Histochemical representation of regional ATP in the brain using a firefly luciferase-immobilized membrane in a multilayer film format.

The enzymatic bioluminescence of firefly luciferase has been used in sensitive pictorial assays of ATP. We describe a method using a membrane with immobilized luciferase in a multilayer film format for the histochemical representation of brain ATP content. The multilayer film consisted of a transparent support, a reagent layer, and a pigment layer. The reagent layer contained all necessary reagents, including immobilized luciferase. The pigment layer was effective for high image resolution. An unfixed slice of frozen brain 16 microns thick was placed on the film. The chemical energy of brain ATP was converted into luminescent energy in the reagent layer and the bioluminescence emitted was recorded photographically with high spatial resolution. A close linear relationship was obtained between the optical density of the bioluminescent images and logarithmic plots of the brain ATP content. With this film, the regional ATP content in fine anatomical structures of gerbil brains was clearly demonstrated in both physiological and pathological states.