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Research on circadian biology over the past decade has paid increasing attention to the photoreceptor mechanisms that align the molecular clock to the 24-h light/dark cycle, and some of the results to emerge are surprising. For example, the rods and cones within the mammalian eye are not required for entrainment. A population of directly light-sensitive ganglion cells exists within the retina and acts as brightness detectors. This article provides a brief history of the discovery of these novel ocular photoreceptors and then describes the methods that have been used to study the photopigments mediating these responses to light. Photopigment characterization has traditionally been based on a number of complementary approaches, but one of the most useful techniques has been action spectroscopy. A photopigment has a discrete absorbance spectrum, which describes the probability of photons being absorbed as a function of wavelength, and the magnitude of any light-dependent response depends on the number of photons absorbed by the photopigment. Thus, a description of the spectral sensitivity profile (action spectrum) of any light-dependent response must, by necessity, match absorbance spectra of the photopigment mediating the response. We provide a step-by-step approach to conducting action spectra, including the construction of irradiance response curves, the calculation of relative spectral sensitivities, and photopigment template fitting, and discuss the underlying assumptions behind this approach. We then illustrate action spectrum methodologies by an in-depth analysis of action spectra obtained from rodless/coneless (rd/rd cl) mice and discuss, for the first time, the full implications of these findings.

Original publication




Journal article


Methods Enzymol

Publication Date





697 - 726


Animals, Biological Clocks, Circadian Rhythm, Humans, Mammals, Mice, Photic Stimulation, Photometry, Photoreceptor Cells, Vertebrate, Rhodopsin, Spectrum Analysis