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SUMMARY Measurement of the width of a thin, cylindrical, transparent object by phase contrast light microscopy has been frustrated by the absence of an established relationship between the true width of the object and its apparent width in the phase contrast image. We have solved this problem by devising a simple method by which individual glass fibres may be measured using both phase contrast light microscopy and electron microscopy. Using this method we have constructed calibration curves relating the diameter measured by phase contrast microscopy to the real diameter of the fibres. These curves are linear in the range 0.10-2.5 μm real diameter, with slopes close to unity and intercepts of about 0.2 μm. Thus widths of such objects are overestimated. The precise value of the intercept is related to the overall numerical aperture of the optical system. Each calibration curve permits the true width of a cylindrical object to be estimated by phase contrast microscopy with an accuracy of better than ±0.05 μm. We have found that greater precision is obtained by taking measurements of light micrographs subjectively using a microcomparator rather than objectively using a microdensitometer.

SUMMARY An electronic interface is described which permits the Vickers M85/M86 microdensitometer to be used with a chart recorder to plot linear distance along a chromosome against (1) the absorbance, (2) the product of the chromosome width and the mean absorbance transverse to the long axis of the chromosome, or (3) the integrated absorbance accumulating during a two-dimensional scan of the specimen.

SUMMARY A Vickers M85A flying-spot microdensitometer has been linked to a standard BBC model B microcomputer with a commercially available interface, and programs developed in which the computer controls step-wise scanning of the specimen. At each image point thirty-two absorbance readings are made and the average value is stored in the computer memory at 8-bit resolution; a complete 48x48 raster takes less than 5 s. An image of the specimen displayed on the monitor uses eight different pseudo-grey-level characters, and the manner in which the 256 possible absorbance values are represented on the screen can be altered by the user. A mask of arbitrary shape and size, drawn on the screen under key-board control, makes it possible to measure the integrated absorbance of even small, irregular and closely packed specimens such as individual bands in Drosophila polytene chromosomes. The coefficient of variation of repeated measurements of a Feulgen-stained frog nucleus is typically better than 1%. Displayed images can be printed on a dot-matrix printer, and data stored on disc for subsequent analysis. The techniques can readily be modified and adapted for other purposes, some of which are briefly discussed.

SUMMARY A film holder is described which allows the Vickers M85/M86 microdensitometer to be used to make transmission and density measurements on images on photographic film. This facility is particularly useful for applications such as making profiles of fluorescent banded chromosomes.

SUMMARY By means of a Vickers M85 microdensitometer, combined with a histogram unit, the distribution of the coloured product of the Feulgen reaction in single nuclei of human lymphocytes was examined by taking the fraction of each nucleus occupied by DNA at different concentrations. Tchacarof's index of homogeneity, derived from these fractions, was found to rise with the mean nuclear DNA concentration, and the calculated linear relation between the index and the concentration was found, in a preliminary experiment, to be independent of the degree of flattening of the nuclei. Further analysis, of the contribution of more and less densely packed areas to differences in mean intranuclear DNA concentration, showed that these differences depended on differences in both denser and less dense areas. The effects of hydrolysis on homogeneity were controlled by the use of Tchacarof's index. The procedure for using the Vickers M86 microdensitometer, with histogram unit, for analyses such as those reported here was developed to become established critically.

SUMMARY A simple and inexpensive interface has been constructed between the Vickers M85 microdensitometer and a BBC model B microcomputer. The interface incorporates three sensitivity ranges and enables the production of pseudocolour images of the specimen using the two-dimensional scanning mode of the M85. The operator can select a 160times256 pixel image with eight colours or a 320times256 display using only four colours. Each colour represents a defined range of transmittance which is software controlled. The image histogram can be displayed and the interval between colours redefined so as to enable contrast stretching. Intervals between colours can be either linear or logarithmic and the images thus obtained can be stored on disc or videotape, or a hard copy can be obtained using a screen dump routine. Two-dimensional absorption images can thus be obtained at any single wavelength from 400 to 700 nm at normal magnifications of the light microscope. In addition, the system can be used to acquire, store and process data from one-dimensional scans to obtain quantitative information about variations in optical density within the specimen, so considerably increasing the usefulness of the instrument. Although obviously limited in its capabilities, the system produces images of very high quality and one-dimensional data of high sensitivity. The interface can be constructed for less than £40. A small modification to one of the M85 circuit boards is necessary to obtain maximum resolution.

