An iterative approach to the analysis of EM autoradiographs

Ma, Williams; Downs, Angela M.

doi:10.1111/j.1365-2818.1978.tb00127.x

Cited by 13 publications

(14 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…At EM level, radiation scatter prevents precise localization of the radioactive molecule (source) according to individual grain position. The cross-fire analysis Parry, 1973, 1977;Downs and Williams, 1978;Salpeter et al, 1978;Williams and Downs, 1978) allows assessment of the source density for each substructure by adjustment of the result of a simulation ("cross-fire" matrix of hypothetical, generated source-grain sets systematically distributed over the tissue) to the distribution ofreal observed grains (observed grain vector). Grains were counted in section areas sampled systematically.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using 125I-alpha scorpion toxin. II. Sodium distribution on postsynaptic membranes

1992

View full text Add to dashboard Cite

A radioiodinated alpha-scorpion toxin (toxin II from Androctonus australis Hector) (alpha ScTx) was used as a probe for EM autoradiography to study the distribution of voltage-dependent sodium channels (Na+ channel) on the postsynaptic side of the mouse neuromuscular junction. Silver grain distribution was analyzed by the cross-fire method to assess the relative Na+ channel density in each membrane domain measured by stereology. This analysis showed that the maximum Na+ channel density was located on the edge of the synaptic gutter, where it reached about twice the mean density in the postsynaptic fold membrane. Na+ channel densities have been calculated using ACh receptor (AChR) density in fold crests as reference. Sodium channel density on the edge of the synaptic gutter was estimated at about 5000/microns 2. Sodium channel distribution in the postsynaptic folds was compared to AChR distribution using density distribution analysis (Fertuck and Salpeter, 1976). The results confirmed that, as already observed by immunogold labeling (Flucher and Daniels, 1989), there are no Na+ channels on fold crests. Na+ channels are located in the rest of the fold membrane (bottom) and may be distributed according to two possible models. In the first, density would be uniformly high, although lower than on the gutter edge. In the second, density would decrease from the crest border, where the value was that of the gutter edge, to the fold end, where the value would be 50% lower. Based on the latter model, which was the “best-fit model,” we propose that the postsynaptic membrane includes two domains. The first is the fold crest, which contains almost exclusively AChRs. This domain is devoted to reception-transduction of the chemical signal. The second includes both the fold bottom membrane and the perisynaptic membrane. Sodium channel density is highest along the crest border and decreases moving away. Its functions are the integration of postsynaptic potentials and generation-conduction of the muscle action potential.

show abstract

Section: Methodsmentioning

confidence: 99%

“…OSD in muscle, axon, axon membrane, and intercellular space was close to zero. Following the "iterative deconvolution procedure" Williams and Downs, 1978), the primary model was then modified by suppression of empty source compartments. These new models were systematically tested.…”

Section: Labeling Of Nkfjsmentioning

confidence: 99%

Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using 125I-alpha scorpion toxin. II. Sodium distribution on postsynaptic membranes

1992

View full text Add to dashboard Cite

show abstract

“…By comparing these expected grain distributions with the observed grain distributions in an autoradiogram, the true sources can be identified, and their specific activities evaluated. Variants of this idea of analyzing EM autoradiograms by successive hypothesis testing have been published by Salpeter and co-workers (15,17,18), Blackett and Parry (1,2), and Williams and Downs (23).…”

mentioning

confidence: 99%

High resolution analysis of the secretory pathway in mammotrophs of the rat anterior pituitary.

