Fluorescence standards allow for quality control and for the comparison of data sets across instr... more Fluorescence standards allow for quality control and for the comparison of data sets across instruments and laboratories in applications of quantitative fluorescence. For example, users of microscopy core facilities expect a homogenous and time-invariant illumination and a uniform detection sensitivity, which are prerequisites for quantitative imaging analysis, particle tracking or fluorometric pH or Ca2+-concentration measurements. Similarly, confirming the three-dimensional (3-D) resolution of optical sectioning micro-scopes prior to volumetric reconstructions calls for a regular calibration with a standardised point source. Typically, the test samples required for such calibration measurements are different ones, and they depend much on the very microscope technique used. Also, the ever-increasing choice among these techniques increases the demand for comparison and metrology across instruments. Here, we advocate and demonstrate the multiple uses of a surprisingly versatile and simple 3-D test sample that can complement existing and much more expensive calibration samples: simple commercial tissue paper labelled with a fluorescent highlighter pen. We provide relevant sample characteristics and show examples ranging from the sub-µm to cm scale, acquired on epifluorescence, confocal, image scanning, two-photon (2P) and light-sheet microscopes. Graphical abstract Pyranine-labeled tissue paper, imaged upon 405-nm epifluorescence excitation through a 455LP LP dichroic and 465LP emission filter. Objective ×20/NA0.25. Overlaid are the normalised absorbance (dashed) and emission spectra (through line), respectively. In the present work we show that this “primitive” and inexpensive three-dimensional (3-D) test sample is a surprisingly versatile and powerful tool for quality assessment, comparison across microscopes as well as routine metrology for optical sectioning techniques, both for research labs and imaging core facilities. Research highlights - highlighter-pen marked tissue paper is a surprisingly powerful and versatile test sample for 3-D fluorescence microscopies - standard tissue paper presents features ranging from 400 nm to centimetres - our sample can simultaneously be used for testing intensity, field homogeneity, resolution, optical sectioning and image contrast - it is easy to prepare, versatile, photostable and inexpensive
Adult neural precursor cells show great promise for the repair of central nervous system (CNS) ti... more Adult neural precursor cells show great promise for the repair of central nervous system (CNS) tissue following injury or disease. Following injury to the CNS, endogenous neural precursor cells (NPCs), found in the neurogenic subventricular zone of the adult forebrain, are activated to proliferate and migrate toward the lesion site. However, this activation is not sufficient for neurorepair. Previous work has demonstrated that murine NPCs are electrosensitive cells that undergo rapid and cathodally directed migration in an applied electric field (EF). Here, we examined the EF induced migration of a clinically relevant human NPC population. With a goal of enhancing cell-based regeneration of the CNS, we explored the role of extracellular matrix (ECM) in human NPC behavior. NPCs plated on matrigel, MaxGel, laminin, and fibronectin were found to migrate in a substrate dependent manner. Common to all substrates was an increase in migration velocity in the presence of an EF. Different, was the direction of migration on the different substrates. The EF application did not induce differentiation on any of the substrates examined, as cells continued to express the precursor markers nestin and SOX2. We determined that secreted factors from neighbouring cells and extracellular pH were not significant factors in EF induced migration, however, substrate stiffness was sufficient to alter the direction of drNPC migration. These findings provide insight into factors that modulate human NPC migration and shed light on considerations for designing cell based therapies for neurorepair.
ABSTRACTElectrocrystallization of the organic superconductor bis(ethylenedithio)tetrathiafulvalen... more ABSTRACTElectrocrystallization of the organic superconductor bis(ethylenedithio)tetrathiafulvalene triiodide, (ET)2I3, on a highly ordered pyrolytic graphite (HOPG) substrate has been visualized using in situ atomic force microscopy (AFM). Previous studies have revealed the formation of a coincident epitaxial monolayer with a structure identical to that of a (001) layer of the superconducting beta phase of this material prior to bulk crystal growth. However, the symmetry of the HOPG substrate leads to domain boundary defects during self assembly of the separately growing domains. The number of defects is significantly reduced after an electrochemical annealing process in which the potential is cycled about the monolayer deposition potential. Annealing of these films is important if they are to be used in electronic devices as the defects may serve as barriers to electron transport in the two-dimensional layers.In addition to (ET)2I3, monolayer growth also has been visualized during ...
