Preparatory methods for DNA hydrolysis, cytochemistry, immunocytochemistry and ploidy analysis. Their application to automated and routine diagnostic cytopathology

A review is presented of some methods used to prepare cytologic specimens for analytical and/or automated studies, with the steps of the procedures detailed in appendices. The preparation of the cell monolayers required for optimal automated cell image analysis and classification, e.g., by the Cytoscan 110, is discussed, as is the preparation of poly-L-lysine-coated slides used in the production of monolayered specimens. These monolayers, which can be prepared from a variety of specimens, are also useful for cytochemical and immunocytochemical studies and DNA ploidy analysis. For DNA analysis, a modified gallocyanin chrome alum staining procedure is described as a stoichiometric alternative to the time-consuming Feulgen reaction. The hydrolysis technique required by the latter method is also detailed. The freeze-fracturing technique for the enhancement of monoclonal antibody immunocytochemical staining of detectable antigens is described, along with an indirect immunoalkaline phosphatase staining method. The use of enzyme cytochemical reactions for glucose 6 phosphate dehydrogenase and lysosomal naphthylamidase is also presented.     

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.     

Computerized tomography using a modified orthogonal tangent correction algorithm.

A modified orthogonal tangent correction algorithm is presented for computerized tomography. The algorithm uses four X-rays scans spaced 45 degrees apart, to reconstruct a transverse axial image. The reconstruction procedure is interative in which image matrix elements are corrected by alternately matching the two sets of orthogonal scan data. The algorithm has been applied to phantom data as well as to video recorded fluoroscopic data.     

The core-microtome: A new tool for surface preparation on cores and time series analysis of varying cell parameters

A microtome designed for the surface preparation of entire increment cores allows cutting plane surfaces on cores up to a length of 40 cm. Compared to the common sanding procedure, the wood cells of the annual rings remain open, not filled with swarf, and the cell walls are smooth and hence clearly visible. This article aims at describing the functionality of the microtome and the procedures needed for an accurate surface preparation to achieve a good contrast for subsequent image analysis. Possible applications for a more detailed analysis of variations in the tracheid structure of conifers and vessel sizes of oak are presented, which can be included in time series analyses.     

A disjunctive analysis of neonatal approach stimulus prepotence

Preliminary results of a disjunctive procedure developed to ascertain the relative attractiveness for domestic chicks of auditory and visual stimuli are promising. A detailed account of the procedure and initial results is presented. Seventy-two Canadian Athens random bred chicks were tested at 24 or 36 h posthatch. A repetitive tone (4 per sec, 50 msec duration, 500 Hz) served as the auditory stimulus, and a flickering light (3.5 flashes per sec, 0.8 foot candle) served as the visual stimulus. An age-dependent change in the attractiveness of auditory and visual stimuli obtained with the disjunctive procedure. No change in stimulus preference obtained when the stimuli were presented individually.     

An image analysis and statistical evaluation program for the assessment of tumour cell invasion in vitro.

Tumour cell invasion is a complex process, which is essential for the formation of metastasis and is therefore of critical clinical importance. For detailed investigations of the invasive process, quantifiable in vitro models of invasion are necessary. In this study we describe an image analysis procedure and a statistical program which facilitate an objective analysis of experiments carried out using the embryonic chick heart invasion model of Mareel. Tumour multicellular spheroids are confronted with embryonic chick heart fragments in culture and are sampled after different time intervals for up to 7 days. Immunohistological sections are then evaluated by an image analysis procedure which provides 9 parameters indicating invasion, proliferation and destruction taking place in the confrontation cultures. The data obtained by image analysis are further evaluated by a statistical program which describes the change with time of each parameter by means of linear regression analysis. Thus the data obtained at various time intervals serve as the source data for a single statistic, namely the slope of the regression line. Confidence intervals and statistical differences between various experiments can be calculated. In order to make the procedure more comprehensible in biological terms, the program provides a full text interpretation of the experimental results. The image analysis procedure in conjunction with statistical evaluation and text interpretation provides a comprehensive tool for the quantitative assessment of experimental invasion in vitro.     

The perceived image: efficient modelling of visual inhomogeneity.

When we fixate on a certain location in a scene, the image that our visual system provides us with is not like a snapshot of the scene. Because of visual inhomogeneity, the image we perceive is sharp and clean only around the fixation location, and it gradually blurs and loses detail with increasing distance from that location. In this paper, a procedure is presented that allows one to obtain the luminance distribution in the perceived image of a scene. The procedure is based on a stack distribution model of spatial visual processing, with parameters obtained experimentally. An application of this procedure is presented which allows an explanation of data on the decrease of visual acuity with eccentricity. The advantages of this procedure for taking visual inhomogeneity into account when seeking explanations for perceptual phenomena are also discussed.     

