BackgroundA cell analysis system was developed to enumerate and differentiate magnetically aligne... more BackgroundA cell analysis system was developed to enumerate and differentiate magnetically aligned cells selected from whole blood. The cellular information extracted is similar to the readout of musical information from a compact disk (CD). Here we describe the optical design and data processing of the system. The performance of the system is demonstrated using fluorescent‐labeled cells and beads.Materials and MethodsSystem performance was demonstrated with 6‐μm polystyrene beads labeled with magnetic nanoparticles and allophycocyanin (APC) and immunomagnetically aligned leukocytes, fluorescently labeled with Oxazine750 and CD4‐APC, CD8‐Cy5.5, and CD14‐APC/Cy7 in whole blood.ResultsThe sensitivity of the system was demonstrated using APC‐labeled beads. With this system, beads containing 333 APC molecules could easily be resolved from the background. This level of sensitivity was not achievable with a commercial flow cytometer. A maximum of 20,000 immunomagnetically labeled cells co...
Sensor Technology in the Netherlands: State of the Art, 1998
All our understanding of how DNA works is based on evidence obtained with bulk methods. Moreover,... more All our understanding of how DNA works is based on evidence obtained with bulk methods. Moreover, information on the detailed conformation of DNA and interactions between DNA and proteins at a high resolution, have been obtained almost entirely under highly nonphysiological conditions (vacuum, low temperature etc.). Recently several methods have become available that make it possible to study individual bio-molecules under physiological conditions. We will describe three of these methods and there application to DNA research: flow analysis of individual molecules, measuring mechanical forces of bio-molecules using optical tweezers and using an AFM to study the dynamics of DNA and DNA protein interactions.
The effects of cooling of a hand on lateral and contralateral digital skin blood flow were invest... more The effects of cooling of a hand on lateral and contralateral digital skin blood flow were investigated in 18 patients with primary or secondary Raynaud's phenomenon. The aim of the study was to compare photoelectrical plethsmog raphy (PhEP) and laser Doppler flowmetry (LDF). PhEP and LDF were used simultaneously for skin blood flow measurements of the third finger of both hands. One hand was cooled in water from 33 ° to 3 ° C in steps of 3 ° C, each step lasting four minutes. It was followed by a recovery period of ten minutes in room air of 24 ° C. During stepwise cooling from 33 ° to 9 ° C the relative PhEP and LDF values, measured on the cooled hand, decreased to 6.2% ± 3.2% and 10% ± 12% respectively. The correlation coefficients between LDF and PhEP varied between 0.79 and 0.99. In the contralateral hand the relative PhEP and LDF values decreased to 38% ± 30.% and 64% ± 7.9% respec tively. The correlation coefficients between LDF and PhEP values were lower on the contralat...
Our research focuses on the development of microscopy techniques for both physical and biological... more Our research focuses on the development of microscopy techniques for both physical and biological applications. With the Confocal Microscope, that has been developed in our lab, single molecules can be imaged with diffraction limited resolution from which spectral information can be obtained as well. With our Near Field Scanning Optical Microscope (NSOM) and Atomic Force Microscope (AFM) it is possible to image single molecules at a resolution of a few nm (AFM) to about 50 nm (NSOM). These techniques provide excellent possibilities to study interactions between DNA and proteins in complex structures.
Confocal Raman microspectroscopy (CRM) is a non-invasive, non-destructive, and sensitive analytic... more Confocal Raman microspectroscopy (CRM) is a non-invasive, non-destructive, and sensitive analytical tool for the study of some aspects of the molecular organization of cells and tissues with high spatial resolution. Filipin, a polyene antibiotic, specifically binds to cholesterol, and its molecular structure predicts it to be Raman-active. The aim of the present study was to assess the potentialities of a combined CRM-filipin approach to study the distribution of cholesterol in the human eye lens. Paraformaldehyde-fixed human lenses were sliced (0.7 mm), incubated with filipin, and analyzed by CRM. Filipin proved to give a specific Raman signal at 1586 cm-1, hardly interfering with signals from lens proteins. The CRM-filipin approach proved to be extremely sensitive, allowing detection of cholesterol in the femtogram range. It has an excellent spatial resolution (0.2-0.5 micron 3) when using point measurements. Due to the intrinsic anisotropy of membranes in the eye lens and therefore of the cholesterol distribution, a line-scan approach has to be adopted when fiber-to-fiber changes in cholesterol are of interest. The distribution of filipin along the optical axis of four human eye lenses was compared with data from the literature. The combined CRM-filipin approach is a highly specific and sensitive method for the study of cholesterol within cells and tissues. The spatial resolution is high and can be adapted to the desired discriminative power. The gross distribution of filipin along the optical axis obtained in this study is similar to that found in biochemical studies.
Focal opacities are signs of early cataractogenesis in the human lens. They progress slowly over ... more Focal opacities are signs of early cataractogenesis in the human lens. They progress slowly over a lifetime and may be precursors of mature cataracts. The authors analyzed changes in proteins, phospholipids, and cholesterol in these opacities using in situ techniques: Raman microspectroscopy, filipin cytochemistry for cholesterol, and transmission electron microscopy (TEM). Human lenses with verified focal opacities were fixed in 1% paraformaldehyde. Slabs with opacities were analyzed using confocal Raman spectroscopy, then filipin Raman analysis of cholesterol, and finally TEM. Compared with normal fibers, opacities consistently showed elevated levels of cholesterol and aliphatic chains, increased phospholipid acyl chain disorder, and changes in phospholipid lateral packing. Disulfide bridges of specific geometry (trans-gauche-trans) were found. Although protein content was unchanged, compared with normal fibers, aromatic amino acid content was significantly lower. The hydrophobici...
