High-throughput film-densitometry: an efficient approach to generate large data sets

A film-handling machine (robot) has been built which can, in conjunction with a commercially available film densitometer, exchange and digitize over 300 electron micrographs per day. Implementation of robotic film handling effectively eliminates the delay and tedium associated with digitizing images when data are initially recorded on photographic film. The modulation transfer function (MTF) of the commercially available densitometer is significantly worse than that of a high-end, scientific microdensitometer. Nevertheless, its signal-to-noise ratio (S/N) is quite excellent, allowing substantial restoration of the output to "near-to-perfect" performance. Due to the large area of the standard electron microscope film that can be digitized by the commercial densitometer (up to 10,000 x 13,680 pixels with an appropriately coded holder), automated film digitization offers a fast and inexpensive alternative to high-end CCD cameras as a means of acquiring large amounts of image data in electron microscopy.     

Effect of constant magnetic field on the liver of guinea pig. Electron microscopic studies.

Twenty guinea pigs were exposed 1 hour daily, for 3 to 7 weeks to constant magnetic field (CMF), the induction of which was 0.005 T, and 0.3 T. Hepatocytes were examined in semithin sections with light microscope and TEM. The negative photographic plates of the TEM were analyzed and measured with densitometer. It was shown that CMF of the induction 0.005 T and 0.3 T exhibited structural changes in hepatocytes, primarily in mitochondria.     

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.     

A note on the use of a scanning laser densitometer to analyse silver-stained lipopolysaccharide patterns on polyacrylamide gels and antigenic profiles on nitrocellulose sheets produced by immunoblotting

Silver-stained patterns of lipopolysaccharide on sodium dodecyl sulphate polyacrylamide gels and microbial antigenic profiles on nitrocellulose sheets produced by electroblotting and immunodetection were analysed with a laser scanning densitometer. Because the patterns fade rapidly it is usual to photograph them immediately to obtain optimum resolution. It has been shown that because of its high resolution the laser densitometer can be used to scan 35 mm photographic negatives without appreciable loss of detail. The method might be of value with whole cell immunoblot antigen profiles as characteristic fingerprints, for example, in taxonomic studies.     

Identification and quantitation of heparins by microelectrophoresis on agarose gel

The microelectrophoresis procedure on agarose devised by Jaques, Ballieux, Dietrich, and Kavanagh (1) for the identification and measurement of heparin provides a quick, versatile, and adaptable procedure. Differentiation of heparin and related polyanions can be accomplished at various stages of the procedure—migration, fixation, staining, destaining, evaluation. Tests such as solubility in solvents, precipitation by chemical reagents, action of enzymes, may be applied to minute amounts of materials.A linear relationship was observed between absorbance and heparin concentration applied and between log of spot area and log concentration so that total optical density of spots can be used to estimate heparin. Visual comparison of microelectrophoresis slides with a series of reference slides with 0.02 to 0.6 units of heparin gave a coefficient of variation of 15.5%. Difficulty was found in providing a densitometer with sufficient sensitivity and reproducibility. Special slide holders were designed to hold the microelectrophoresis slide in the Chromoscan densitometer and in the Beckman DK-2 spectrophotometer. By specifying carriage position for the slide, slit size in the optical system, baseline adjustment, background correction, light filter, reproducibility of measurement could be achieved with the Chromoscan densitometer with 10% maximum error with low counts. The Coefficient of Variation was 6.2% when all precautions were taken.     

Evaluation of densitometry data using interactive computer graphics: Application to DNA agarose gels

A method of analyzing DNA agarose gels using interactive computer graphics is described. After electrophoresis in an alkaline agarose gel, DNA is neutralized, stained with ethidium bromide and excited with ultraviolet radiation. The resulting fluorescent distribution on the gel is photographed, and the negative scanned by a digitizing densitometer. The data is subsequently analyzed using a computer program developed to facilitate manipulation and selection of data from the densitometer trace. The method has been applied to determine pyrimidine dimer yields in DNA from human lymphocytes exposed to UV radiation. The technique significantly reduces the time required to analyze such data, while also providing greater accuracy. The method could be easily adapted to assist in similar analyses of other macromolecules such as RNA or proteins.     

A spectrophotometric microassay for sulfated glycosaminoglycans using a laser densitometer

The absorption spectrum of the dye 1,9-dimethylmethylene blue shifts if complexed with sulfated glycosaminoglycans. The present method uses the decrease in A633 rather than the increase in A535, described in a recent method, to measure the sulfated glycosaminoglycan content of biological samples. A conventional spectrophotometer was used to estimate the levels of sulfated glycosaminoglycan in papain extracts from intestinal wall tissue, by measuring both the A535 and the A633 and comparing them with a chondroitin sulfate standard: a highly significant correlation (r = 0.974, n = 17) was obtained. Also, interference by substances like RNA, DNA, and hyaluronic acid was similar for both methods. These results allowed us to employ a laser densitometer with a helium/neon laser emitting at 633 nm to improve the sensitivity and the capacity of the assay. The combination of a small reaction volume and a high-intensity light source allows the detection of less than 0.1 microgram chondroitin sulfate, a 40-fold improvement in sensitivity as compared with the original method. A very significant correlation (r = 0.885, n = 17) existed between results obtained with the macroassay, using a spectrophotometer, and those found by employing the microassay, using the laser densitometer. The use of microtiter plates and the screening potential of the densitometer yields an assay which is fast, very sensitive, and suitable for processing large numbers of samples.     

Quantitative autoradiography of dot blots using a microwell densitometer

We have established conditions for the quantitation of DNA hybridization by "reading" dot blot autoradiographs with a microwell plate densitometer. This method is more convenient, as accurate, and more sensitive than counting the spots in a liquid scintillation counter.     

