Resolution-limiting factors in 2-deoxyglucose autoradiography. I. Factors other than diffusion

We measured the extent to which factors other than the diffusion of the radioactive label during tissue preparation limits the spatial resolving power of 2-deoxyglucose (2-DG) autoradiography. Radioactive swept frequency gratings were created using microcircuit lithography. The gratings consisted of alternating equal width radioactive and non-radioactive bars in groups of narrowing bar width (effective range 500-20 mum). The vertical thickness of the gratings ranged from 2.25 to 20 mum. The isotope in the radioactive bars was either 14C or 3H. A variety of X-ray films were exposed to these gratings and the resulting images scanned with microdensitometers or video digitizers to determine the fall off in image contrast (dark-bar values minus light-bar values) as a function of the number of dark bars (lines) per millimeter. The power of the isotope was the resolution limiting factor. Grating thickness and type of film made little difference. The limit of resolution with 14C was 10 lines/mm; with 3H, it was 25 lines/mm. The microdensitometer itself is apt to be a resolution limiting factor; the resolving power of those commonly used in autoradiography is unlikely to exceed 10 lines/mm. From measurements of the steepness of gray-matter to white-matter transitions in the image from a tissue section, we conclude that the resolution in the image is no worse than 1.6-3.2 lines/mm. Either the isotope or diffusion of the 2-DG during tissue preparation must be the factor that limits resolution.