmentation performance. Because segmentation involves the classification of pixels belonging to regions within the cell or belonging to background, these algorithms are evaluated based on their total misclassification error. Misclassification error is particularly relevant in the analysis of quantitative descriptors of cell morphology involving pixel counts, such as projected area, aspect ratio and diameter. Since the cumulative distribution function captures completely the stochastic properties of a population of misclassification errors it is used to compare segmentation performance.
Cytometry Part A, 2007
To accurately interpret the data from fluorescent proteins as reporters of gene activation within... more To accurately interpret the data from fluorescent proteins as reporters of gene activation within living cells, it is important to understand the kinetics of the degradation of the reporter proteins. We examined the degradation kinetics over a large number (>1,000) of single, living cells from a clonal population of NIH3T3 fibroblasts that were stably transfected with a destabilized, enhanced green fluorescent protein (eGFP) reporter driven by the tenascin-C promoter. Data collection and quantification of the fluorescence protein within a statistically significant number of individual cells over long times (14 h) by automated microscopy was facilitated by culturing cells on micropatterned arrays that confined their migration and allowed them to be segmented using phase contrast images. To measure GFP degradation rates unambiguously, protein synthesis was inhibited with cycloheximide. Results from automated live cell microscopy and image analysis indicated a wide range of cell-to-cell variability in the GFP fluorescence within individual cells. Degradation for this reporter was analyzed as a first order rate process with a degradation half-life of 2.8 h. We found that GFP degradation rates were independent of the initial intensity of GFP fluorescence within cells. This result indicates that higher GFP abundance in some cells is likely due to higher rates of gene expression, because it is not due to systematically lower rates of protein degradation. The approach described in this study will assist the quantification and understanding of gene activity within live cells using fluorescent protein reporters. Published 2007 Wiley-Liss, Inc.
This paper presents preliminary results on the use of transferrin protein nanospheres (TfpNS) for... more This paper presents preliminary results on the use of transferrin protein nanospheres (TfpNS) for targeting cancer cells in vitro. Protein nanospheres represent an easily prepared and modifiable nanoplatform for receptor-specific targeting, molecular imaging and gene delivery. Rhodamine B isothiocyanate conjugated TfpNS (RBITC-TfpNS) show significantly enhanced uptake in vitro in SK-MEL-28 human malignant melanoma cells known to overexpress transferrin receptors compared to controls. RBITCTfpNS labeling of the cancer cells is due to transferrin receptor-mediated uptake, as demonstrated by competitive inhibition with native transferrin. Initial fluorescence microscopy studies indicate GFP plasmid can be transfected into melanoma cells via GFP plasmid encapsulated by TfpNS.
Analytical Chemistry, 2009
Oxygen tension in mammalian cell culture can profoundly affect cellular differentiation, viabilit... more Oxygen tension in mammalian cell culture can profoundly affect cellular differentiation, viability, and proliferation. However, precise measurement of dissolved oxygen in real time remains difficult. We report a new noninvasive sensor that can accurately measure oxygen concentration during cell culture while being compatible with live-cell imaging techniques such as fluorescence and phase contrast microscopy. The sensor is prepared by integrating the porphyrin dye, Pt(II) meso-tetrakis(pentafluorophenyl)porphine (PtTFPP) into polydimethylsiloxane (PDMS) thin films. Response of the sensor in the presence of oxygen can be characterized by the linear Stern-Volmer relationship with high sensitivity (K(SV) = 584 +/- 71 atm(-1)). A multilayer sensor design, created by sandwiching the PtTFPP-PDMS with a layer of Teflon AF followed by a second PDMS layer, effectively mitigates against dye cytotoxicity while providing a substrate for cell attachment. Using this sensor, changes in oxygen tension could be monitored in real-time as attached cells proliferated. The oxygen tension was found to decrease due to oxygen consumption by the cells, and the data could be analyzed using Fick's law to obtain the per-cell oxygen consumption rate. This sensor is likely to enable new studies on the effects of dissolved oxygen on cellular behavior.