Apparatus for discontinuous electrophoresis in polyacrylamide gel slabs

Construction of an inexpensive slab discontinuous electrophoresis apparatus is described. Using this apparatus 23 human serum proteins were resolved and the gel could be scanned with a standard densitometer to yield a trace with discrete peaks or pronounced shoulders for each protein band. The advantages of a single homogeneous slab for comparative studies are indicated.     

双能X线吸收法骨密度仪测试原理浅析

骨质疏松症是当前越来越受到人们重视的一种病。本文简要的介绍了骨密度仪的种类和双能X线的检测工作原理。     

Population parameters. Acquisition and analysis of electrophoretic brain protein patterns by minicomputer-coupled densitometry.

Interfacing a minicomputer with a high resolution densitometer permits the online acquisition and statistical analysis of population parameters for experimental and control sample patterns of various macromolecules obtained by polyacrylamide gel electrophoresis. This technique, illustrated in this paper using protein from the brains of Japanese quail, enables the experimenter to make comparisons which were previously not readily available for electrophoresis.     

A specific assay for subnanogram concentrations of reserpine in human plasma

A sensitive analytical procedure was developed for the measurement of reserpine in human plasma. The method is based on thin layer chromatography followed by development of a fluorophor with acetic acid vapor. The thin layer plates were scanned with a Vitatron “flying spot” densitometer. Use of an internal standard minimized errors in quantitative spotting. Plasma levels were measured in healthy subjects after single oral and intramuscular doses of 1 mg of reserpine.     

Densitometric quantitation of individual phospholipids from natural sources separated by one-dimensional thin-layer chromatography

A method for the quantitation of small amounts of phospholipids derived from biological sources is described. Total phospholipid is determined by mineralization followed by the estimation of liberated phosphate by means of malachite green. The main phospholipid species are separated by one-dimensional thin-layer chromatography. The individual phospholipids are detected by charring with CuSO4/H3PO4. They may be directly quantitated by scanning the thin-layer chromatography plates with a laser densitometer.     

Adaptation of the Nelson-Somogyi reducing-sugar assay to a microassay using microtiter plates

The Nelson-Somogyi assay for reducing sugars was adapted to microtiter plates. The primary advantages of this modified assay are (i) smaller sample and reagent volumes, (ii) elimination of boiling and filtration steps, (iii) automated measurement with a dual-wavelength scanning TLC densitometer, (iv) increased range and reproducibility, and (v) automated colorimetric readings by reflectance rather than absorbance.     

A sensitive assay for allantoin

Allantoin is separated by thin-layer chromatography (TLC) and sprayed with an acidic solution ofp-dimethylaminobenzaldehyde. The yellow color produced is read in a densitometer and compared with that of a standard. The lower limit of quantitation is 0.1 μg per spot (0.5 mg/100 ml). The method can be utilized for the estimation of allantoin in serum, lymph, and urine.     

Thin-layer chromatography of urinary 17-oxosteroids using dansylhydrazine as a prelabelling reagent

This paper describes a modification of a high-performance liquid chromatographic method for measurement of 17-oxosteroids in biological fluids for use with thin-layer chromatography and fluorometric scanning detection. After extraction from urine samples with Separon-C₁₈ microcolumns, free oxosteroids were labelled with dansylhydrazine in acetonitrile-acetic acid and chromatographed on silica gel F-254 plates with the solvent system chloroform—methanol (97:3). Linearity of fluorescence detection (Shimadzu CS-9000 densitometer) was obtained between 30 and 1000 ng.     

A rapid and quantitative determination of amino acid phenylthiohydantoins by direct densitometry on thin-layer chromatogram.

Thin-layer chromatograms on silica gel plate of amino acid phenylthiohydantoins were simultaneously used for identification and quantitation. With a scanning, two-wavelength densitometer, linear calibration curves have been obtained for 15 different derivatives, which can be employed for a rapid and simple quantitation of sample spots. A satisfactory application of the method to the determination of amino-terminal residues of bovine α-chymotrypsin is described.     

A rapid, inexpensive, quantitative, general-purpose densitometer and its application to one-dimensional gel electrophoretograms

We describe the construction of a densitometer based on a TV camera linked to an IBM personal computer (PC) using commercially available components. We discuss some of the problems of accurate quantitative densitometry using low-cost video systems, and describe the manner in which absolute optical density values may be recorded. We describe a digital analysis routine designed to quantitate unresolved bands on noisy one-dimensional electrophoretograms.     

Detection of growth of Escherichia coli on two-dimensional diffusion gradient plates

One drawback of using two-dimensional diffusion gradient plates, the subjective visual assessment of microbial growth, has been overcome. Growth of Escherichia coli was detected with pH indicators in the medium or by staining growth with a biochemical stain, L-alanine- p -nitroanilide, or a respiratory dye, 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride. Stained growth was scanned with a laser densitometer and traces combined in a computer to give a three-dimensional semi-quantitative representation of growth over the gradient plate.     

Assay of matrix metalloproteinases in substrate impregnated gels in multiwells.

A zymographic method for the assay of matrix metalloproteinases in substrate impregnated gels in multiwells has been developed for the analysis of a large number of samples at a time. Enzyme was copolymerized with 300 microliters of 10% acrylamide impregnated with gelatin substrate and incubated for 16 hr. The gels were stained with coomassie blue, destained with water and the intensity measured in a densitometer. This method was tested with pure bacterial collagenase and three different gelatinases purified from rat mammary gland. The characteristics of these enzymes such as cation dependence, inhibition and concentration dependence have been examined by this method.