Bioimaging最新文献

Reversibly binding silicone cartridges have been developed to form reaction containers in which ‘single molecule chemistry’ can be performed. We use an optical trap to drive 1μm streptavidin-coated beads into a region containing biotinylated DNA until binding of a strand of DNA occurs. A quadrant detector is used in reflective mode to track the lateral position of trapped beads. Relative motion between the bead and the solution causes a viscous drag force which is increased when a single strand of DNA is attached to the bead; DNA-bead attachment is done in minutes with less than a femtomole of DNA. The method allows the study of single molecule digestion.

The resolution along the optical axis is much less than the lateral resolution in both confocal and conventional fluorescence light microscopy. To correct for the anisotropy in resolution one may generate tilted views from the same object and subsequently merge these tilted views into one image with improved isotropic resolution. It has been suggested to obtain tilted views in confocal theta microscopy with double observation by illuminating the object from one side and simultaneously detecting the emitted light in the direction parallel to the illumination direction and in a direction with an angle theta to the illumination direction. Alternatively, one might use the usual optical set-up of a light microscope together with a tilting device to rotate the object under the microscope. With this method the object can be viewed from all directions (axial tomography). In this paper we investigate two methods for merging tilted views. If the image sets obtained from the different tilt angles are interpreted as statistic samples of one and the same object, a gain in resolution along the optical axis is accompanied by a significant trade-off in lateral resolution. An optical interpretation of the image formation in each single object point by retaining the frequencies with the highest amplitudes in the different tilted views provides a gain in axial resolution without significant decrease in lateral resolution. We will describe a quantitative method to determine the gain in resolution dependent on the number and geometry of the tilted views. It will be demonstrated that a previously suggested reconstruction method implying a multiplication of tilted views in the spatial domain does not improve resolution. Simulated reconstructions based on measured point spread functions in axial tomography and confocal theta microscopy with double observation will demonstrate the validity of our reconstruction methods.

A method is presented for measuring 3-D plant growth using the optical flow computed on an image sequence of a growing corn seedling. Each image in the sequence consists of two views of the same seedling; one view of the corn seedling is front-on while the second view is a orthogonal view (at 90°) of the seedling made by projecting the plant's orthogonal image onto a mirror oriented at 45° with respect to the camera. We compute 3-D velocity (motion) of the corn seedling's tip by using a simple extension of the 2-D motion constraint equation used in optical flow analysis. This method is an extension of the work presented by Barron and Liptay where optical flow was used to measure the 2-D growth (in the vertical plane) of a corn seedling.

Fluorescence correlation spectroscopy (FCS) provides information about two important parameters in molecular biology. These are the average number of molecules in the detection volume, and the translational diffusion coefficient of the molecules. Although the properties of the translational diffusion reflect the molecular weight and shape, our work focuses on the analysis of the number of cleaved DNA fragments in solution. The cleavage of fluorescently labelled M13 DNA and pUC19 DNA was carried out with three kinds of restriction enzymes (HaeIII, HgaI and BsmAI), and the number of DNA fragments was monitored using FCS. The reciprocal value of the autocorrelation function at time τ = 0 is consistent with the number of fragments that are expected from restriction enzyme maps of the DNA. Our study indicates the capability of FCS as a tool for finding genetic differences in DNA sequences.

The effect of the Fresnel diffraction on the imaging properties of confocal microscopy with one circular aperture and one annular aperture is investigated. The results show that the Fresnel diffraction patterns alter the axial imaging properties more seriously than the transverse imaging properties. The axial response of such a confocal microscope is sharpened, but severely distorted.

Multifluorescence labeling is routinely performed to detect the spatial coincidence between several markers within biological specimens. We have recently developed image correlation methods to identify double fluorescent structures by virtue of local similarities between fluorescence distributions. We extend this approach here to analyze statistically the fluorescence distribution of structures of interest on micrographs. This digital cytometry relies mainly on the segmentation of multifluorescence images. Once identified, all objects are analyzed through a range of attributes estimating size, morphology, fluorescence content and mean colocalization level between fluorescence channels. The data sets which are saved in flow cytometry standard (FCS) files, allow multiparameter classification of objects and subpopulation counting. The combination of fluorescence, morphometric and local image correlation attributes has been applied here to compare the frequency of single- and multiple-labeled structures at the subcellular level.

This paper describes a modelling method for continuous closed contours. The initial input data set consists of two-dimensional (2-D) points, which may be represented as a discrete function in a polar coordinate system. The method uses the Shannon interpolation between these data points to obtain the global continuous contour model. A minimal description of the contour is obtained using the link between the Shannon interpolation kernel and the Fourier series of polar development (FSPD) for periodic functions. The Shannon interpolation kernel allows the direct interpretation of the contour smoothness in terms of both samples and Fourier frequency domains.

In order to deal with deformation point sources, often encountered in active modelling techniques, a method of local deformation is proposed. Each local deformation is performed in an angular sector centred on the deformation point source. All the neighbouring characteristic samples are displaced in order to minimize the oscillations of the newly created model outside the deformation sector. This deformation technique preserves the frequency characteristics of the contour, regardless of the number and the intensity of deformation sources. In this way, the technique induces a frequency modelling constraint, which may be subsequently used in an active detection and modelling environment.

Experiments on synthetic and real data prove the efficiency of the proposed technique. The method is currently used to model contours of the left ventricle of the heart obtained from ultrasound apical images. This work is part of a larger project, the aim of which is to analyse the space and time deformations of the left ventricle. The 2-D Fourier–Shannon model is used as a basis for more complex three-dimensional and four-dimensional Fourier models, able to recover automatically the movement and deformation of the left ventricle of the heart during a cardiac cycle.

Spatially resolved single molecule detection in solution is a prerequisite for molecule tracking and provides new opportunities in single molecule manipulation, for example in a sample flow. A detector with fast timing properties and high spatial resolution is required for these purposes. We introduce a concept for spatially resolved optical single molecule detection in an epi-illuminated microscope using a novel kind of detector and a new algorithm in configurable hardware for intelligent data processing. The analysis is performed on a parallel hardware interface and includes burst detection. It can be extended to on-line spatial and temporal data analysis. This detector will be used in combination with a molecular sorter where molecules are sorted in a microstructured flow device made of silicon. In a first step towards such sorting in these microstructures, we show that a reliable detection of single molecules in silicon microstructures is possible in zero-dimensional detection volumes. The noise structure of the data is analysed in terms of Poissonian statistics and it is shown that in the smallest structure used (depth 20 μm) a signal-to-noise ratio of 40 is achieved.