Ian T. Young

Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy

An investigation on the photobleaching behavior of fluorescein in microscopy was carried out through a systematic analysis of photobleaching mechanisms. The individual photochemical reactions of fluorescein were incorporated into a theoretical analysis and mathematical simulation to study the photochemical processes leading to photobleaching of fluorescein in microscopy. The photobleaching behavior of free and bound fluorescein has also been investigated by experimental means. Both the theoretical simulation and experimental data show that photobleaching of fluorescein in microscopy is, in general, not a single-exponential process. The simulation suggests that the non-single-exponential behavior is caused by the oxygen-independent, proximity-induced triplet-triplet or triplet-ground state dye reactions of bound fluorescein in microscopy. The single-exponential process is a special case of photobleaching behavior when the reactions between the triplet dye and molecular oxygen are dominant.

Recursive implementation of the Gaussian filter

Abstract In this paper we propose a recursive implementation of the Gaussian filter. This implementation yields an infinite impulse response filter that has six MADDs per dimension independent of the value of σ in the Gaussian kernel. In contrast to the Deriche implementation (1987), the coefficients of our recursive filter have a simple, closed-form solution for a desired value of the Gaussian σ. Our implementation is, in general, faster than (1) an implementation based upon direct convolution with samples of a Gaussian, (2) repeated convolutions with a kernel such as the uniform filter, and (3) an FFT implementation of a Gaussian filter.

Spectral imaging: principles and applications.

Spectral imaging extends the capabilities of biological and clinical studies to simultaneously study multiple features such as organelles and proteins qualitatively and quantitatively. Spectral imaging combines two well‐known scientific methodologies, namely spectroscopy and imaging, to provide a new advantageous tool. The need to measure the spectrum at each point of the image requires combining dispersive optics with the more common imaging equipment, and introduces constrains as well.

SPECTRAL IMAGE ANALYSIS FOR MEASURING RIPENESS OF TOMATOES

The present invention provides transport apparatus for transporting and articulating an article into and out of a housing having limited access, and includes a lifting beam having a curved shape to which is attached a lifting assembly. A hoist device is connected to the lifting assembly for raising and lowering the entire transport apparatus and also includes a worm-drive assembly for moving the lifting assembly relative to the lifting beam to thereby articulate an article about a first axis. In addition, one end of the lifting beam includes a rotational mechanism which is connected to a second worm-drive assembly. Operation of the second worm-drive assembly allows the article to be rotated relative to the lifting beam which allows the article to be rotated relative to a second axis. The combined operations of hoisting and movement about the two axes allows the article to be articulated in any direction for insertion or removal from a housing having limited access.

Recursive Gaussian derivative filters

We propose a strategy to design recursive implementations of the Gaussian filter and Gaussian regularized derivative filters. Each recursive filter consists of a cascade of two stable Nth-order subsystems (causal and anti-causal). The computational complexity is 2N multiplications per pixel per dimension independent of the size (/spl sigma/) of the Gaussian kernel. The filter coefficients have a closed-form solution as a function of scale (/spl sigma/) and recursion order N (N=3, 4, 5). The recursive filters yield a high accuracy and excellent isotropy in n-D space.

A nonlinear laplace operator as edge detector in noisy images

Abstract An edge detection scheme is developed robust enough to perform well over a wide range of signal-to-noise ratios. It is based upon the detection of zero crossings in the output image of a nonlinear Laplace filter. Specific characterizations of the nonlinear Laplacian are its adaptive orientation to the direction of the gradient and its inherent masks which permit the development of approximately circular (isotropic) filters. We have investigated the relation between the locally optimal filter parameters, smoothing size, and filter size, and the SNR of the image to be processed. A quantitative evaluation shows that our edge detector performs at least as well—and in most cases much better—than edge detectors. At very low signal-to-noise ratios, our edge detector is superior to all others tested.

