Measuring the wavelength-dependent divergence of transmission through sub-wavelength hole-arrays by spectral imaging.

We present a study on the far-field patterns of light transmitted through sub-wavelength metallic hole-arrays. Spectral imaging measurements are used here on hole arrays for the first time. It provides both spatial and spectral information of the transmission in far-field. The visibility of the images, measured in two illumination modes: Köhler and collimated, is calculated for different planes in and out of focus. The transmission under collimated illumination reveals that 75% of the beam if non-divergent. The results are in agreement with the low divergence measured by Lezec [Science 297, 820 (2002)].

Characterizing the three-dimensional organization of telomeres.

BACKGROUND: Quantitative analysis can be used in combination with fluorescence microscopy. Although the human eye is able to obtain good qualitative results, when analyzing the spatial organization of telomeres in interphase nuclei, there is a need for quantitative results based on image analysis. METHODS: We developed a tool for analyzing three-dimensional images of telomeres stained by fluorescence in situ hybridization in interphase nuclei with DNA counterstained with 4',6-diamidino-2-phenylindole. After deconvolution of the image, we segmented individual telomeres. From the location of the telomeres we derived a distribution parameter rhoT, which indicated whether the telomeres were in a disk (rhoT >> 1) or not (rhoT approximately 1). We sorted mouse lymphocyte nuclei and measured rhoT. We also performed a bromodeoxyuridine synchronous cell sorting experiment on live cells and measured rhoT at several instances. RESULTS: Measuring rhoT for nuclei in G0/G1, S, and G2 produced 1.4 +/- 0.1, 1.5 +/- 0.2, and 14 +/- 2, respectively, showing a significant difference between G2 and G0/G1 or S. For the bromodeoxyuridine synchronous cell sorting experiment, we found a cell cycle dependency of rhoT and a correlation between rhoT and an observer. CONCLUSIONS: In this study we present a quantitative method to characterize the organization of telomeres using three-dimensional imaging, image processing, and image analysis.

3D restoration with multiple images acquired by a modified conventional microscope.

A problem in high magnification microscopy is the blurring in the imaging of an object. In this article, we demonstrate a restoration technique that simultaneously makes use of the confocal image and the wide-field image. These images can be acquired by a modified conventional microscope. In front of the light-source, there is an array of pinholes. There are no pinholes at the detection plane. Instead, one or more pixels from the CCD camera are used, where the pinholes would have been. Using all pixels gives the wide-field image, but using a selected subset can give a confocal image. The array is used to speed up the process of acquiring the image. Note that the speed of acquisition is proportional to the number of pinholes. We show that the restoration from the two images can lead to a better result than using only one of the images. If this is the case, we show that a distance of 5 times the diameter of the pinholes can give the same results as a distance of 20 times after deconvolution. This offers an increase in acquisition time of a factor 16.

Monitoring enzymatic reactions in nanolitre wells.

We have developed a laboratory-on-a-chip microarray system based on nanolitre-capacity wells etched in silicon. We have devised methods for dispensing reagents as well as samples, for preventing evaporation, for embedding electronics in each well to measure fluid volume per well in real-time, and for monitoring the fluorescence associated with the production or consumption of NADH in enzyme-catalysed reactions. Such reactions can be found in the glycolytic pathway of yeast. We describe the design, construction and testing of our laboratory-on-a-chip. We also describe the use of these chips to measure both fluorescence (such as that evidenced in NADH) as well as bioluminescence (such as evidenced in ATP assays). We show that our detection limit for NADH fluorescence is 5 micro m with a microscope-based system and 100 micro m for an embedded photodiode system. The photodiode system also provides a detection limit of 2.4 micro m for ATP/luciferase bioluminescence.

Shading correction: compensation for illumination and sensor inhomogeneities.

