The statistical analysis of autoradiographs: I. Grain count distributions over uniformly labelled sources

SUMMARY The present study was designed to investigate the cellular uptake of technetium‐99m (99mTc) and chromium‐51 (51Cr) by autoradiographic methods and to compare these data to those obtained by gamma scintillation counting. HeLa cells, a fibrosarcoma of murine origin (Sarcoma I) and human peripheral blood lymphocytes were labelled with either 99mTc‐sodium pertechnetate or 51Cr‐sodium chromate. Cells were non‐uniformly labelled with both radionuclides. Nevertheless, uptake as determined by both autoradiography and gamma scintillation counting revealed that each cell type had a characteristic labelling pattern which appeared to have a relationship to cell surface area. This was determined to be linear for 99mTc when counts per minute were plotted as a function of area (μm2). Grains were randomly scattered over the entire cell surface and occasionally tracks could be seen as a trail radiating out from the periphery. HeLa cells had the largest surface area, the highest grain counts and the greatest uptake of both 99mTc and 51Cr as determined by gamma counting. Lymphocytes had the smallest surface area, the lowest grain counts and the least uptake. Both radionuclides are non‐specific cell labels which are taken up independently of mitosis and protein synthesis. The favourable chemical properties of 99mTc‐sodium pertechnetate have led to its use as a label for a wide variety of inorganic and organic compounds which are employed as radiopharmaceuticals. Autoradiographic methods could be useful in determining the cellular localization of these compounds, as well as any protein molecule with which pertechnetate forms chelates. Although the 6 h half life of 99mTc limits its use to short term experiments, it does permit brief exposure times prior to development.

Section: Autoradiographymentioning

confidence: 99%

Autoradiographic studies on the cellular uptake of Technetium‐99m and Chromium‐51

Barth

Clancy

Pugh

1977

“…The use of photometry in incident light might introduce an added variance into the measurements. This aspect has been examined (England & Rogers, 1970) by making repeated grain counts, both visually and photometrically, over a section of 3H-methacrylate. The observed scatter of counts about the mean value (Fig.…”

Section: O B T a I N I N G A S U I T A B L E S I G N A L F R O M T H mentioning

confidence: 99%

Photometrie measurements of grain density in autoradiographs

Rogers

1972

Self Cite

SUMMARY A technique is described for measuring the grain densities in areas of emulsion from 10 to 200 μm2, using vertical incident illumination which converts the silver grains into bright specks on a black background. The light reflected by the silver grains in the measuring area is integrated with a photometer to give a reading that is directly proportional to the number of grains present over a wide range of densities. Speeds of 200 counts/h can be maintained on suitable material throughout a working day without loss of accuracy. The characteristics of the method, the selection of equipment, and some sources of artefacts are discussed.

“…Silver grains or tracks overlying the tissue sections are often counted by direct visual methods using a microscope with an eyepiece grid, expressing the results as grains/unit area of emulsion or as grains/unit volume of emulsion (Boyd & Levi, 1950;Gross et al, 1951;Pelc & Spear, 1951). Under ideal conditions the grain or the track density is directly proportional to the radioactivity of the underlying tissue (Levi & Rogers, 1963;Levi, 1964) and repeated measurements are random in distribution (England & Rogers, 1970). This is because the random or Poisson distribution is the only function which is appropriate if grains in the emulsion accurately reflect the underlying radioactivity, because emissions from the radioactive section are themselves random.…”

Section: Visual Estimation Of Grain Densitymentioning

confidence: 99%

“…The average number of grains in area A is Ave(n) = AA, and the variance is Var(n) = AA. This relationship has been shown to apply experimentally to autoradiographic grain-density determinations (England & Rogers, 1970), though when grain density is not proportional to tissue radioactivity other distributions may be more appropriate (Chernick & Evans, 1968).…”

Section: Visual Estimation Of Grain Densitymentioning

confidence: 99%

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The statistical analysis of autoradiographs: II. Theoretical aspects including methods for optimal allocation of measurement effort

England

Miller

1970

Self Cite

SUMMARY Under carefully controlled conditions autoradiographic grain densities vary in accordance with the Poisson distribution. The present paper derives several different formulæ and presents graphs to enable the experimenter to measure such grain densities to any specified accuracy after making the appropriate correction for radioactive background. Emphasis is placed upon the allocation of counting effort between one or more labelled sources and the unlabelled background so that the total area of emulsion to be counted is minimized. This technique of optimal measurement allocation has been considered for grain‐density measurements both by visual counting and by photometric methods. This approach has also been applied to the problem of accurately determining the percentage of labelled cells by sampling from a large population.