The objective of this work was to develop and improve technologies in cytometry and molecular biology for the specific in situ detection of viral nucleic acids. The major application for this system was the detection and measurement of individual cells stained specifically for the Human Immunodeficiency Virus (HIV) in patients with Acquired Immune Deficiency Syndrome (AIDS). Staining procedures used nucleic acid either directly or indirect labeled with enzymes or fluorescent probes. A cytometry system was used to acquire digitized images of labeled cells and determine their individual staining density or intensity. Efforts are underway to improve the sensitivity of these assays using time-resolved methods.
Autonomous learning modules in a diagnostic expert system serve to reveal distributional properties of the diagnostic clue values. This leads to more exhaustive utilization of the collected information. It also results in matching the design granularity for the diagnostic discrimination to the true structure of the diagnostic data. Additional conceptual entities augment the knowledge base.
Confocal microscopy, in comparison to conventional microscopy, offers a narrower point-spread function, the rendition of phase structures in contrast, and high axial resolution. Of these, the capabilities of optical sectioning and the three-dimensional imaging of cells and tissues have attracted the most interest. The literature offers a number of studies exploring the factors affecting depth of field in confocal microscopy. In this article, the three-dimensional representation of fluorescence imagery is examined and the design of a laser scanning confocal fluorescence microscope with enhanced depth discrimination is described.
Algorithms and procedures employed to attain reliable and exhaustive segmentation in histopathologic imagery of colon and prostate sections are detailed. The algorithms are controlled and selectively called by a scene segmentation expert system as part of a machine vision system for the diagnostic interpretation of histopathologic sections. At this time, effective segmentation of scenes of glandular tissues is produced, with the system being conservative in the identification of glands; for the segmentation of overlapping glandular nuclei an overall success rate of approximately 90% has been achieved.
Identification of cell subpopulations is of interest in the context of both flow cytometry and image analysis. Flow cytometry makes it possible to examine very large cell populations rapidly and to record a measurement vector for each cell. Thus flow cytometry, as a methodology, is ideally suited to the identification of small subpopulations of cells, which is particularly of interest in analyzing lymphoid cell populations.
The performance of a multiprocessor computer system in histopathology applications was studied. Specifically, timing studies have been done on an operational multiprocessor, the Heidelberg Polyp, running expert system-guided scene segmentation and image analysis software. A number of actual and potential system bottlenecks and methods of attack for removing them are discussed.
Three-dimensional (3D) data are obtained on biological cells by collecting optical slices at different depths in the cell using confocal laser scanning microscopy. A method has been developed which allows such three-dimensional data to be automatically segmented so that objects within the cell can be identified and labelled. Segmentation allows an object in a field to be recognized as distinct from other objects and from the background. This is done using an algorithm which performs a cell-dependent sequence of image processing functions on each slice of the image data (2D). Identification requires that a single object be recognized as one entity through all of the slices of data (3D). This is done with a 3D labelling routine. In this application, cell nuclei are first defined in 3D, and then structures within them (i.e. chromosome domains) can be identified. Measurements are made on these domains for the purpose of determining the relationship between chromosomal locations and the possibility that they will exchange genetic information.
Preliminary work on a new approach to sequencing polynucleotides by the Sanger procedure is presented. The method is based on a mass spectrometric determination of the four component terminal nucleotide residues, where the information regarding the identity of the individual nucleotides is contained in the mass of a stable nuclide marker. Specifically, the four stable isotopes of sulfur (32, 33, 34, 36) will be used to identify the four terminal bases (A, T, G, C ) where the label is incorporated as a thiophosphate bridge. The nucleotide fragments are separated by capillary electrophoresis. The identity of the isotope marker for the terminal base is then determined by combustion, and mass spectrometric analysis of the resulting isotopically labeled sulfur dioxide.