Salpeter¹,

Farquhar²

1981

The Journal of Cell Biology

View full text Add to dashboard Cite

The secretory process in pituitary mammotrophs was analyzed by quantitative electron microscope autoradiography. Dispersed pituitary cells from estrogen-treated female rats were subjected to pulse- labeling with [3H]leucine (5 min) followed by a chase incubation of up to 4 h. Autoradiograms were prepared using fine-grained emulsion (Kodak 129-01), and analyzed using a three-step "mask analysis' procedure: (a) the distribution of autoradiographic grains is determined as in a simple grain density analysis; (b) masks (transparent overlays) are used to generate expected grains from assumed sources; and (c) a computer program compares these two distributions and varies the expected distribution to match the observed distribution, thereby identifying the radioactive sources in the tissue. The overall route of intracellular transport of prolactin from rough endoplasmic reticulum (ER) leads to Golgi complex leads to immature secretory granules leads to mature secretory granules was as established in previous studies. However, by use of the high resolution emulsion and method of analysis, the precision with which label could be localized within individual source compartments was much greater and the time resolution was much sharper than achieved previously using Ilford L4 emulsion and simple grain density analysis. The main new findings were as follows: (a) the ER was essentially drained of radioactivity by 30 min, the Golgi complex by 1 h, and the immature secretory granules by 2h postpulse. This indicates that the secretory product (prolactin) is rapidly and efficiently transported out of these compartments. (b) approximately 30% of the total radioactivity remains located in the ground cytoplasm over the entire postpulse period examined (up to 4 h), and by 30 min postpulse the grain density in the ground cytoplasm exceeded that of the ER. This indicates the ability to resolve ER-associated label (presumably associated mainly with secretory products) from the cytoplasmic label (presumably associated with nonsecretory proteins). (c) the specific activity of immature secretory granules was much greater than previously appreciated; at 1 h postpulse it was greater than 200 times that of the adjacent Golgi complex cisternae. This large dynamic range in observed grain density demonstrates the ability to effectively correct for radiation spread and thus to detect with great accuracy high concentration of label even from very small structures (20-100 nm) which constitute a small percentage (less than 1%) of the total cell area.

show abstract

“…It is also supposed that all technical and developmental conditions in preparing the autoradiographs are rigorously standardized so as to minimize background, chemography , multiple hits and departures from the required geometric characteristics of the specimens. It is in this case that the number of silver grains produced by the radioactivity in the labelled cell organelle profiles is directly proportional to the number of distintegrations occurring (Downs & Williams , 1978).…”

Section: Theoretical Principle and Basic Computationsmentioning

confidence: 99%

“…Thus, a considerable proportion of the silver grains are formed over cell structures adjoining to those where the disintegration events occurred. This phenomenon known as cross-fire (Williams, , 1977Downs & Williams, 1978) takes place over the junctional regions between neighbouring cell features. Its extent depends on the intracellular organization and the factors governing resolution (Rogers, 1979).…”

Section: Introductionmentioning

confidence: 99%

Quantitative electron microscope autoradiography: application of multiple linear regression analysis

Markov

1986

Journal of Microscopy

View full text Add to dashboard Cite

A new method for the analysis of high resolution EM autoradiographs is described. It identifies labelled cell organelle profiles in sections on a strictly statistical basis and provides accurate estimates for their radioactivity without the need to make any assumptions about their size, shape and spatial arrangement. The radioactivity in interfacial membranes and transitional junctional regions between pairs of adjoining cell structures can be determined without any additional measurements. The uniformity of internal labelling of large cell organelles can be also assessed. Correcting for cross-fire does not need any predictive information from a frequency distribution function describing the image spread about a radioactive point source. Instead, the section area is subdivided into regions in such a way that each region with crossfired silver grains over it includes also the sites of the radioactive disintegrations that have produced them. The method is based on the quantitative regression relationship between the number of developed silver grains overlying any given region and the size of the cell organelle profiles included. A transparent overlay screen bearing a regular array of circles and a pointcounting procedure are applied to divide the section area into regions and to measure their area and the size of the cell organelle profiles. The least squares method is used for the simple and rapid calculation of specific activity estimates and exact standard errors to be attached to them. A modest computing facility and a standard library program are required. Simulation modelling and analysis of residuals were used to lend support to the validity of the method.

show abstract

An iterative approach to the analysis of EM autoradiographs

Cited by 13 publications

References 12 publications

Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using 125I-alpha scorpion toxin. II. Sodium distribution on postsynaptic membranes

Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using 125I-alpha scorpion toxin. II. Sodium distribution on postsynaptic membranes

High resolution analysis of the secretory pathway in mammotrophs of the rat anterior pituitary.

Quantitative electron microscope autoradiography: application of multiple linear regression analysis

Contact Info

Product

Resources

About