The interaction between objects in real space, the illumination, and the camera frequently leads to a situation in which a microscope image exhibits significant shading across the field of view. In general this shading effect is undesirable and requires elimination, especially for quantitative microscopy. Starting with a simple mathematical model, this unit develops procedures to correct images for the shading thus introduced in the image-formation process. Two cases are distinguished: the a priori, which assumes the availability of calibration images in addition to the images of interest; and the a posteriori, which assumes that only the recorded images of interest are available.

Calibration: sampling density and spatial resolution.

This unit presents a discussion of procedures for measuring the sampling density and spatial resolution of a quantitative microscope system. These two independent quantities are fundamental characteristics of a system that must be known for proper processing and interpretation of digitized microscope images and of measurements extracted from such images.

Model-based resolution: applying the theory in quantitative microscopy.

Model-based image processing techniques have been proposed as a way to increase the resolution of optical microscopes. Here a model based on the microscope's point-spread function is analyzed, and the resolution limits achieved with a proposed goodness-of-fit criterion are quantified. Several experiments were performed to evaluate the possibilities and limitations of this method: (a) experiments with an ideal (diffraction-limited) microscope, (b) experiments with simulated dots and a real microscope, and (c) experiments with real dots acquired with a real microscope. The results show that a threefold increase over classical resolution (e.g., Rayleigh) is possible. These results can be affected by model misspecifications, whereas model corruption, as seen in the effect of Poisson noise, seems to be unimportant. This research can be considered to be preliminary with the final goal being the accurate measurement of various cytogenetic properties, such as gene distributions, in labeled preparations.

FISH and chips: automation of fluorescent dot counting in interphase cell nuclei.

Fluorescence in situ hybridization allows the enumeration of chromosomal abnormalities in interphase cell nuclei. This process is called dot counting. To estimate the distribution of chromosomes per cell, a large number of cells have to be analyzed, especially when the frequency of aberrant cells is low. Automation of dot counting is required because manual counting is tedious, fatiguing, and time-consuming. We developed a completely automated fluorescence microscope system that can examine 500 cells in approximately 15 min to determine the number of labeled chromosomes (seen as dots) in each cell nucleus. This system works with two fluorescent dyes, one for the DNA hybridization dots and one for the cell nucleus. After the stage has moved to a new field, the image is automatically focused, acquired by a Photometrics KAF 1400 camera (Photometrics Ltd., Tuscon, AZ, USA), and then analyzed on a Macintosh Quadra 840AV (Apple Computer, Inc., Cupertino, CA, USA) computer. After the required number of cells has been analyzed, the user may interact to correct the computer by working with a gallery of the cell images. The automated dot counter has been tested on a number of normal specimens where 4,'6-diamidino-2-phenylindole (DAPI) was used for the nucleus counterstain and a centromeric 8 probe was used to mark the desired chromosome. The slides contained lymphocytes from cultured blood. We compared the results of the dot counter with manual counting. Manually obtained results, published in the literature, were used as the "ground truth." For a normal specimen, 97.5% of cells will have two dots. Fully automated scanning of 13 slides showed that an average of 89% of all nuclei were counted correctly. In other words, an average of 11% has to be interactively corrected, using a monitor display. The machine accuracies, after interactive correction, are comparable to panels of human experts (manual). The fully automatically obtained results are biased with respect to manual counting. An error analysis is given, and different causes are discussed.

Influence of fluorochrome labeling density on the photobleaching kinetics of fluorescein in microscopy.

The objective of this study was to identify, through kinetic analysis of individual elementary reactions, the conditions under which a simple first-order photobleaching kinetic model is sufficient for quantitative fluorescence measurements, and those under which more complex photobleaching kinetics must be considered. Three model systems of various fluorophore densities and distributions were employed to verify the kinetic analysis. The results showed that the photobleaching kinetics of free fluorescein at concentrations lower than 5 microM corresponded closely to a single exponential function and therefore involved predominantly simple unimolecular or pseudounimolecular photochemical reactions. When fluorescein was bound to polyvinyl alcohol (PVA) molecules, the photobleaching kinetics of the densely labeled PVA deviated more from a single-exponential function than sparsely labeled PVA. When fluorescein was bound to a DNA probe, the photobleaching kinetics were more complex and deviated significantly from a single-exponential function, due to one or more bimolecular processes with apparent concentration-dependent photobleaching rate constants. The practical applications of time-integrated fluorescence emission are discussed in the context of simple and complex photobleaching kinetics.

