Medical Physics at University of Naples, Italy

Autoradiography



Table of Contents:

1. Autoradiography with silicon microstrip detectors: cell clusters.
2. Autoradiography with GaAs pixel detectors:
cell clusters and Southern blotting (Omega electronics)
3. Autoradiography with GaAs pixel detectors : genetics and dynamic studies (Medipix electronics)
4. Researchers involved.




1. Digital Autoradiography with double sided silicon microstrips

As a part of MEDIM Project (INFN) we developed a digital autoradiography system based on double-sided silicon strip detectors. We tested the system with beta emitting markers. The system is based on a 1.6x1.6 mm2 active detector unit, with 100 micron read-out pitch: the radioactive sample is moved on the detector by a PC-controlled scanning system, in order to store images of the sample in the form of tiles of a mosaic. Interaction data are stored in memories linked to transputers, which perform image parallel reconstruction. The off- line reconstruction allows for electronic collimation of the beta tracks, thus reducing the image blurring due to long, non-perpendicular tracks in the detector. Images were taken of human mammary epithelial cells, marked with 32P.

You can see here a 5x5 mm2 autoradiography of mammary cell clusters, obtained in a 10 minute exposure time, using 32P-orthophosphate in acqueous solution as a marker: we estimated about 77 nCi total activity of the clusters.


Figure 1

2. Digital Autoradiography with Silicon and GaAs pixel detectors (Omega electronics)

Solid state detectors made of Si (5x8 mm2) and GaAs (6.4x8 mm2) pixel matrices bump-bonded to the OMEGA2 and OMEGA3 systems, developed for high energy physics (HEP) experiments, have been used to obtain images of clusters of human epithelial cells, labeled with 32P, a b- emitting tracer. The system has shown high sensitivity (10- 4 cps mm-2), and has proved linear down to the very low activity range 0.2-20 Bq, typical of autoradiography.
Our present set-up is based on a GaAs pixel array (64x64 pixels, 170 micron size) and bump-bonded low-noise electronics (Medipix), developed within an international collaboration for Digital Radiography. This allows for better spatial resolution, better sensitivity, better pixel uniformity, lower energy markers, larger areas. See here for details.

2.1 Imaging of radiolabelled cell clusters

We have obtained images of different samples of cells marked with different tracer concentrations. The dimensions of such cells are of the order of 10 µm, beyond the resolution capability of the system that has a pixel size of 50x500 µm2 (GaAs-Omega3 detector). Nevertheless the system can clearly localize clusters of cells which have incorporated the radioactive tracer.


Figure 2


2.2 Extension of this investigation to the study of DNA sequences by the "blotting" technique

Localization of particular sequences within genomic DNA can be accomplished by the transfer techniques called Southern Blotting. Genomic DNA is digested with one or more restriction enzymes, and the resulting positively charged fragments are separated according to their size by electrophoresis through an agarose gel. The DNA is then denaturated in situ and transferred from the gel to a solid support (usually a nitrocellulose filter o nylon membrane). The relative position of the DNA fragments are preserved during their transfer to the filter. The DNA attached to the filter is hybridized (selectively linked) to radiolabeled (32P) DNA, following standard procedure. In our test we prepared several DNA sequences using the standard Southern technique and exposed the 4.8x8 mm2 silicon detector to the blotting positioned at a distance of 1 mm). A 5-position scanning sequence was needed to cover the total blotting area. In figure 3 it is shown a 24 x 8 mm2 image of a DNA sequence, obtained with our silicon detector with a 10 h exposure. Two 32P- labelled DNA fragments are easily localized as two "hot" spots shifted along the vertical direction.


Figure 3


In order to have a more quantitative localization of the sequences, a line profile along the electrophoresis direction was calculated (in order to increase the statistics we added all pixels' content of each row), and in figure 4 the result is shown and compared with the line profile obtained with a commercial storage phosphor imaging plate (Molecular Dynamics PhosphorImager SF, scanned at 200 µm resolution), with a 20 h exposure time. under way: the size of the pixel small side would allow for a very good spatial resolution.


Figure 4


3. Digital Autoradiography with Silicon and GaAs pixel detectors (Medipix electronics)


Our present set-up is based on a GaAs pixel array (64x64 pixels, 170 micron size) and bump-bonded low-noise electronics (Medipix), developed within an international collaboration for Digital Radiography.
We are planning to use our devices for imaging of animal tissue (for pharmaceutical studies), dynamic studies in small organisms and genetic studies. Look here for a closer view of our projects concerning DNA studies.

The autoradiographys apparatus with a 3D scanning system for sample positioning

A close-up view of the chipboard with the detector (about 1 cm**2)



Preliminary results: an image of a P32 spot : 2.5 mm diameter, 30 min acquisition time, 300 Bq total activity.

4. Researchers and Institutions involved:

L.Abate, E. Bertolucci, M. Conti, C. Montesi, P. Russo,
INFN and University of Naples.

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