The mitochondrial glycerophospholipid cardiolipin plays important roles in mitochondrial biology.... more The mitochondrial glycerophospholipid cardiolipin plays important roles in mitochondrial biology. Most notably, cardiolipin directly binds to mitochondrial proteins and helps assemble and stabilize mitochondrial multi-protein complexes. Despite their importance for mitochondrial health, how the proteins involved in cardiolipin biosynthesis are organized and embedded in mitochondrial membranes has not been investigated in detail. Here we show that human PGS1 and CLS1 are constituents of large protein complexes. We show that PGS1 forms oligomers and associates with CLS1 and PTPMT1. Using super-resolution microscopy, we observed well-organized nanoscale structures formed by PGS1. Together with the observation that cardiolipin and CLS1 are not required for PGS1 to assemble in the complex we predict the presence of a PGS1-centered cardiolipin-synthesizing scaffold within the mitochondrial inner membrane. Using an unbiased proteomic approach we found that PGS1 and CLS1 interact with multi...
Molecular combing of DNA fibers is a powerful technique to monitor origin usage and DNA replicati... more Molecular combing of DNA fibers is a powerful technique to monitor origin usage and DNA replication fork progression in the budding yeast Saccharomyces cerevisiae. In contrast to traditional flow cytometry, microarray, or sequencing techniques, which provide population-level data, DNA combing provides DNA replication profiles of individual molecules. DNA combing uses yeast strains that express human thymidine kinase, which facilitates the incorporation of thymidine analogs into nascent DNA. First, DNA is isolated and stretched uniformly onto silanized glass coverslips. Following immunodetection with antibodies that recognize the thymidine analog and the DNA, the DNA fibers are imaged using a fluorescence microscope. Finally, the lengths of newly replicated DNA tracks are measured and converted to base pairs, allowing calculations of the speed of the replication fork and of interorigin distances. DNA combing can be applied to monitor replication defects caused by gene mutations or by...
The faithful replication of eukaryotic chromosomal DNA occurs during S phase once per cell cycle.... more The faithful replication of eukaryotic chromosomal DNA occurs during S phase once per cell cycle. Replication is highly regulated and is initiated at special structures, termed origins, from which replication forks move out bidirectionally. A wide variety of techniques have been developed to study the features and kinetics of replication. Many of these, such as those based on flow cytometry and two-dimensional and pulsed-field gel electrophoresis, give a population-level view of replication. However, an alternative approach, DNA fiber analysis, which was originally developed more than 50 years ago, has the advantage of revealing features of replication at the level of individual DNA fibers. Initially based on autoradiography, this technique has been superseded by immunofluorescence-based detection of incorporated halogenated thymidine analogs. Furthermore, derivations of this technique have been developed to distribute and stretch the labeled DNA fibers uniformly on optically clear ...
Fluorescence standards allow for quality control and for the comparison of data sets across instr... more Fluorescence standards allow for quality control and for the comparison of data sets across instruments and laboratories in applications of quantitative fluorescence. For example, users of microscopy core facilities expect a homogenous and time-invariant illumination and a uniform detection sensitivity, which are prerequisites for quantitative imaging analysis, particle tracking or fluorometric pH or Ca2+-concentration measurements. Similarly, confirming the three-dimensional (3-D) resolution of optical sectioning micro-scopes prior to volumetric reconstructions calls for a regular calibration with a standardised point source. Typically, the test samples required for such calibration measurements are different ones, and they depend much on the very microscope technique used. Also, the ever-increasing choice among these techniques increases the demand for comparison and metrology across instruments. Here, we advocate and demonstrate the multiple uses of a surprisingly versatile and simple 3-D test sample that can complement existing and much more expensive calibration samples: simple commercial tissue paper labelled with a fluorescent highlighter pen. We provide relevant sample characteristics and show examples ranging from the sub-µm to cm scale, acquired on epifluorescence, confocal, image scanning, two-photon (2P) and light-sheet microscopes. Graphical abstract Pyranine-labeled tissue paper, imaged upon 405-nm epifluorescence excitation through a 455LP LP dichroic and 465LP emission filter. Objective ×20/NA0.25. Overlaid are the normalised absorbance (dashed) and emission spectra (through line), respectively. In the present work we show that this “primitive” and inexpensive three-dimensional (3-D) test sample is a surprisingly versatile and powerful tool for quality assessment, comparison across microscopes as well as routine metrology for optical sectioning techniques, both for research labs and imaging core facilities. Research highlights - highlighter-pen marked tissue paper is a surprisingly powerful and versatile test sample for 3-D fluorescence microscopies - standard tissue paper presents features ranging from 400 nm to centimetres - our sample can simultaneously be used for testing intensity, field homogeneity, resolution, optical sectioning and image contrast - it is easy to prepare, versatile, photostable and inexpensive
Adult neural precursor cells show great promise for the repair of central nervous system (CNS) ti... more Adult neural precursor cells show great promise for the repair of central nervous system (CNS) tissue following injury or disease. Following injury to the CNS, endogenous neural precursor cells (NPCs), found in the neurogenic subventricular zone of the adult forebrain, are activated to proliferate and migrate toward the lesion site. However, this activation is not sufficient for neurorepair. Previous work has demonstrated that murine NPCs are electrosensitive cells that undergo rapid and cathodally directed migration in an applied electric field (EF). Here, we examined the EF induced migration of a clinically relevant human NPC population. With a goal of enhancing cell-based regeneration of the CNS, we explored the role of extracellular matrix (ECM) in human NPC behavior. NPCs plated on matrigel, MaxGel, laminin, and fibronectin were found to migrate in a substrate dependent manner. Common to all substrates was an increase in migration velocity in the presence of an EF. Different, was the direction of migration on the different substrates. The EF application did not induce differentiation on any of the substrates examined, as cells continued to express the precursor markers nestin and SOX2. We determined that secreted factors from neighbouring cells and extracellular pH were not significant factors in EF induced migration, however, substrate stiffness was sufficient to alter the direction of drNPC migration. These findings provide insight into factors that modulate human NPC migration and shed light on considerations for designing cell based therapies for neurorepair.
