Immunofluorescence localization of Z-DNA in chromosomes: quantitation by scanning microphotometry and computer-assisted image analysis

Anti-Z-DNA polyclonal and monoclonal immunoglobulins raised against left-handed polynucleotides show various degrees of specificity for base sequence and substitution. Class 1 IgGs recognize all Z-DNA with equal affinity; class 2 IgGs show a preference for d(G-C)n sequences and class 3 IgGs for d(G-C)n sequences with substitutions at the C5 position of the pyrimidine. These antibodies served as probes for the localization of Z-DNA in polytene and metaphase chromosomes and in interphase chromatin by indirect immunofluorescence. A quantitative assessment of the binding of anti-Z-DNA IgGs to polytene chromosomes of Chironomus and Drosophila was made by scanning microphotometry and by computer-assisted image analysis of double immunofluorescence and DNA-specific dye fluorescence images. The three classes of antibodies bind to most of the bands in acid fixed polytene chromosomes of C. thummi; however, preferential binding of one class of antibody over another can be observed in certain regions. These differences can be quantitated by arithmetic division or subtraction of the normalized digital images. If a class 2 antibody is first bound at saturating concentrations the binding of class 1 antibody is reduced throughout most bands by 40-50%. However, the telomeres of the three large chromosomes bind greater than 10 times as much class 1 antibody as class 2 antibody, indicating that the Z-DNA tracts in these regions are comprised largely of alternating sequences containing the A X T basepair, e.g., A-C. High-resolution image analysis of class 1 and class 2 immunofluorescence patterns and the total DNA distribution from polytene chromosomes of D. melanogaster show that the two antibody distributions are very similar in a large majority of the bands, but they often deviate from the mean DNA distribution profile. Z-DNA sequences of both G-C and A-C type are detectable at all levels of ploidy from 2n to 2(13)n and in species as diverse as insects and man. We conclude that the vast majority of polytene chromosome bands (genes) contain one or a few DNA sequences with potential for undergoing the B----Z transition and contain both alternating purine-pyrimidine G-C and A-C tracts or mixed sequences. Highly heterochromatic bands and telomeres have more Z potential sequences than do other bands.     

An automated quantitative image analysis tool for the identification of microtubule patterns in plants

High throughput confocal imaging poses challenges in the computational image analysis of complex subcellular structures such as the microtubule cytoskeleton. Here, we developed CellArchitect, an automated image analysis tool that quantifies changes to subcellular patterns illustrated by microtubule markers in plants. We screened microtubule‐targeted herbicides and demonstrate that high throughput confocal imaging with integrated image analysis by CellArchitect can distinguish effects induced by the known herbicides indaziflam and trifluralin. The same platform was used to examine 6 other compounds with herbicidal activity, and at least 3 different effects induced by these compounds were profiled. We further show that CellArchitect can detect subcellular patterns tagged by actin and endoplasmic reticulum markers. Thus, the platform developed here can be used to automate image analysis of complex subcellular patterns for purposes such as herbicide discovery and mode of action characterisation. The capacity to use this tool to quantitatively characterize cellular responses lends itself to application across many areas of biology.      

A technique for section thickness evaluation for microphotometry and image analysis of sectioned nuclei.

The exact knowledge of the section thickness is a requisite for making the necessary corrections on DNA measurements in tissue sections. Several methods have been proposed to evaluate section thickness, each of them with advantages and disadvantages depending on the type of specimen and equipment available. We herein report another method based on preparation of standard material whose optical density varies as a function of its thickness and is sectioned and measured alongside the tissue specimen. The standards consist of celloidin cylinders stained with the PAS reaction and embedded in paraffin. For prior characterization of the cylinders, sections of different thickness were obtained and mounted. The optical density of each section was measured by direct microphotometry or image analysis. The actual thickness of each section was evaluated following re-embedding of piled groups of sections in a paraffin block and transversal sectioning. The thickness was then measured with a micrometric eye-piece. Optical density and actual thickness of each section were plotted on a normogram curve. Once a given tissue is sectioned alongside with the reference cylinder, the actual thickness is determined by its optical density on the normogram curve.     

