Bioimaging最新文献

The potential for the application of fluorescence lifetime imaging (FLIM) microscopy to studies of photosensitization mechanisms in photodynamic therapy (PDT) has been investigated. The fluorescence microscope incorporates a standard inverted optical microscope, a picosecond pulsed dye-laser excitation source, and an intensified CCD camera detector capable of being gated on a sub-nanosecond timescale. Fluorescence lifetime images resulting from multi-component analysis of sub-nanosecond gated fluorescence images of monolayer V79-4 Chinese hamster lung fibroblasts stained with disulphonated aluminium phthalocyanine (AlPcS2), a photosensitizer used in PDT, are presented. The results of these measurements are discussed in terms of the intracellular localization of the sensitizer. Preliminary results from multi-component FLIM of V79-4 cells multiply stained with AlPcS2 and a potential intracellular pH lifetime probe, 5(+6)-carboxynaphthofluorescein, are also presented.

Time-resolved optical computer tomographic imaging has received considerable interest recently because of its non-invasiveness and non-destructiveness to biological tissue, and several attempts have been made for its implementation. The image recovery algorithm described in this paper is based on the finite-element method solution to the diffusion equation as the photon transport model. By formulating image reconstruction as an optimization problem with the objective function expressed by the error norm between actual and modeled measurement sets, this algorithm incorporates a direct search optimization method, the rotating coordinates method, into the solution strategy to avoid excessive computation of the time-consuming forward problem. Several numerically simulated results for the images of the absorption and scattering coefficient distribution within a circular tissue, reconstructed from the integrated intensity, the mean time of photon flight or their weighted sum, are given. The influence of the numbers of stimulating sources and boundary detection points on the resulting images is discussed. Finally, this paper concludes that the reconstruction algorithm used is feasible but needs to be improved in its accuracy and efficiency.

Differential interference contrast (DIC) microscopy is the preferred imaging mode for studying live unstained cells and embryos. This is mainly attributed to the optical sectioning capability which makes DIC suitable for three-dimensional microscopy. However, DIC images are not convenient for standard computerized image interpretation. We present here a processing algorithm that converts DIC images into a form which is much more amenable for such analysis. The algorithm is based on computational inversion of the directional gradient performed optically by DIC, namely path integration (line integrated DIC, or LID). LID images relate to the refractive index of the specimen, and are inherently positive in value, thus many powerful algorithms developed for fluorescent images can be applied to them. The method is demonstrated here for identifying and tracking nuclei in developing zebrafish embryos, and for reconstructing the shape of live leukocytes.

The fractal dimension is a measure of the space filling characteristics of a particular object. In this paper we describe a method to quantify the fractal dimension (D) of three-dimensionally reconstructed samples from serial sections based on the two-dimensional mass–radius relation of the object embedded in the sections. The advantage of performing the analysis in this way is that it requires little computer memory to hold the data in comparison to the procedure that calculates the mass–radius dimension from the three-dimensional data array. In this case, the test datum is a human first lumbar vertebra embedded in black resin, then horizontally sectioned 256 times and each section photographed using a digital imaging system. The captured images were converted to binary images and then analysed using the 2-D and 3-D mass–radius relation to determine the fractal dimension (D) of the trabecular bone. D was 3.01 using the cubic structuring element and 2.92 using the spherical structuring element. The method may be used to quantify in an objective manner the complex structure of trabecular bone, to model and design new materials (bone substitutes), to understand the physical properties of bone and model the patterns of radiographic images.

The development of fractal geometry has prompted the use of fractal dimensions of objects as measures of morphological complexity. Many biological specimens show fractal scaling, but within limited scale ranges. Beyond those limits, the specimens are Euclidean. Such objects are called asymptotic fractals and alternative models have been proposed to describe them. These approaches rely on fitting data gathered with length-resolution techniques (for example the ‘yardstick’ method) to theoretical models of asymptotic fractal scaling. Unfortunately, data produced with the so-called ‘box counting’ method cannot be used with these models. We report a new approach to estimate the asymptotic fractal behaviour (at low resolution) of asymptotic fractals based on the single assumption that the specimen approaches a Euclidean object at high resolutions. The procedure described can be applied using length-resolution methods as well as the box counting method and opens the possibility for estimating asymptotic fractal behaviour in both cluster and branching structures.