BackgroundA cell analysis system was developed to enumerate and differentiate magnetically aligne... more BackgroundA cell analysis system was developed to enumerate and differentiate magnetically aligned cells selected from whole blood. The cellular information extracted is similar to the readout of musical information from a compact disk (CD). Here we describe the optical design and data processing of the system. The performance of the system is demonstrated using fluorescent‐labeled cells and beads.Materials and MethodsSystem performance was demonstrated with 6‐μm polystyrene beads labeled with magnetic nanoparticles and allophycocyanin (APC) and immunomagnetically aligned leukocytes, fluorescently labeled with Oxazine750 and CD4‐APC, CD8‐Cy5.5, and CD14‐APC/Cy7 in whole blood.ResultsThe sensitivity of the system was demonstrated using APC‐labeled beads. With this system, beads containing 333 APC molecules could easily be resolved from the background. This level of sensitivity was not achievable with a commercial flow cytometer. A maximum of 20,000 immunomagnetically labeled cells co...
Sensor Technology in the Netherlands: State of the Art, 1998
All our understanding of how DNA works is based on evidence obtained with bulk methods. Moreover,... more All our understanding of how DNA works is based on evidence obtained with bulk methods. Moreover, information on the detailed conformation of DNA and interactions between DNA and proteins at a high resolution, have been obtained almost entirely under highly nonphysiological conditions (vacuum, low temperature etc.). Recently several methods have become available that make it possible to study individual bio-molecules under physiological conditions. We will describe three of these methods and there application to DNA research: flow analysis of individual molecules, measuring mechanical forces of bio-molecules using optical tweezers and using an AFM to study the dynamics of DNA and DNA protein interactions.
The effects of cooling of a hand on lateral and contralateral digital skin blood flow were invest... more The effects of cooling of a hand on lateral and contralateral digital skin blood flow were investigated in 18 patients with primary or secondary Raynaud's phenomenon. The aim of the study was to compare photoelectrical plethsmog raphy (PhEP) and laser Doppler flowmetry (LDF). PhEP and LDF were used simultaneously for skin blood flow measurements of the third finger of both hands. One hand was cooled in water from 33 ° to 3 ° C in steps of 3 ° C, each step lasting four minutes. It was followed by a recovery period of ten minutes in room air of 24 ° C. During stepwise cooling from 33 ° to 9 ° C the relative PhEP and LDF values, measured on the cooled hand, decreased to 6.2% ± 3.2% and 10% ± 12% respectively. The correlation coefficients between LDF and PhEP varied between 0.79 and 0.99. In the contralateral hand the relative PhEP and LDF values decreased to 38% ± 30.% and 64% ± 7.9% respec tively. The correlation coefficients between LDF and PhEP values were lower on the contralat...
Our research focuses on the development of microscopy techniques for both physical and biological... more Our research focuses on the development of microscopy techniques for both physical and biological applications. With the Confocal Microscope, that has been developed in our lab, single molecules can be imaged with diffraction limited resolution from which spectral information can be obtained as well. With our Near Field Scanning Optical Microscope (NSOM) and Atomic Force Microscope (AFM) it is possible to image single molecules at a resolution of a few nm (AFM) to about 50 nm (NSOM). These techniques provide excellent possibilities to study interactions between DNA and proteins in complex structures.
Confocal Raman microspectroscopy (CRM) is a non-invasive, non-destructive, and sensitive analytic... more Confocal Raman microspectroscopy (CRM) is a non-invasive, non-destructive, and sensitive analytical tool for the study of some aspects of the molecular organization of cells and tissues with high spatial resolution. Filipin, a polyene antibiotic, specifically binds to cholesterol, and its molecular structure predicts it to be Raman-active. The aim of the present study was to assess the potentialities of a combined CRM-filipin approach to study the distribution of cholesterol in the human eye lens. Paraformaldehyde-fixed human lenses were sliced (0.7 mm), incubated with filipin, and analyzed by CRM. Filipin proved to give a specific Raman signal at 1586 cm-1, hardly interfering with signals from lens proteins. The CRM-filipin approach proved to be extremely sensitive, allowing detection of cholesterol in the femtogram range. It has an excellent spatial resolution (0.2-0.5 micron 3) when using point measurements. Due to the intrinsic anisotropy of membranes in the eye lens and therefore of the cholesterol distribution, a line-scan approach has to be adopted when fiber-to-fiber changes in cholesterol are of interest. The distribution of filipin along the optical axis of four human eye lenses was compared with data from the literature. The combined CRM-filipin approach is a highly specific and sensitive method for the study of cholesterol within cells and tissues. The spatial resolution is high and can be adapted to the desired discriminative power. The gross distribution of filipin along the optical axis obtained in this study is similar to that found in biochemical studies.
Focal opacities are signs of early cataractogenesis in the human lens. They progress slowly over ... more Focal opacities are signs of early cataractogenesis in the human lens. They progress slowly over a lifetime and may be precursors of mature cataracts. The authors analyzed changes in proteins, phospholipids, and cholesterol in these opacities using in situ techniques: Raman microspectroscopy, filipin cytochemistry for cholesterol, and transmission electron microscopy (TEM). Human lenses with verified focal opacities were fixed in 1% paraformaldehyde. Slabs with opacities were analyzed using confocal Raman spectroscopy, then filipin Raman analysis of cholesterol, and finally TEM. Compared with normal fibers, opacities consistently showed elevated levels of cholesterol and aliphatic chains, increased phospholipid acyl chain disorder, and changes in phospholipid lateral packing. Disulfide bridges of specific geometry (trans-gauche-trans) were found. Although protein content was unchanged, compared with normal fibers, aromatic amino acid content was significantly lower. The hydrophobici...
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