Quantitative assessment of autoradiographs by photometric reflectance microscopy. An improved method using polarized light

Synopsis

The use of polarized light in the quantitation of biological autoradiographs by photometric reflectance microscopy was investigated. Crossed polars greatly reduced the intensity of reflections from various surfaces in the specimen and optical system, including melanin granules, extraneous crystalline deposits and stained cellular material. The signal/noise ratio of measurements of autoradiographic silver grains was significantly improved with crossed polars, and many previously unusable preparations became measurable. Suitable staining methods included Neutral Red, Haemalum, Methyl Green-Pyronin, New Methylene Blue and the chloroacetate esterase method, but fluorescence from tissues stained with Eosin or Eosin-containing mixtures such as Giemsa was not diminished by the use of polarized light.

A method, involving successive measurements of the reflectance with crossed and parallel polars, is described for quantitating autoradiographs in the presence of material (such as melanin granules) the reflectance of which is incompletely extinguished by crossed polars.

The use of polarized light significantly extends the useful range of the reflectance technique, since an approximately linear relationship between reflectance and absorbance (and hence between reflectance and exposure) is maintained to higher grain densities than with unpolarized light.