Using a computer-controlled fluorescence microscope system, spatial arrangement of homologous chromosomes was analyzed in fixed and living embryos of Drosophila melanogaster at the syncytial blastoderm stage. In fixed embryos, chromosomes were stained with a DNA-specific fluorescent dye, 4′,6-diamidino-2-phenylindole; the arrangement of anaphase chromosomes was determined with their identification by high-resolution analysis. In living embryos, the behavior of chromosomes during anaphase was examined by the use of a strain carrying a long translocated chromosome as a cytological marker to identify the chromosome while the chromosomes were stained by microinjection of rhodamine-conjugated histones into the embryos. Chromosome arrangement was also examined in nuclei that had swollen artificially under anoxic conditions for better spatial separation of individual chromosomes in such nuclei. All these experiments consistently showed that homologous chromosomes were not associated with each other in syncytial blastoderm embryos of Drosophila melanogaster. Our studies also demonstrated that cytological tools greatly facilitate the dissection of nuclear structures when used in combination with imaging technology.

Imaging in fluorescence microscopy is analysed using a vectorial diffraction theory. Both conventional and confocal microscopy are considered. A fluorescent molecule is modelled as a radiating electric dipole. Images for particular orientations in fluorescence polarization microscopy are considered. We also average over all dipole orientations. In this case, two particular limiting cases are considered, corresponding to different depolarization relaxation times: the dipole can either freely rotate in space between excitation and emission, or is fixed in space. The image is different in each limiting case. If the dipole can freely rotate, the image after averaging is identical to that calculated assuming an isotropic point object.

An overview of our recent results in modeling single molecule detection in fluid flow is presented. Our mathematical approach is based on a path integral representation. The model accounts for all experimental details, such as light collection, laser excitation, hydrodynamics and diffusion, and molecular photophysics. Special attention is paid to multiple molecule crossings through the detection volume. Numerical realization of the theory is discussed. Measurements of burst size distributions in single B-phycoerythrin molecule detection experiments are presented and compared with theoretical predictions.

The efficiency of detecting a single fluorescent coumarin dye molecule in aqueous solution by one-photon excitation (OPE) at 350 nm as well as by coherent two-photon excitation (TPE) at 700 nm is studied. The photostability, which is crucial for single molecule detection (SMD), is determined at a low irradiance for various coumarin derivatives using a ‘cell-bleaching’ method. The yields of photobleaching for these coumarins in aqueous solution are in the order of 10⁻³ to 10⁻⁴. Thus, most of the dyes are sufficiently stable to allow SMD. However, for SMD in a fluorescence microscope a high quasi-CW irradiance (at least 10⁴ W cm⁻²) is necessary for efficient OPE by a pulsed, frequency doubled titanium:sapphire laser. Detailed investigations on the dye Coumarin-120 using fluorescence correlation spectroscopy (FCS), different repetition rates of the laser and transient absorption spectroscopy (TRABS) gave clear evidence that OPE at a high irradiance results in two-step photolysis via the first electronic excited singlet and triplet state, S₁ and T₁, producing dye radical ions and solvated electrons. Hence, this additional photobleaching pathway limits the applicable irradiance for OPE. Using coherent TPE for single molecule detection, saturation of the fluorescence was observed for a high quasi-CW irradiance (10⁸ W cm⁻²), which may also be caused by photobleaching. Furthermore, TPE is deteriorated by other competing nonlinear processes (e.g. continuum generation in the solvent), which only occur above a threshold irradiance (7 × 10⁷ W cm⁻²). Nevertheless, TPE allows an efficient detection of single Coumarin-120 molecules in water. Using a maximum likelihood estimator, we are also able to identify single dye molecules via their characteristic fluorescence lifetime of 4.8 ± 1.2 ns.

We demonstrate efficient detection of single fluorescent molecules eluting off a polystyrene microsphere optically trapped in a flowing sheath stream. A 1μm diameter analyte doped microsphere was positioned ∼20 μm upstream of a 16 μm diameter probe laser without significant degradation of the detection signal-to-noise ratio due to scattered laser light and fluorescence from the microsphere. In comparison to more standard capillary sample introduction, the microsphere causes only small perturbations to the sheath fluid flow. The small diameter of the analyte stream eluting from the microsphere results in a greater than 90% detection efficiency for single rhodamine-6G molecules, limited primarily by the photostability of the dye.