The persistence length of double stranded DNA determined using dark field tethered particle motion

The wormlike chain model describes the micromechanics of semiflexible polymers by introducing the persistence length. We propose a method of measuring the persistence length of DNA in a controllable near-native environment. Using a dark field microscope, the projected positions of a gold nanoparticle undergoing constrained Brownian motion are captured. The nanoparticle is tethered to a substrate using a single double stranded DNA (dsDNA) molecule and immersed in buffer. No force is exerted on the DNA. We carried out Monte Carlo simulations of the experiment, which give insight into the micromechanics of the DNA and can be used to interpret the motion of the nanoparticle. Our simulations and experiments demonstrate that, unlike other similar experiments, the use of nanometer instead of micrometer sized particles causes particle-substrate and particle-DNA interactions to be of negligible effect on the position distribution of the particle. We also show that the persistence length of the tethering DNA can be estimated with a statistical error of 2 nm, by comparing the statistics of the projected position distribution of the nanoparticle to the Monte Carlo simulations. The persistence lengths of 45 single molecules of four different lengths of dsDNA were measured under the same environmental conditions at high salt concentration. The persistence lengths we found had a mean value of 35 nm (standard error of 2.8 nm), which compares well to previously found values using similar salt concentrations. Our method can be used to directly study the effect of the environmental conditions (e.g., buffer and temperature) on the persistence length.

The nuclear lamina promotes telomere aggregation and centromere peripheral localization during senescence of human mesenchymal stem cells

Ex vivo, human mesenchymal stem cells (hMSCs) undergo spontaneous cellular senescence after a limited number of cell divisions. Intranuclear structures of the nuclear lamina were formed in senescent hMSCs, which are identified by the presence of Hayflick-senescence-associated factors. Notably, spatial changes in lamina shape were observed before the Hayflick senescence-associated factors, suggesting that the lamina morphology can be used as an early marker to identify senescent cells. Here, we applied quantitative image-processing tools to study the changes in nuclear architecture during cell senescence. We found that centromeres and telomeres colocalised with lamina intranuclear structures, which resulted in a preferred peripheral distribution in senescent cells. In addition, telomere aggregates were progressively formed during cell senescence. Once formed, telomere aggregates showed colocalization with γ-H2AX but not with TERT, suggesting that telomere aggregates are sites of DNA damage. We also show that telomere aggregation is associated with lamina intranuclear structures, and increased telomere binding to lamina proteins is found in cells expressing lamina mutants that lead to increases in lamina intranuclear structures. Moreover, three-dimensional image processing revealed spatial overlap between telomere aggregates and lamina intranuclear structures. Altogether, our data suggest a mechanical link between changes in lamina spatial organization and the formation of telomere aggregates during senescence of hMSCs, which can possibly contribute to changes in nuclear activity during cell senescence.

Human chromosome classification based on local band descriptors

In this paper several new techniques for automated chromosome analysis are described: one for piecewise-linear chromosome stretching and projection, two for accurately localizing the centromere and one for two-dimensional local band pattern description. A classification procedure is described that is based upon local band descriptors. Classification results obtained with this method are compared with results obtained with the global band description method (WDD functions). Data sets from two different laboratories are used to investigate the influence of the preparation. Results show the suitability of the local description method in its ability to visualize the image processing technique at the level of the chromosome image.

Calibration and characterisation of imaging spectrographs

Spectrograph-based spectral imaging systems provide images with a large number of contiguous spectral channels per pixel. This paper describes the calibration and characterisation of such systems. The relation between pixel position and measured wavelength has been determined using three different wavelength calibration sources. Results indicate that for spectral calibration, a source with very narrow peaks, such as a HgAr source, is preferred to narrow band filters. A third-order polynomial model gives an appropriate fit for the pixel to wavelength mapping. The signal-to-noise ratio (SNR) is determined per wavelength. In the blue part of the spectrum, the SNR is lower than in the green and red part. This is due to a decreased quantum efficiency of the sensor, a smaller transmission coefficient of the spectrograph, as well as low output power of the illuminant. Increasing the amount of blue light, using an additional fluorescent tube with a special coating considerably increases the SNR. Furthermore, the spatial and spectral resolution of the system has been determined in relation to the wavelength. These can be used to choose appropriate binning factors to decrease the image size without losing information. In our case this could reduce the image size by a factor of 60 or more.