Schwarzschild-Villiger Effect in Microspectrophotometry

Lison, L

doi:10.1126/science.118.3066.382

Cited by 14 publications

(6 citation statements)

References 6 publications

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“…In previous communications (32,34), Naora has pointed out the possible existence of the Schwarzschild-Villiger effect in the equipment hitherto employed and he constructed the improved apparatus in which this effect is eliminated. It should be noted that the involvement of Schwarzschild-Villiger effect was challenged by Ornstein and Pollister (42) who were, in turn, critically answered by Lison (28), and Naora (35).…”

Section: A Estimation Of Relative Dna Content Of Individual Nudeimentioning

confidence: 99%

Microspectrophotometry of Cell Nuclei Stained With the Feulgen Reaction

Naora

1957

The Journal of Cell Biology

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1. DNA contents of the individual parenchymal nuclei of rat livers during postnatal growth were estimated by microspectrophotometric apparatus, and different ploidy classes of nuclei were classified by their DNA contents. With the same material the total number of parenchymal nuclei in the liver was counted microscopically. 2. If the DNA content of nuclei encountered most frequently in several tissues represents the diploid class, the ploidy classes of the rat liver cell nuclei correspond to di-, tri-, tetra-, and octoploid, with the di- and tetraploid ones predominating considerably. 3. In suckling rats (below 25 gm. of body weight) the liver parenchyma is composed almost exclusively of cells with diploid nuclei, whereas in young rats (above 80 gm.), of tetraploid nuclei. In the growth stage between 25 and 80 gm., there is a remarkable replacement of the diploid nuclei by the tetraploid ones. However, in the liver of adult rats weighing more than 150 gm., any increase or decrease in the frequency of diploid and tetraploid nuclei is hardly observable. In such rats, the nuclear population of the liver parenchyma seems to reach a cell-ecological equilibrium which is considered to be a stable one. 4. It is shown that such nuclear populations and the total number of nuclei in a liver are controlled by the growth state, and not by the age. 5. The decrease in the total number of diploid nuclei and the increase in tetraploid nuclei in the growing livers of rats weighing from 40 up to 130 gm. can both be explained by the hypothesis that the tetraploid nuclei originate from the interphase diploid nuclei without involving mitosis. This hypothesis implies that mitosis is confined to the reproduction of diploid cells alone. 6. It is suggested that, in general, the synthesis of DNA does not necessarily result in the formation of visible mitotic chromosomes. 7. Mitotic time and generation time of diploid nuclei and the percentage of the tetraploidization from diploid nuclei are calculated and discussed.

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Section: A Estimation Of Relative Dna Content Of Individual Nudeimentioning

confidence: 99%

Microspectrophotometry of Cell Nuclei Stained With the Feulgen Reaction

Naora

1957

The Journal of Cell Biology

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“…identical to one given without derivation by Lison (1953), would also be valid if the specimen almost filled the finite field illuminated, but the glare would be reduced. If the intensity of the glare falling on the image of the specimen is assumed to be proportional to the illuminated, specimen-free area from which the glare originates, the glare Ff with a finite illuminated area would be related to the glare F, found with an infinitely large illuminated area (or an infinitesimally small object) by the approximate expression.…”

Section: -Fmentioning

confidence: 84%

“…Reflections and scattering at glass-air surfaces, and hence glare, can be minimized by attention to the cleanliness of lenses, microscope slides and coverslips (Caspersson, in discussion of paper by Exner, 1960). Lison (1953) further notes that under similar conditions of illumination different objectives can give very different amounts of glare (Table l), so that selection of suitable lenses can reduce the error considerably. Keohane & Metcalf (1959) and Chamberlain & Walker (1965) found that in general reflecting objectives give less glare than do refracting lenses, which have a greater number of glass-air surfaces.…”

Section: Introductionmentioning

confidence: 99%

“…The effect on glare of the condenser aperture is much less than that of the field stop. Naora (1955) found that varying the condenser NA between 0.8 and 1.25 had a negligible effect on the amount of glare, and Lison (1953) observed no appreciable difference on reducing the NA from 0.9 to 0.66. Reducing the aperture still further does however have some effect (Pogo & Funes, 1959, see Table I) In scanning microdensitometers where the specimen itself is moved by a special mechanical stage, as in the Caspersson instrument produced commercially as the 'UMSP' by Messrs Zeiss (West Germany), it is possibly in principle to minimize glare error by severe restriction of the area of specimen illuminated.…”

Section: Introductionmentioning

confidence: 99%

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Aspects of scanning microdensitometry I. Stray light (glare)

Goldstein

1970

Journal of Microscopy

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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.

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“…This may account for some of the discrepancies in the ]iterature concerning the effect of condenser angle on flare error (8,11,22).…”

Section: Transmission Of Convergent Light Through a Parallel-sided Sementioning

confidence: 99%

The Nature, Significance, and Evaluation of the Schwarzschild-Villiger (SV) Effect in Photometric Procedures

Howling

Fitzgerald

1959

The Journal of Cell Biology

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The Schwarzschild-Villiger effect has been experimentally demonstrated with the optical system used in this laboratory. Using a photographic mosaic specimen as a model, it has been shown that the conclusions of Naora are substantiated and that the SV effect, in large or small magnitude, is always present in optical systems. The theoretical transmission error arising from the presence of the SV effect has been derived for various optical conditions of measurement. The results have been experimentally confirmed. The SV contribution of the substage optics of microspectrophotometers has also been considered.A simple method of evaluating a flare function f(A) is advanced which provides a measure of the SV error present in a system. It is demonstrated that measurements of specimens of optical density less than unity can be made with less than I per cent error, when using illuminating beam diameter/specimen diameter ratios of unity and uncoated optical surfaces.For denser specimens it is shown that care must be taken to reduce the illuminating beam/specimen diameter ratio to a value dictated by the magnitude of a flare function f(A), evaluated for a particular optical system, in order to avoid excessive transmission error. It is emphasized that observed densities (transmissions) are not necessarily true densities (transmissions) because of the possibility of S V error.The ambiguity associated with an estimation of stray-light error by means of an opaque object has also been demonstrated.The errors illustrated are not necessarily restricted to microspectrophotometry but may possibly be found in such fields as spectral analysis, the interpretation of x-ray diffraction patterns, the determination of ionizing particle tracks and particle densities in photographic emulsions, and in many other types of photometric analysis.

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Schwarzschild-Villiger Effect in Microspectrophotometry

Cited by 14 publications

References 6 publications

Microspectrophotometry of Cell Nuclei Stained With the Feulgen Reaction

Microspectrophotometry of Cell Nuclei Stained With the Feulgen Reaction

Aspects of scanning microdensitometry I. Stray light (glare)

The Nature, Significance, and Evaluation of the Schwarzschild-Villiger (SV) Effect in Photometric Procedures

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