SUMMARY Microdensitometric errors can result from various factors associated with the monochromator system, including imperfect monochromaticity of the light, incorrect setting of the wavelength, and non-uniform illumination of either the microscopic field or the objective aperture. Certain types of potential error are characteristic of particular instruments. Thus in the Vickers M85 microdensitometer, where the flying spot is the reduced image of a hole situated at the monochromator exit aperture, the interpretation of results obtained with different spot sizes is complicated by the fact that the hole size affects both the spatial resolution and the spectral bandwidth of the system. Similarly, in instruments in which the monochromator exit slit lies in an aperture plane the numerical aperture of the whole system may be affected by the spectral bandwidth and vice versa. Overall instrumental sensitivity is mainly limited at the blue and red ends of the spectrum respectively by the lamp output and the photomultiplier tube sensitivity. Quartz-iodine lamps are slightly brighter than conventional tungsten sources, especially at short wavelengths, but tend to be less stable photometrically and are more expensive. Simple refracting monochromators and graded-spectrum interference filters in general pass more light, in the visible spectrum, than do grating monochromators of similar bandwidth. Most errors of wavelength setting can be avoided by routinely measuring at that wavelength, λmax, found empirically to give the maximum absorbance or integrated absorbance. Off-peak wavelengths can be set reproducibly with the aid of an eyepiece spectroscope, or by adjusting the wavelength so that the absorbance of a given specimen is some precise fraction of that at λmax. The monochromator bandwidth affects the apparent absorbance spectrum of a given specimen, but spectra are not very helpful in assessing possible microdensitometric errors at a fixed wavelength. In probably the best single test of monochromator performance, advisable before any microdensitometry using unfamiliar instrumental settings or staining methods, the apparent absorbance at a given wavelength is plotted against pathlength through a solution having an absorbance spectrum identical with or very similar to, that of the microscopic specimens of interest. Deviations from proportionality between pathlength and apparent absorbance have been found using the graded-spectrum interference filter of the Barr & Stroud GN2 microdensitometer, but have not so far been demonstrated with the refracting monochromator of the Vickers M85 microdensitometer. Such deviations could result in systematic errors in the comparison of densely- and lightly-stained specimens, and two methods to correct these errors are described. Where the error is a known function of the monochromator slit-width it may sometimes be possible to use data obtained with two slit-widths to estimate the ideal result corresponding to monochromatic light. More generally, non-linearity can be corrected by electronic off-setting of the photomultiplier tube output. This procedure is analogous with, and if necessary should be carried out together with, a method previously described for the correction of error due to glare. Provided the pathlength and apparent absorbance are proportional over the range of absorbances of interest, results obtained with different monochromator bandwidths can be standardized by multiplication by an empirical calibration factor.

Summary In Scanning Transmission Electron Microscopy (STEM) details at the top of thick specimens can be imaged with a better resolution than those at the bottom, because the scanning electron probe is broadened by multiple scattering. For quantitative measurement we used a test specimen consisting of a substrate film with an island-structured indium film and coated with polystyrene spheres of different diameters up to 1.1 μm. The fine detail contained in the indium film was observed and the beam broadening was that caused by the polystyrene spheres. This specimen was imaged with the spheres both above and below the layer, in a JEOL 100B electron microscope with a scanning device. Electron energies from 20 to 100 keV were used. Images with the sphere below show a higher resolution than those with the sphere above and also those of conventional electron microscopical images at 100 kV and 200 kV. The broadening of the edges on the photographic film was recorded with a microdensitometer. The results can be compared with theoretical calculations by a Monte Carlo method. There is a good agreement with the experiments if one uses an effective aperture obtained by transmission experiments.

SUMMARY A simple device for densitometering electron micrographs or other transparencies by one-dimensional scanning is described. Micrographs of crystalline materials are oriented by means of their optical diffraction patterns displayed with a slightly modified microscope.

SUMMARY Glare or stray light, an important source of error in microdensitometry, is mainly due to reflections at glass-air surfaces in the microscope objective, and much less to imperfections in other parts of the optical system. It is only slightly affected by the numerical aperture of the microscope condenser, but is closely related to the area of specimen-free field illuminated. If Ff is the amount of glare (expressed as a fraction of the intensity of the light incident on the specimen) with a given object and area of field illuminated, and F∞ is the glare with an infinite field size or an infinitesimally small object, F∞ approximately equals FfA/(A - a), where A is the area of the whole field illuminated and a is the area of the specimen itself. Glare with a given specimen and under given conditions of illumination may be taken to equal the apparent transmittance of an opaque object of the same size as the specimen to be measured, provided the opaque object is light absorbing (of low reflectance) and not too small (at least 5–10 μm diameter). The true absorbance Et of the specimen equals log [(1 - F)/(Ip - F)] where the intensity of the incident light is unity, and Ip is the apparent transmittance of the specimen in the presence of F glare. The true absorbance of a specimen is never less than F% higher than its apparent absorbance, in the presence of F% glare, and the error rises sharply with increasing absorbance. Apparent differences in the amount of dye taken up by nuclei of different sizes, stained by the Feulgen method, which have been reported by various workers and attributed to differences in DNA content, are of an order of magnitude which could be due to the presence of glare in the system. This factor should be allowed for in critical work. Two methods of correcting the error due to glare in measurements of integrated absorbance are described. The first method utilizes data on the area of specimen containing material with an absorbance greater than a set threshold value, as provided for example by the area-measurement facility on the Vickers M85 integrating microdensitometer. In the second, and generally preferable method, glare is compensated for electronically in a way analogous to the offsetting of the dark-current of the photomultiplier tube of the instrument.