Automation of spot counting in interphase cytogenetics using brightfield microscopy.

In situ hybridization techniques allow the enumeration of chromosomal abnormalities and form a great potential for many clinical applications. Although the use of fluorescent labels is preferable regarding sensitivity and colormultiplicity, chromogenic labels can provide an excellent alternative in relatively simple situations, e.g., where it is sufficient to use a centromere specific probe to detect abnormalities of one specific chromosome. When the frequency of chromosomal aberrations is low, several hundreds or even thousands of cells have to be evaluated to achieve sufficient statistical confidence. Since manual counting is tedious, fatiguing, and time consuming, automation can assist to process the slides more efficiently. Therefore, a system has been developed for automated spot counting using brightfield microscopy. This paper addresses both the hardware system aspects and the software image analysis algorithms for nuclei and spot detection. As a result of the automated slide analysis the system provides the frequency spot distribution of the selected cells. The automatic classification can, however, be overruled by human interaction, since each individual cell is stored in a gallery and can be relocated for visual inspection. With this system a thousand cells can be automatically analyzed in approximately 10 min, while an extra 5-10 min is necessary for visual evaluation. The performance of the system was analyzed using a model system for trisomy consisting of a mixture of male and female lymphocytes hybridized with probes for chromosomes 7 and Y. The sensitivity for trisomy detection in the seeding experiment was such that a frequency of 3% trisomic cells could be picked up automatically as being abnormal according to the multiple proportion test, while trisomy as low as 1.5% could be detected after interaction.

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.

3D base: a geometrical data base system for the analysis and visualisation of 3D-shapes obtained from parallel serial sections including three different geometrical representations.

In this paper we discuss a geometrical data base that includes three different geometrical representations of one and the same reconstructed 3D shape: the contour-pile, the voxel enumeration, and the triangulation of a surface. The data base is tailored for 3D shapes obtained from plan-parallel serial sections. It is explained how this geometrical data base is useful with the different processing approaches of a 3D shape, such as analysis and visualisation. Methods of conversion between the geometrical representations are discussed. Examples of the operation of the data base as it is embedded in a data base management system are given by illustrations of retrieval of geometrical information.

Towards a quantitative grading of bladder tumors.

The histological inspection of tumor tissue for the purpose of reporting a tumor grade is a problem of significant clinical importance. The grading by a pathologist is only partly reproducible due to vaguely defined, subjective criteria. In this article we describe and evaluate a set of measurable features that quantitate the differences in tumor tissue. Different aspects of the reproducibility of the measurements under varying conditions of image selection, focus, and noise have been investigated. Three hundred thirty-three images were digitized from 111 bladder tissue sections (4 microns thick, Feulgen stained), using the ICAS microscope-camera platform. A segmentation routine was developed to segment the images into nuclei and background without any user interaction. Size, shape, optical density, and texture features were measured on and among the objects found by this segmentation routine using the image analysis package Acuity. The results of the measurements showed that there is a significant quantitative difference between grade 1 and grade 3 tumors. Grade 2 tumors can be described as "in between grade 1 and grade 3" and falling somewhere on an increasing scale between grades 1 and 3. Grade 2 tumors do not seem to represent a statistically distinct population. The procedure described here is shown to be quite reproducible in the presence of noise, reasonably reproducible in the event of a modest amount of defocusing (with grade 3 tumors exhibiting the most sensitivity), and less reproducible in the context of which fields-of-view are chosen for analysis.

The Athena semi-automated karyotyping system.