An automated differential scanning dilatometer.

A sensitive scanning dilatometer has been constructed which continuously records the apparent partial specific volume of small biological samples (10–100 mg) as a function of temperature. Changes in volume of 0.02 μl can be resolved. Tedious calibrations with their inherent errors are eliminated by employing a differential approach in which a recording electrobalance responds to the difference in buoyancy of identical sample and reference tubes. Agreement with the literature values for the specific volume of n-eicosane and the apparent partial specific volume of KCl was excellent. Apparent partial specific volumes obtained for dipalmitoyl lecithin, egg albumin, and bovine serum albumin agreed well with published values.     

An automated microscope for cytologic research a preliminary evaluation.

A research-oriented system for automated microscopy is described from an operational point of view. The system consists of a microscope, a TV camera, an automatic cell finder and a servo-driven computer controlled stage. The system is interfaced to a NOVA 840 computer having 112,000 words of 16-bit core memory and extensive peripherals. It is capable of performing a wide variety of image processing tasks and is being used to study various aspects of automated microscopy, with applications in, but not limited to, cytology. Results of preliminary performance evaluations are given.     

Software for quantification of labeled bacteria from digital microscope images by automated image analysis

Automated image analysis software, CellC, was developed and validated for quantification of bacterial cells from digital microscope images. CellC enables automated enumeration of bacterial cells, comparison of total count and specific count images [e.g., 4',6-diamino-2-phenylindole (DAPI) and fluorescence in situ hybridization (FISH) images], and provides quantitative estimates of cell morphology. The software includes an intuitive graphical user interface that enables easy usage as well as sequential analysis of multiple images without user intervention. Validation of enumeration reveals correlation to be better than 0.98 when total bacterial counts by CellC are compared with manual enumeration, with all validated image types. The software is freely available and modifiable: the executable files and MATLAB source codes can be obtained at www. cs. tut.fi/sgn/csb/cellc.     

An image analysis system for cervical cytology automation using nuclear DNA content.

An experimental computer/image analysis system has been used to investigate cytology automation techniques based on nuclear DNA measurement and morphological artefact rejector tests. The system automatically measures and normalizes the integrated optical density of cell nuclei in specially prepared cervical cytology specimens, and selects any objects with abnormally high values for further analysis. These are then analyzed by morphological and densitometric tests designed to eliminate false positive signals caused by non-nuclear artefacts. The coordinates of the remaining abnormal nuclei are recorded so that they can subsequently be relocated and examined by a cytotechnician. Preliminary results are given showing the measurement accuracy of the system and the performance of the artefact rejection tests.     

激光共聚焦显微镜在微痕定量分析中的应用综述

微痕分析是石器、骨器、牙齿等工艺、功能研究的常用方法,但无论是高倍还是低倍等常用方法,受限于体式光学显微镜、扫描电镜等观察方法,微痕分析通常很难进行原位定量分析,因此微痕分析的定量化是目前学术界遇到的主要问题。近年来随着激光共聚焦显微镜（LSCM）在微痕分析中的应用,微痕定量化在西方学术界得到了新的实践和进展。本文主要介绍激光共聚焦显微镜的原理及其在微痕分析中的应用案例,以期促进微痕分析在中国的进一步发展。     

An automated method for scanning LC-MS data sets for significant peptides and proteins, including quantitative profiling and interactive confirmation

Differential quantification of proteins and peptides by LC-MS is a promising method to acquire knowledge about biological processes, and for finding drug targets and biomarkers. However, differential protein analysis using LC-MS has been held back by the lack of suitable software tools. Large amounts of experimental data are easily generated in protein and peptide profiling experiments, but data analysis is time-consuming and labor-intensive. Here, we present a fully automated method for scanning LC-MS/MS data for biologically significant peptides and proteins, including support for interactive confirmation and further profiling. By studying peptide mixtures of known composition, we demonstrate that peptides present in different amounts in different groups of samples can be automatically screened for using statistical tests. A linear response can be obtained over almost 3 orders of magnitude, facilitating further profiling of peptides and proteins of interest. Furthermore, we apply the method to study the changes of endogenous peptide levels in mouse brain striatum after administration of reserpine, a classical model drug for inducing Parkinson disease symptoms.     