Biointerphases, 2008
While it is well-appreciated that the extracellular matrix plays a critical role in influencing c... more While it is well-appreciated that the extracellular matrix plays a critical role in influencing cell responses, well-defined and reproducible presentation of extracellular matrix proteins poses a challenge for in vitro experiments. Films of type 1 collagen fibrils assembled on alkanethiolate monolayers formed at gold-coated surfaces have been shown to elicit a cellular response comparable to collagen gels, but with the advantages of excellent optical properties, and high reproducibility and robustness. To make this collagen matrix more accessible to laboratories that do not have access to gold film deposition the authors have examined the use of untreated polystyrene as a substrate for forming fibrillar collagen films. Direct comparison of films of fibrillar collagen fibrils formed at polystyrene with those formed at alkanethiolate monolayers indicates that films of collagen formed on these two surfaces compare very favorably to one another, both in their supramolecular structural characteristics as well as in the cell response that they elicit. Both substrates exhibit a dense covering of fibrils approximately 200 nm in diameter. The spreading of fibroblasts and activation of the tenascin-C gene promoter are statistically equivalent as determined by a metric derived from the D-statistic normally used in the Kolmogorov-Smirnov statistical test. The results of this study suggest that biologically relevant, robust thin films of collagen fibrils can be formed in any laboratory in untreated polystyrene dishes and multi-well polystyrene plates.
Biophysical Journal, 2010
Assembly of a high numerical objective platform for surface plasmon resonance imaging (SPRI) was ... more Assembly of a high numerical objective platform for surface plasmon resonance imaging (SPRI) was used to investigate a high resolution diffraction limited refractive index image of cell-substrate contacts. SPRI is a highly sensitive biochemical surface sensor measurement technique that has only recently been applied to the field of cell-biology. The nature of the refractive index measurement made at the cell-surface interface is investigated. We present SPRI measurements of several different cell types as well as multimode image comparisons with fluorescence and transmission mode microscopies to scrutinize the signal composition of the SPRI refractive index signal. While the primary correlation of the SPRI signal appears to be the cellular membrane distance to substrate, depending on the cell type, cell focal contacts, and cellular organelles can also contribute to subcellular refractive index changes at the cell-substrate interface.
Cytometry Part A, 2010
Spatially resolved details of the interactions of cells with a fibronectin modified surface were ... more Spatially resolved details of the interactions of cells with a fibronectin modified surface were examined using surface plasmon resonance imaging (SPRI). SPRI is a label-free technique that is based on the spatial measurement of interfacial refractive index. SPRI is sensitive to short range interactions between cells and their substratum. The high contrast in SPR signal between cell edges and substratum facilitates identification of cell edges and segmentation of cell areas. With this novel technique, we demonstrate visualization of cell-substratum interactions, and how cell-substratum interactions change over time as cells spread, migrate, and undergo membrane ruffling. Published 2010 Wiley-Liss, Inc.
Journal of Theoretical Biology, 2009
A population of cells in culture displays a range of phenotypic responses even when those cells a... more A population of cells in culture displays a range of phenotypic responses even when those cells are derived from a single cell and are exposed to a homogeneous environment. Phenotypic variability can have a number of sources including the variable rates at which individual cells within the population grow and divide. We have examined how such variations contribute to population responses by measuring cell volumes within genetically identical populations of cells where individual members of the population are continuously growing and dividing, and we have derived a function describing the stationary distribution of cell volumes that arises from these dynamics. The model includes stochastic parameters for the variability in cell cycle times and growth rates for individual cells in a proliferating cell line. We used the model to analyze the volume distributions obtained for two different cell lines and one cell line in the absence and presence of aphidicolin, a DNA polymerase inhibitor. The derivation and application of the model allows one to relate the stationary population distribution of cell volumes to extrinsic biological noise present in growing and dividing cell cultures.
Assay and Drug Development Technologies, 2009
Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are... more Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are important for characterizing decontamination strategies and developing detection technologies for field use. We report here an assay for ricin that provides a response that is relevant to the mechanism of ricin activity and permits a much faster readout than the commonly used assays for cytotoxicity. The assay relies on the response of an engineered reporter cell line that was produced by stably transfecting Vero cells to express green fluorescent protein (GFP) under the control ofa cytomegalovirus (CMV) promoter. The results of the GFP-based assay were compared with the assay results from three commercially available cytotoxicity assays. The GFP assay reports a sensitive response to ricin after 6 h of treatment while the other assays require a 24-h incubation. Unlike the other assays, monitoring cellular GFP on a per-cell basis allows detection of reduced ribosome activity before significant cell death occurs, and the results are not convoluted by the numbers of cells being assayed.