ABSTRACTElectrocrystallization of the organic superconductor bis(ethylenedithio)tetrathiafulvalen... more ABSTRACTElectrocrystallization of the organic superconductor bis(ethylenedithio)tetrathiafulvalene triiodide, (ET)2I3, on a highly ordered pyrolytic graphite (HOPG) substrate has been visualized using in situ atomic force microscopy (AFM). Previous studies have revealed the formation of a coincident epitaxial monolayer with a structure identical to that of a (001) layer of the superconducting beta phase of this material prior to bulk crystal growth. However, the symmetry of the HOPG substrate leads to domain boundary defects during self assembly of the separately growing domains. The number of defects is significantly reduced after an electrochemical annealing process in which the potential is cycled about the monolayer deposition potential. Annealing of these films is important if they are to be used in electronic devices as the defects may serve as barriers to electron transport in the two-dimensional layers.In addition to (ET)2I3, monolayer growth also has been visualized during ...
The mitochondrial glycerophospholipid cardiolipin plays important roles in mitochondrial biology.... more The mitochondrial glycerophospholipid cardiolipin plays important roles in mitochondrial biology. Most notably, cardiolipin directly binds to mitochondrial proteins and helps assemble and stabilize mitochondrial multi-protein complexes. Despite their importance for mitochondrial health, how the proteins involved in cardiolipin biosynthesis are organized and embedded in mitochondrial membranes has not been investigated in detail. Here we show that human PGS1 and CLS1 are constituents of large protein complexes. We show that PGS1 forms oligomers and associates with CLS1 and PTPMT1. Using super-resolution microscopy, we observed well-organized nanoscale structures formed by PGS1. Together with the observation that cardiolipin and CLS1 are not required for PGS1 to assemble in the complex we predict the presence of a PGS1-centered cardiolipin-synthesizing scaffold within the mitochondrial inner membrane. Using an unbiased proteomic approach we found that PGS1 and CLS1 interact with multi...
Molecular combing of DNA fibers is a powerful technique to monitor origin usage and DNA replicati... more Molecular combing of DNA fibers is a powerful technique to monitor origin usage and DNA replication fork progression in the budding yeast Saccharomyces cerevisiae. In contrast to traditional flow cytometry, microarray, or sequencing techniques, which provide population-level data, DNA combing provides DNA replication profiles of individual molecules. DNA combing uses yeast strains that express human thymidine kinase, which facilitates the incorporation of thymidine analogs into nascent DNA. First, DNA is isolated and stretched uniformly onto silanized glass coverslips. Following immunodetection with antibodies that recognize the thymidine analog and the DNA, the DNA fibers are imaged using a fluorescence microscope. Finally, the lengths of newly replicated DNA tracks are measured and converted to base pairs, allowing calculations of the speed of the replication fork and of interorigin distances. DNA combing can be applied to monitor replication defects caused by gene mutations or by...
The faithful replication of eukaryotic chromosomal DNA occurs during S phase once per cell cycle.... more The faithful replication of eukaryotic chromosomal DNA occurs during S phase once per cell cycle. Replication is highly regulated and is initiated at special structures, termed origins, from which replication forks move out bidirectionally. A wide variety of techniques have been developed to study the features and kinetics of replication. Many of these, such as those based on flow cytometry and two-dimensional and pulsed-field gel electrophoresis, give a population-level view of replication. However, an alternative approach, DNA fiber analysis, which was originally developed more than 50 years ago, has the advantage of revealing features of replication at the level of individual DNA fibers. Initially based on autoradiography, this technique has been superseded by immunofluorescence-based detection of incorporated halogenated thymidine analogs. Furthermore, derivations of this technique have been developed to distribute and stretch the labeled DNA fibers uniformly on optically clear ...
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Papers by Christopher Yip