3-(p-hydroxyphenyl)propionic acid as a new fluorogenic reagent for amine oxidase assays

A detailed procedure of a new and extremely sensitive fluorometric assay for amine oxidases is presented. Hydrogen peroxide, produced by the oxidase reaction, reacted with 3-(p-hydroxyphenyl)propionic acid in the presence of peroxidase to yield a fluorescent compound by which enzyme activity could be determined. The enzyme reaction was terminated by NaOH solution, which increased the fluorescence intensity three- to fivefold. The detection limit thus obtained was as little as 0.02 nmol. The alkalinization also contributed to stopping the enzyme reaction and to the clarification of assay mixtures containing turbid enzyme preparations.     

Prostaglandin F2 radioimmunoassay utilizing polyethylene glycol separation technique

A radioimmunoassay for Prostaglandin F which utilizes polyethylene glycol to separate antibody-bound and free prostaglandin is discribed. The detailed procedure for extraction and chromatographic fractionalization of the "E" and "F" series is presented. The recoveries of labeled prostaglandin obtained at each step of the procedure compared favorably with the 68% overall recovery figure for the complete assay. Data on the sensitivity, specificity, accuracy and reproducibility of the assay are presented. PgF levels in plasmas obtained from normal males and females, pregnant females in the third trimester and females in labor have been determined. Ovarian and uterine tissue levels of Prostaglandin F are also included.     

14C]acetate metabolism in the peripheral nervous system

A method for the complete and specific removal of histones H2A and H2B from nucleosome core particles is presented. Reconstitution of the separated products of depletion form a particle which has the same structure as native core particles as judged by a number of physical and biochemical criteria. The technique described also minimises the possibility of the formation of reconstituted core particles with different histone stoichiometries. These experiments are important as they demonstrate a procedure which can be extended to prepare core particles with selectively deuterated components while maintaining complete integrity of structure. When prepared, and studied by neutron scattering, selectively-deuterated core particles can give detailed information with respect to the relative positions and structure of the histone fractions within the core particle.     

Progress in application of the image analysing system QUANTIMET 720 in the histomorphometry of the microcirculatory system

The application of automated image analysing methods in the field of histomorphometry has been improved in the last decades. In this paper a new method is presented for the investigation of morphological reactions of the microcirculatory system. It is based on the measurement of quantitative changes in microvessel wall tissue by using the image analysing system QUANTIMET 720. In the measurement model, a microvessel intersection figure is considered as an ellipsis. A procedure is presented allowing the estimation of the wall and lumen area of the microvessel section and the calculation of its radius for a totally collapsed state. From this data, further morphometric parameters are derived. Using this method, the analysis of 500 microvessels including classification and parameter derivation takes about 10 min.     

Hydrodynamic properties of rigid macromolecules composed of ellipsoidal and cylindrical subunits.

A procedure is devised for the calculation of hydrodynamic properties of rigid macromolecules composed subunits that are modeled as ellipsoids of revolution and cylinders. Owing to the axial symmetry of these shapes, smooth shell models can be constructured for the subunit structure. The bead shell model so constructed is employed for the calculation of the properties. A computer program, HYDROSUB, has been written implementing both the model building and the hydrodynamic calculation. A detailed example of the use of this methodology is presented for the case of the solution properties of the human antibody molecule immunoglobulin G3 (IgG3). Finally, hints are given on other uses and applications of the procedure.     

Primedin situ labeling (PRINS)

PRimedIn Situ labeling (PRINS) is a fast and sensitive alternative to fluorescencein situ hybridization (FISH) for identification of chromosome aberrations. In this article, we present the detailed protocols for detection of repeat sequences using oligonucleotides or fragments of cloned probes as primers for PRINS. We describe a multicolor PRINS procedure for simultaneous visualization of more probes in different colors on a metaphase preparation, and a PRINS-painting procedure, which combines PRINS and chromosome painting. Finally, a protocol for detection of single-copy genes is presented.     

Quantification of strains in biaxially tested soft tissues

A technique for the quantification of the strain field in the central region of biaxially tested planar soft tissues is presented. A vidicon-based image analysis system interfaced to a PDP 11/34 minicomputer is employed to track particles affixed to the specimen surface in real-time, from which the strains are inferred. Illustrative results are given for tests on excised canine pleural specimens on which four particles were affixed. The technique is applicable, however, to any planar soft tissue and any number of tracking particles. This procedure is recommended over previously used methods when testing anisotropic tissues.     