Fluorescent and chemiluminescent detection of DNA hybrids on polymer membranes has been investigated using a cryogenically cooled, slow readout, two dimensional CCD camera in an imaging mode. The fluorescent background characteristics of commercially available nylon blotting membranes and a polypropylene membrane modified to bind DNA have been studied. The polypropylene membrane exhibits a 15-fold increase in DNA binding, 3-fold less background fluorescence and less background noise than nylon blotting membranes. However the detection limits determined from vacuum slot blots of crosslinked fluoresceinlabelled oligonucleotides show that the improved qualities of the polypropylene support do not result in a lower detection limit. This is mainly due to background noise arising from sources other than the membrane itself during the blotting/washing procedure and to a low signal-to-amount of DNA ratio with the polypropylene membrane. The lowest amount of fluorescein-labelled oligonucleotide detectable is 1.4 femtomol, with a typical exposure time of 10 minutes to image a 6x9 cm area of the membrane. The detection of chemiluminescence was done using a biotin-avidin complex in combination with an enzymatic assay. The assay was carried out after hybridization with biotin-labelled probes on vacuum slot blots with crosslinked target DNA. The detection limit is 0.12 femtomol of DNA target, a result obtained after 30 mm exposure. Further improvements are necessary to image sequencing blots with typically 1 femtomol or less of DNA per band in an acceptable exposure time.
Individual ethidium stained DNA molecules, 50 kbp to 2Mbp in length, are observed with a fluorescence microscope, image intensifier, and video camera. The microscope slide is equipped with electrodes to . introduce an electric field across the sample. An array of electrodes, controlled by computer, can select both field strength and direction. When embedded in a thin (10-20 micron) layer of agarose and electrophoresis buffer, linear DNA molecules snake through the gel toward the positive electrode. The linear polymers alternately stretch and contract as they become hooked around obstacles and then slip off. Mechanisms responsible for size separation in pulse field gel electrophoresis are explored. Open circular plasmids become permanently trapped on gel obstructions in a strong electric field. They behave like negatively charged rubber bands as they elongate in a strong field and contract as the field is reduced. Measurement of plasmid elongation in various fields allows calculation of the effective electrophoretic charge on DNA. Measurement of random coil diameters permits calculation of DNA's persistence length.
Flow Cytometry has become an accepted technique in the clinical laboratory for rapid immunophenotyping of patient blood samples. Multiple, fluorescent labeled monoclonal antibodies are used to tag the cells, which are then analyzed one at a time at rates of several thousand cells a second. Patient samples are processed through the flow cytometer at more than one a minute. Clinicians are being overwhelmed by the large amount of data that must be analyzed to provide the information needed to assist in disease diagnosis. An expert system is being developed to assist clinicians in analyzing this multivariate flow cytometry data. The data from each sample are processed by a clustering algorithm, which finds the means of the distinct cell subpopulations in a sample. These mean values of fluorescence are translated into words such as "negative", "dim" and "bright" and the words are combined into patterns that are matched against the premises on the left hand side of the rules used to identify the disease categories. This is a report of work in progress.
The use of protein-based biological pigments in combination with low molecular weight organic dye molecules allows the simultaneous detection of up to six independent components of a cell surface by antibody dependent fluorescence (immunofluorescence) excited with a single 488nm laser line. This paper describes the state of the art in multi-color immunofluorescence with 488nm Ar-ion laser excitation. It provides the data to design systems for specialized applications.
Eigenanalysis is a mathematical approach used to obtain characteristic roots and vectors from a matrix and is an important method for extracting information from digital images in microscopy. To use multiple images as input data, the images must be correctly aligned and the pattern common for each input image must not be geometrically distorted. Otherwise, the different geometries produce blurring. Alignment and removal of geometric distortion from digital images of random examples of 20 high resolution preparations of human karyotypes has been achieved by the eigenanalysis performed in the Fourier spectral domain on the phase and the amplitude of the Fourier transform. Our results show that eigenanalysis in the frequency & un provides a better resolution of features than does eigenanalysis in the spatial domain and makes it possible to construct statistically based prototypes.