Bioimaging最新文献

The transmittance of an evanescent wave generated from a prism coated with a double-layer thin-film structure is optimized with respect to various physical parameters including the incident angle, the refractive index and the thickness of the layers, and the illumination wavelength. A prism coated with an optimized double-layer stack produces an enhanced evanescent wave with a transmittance of 963, which is 17% higher than the reported result.

A modified optical system for the light microscope has been devised in order to remotely shift the focal plane and to manipulate the point spread function for any given objective lens. An adjustable telescope system is inserted into the microscope tube so as to move the intermediate image position, thus achieving two goals of fundamental importance for the three-dimensional imaging of biological samples. First, it allows the focus to be rapidly varied without actually moving the objective lens. This permits high throughput three-dimensional microscopy of living specimens. Secondly, it makes possible the compensation of objective lens spherical aberration. This distortion is especially significant when high numerical aperture objectives are utilized to image deep into thick specimens.

In this note we will present methods based on image processing techniques to evaluate the performance of light microscopes. These procedures are applied to three different ‘high-end’ light microscopes. Tests are carried out to measure the homogeneity of the illumination system. From these tests it follows that Köhler illuminated images can have an exceedingly high amount of shading. Another result found from the illumination calibration is that traditional lamp housings are not designed to make fine-tuning easy. Next, the (automated) stage is considered. Several tests are performed to measure the stage motion in the xy-plane and in the axial direction to address accuracy, precision, and hysteresis effects. Finally, the entire electro-optical system is characterized by measuring the optical transfer function (OTF) at wavelengths 400 nm, 500 nm, 600 nm, and 700 nm. The results of these experiments show that there is a consistent deviation from the theoretical OTF at wavelengths around 400 nm. The final conclusion is that modern light microscopes perform better than their five-to-ten-year-old predecessors.

The cleaving process of rhodamine labeled DNA with T7-exonuclease was monitored using the fluorescence correlation method. The fluorescence correlation function, formulated so as to take into account the fluorescence efficiency of the fragment, was derived to allow analysis of the two-component system. The reaction was well represented by the two-component model, allowing monitoring of the progressive cleavage of DNA. The analysis yielded not only the size of the polymer fragment but also the efficiency of the incorporation of rhodamine and the ratio of its quantum yield in DNA and in solution.

Multifocal multiphoton microscopy (MMM) is an efficient and technically simple method for generating multiphoton fluorescence images. Featuring the high axial resolution of confocal and multiphoton scanning microscopes, MMM also achieves high speed in 3-D microscopy. In this paper, examples of fast-mode 3-D microscopy are given including imaging of fixed brain tissue as well as living PC12 cells. The imaging speed of MMM is solely determined by the fluorescence photon flux, so that in practice, for brightly fluorescent specimens, a whole stack of about 50 images of 30–70 μm diameter can be acquired within a few seconds. MMM represents a significant advance towards high speed nonlinear optical tomography of fluorescent specimens.

Laser-induced fluorescence (LIF) from single molecules at room temperature displays rich dynamics of reversible and irreversible transitions within the photodestruction lifetime of the molecule. In contrast to ensemble studies, these transitions are directly resolved when the fluorescence signal is monitored in time. We present a strategy and describe the instrumentation needed for spectroscopic studies on a large number of individual molecules. It is based on a computer controlled optical system which automatically and rapidly positions single molecules in the excitation volume of a confocal microscope and subsequently performs spectroscopic measurements. Examples for such spectroscopies are presented.

Computer programs have been developed to determine decay time constants from a temporal sequence of digitized images with decaying intensities characterized by either single or double exponentials plus a constant background term. The very fast algorithms are evaluated at every pixel position. A non-iterative Prony-like method provides high quality initial estimates that are used for the subsequent non-linear least-squares procedure in which the normal equations are solved directly. Error analysis routines enable a pixel-by-pixel estimation of the quality of the experimental data. The stand-alone programs were fully integrated into a commercial image-processing environment (SCIL-Image) for a convenient and optimal display of the decay analysis. The features of the programs are illustrated by the analysis of simulated image data. With current workstations, the fitting routines (including reading of data, initial estimate and error analysis) require 0.13 ms/pixel using the single exponential algorithm applied to 50 time points, and 1.37 ms/pixel for 100 time points and the double exponential algorithm. The programs are of general applicability and have been used to analyse data from time-resolved fluorescence, phosphorescence, and photobleaching-based microscopy. Two examples of the latter case are shown, illustrating the utility of the programs for the quantitative evaluation of spatially resolved fluorescence resonance energy transfer (FRET) and for generating contrast by allocating specific cellular structures to particular decay components in a fluorescence image.