Biomedical subjects

Jan Greve

Publications and source records attributed to Jan Greve.

8 recordsLinked to original sources

PubMed2005

Drift correction in a multichannel integrated optical young interferometer.

We demonstrate that in a sensor based on a multichannel Young interferometer, the phase information obtained for different pairs of channels can be used to correct the long-term instability (drift) due to temperature differences between measuring and reference channels, the drift in the alignment of the setup, etc. Experiments show that the nature of a major part of the drift is such that the drift present in one of the channels can be determined by interpolation of the drift measured in the two adjacent channels. It is shown that a drift reduction of 10 times can be achieved as compared with the situation in which no correction is applied. We anticipate that these findings will permit the exploitation of the extreme sensitivity of interference-based sensors to a much greater extent.

Journal Article↗

PubMed2004

Unexpected binding motifs for subnucleosomal particles revealed by atomic force microscopy.

The structure of individual nucleosomes organized within reconstituted 208-12 arrays at different levels of compaction was examined by tapping mode atomic force microscopy in air and liquid. Reconstitution at lower histone octamer to DNA weight ratios showed an extended beads-on-a-string morphology with less than the expected maximum of 12 nucleosome core particles per array, each particle located in the most favored positioning site. A correlation of the contour lengths of these arrays with the number of observed particles revealed two distinct populations of particles, one with approximately 50 nm of bound DNA and a second population with approximately 25 nm. The measured nucleosome center-to-center distances indicate that this approximately 25 nm is not necessarily symmetrically bound about the dyad axis, but can also correspond to DNA bound from either the entry or exit point of the particle to a location at or close to the dyad axis. An assessment of particle heights suggests that particles wrapping approximately 25 nm of DNA are most likely to be subnucleosomal particles, which lack either one or both H2A-H2B dimers. At a higher reconstitution ratio, folded compact arrays fully populated with 12 nucleosome core particles, were observed. Liquid measurements demonstrated dynamic movements of DNA loops protruding from these folded arrays.

Binding Sites↗

PubMed2003

Realization of a multichannel integrated Young interferometer chemical sensor.

We report on the design, realization, and characterization of a four-channel integrated optical Young interferometer device that enables simultaneous and independent monitoring of three binding processes. The generated interference pattern is recorded by a CCD camera and analyzed with a fast-Fourier-transform algorithm. We present a thorough theoretical analysis of such a device. The realized device is tested by monitoring glucose solutions that induce well defined phase changes between output channels. The simultaneous measurement of three different glucose concentrations shows the multipurpose feature of such devices. The observed errors, caused by the mismatching of spatial frequencies of individual interference patterns with those determined from the CCD camera, are reduced with different reduction schemes. The phase resolution for different pairs of channels was approximately 1 x 10(-4) fringes, which corresponds to a refractive-index resolution of approximately 8.5 x 10(-8). The measured sensitivity coefficient of the phase change versus refractive-index change of approximately 1.22 x 10(3) x 2pi agrees well with the calculated coefficient of approximately 1.20 x 10(3) x 2pi.

Interferometry↗

PubMed2002

Resonance CARS study of the structure of "green" and "red" chromophores within the red fluorescent protein DsRed.

Vibrational spectra of red fluorescent protein DsRed have been studied for the first time by polarization-sensitive multiplex coherent anti-Stokes Raman scattering at two excitation wavelengths, 545 and 583 nm, in resonance with the absorption bands of the immature "green" and mature "red" protein chromophores. Overall vibrational patterns of both DsRed chromophores were found to be similar to each other and to differ from that of S65T-GFP at pH8. The combined analysis of our CARS data and published structural information suggest that both "green" and "red" DsRed species possess an extended chromophore structure. Consequently, our data suggest that pi-bonding system extension during isomerization around the cis peptide bond between Phe 65 and Gln 66 is a necessary but not sufficient step in DsRed chromophore maturation.

Animals↗

PubMed2002

Imaging technique implemented in CellTracks system.

