Abstract: Accurate quantitative determination of signals produced on film by biological molecules of interest, such as DNA and RNA, identified by hybridization with radioactively-labeled probes comprising homologous sequences (DNA or RNA) is assured by means of preimaging of a density gradient reference series on the film to calibrate the linear range of the film's response. The reference may include a gradient of incremented density levels or it may include multiple steps produced by differing amounts of a standard radiant material. The film may be preimaged with a step-wedge density gradient by means of a film holder with arrangements to optically isolate a substantial portion of the film. Densitometric tracings of the signals generated by the radioactively-identified molecules can be adjusted to lie within the linear range of response of the film defined by the tracing of the step-wedge image by adjustment of the film exposure or sample concentrations.