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Biomedical subjects

J W Prothero

Publications and source records attributed to J W Prothero.

At least 19 recordsLinked to original sources

Three-dimensional reconstruction from serial sections. V. Calibration of dimensional changes incurred during tissue preparation and data processing.

Three-dimensional (3-D) reconstructions from serial tissue sections produce a table of x, y, z coordinates (i.e., a numerical description of the object) that will support 3-D computer graphics displays and morphometric analyses. While measures such as volume or surface area can be generated interactively, almost instantaneously, they are usually of unknown accuracy due to artifacts that may be introduced at two different stages: (1) tissue shrinkage during dehydration and polymerization of a plastic and compression or expansion during sectioning and mounting and (2) geometric and intensity distortions during image capture and data processing. This paper describes simple methods for (1) introducing fiducials (reference marks) that allow measurement of the net distortion incurred during tissue preparation and (2) estimating the amount of geometric distortion arising during image capture and data processing. Application of these methods showed that the net areal change introduced in dog and sheep hearts during tissue processing amounted to +/- 5%. Apparently, the substantial shrinkage that occurs during tissue processing is largely compensated for by the expansion during tissue sectioning and mounting. The methods described may have application to other semisolid tissues.

Animals

Evidence for a universal process underlying clonal attenuation.

It is shown by computer simulation that an established commitment model of clonal attenuation can account for clone size distribution data obtained from three vertebrate species--chick, hamster and human--from two evolutionarily divergent classes. The different in vitro replicative lifespans of each cell strain can be explained by differences in cell kinetics. These results suggest that the process of clonal attenuation is qualitatively similar in fibroblasts from all vertebrate species.

Animals

Three-dimensional reconstruction from serial sections. IV. The reassembly problem.

In many fields of biology and medicine there is a pressing need for quantitative descriptions of biological structures at a resolution of micrometers. This need is currently met best by three-dimensional reconstruction from serial sections. The preliminary steps in three-dimensional reconstruction include fixation, embedding in plastic, introduction of fiducials, serial sectioning, and staining. At the light microscope level, with which we are chiefly concerned, one will usually want to do photomicrography (or videomicrography) of adjacent fields within individual tissue sections. The resultant images are projected onto a digitizer pad and the contours of interest manually digitized. From the digitized coordinates generated thereby, one wishes to generate a likeness of the original object, using computer graphic displays, and to then do interactive morphometrics. The problem of combining the digitized coordinates so as to produce a numerically faithful representation of the original object (i.e., the reassembly problem) is, as a practical matter, nontrivial. A technical description of the reassembly problem is presented. The main factors entering into a solution of the problem are discussed and a mathematical statement of the solution is given.

Anatomy

Three-dimensional reconstruction from serial sections III. AUTOSCAN, a software package in FORTRAN for semiautomated photomicrography.

A FORTRAN program, called AUTOSCAN, is described. This program permits the collection of photomicrographic data from serial sections to be semiautomated. In essence the user defines a box around a microscopic field of interest. Then the program drives the stage incrementally in the x and y directions, taking photographs of contiguous subfields. The box is defined by the use of a joystick and the "return" key. That is, movements of a joystick cause the stage to translate in the x and y directions. When a corner of the object is reached, as defined by cross-hairs in the microscope eyepiece, the user hits the return key. Repetition of this process at each corner defines a "box" within which photographs are to be taken. AUTOSCAN then calculates the step size and the number of frames to be taken from the user-defined values for the magnification. The actual movements of the stage in the x and y directions and the photography are fully automated. Each frame of film has the x and y coordinates of the center of the subfield being photographed imprinted in one corner, along with other relevant data. The x and y coordinates permit the resultant information to be assembled correctly into a two-dimensional montage.

Computers

TOPPER, a software package in FORTRAN for scaling studies.

Scaling studies are concerned with the differences in structure, function, behaviour and life-history associated with differences in size in broadly similar organisms. These studies make extensive use of bivariate regression analysis. Often it is of interest to carry out such analyses on many different subpopulations within a given sample population. The programs described here allow these analyses to be carried out efficiently. The output gives a summary of taxonomic parameters in addition to the usual statistical parameters, such as standard deviations and mean percent deviation. Some improvements in the software which are currently being implemented are discussed briefly.

Animals

Clonal attenuation in chick embryo fibroblasts. Experimental data, a model and computer simulations.