Accurate automated quantitative imaging of tortoise erythrocytes using the nis image analysis system

Abstract

The standard method for assessing blood cell characteristics using an ocular micrometer is time-consuming and limited. We used the Nikon NIS Elements imaging software and May- Grünwald-Giemsa staining to determine whether automated image analysis is suitable for rapid and accurate quantitative morphometry of erythrocytes. Blood was collected during four seasons from 126 geometric tortoises and the blood smears were evaluated for cell (C) and nuclear (N) characteristics of the erythrocytes. We measured area, length (L), width (W), perimeter, elongation and pixelation intensity, and calculated L/W and N/C areas.

Erythrocyte size differed among cohorts; females, the larger sex, had smaller erythrocytes than either males or juveniles. Males had more elongated erythrocytes than females and erythrocytes of adults were more elongated than those of juveniles. Erythrocyte size and shape influence the efficiency of gas exchange owing to surface area to volume ratios, which are greater for small, elongated cells than for large, round cells. The high N/C ratio and low pixelation intensities of males and juveniles indicate that they may have had more immature erythrocytes in their circulation than females. The use of pixelation intensity to indicate the presence of immature erythrocytes was validated by seasonal differences that corresponded to the biology of the tortoises. Pixelation intensity was lowest in winter. We found that automated image analysis is a rapid and reliable method for determining cell size and shape, and it offers the potential for distinguishing among developmental stages that differ in staining intensity. The method should be useful for rapid health assessments, particularly of threatened species, and for comparative studies among different vertebrates.     

An automated quantitative assay for fungal growth inhibition

Abstract A simple technique which enables the monitoring of fungal growth with the aid of a microplate reader is described. In the absorbance range of 0 to 0.6 units, a straight-line relationship exists between absorbance at 595 nm and dry weight of microplate cultures, indicating that culture absorbance is an accurate indicator of fungal biomass. The relative standard deviation of the absorbance measurements was low (typically between 2 and 6%) when spores were used for inoculum. With inoculi consisting of mycelial fragments, slightly higher standard deviations (ca. 10%) were found. The microplate reader technique is particularly suited for determination of growth inhibition curves, since it is extremely fast, reliable, and requires as little as 75 μl of total culture volume.     

An automated analysis of chemotaxis in vitro using a computer-assisted scanning densitometer

The quantitation of chemotaxis in vitro was developed with a computer-assisted scanning densitometer. The method of estimating the number of cells on a filter was based on the photo-reflection from the nuclei of stained cells. Samples obtained from a 48-well micro chemotaxis assembly were successfully analyzed by this method. This assay system could quantitate chemotaxis much faster and more accurately than by cell counting under the microscope. It was sensitive enough to determine the responsiveness of SMCs and fibroblasts to various chemoattractants. This system could be applied to medical and biological screening tests for drugs and clones in laboratories.     

Acriflavine-Feulgen stilbene staining: a procedure for automated cervical cytology with a television based system (LEYTAS).

A sample preparation and staining procedure for automated cytology with a TV based system (LEYTAS) is described. It consists of a centrifugation technique and automated acriflavine-Feulgen stilbene staining of cervical specimens. The advantages of using both the fluorescence and the absorption image of acriflavine-Feulgen stilbene stained cervical cells for a television based system are discussed.     

Effect of colchicine on atherosclerosis. III. Study of dermal elastic fibers by quantitative histochemistry, automated image analysis

Computerized automatic-image analytical procedure was applied on dermal biopsies stained for elastin by a new procedure giving a completely white background and staining only the elastic fiber system. In arteriosclerotic hypertensive patients, a 3-4 months' treatment with 1 mg colchicine per day resulted in a significant (60-80%, p less than 0.01) increase of the dermal elastic fiber density both in the superficial papillary dermis and in the deep dermis. This result shows that the age-dependent increase of elastic fibers can be influenced by pharmacological means. The inhibition by colchicine of the synthesis and secretion of the fibroblast-derived metalloelastase-type protease could be a plausible explanation of this finding.