Synthesis of pyrophosphates for in vitro selection of catalytic RNA molecules

Reported here are synthetic routes to pyrophosphates linking riboflavin with various nucleosides. The focus is on a flavin-uracil dinucleotide having a biotin tag on the uracil, a molecule that has potential value in the selection of RNA enzymes that catalyze the template-directed polymerization of RNA in the 3'-to-5' direction, which is the direction opposite that catalyzed by standard protein polymerases. Two detailed procedures are presented to prepare this new compound, as well as one procedure to prepare the new flavin-2,6-diaminopurine dinucleotide.     

Simple Method for Determination of Biodegradable Dissolved Organic Carbon in Water

Determination of biodegradable dissolved organic carbon in waters is of particular importance for the water treatment industry. A simple method for determining biodegradable dissolved organic carbon which is applicable to surface and drinking water is proposed. It consists of sterilizing the water sample, inoculating it with autochthonous bacteria, and measuring the decrease in dissolved organic carbon concentration due to the carbon oxidization by bacteria. The detailed experimental procedure is discussed, and validation of the method is presented. The method has been used for studying river waters and for drinking water treatment plant design.     

A new method for quenching correction leads to revisions of data in receptor autoradiography

Differential quenching of beta-emission affects strongly the analysis of receptor distribution patterns in quantitative receptor autoradiography with tritiated ligands. Different methods for the quenching correction have been described in the past, but some of these are of limited value, if a detailed anatomical parcellation is necessary. Other methods correct exclusively local variations in lipid concentration, which is an important, but only one of several factors causing quenching. A new method for the measurement of quenching (or autoradiographic efficiency) is presented, which permits an anatomically detailed and direct determination of the total quenching without lipid extraction procedures. This method is based on the measurement of autoradiographic efficiency in cryostat sections homogeneously labeled with tritiated formaldehyde by an underlying gelatine section containing this labeled compound. Regional and layer specific measurements of autoradiographic efficiency in cortical and subcortical regions of the human and rat brain are reported. A significant correlation was found between the density of myelin and autoradiographic efficiency but other factors were also shown to influence differential quenching. The use of the here presented correction procedure leads to revisions of the laminar distribution patterns reported for different receptors in human and rat cortical areas. Our results show, that a complete quenching correction is necessary for the mapping of receptor distributions with tritiated ligands.     

Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi

Gene replacement via homologous double crossover in filamentous fungi requires relatively long (preferentially >0.5 kb) flanking regions of the target gene. For this reason, gene replacement cassettes are usually constructed through multiple cloning steps. To facilitate gene function studies in filamentous fungi avoiding tedious cloning steps, we have developed a PCR-assisted DNA assembly procedure and applied it to delete genes in filamentous fungi. While the principle of this procedure is essentially the same as other recently reported PCR-based tools, our technique has been effectively used to delete 31 genes in three fungal species. Moreover, this PCR-based method was used to fuse more than 10 genes to a controllable promoter. In this report, a detailed protocol for this easy to follow procedure and examples of genes deleted or over-expressed are presented. In conjunction with the availability of genome sequences, the application of this technique should facilitate functional characterization of genes in filamentous fungi. To stream line the analysis of the transformants a relatively simple procedure for genomic DNA or total RNA isolation achieving approximately 100 samples/person/day is also presented.     

Weed Mapping in Early-Season Maize Fields Using Object-Based Analysis of Unmanned Aerial Vehicle (UAV) Images

The use of remote imagery captured by unmanned aerial vehicles (UAV) has tremendous potential for designing detailed site-specific weed control treatments in early post-emergence, which have not possible previously with conventional airborne or satellite images. A robust and entirely automatic object-based image analysis (OBIA) procedure was developed on a series of UAV images using a six-band multispectral camera (visible and near-infrared range) with the ultimate objective of generating a weed map in an experimental maize field in Spain. The OBIA procedure combines several contextual, hierarchical and object-based features and consists of three consecutive phases: 1) classification of crop rows by application of a dynamic and auto-adaptive classification approach, 2) discrimination of crops and weeds on the basis of their relative positions with reference to the crop rows, and 3) generation of a weed infestation map in a grid structure. The estimation of weed coverage from the image analysis yielded satisfactory results. The relationship of estimated versus observed weed densities had a coefficient of determination of r²=0.89 and a root mean square error of 0.02. A map of three categories of weed coverage was produced with 86% of overall accuracy. In the experimental field, the area free of weeds was 23%, and the area with low weed coverage (<5% weeds) was 47%, which indicated a high potential for reducing herbicide application or other weed operations. The OBIA procedure computes multiple data and statistics derived from the classification outputs, which permits calculation of herbicide requirements and estimation of the overall cost of weed management operations in advance.