When cells from mass cultures of chick embryo fibroblasts are grown at very low density, some cells yield large clones while others produce smaller clones, and some cells fail to divide at all. The distribution of clone sizes is related to the number of population doublings which the donor mass culture has undergone: the more doublings which have occurred, the smaller the average clone size. In this report we describe a model which analyses this phenomenon, referred to as 'clonal attenuation', in detail. The model is based on the concept that a cell with hypothetically unlimited replicative potential--i.e. a 'stem' cell--can become 'committed' to a programme of limited replicative potential. This event is assumed to be stochastic and to have a fixed probability per stem cell division. The parameters of the model are: Pc, the probability of commitment; N, the number of differentiative divisions; and Tc, the cell-cycle times. By computer simulation, it is shown that Pc increases roughly exponentially at each successive stem cell division. According to the model, when the daughter of a stem cell becomes committed, its progeny proceed through N obligatory divisions before becoming terminally differentiated (post-mitotic). The best-fit value of N was found to be seven. The simulations also reveal that the absolute number of stem cells in the total population increases for most of the lifespan of the culture. When Pc becomes much greater than 0.5, the number of stem cells declines rapidly to zero, and the culture nears senescence. Sensitivity analysis shows that Pc can assume only a limited range of values at each stem-cell division.

Animals

Folding of the cerebral cortex in mammals. A scaling model.

A model of cortical folding in mammals is presented. The model consists of a cube, superimposed on which are straight close-packed gyri, running the length of the cube. The cortex is represented by a thin layer of constant thickness. It proves possible, by adjusting the length, height, and width of the 'gyri' and the thickness of the 'cortex', to obtain a reasonable fit to the available empirical data (which extend over three to four orders of magnitude in brain weight). The model directs attention to possible features of the macroscopic organization of the mammalian brain which are novel and hitherto unremarked.

Animals

Three-dimensional reconstruction from serial sections: II. A microcomputer-based facility for rapid data collection.

A microcomputer-based facility is described that permits the data required for three-dimensional reconstructions to be collected quickly and inexpensively from serial sections. The facility consists of a microcomputer, a digitizer tablet, a graphics terminal, a printer, a plotter, and telephone coupler. Images of serial sections are superimposed on the digitizer tablet. Contours of interest on each section are digitized and the coordinates are stored on "floppy" disks. The problems of putting successive sections in correct register and of taking into account magnification factors are discussed briefly. Use of the facility for high-resolution applications is also considered.

Animals

Organ scaling in mammals: the liver.

1. Values for liver weight, in growing and adult male and female mammals, both terrestrial and aquatic, as well as values for hepatic blood flow, blood volume and oxygen consumption are submitted to linear (log-log) regression analysis. 2. The slope of the regression line for liver weight on adult body weight in adult mammals was found to be 0.886. No statistically significant difference was found between male and female, nor between terrestrial and aquatic mammals (at the 1% confidence level). 3. Over about four orders of magnitude there is (on present evidence) a tendency for the mammalian liver to grow as about the 0.94 power of body weight (pre- and post-natal). 4. The slopes of the regression lines for hepatic blood flow, blood volume and oxygen consumption were found to be 0.91, 0.86 and 0.69, respectively. 5. The mean hepatocyte size in fixed tissue of rats was found to be 7400 micrometers 3. 6. It is argued that the slope of the regression line for hepatic oxygen consumption in mammals generally is likely to fall in the range of 0.67-0.77.

Animals

Evidence for clonal attenuation of growth potential in HeLa cells.

The growth of primary clones and serial subclones of HeLa cells and of diploid human fibroblast-like cells were compared both in the presence and absence of feeder layers; the latter had no significant effects upon the results. Clones and subclones of both cell types displayed great heterogeneity in growth rates, typically with a bimodality of growth distributions. Serial passages of clones selected on the basis of superior rates of proliferation showed attentuation of growth potentials; the extent of such attentuations was much less in the case of HeLa cells, suggesting at least one possible basis for the differences in long-term growth potential between these two classes of cell lines.

Cell Division

The blastomere pattern in echinoderms: cleavages one to four.

The results of a longitudinal study of the blastomere pattern in six embryos during the first four cleavages are reported. At each cleavage stage optical sections through an embryo, taken at vertical intervals of 5 or 10 micron, were recorded on 35 mm film: digitization of the blastomere contours and computer analysis allow calculation of the center, radius, surface area and volume of each blastomere. The subjective impression of exquisite regularity seen in normal echinoderm blastulae acquires a quantitative dimension from the present study. For example, the individual angles formed by the various quartets of blastomeres depart from right angles by at most a few degrees. The egg volume was found to be conserved up to the fourth cleavage. At the 16-cell stage, unlike the earlier stages, the blastomere positions cannot be ascribed solely to the position and orientation of the respective cleavage planes. Finally, a few features of a formal model of these early cleavages are sketched.

Animals