BACKGROUND: We developed the CellTracks cell analysis system that, similar to flow cytometry, yields multiparameter information by which the cells can be differentiated. We describe the implementation of a laser scanning imaging method in the system. Image analysis of the cells improves the specificity of cell classification, especially in cases where the particular cells are found relatively infrequently and one has to discriminate between artifacts and real events. METHODS: Fluorescent images of immunomagnetically labeled and aligned cells are obtained by passing the cells through a laser focus. The laser focus is smaller than the objects and subsequent frames captured by a regular surveillance CCD camera with a frame grabber board represent different parts of the cells. Complete images of the cells are constructed by shifting each image with respect to each other and adding individual pixel values. RESULTS: The power of combining a fluorescent image with multiparametric data is demonstrated by imaging fluorescent and magnetically labeled beads and cells. The image gives additional information about the dye distribution across the objects. Changes in dye distribution as a function of time were observed in leukocytes labeled with the red fluorescent label, Oxazine750, which are imaged at different time intervals. CONCLUSIONS: An imaging technique implemented in the CellTracks system provides high-resolution fluorescent images of events previously identified by the system. The images of the fluorescent cells enhance the ability to classify rare events.

Blood Cells↗

PubMed2002

Magnetic field design for selecting and aligning immunomagnetic labeled cells.

BACKGROUND: Recently we introduced the CellTracks cell analysis system, in which samples are prepared based on a combination of immunomagnetic selection, separation, and alignment of cells along ferromagnetic lines. Here we describe the underlying magnetic principles and considerations made in the magnetic field design to achieve the best possible cell selection and alignment of magnetically labeled cells. Materials and Methods Computer simulations, in combination with experimental data, were used to optimize the design of the magnets and Ni lines to obtain the optimal magnetic configuration. RESULTS: A homogeneous cell distribution on the upper surface of the sample chamber was obtained with a magnet where the pole faces were tilted towards each other. The spatial distribution of magnetically aligned objects in between the Ni lines was dependent on the ratio of the diameter of the aligned object and the line spacing, which was tested with magnetically and fluorescently labeled 6 microm polystyrene beads. The best result was obtained when the line spacing was equal to or smaller than the diameter of the aligned object. CONCLUSIONS: The magnetic gradient of the designed permanent magnet extracts magnetically labeled cells from any cell suspension to a desired plane, providing a homogeneous cell distribution. In addition, it magnetizes ferro-magnetic Ni lines in this plane whose additional local gradient adds to the gradient of the permanent magnet. The resultant gradient aligns the magnetically labeled cells first brought to this plane. This combination makes it possible, in a single step, to extract and align cells on a surface from any cell suspension.

Algorithms↗

PubMed2002

Cell analysis system based on compact disk technology.

BACKGROUND: A 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 Methods System performance was demonstrated with 6-microm 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. RESULTS: The 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 could be aligned and analyzed in between 0.6 m of Ni lines, distributed over a surface area of 18 mm(2) and extracted from a blood volume that depended on the height of the chamber. The utility of the system was demonstrated by performing a three-color CD4-CD8-CD14 assay. CONCLUSIONS: We built a cell analysis system based on immunomagnetic cell selection and alignment and analysis of fluorescent signals employing CD-technology that is as good or better than current commercial analyzers. The cell analysis can be performed in whole blood or any other type of cell suspension without extensive sample preparation.

Animals↗

PubMed2002

Optical tweezers stretching of chromatin.

Recently significant success has emerged from exciting research involving chromatin stretching using optical tweezers. These experiments, in which a single chromatin fibre is attached by one end to a micron-sized bead held in an optical trap and to a solid surface or second bead via the other end, allows manipulation and force detection at a single-molecule level. Through force-induced stretching of chromatin, mechanical properties, specific intermolecular bond strengths and DNA-protein association and dissociation kinetics have been determined. These studies will be extremely fruitful in terms of understanding the function of chromatin structure and its dynamics within the cell.

Animals↗