Autoradiography: its methodology at the present time

Abstract

SUMMARY The autoradiographic methods used in biological and medical research are reviewed from an historical viewpoint. It is pointed out that early methodological research was directed at the selection of suitable detector substances for radiation (nuclear emulsions) and at the preparation and application of suitable layers of these detectors to biological samples. On the other hand, later research was more concentrated on aspects of the preparation of the biological components of the specimens. In particular, the retention of biochemicals in the tissue sample was studied in detail. Research in the quantification of the emulsion response to α and β radiation paved the way for certain quantitative applications of autoradiography. This has been followed by the derivation of models for the measurement of image spread and the evolution of image analysis methods which allow refined determinations of the specific radioactivity of fine structures taking into account losses and gains of silver grains by image spread. The sum of all these advances is a methodology capable of localizing and quantifying radioactivity and hence marking chemical processes from an organism and organ level down almost to the present limits of anatomical delineation.

Cortical Glutamatergic Markers in Schizophrenia

“…Grayscale values were obtained for isodense bands identified for each probe, corrected for tissue background, and then converted to optical density (OD). OD values were then converted to amount of radioactivity bound (in nCi/g), determined from calibrated [ 14 C]microscale standards (Amersham Biosciences, Piscataway, NJ) (Akbarian et al, 1996;Downs and Williams, 1984;Williams, 1982), which had been exposed alongside the slides for each probe. Using the number of uridine residues contained in each subclone and specific activity of [ 35 S]UTP, bound radioactivity was converted to concentration of mRNA per isodense band, expressed as femtomoles of mRNA per gram of tissue (fmol/g) (Clinton et al, 2003;McCullumsmith et al, 2003).…”

Expression of transcripts for myelination-related genes in the anterior cingulate cortex in schizophrenia☆

“…The sensitivity and reproducibility of in situ hybridization has previously been well characterized (Williams, 1982;Downs and Williams, 1984;Stolz et al, 1989). mRNA expression was measured by in situ hybridization using subclones that were generated by amplifying unique segments of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (Abbreviation: CNP, Genebank accession number: NM_03313, region used for probe: 523-1066), myelinassociated glycoprotein (MAG, XM_01287, 305-835), transferrin (TF, XM_00279, 1172-1940, quaking (QKI, AF142421, 508-715), gelsolin (GSN, XM_01654, 1249-1649, myelin oligodendrocyte glycoprotein (MOG, U18800, 109-263), v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ErbB3, NM_00198, 3081-3325), erbb2 interacting protein (ErbB2IP, NM_01869, 3531-3858), motility-related protein-1 (CD9, M38690, 330-666), SRY-box containing gene 10 (SOX10, BL007595, 1337-1560), oligodendrocyte transcription factor 2 (OLIG2, NM_00580, 423-628), peripheral myelin protein 22 (PMP22, BC019040, 293-637), and Claudin-11 (CLDN11, NM_00560, 610-821) from a human cDNA brain library (Human Adult Brain Unamplified cDNA Library, Edge Biosystems; Gaithersburg, MD) and Polymerase Chain Reaction (PCR).…”

Video microscopic image processing facilitates the evaluation of light microscopic autoradiography at high magnification

“…At low magnification the depth of focus of the objective is in general sufficient to document the structure of the section and the layer of silver grains. However, an intense stain of the section can cause difficulties in the recognition of subcellular structures and thus in the interpretation of the autoradiographic results (Williams, 1977(Williams, , 1982Rogers, 1979). The highly recommended technique of photographing LM autoradiographs by the double exposure technique, such as combining a dark-field image of a silver grain layer with a transmitted light picture of the specimen, is not possible for many types of tissue sections.…”

“…Individual silver grains and structural details of a sectioned embryonic nucleus are now clearly documented. Considering the particularly good resolution of 'H-thymidine autoradiography (Salpeter et al, 1974;Fakan, 1976;Williams, 1977Williams, , 1982Rogers, 1979), video microscopic evaluation of contrast image obtained by high-magnification conventional light microscopy. The outlines of the nuclear structure can easily be recognized, whereas the grains can hardly be identified.…”

“…However, a major technical problem exists in establishing a precise correlation of labelled subcellular structure with the overlay of silver grains following the development of the autoradiograph. This holds in particular if dynamic processes in small somatic cells are to be analysed (Williams, 1982).…”