In this article we describe Athena, a system that provides for semi-automated karyotyping of metaphase spreads. The system is based upon the Macintosh II computer. It uses software that is written entirely in C and consists of approximately 200 Kbytes of executable code. Athena provides automated segmentation of metaphase images into individual chromosomes, automated measurements on each banded chromosome, and automated classification into the standard Paris-convention karyotype. Furthermore, the system provides the ability to construct one or more chromosome data bases to represent the types of metaphase spreads and staining techniques that may be used in a given laboratory. Because we believe that it is impossible to construct a system that can achieve perfect segmentation, perfect separation of touching and overlapping chromosomes, perfect localization of the centromeres, and perfect classification, the system offers the possibility for interaction at each of the above stages using the well-accepted Macintosh user interface.

Experience with the Athena semi-automated karyotyping system.

The traditional analysis and assembly of metaphase chromosomes into a karyogram is a slow and tedious process requiring intermediate photographic steps and manual manipulation of the chromosome images. Much of this task is highly repetitive and readily lends itself to partial automation. Semi-automated karyotyping systems now are being used increasingly in both clinical and research cytogenetic laboratories. Digital image processing techniques are used to capture, manipulate, and make an initial classification of chromosome images. The Athena system uses commercially available components based on a Macintosh II personal computer. Digital image processing procedures automatically isolate chromosome images from the metaphase and arrange them into a karyogram, using information about relative chromosome length, centromeric index, and banding pattern. The operator uses the intuitive graphics interface of the Macintosh computer to monitor each phase of the analysis, to resolve any problems in isolating chromosome images, and to rearrange the individual chromosome images while assembling the final karyogram. Athena is designed as a semi-automated karyotyping system that is easy to learn and has sufficient power and versatility for routine use in the analysis of human metaphase chromosomes.

Sampling density and quantitative microscopy.

The sampling densities required for the quantitative analysis of digitized microscope images is discussed. It is shown that the Nyquist sampling theorem is not the proper reference point for determining the sampling density when the goal is measurement, although it may be a proper reference point when the goal is image filtering and reconstruction. The problems associated with signal truncation--the use of a finite amount of data--and the finite amount of time available for computation make it impossible to reconstruct an arbitrary image, even if it is bandlimited. Two examples taken from straightforward measurement problems exhibit the fundamental problems associated with the measurement of analog quantities from digital data and the role played by the sampling density.

Characterization of chromatin distribution in cell nuclei.

In this paper we develop four measures to describe the distribution of nuclear chromatin. These measures attempt to describe in an objective and meaningful way the heterogeneity, granularity, condensation, and margination of chromatin in cell nuclei. Starting with a high-resolution digitized image of a cell where the nuclear pixels have been identified, the four measures may be rapidly estimated. The range of each is derived and the interpretation of the measures in the context of chromatin compaction and distribution is developed. Implementation issues such as sampling density, thresholding and subsequent pre-processing, and algorithmic complexity are discussed.

A comparison of different focus functions for use in autofocus algorithms.

A number of functions for the autofocusing of microscopes and other optical instruments are to be found in the literature. In this article we compare 11 of them to determine, in an objective manner, which functions are most suitable for implementation with real-time video acquisition systems. Three different images, each representing a typical class of image, are used in the comparison. Among the best focus functions found in our study are the squared magnitude gradient, the squared Laplacian, and the normalized image standard deviation.

Morphologic changes in rat urothelial cells during carcinogenesis: II. Image cytometry.

Improved early detection of neoplasia by screening of urothelial cells requires an understanding of the features distinguishing normal and neoplastic cell populations. We have begun a program of study based upon a rat model system for the controlled observation of early-stage lesions produced by the carcinogen N-butyl-N-(4-hydroxybutyl)- nitrosamine. Cells dissociated directly from normal and malignant urothelium were characterized by conventional cytopathology techniques and by quantitative microscopy (for nuclear texture and nuclear and cytoplasmic size, shape, and stain content) to derive a comprehensive picture of bladder tumor development. By following the changes that occur in the dissociated urothelial cells we have found that the nuclear area, total nuclear stain, nuclear shape, and the nuclear chromatin change significantly over a 48-wk interval as the lesions progress toward malignancy.