Measurement of the Optical Properties of Leaves Under Diffuse Light Holly L. Gorton* 1 , Craig R. Brodersen 2† , William E. Williams 1 and Thomas C. Vogelmann 2 1 Department of Biology, St Mary’s College of Maryland, St Mary’s City, MD 2 Department of Plant Biology, University of Vermont, Burlington, VT Received 4 February 2010, accepted 30 April 2010, DOI: 10.1111/j.1751-1097.2010.00761.x ABSTRACT Measuring leaf light absorptance is central to many areas of plant biology including photosynthesis and energy balance. Absorptance is calculated from measured values of transmittance and reflectance, and most such measurements have used direct beam light. However, photosynthesis and other processes can differ under direct and diffuse light. Optical properties under diffuse light may be different, but there have been technical difficulties involved in measuring total reflectance of diffuse light. We developed instrumentation to measure this reflectance using a chopped measuring beam delivered alternately to sample and reference integrating spheres, and lock-in detection. We also built instrumentation for measuring transmittance of diffuse light. We developed standards to calibrate our instruments and correct for substitution error, a known systematic error with integrating sphere-based measurements. Helianthus annuus leaves measured under diffuse light reflected 5–10% more and transmitted a few percent less 400–700 nm light than under direct light. Overall absorptance was only a few percent higher under direct light, but leaves may utilize absorbed direct and diffuse light differently. For example, of the light entering the leaf, significantly more direct light than diffuse light is trans- mitted through the leaf, suggesting differences in localization of absorption within the leaf. INTRODUCTION Knowledge of leaf light absorptance is fundamental to understanding a host of plant processes. For example, photo- synthetic responses to light are best expressed on an absorbed- photon basis, and knowledge of leaf absorptance is essential for calculating leaf energy balance and for modeling global terrestrial net primary productivity. However, the vast major- ity of leaf optics and photosynthesis measurements have been made by irradiating samples with direct beam (collimated) light. Although light like this occurs naturally on a clear day in the direct beam of the sun, light directional quality varies on a continuum from fully collimated light with parallel rays to completely diffuse light with randomly traveling rays. Even on a clear day Rayleigh scattering by the atmosphere causes about 15% of sunlight to be diffuse sky light (1). In addition, there are many times when clouds or particulates obscure the sun, scatter sunlight to varying degrees and increase the proportion of diffuse light striking the earth. The directional quality of light has biological consequences. Some photoresponses, such as phototropism or phototaxis, are potentiated by unidirectional light and clearly would not occur normally if light striking the organism were completely diffuse. Solar tracking in many leaves also requires a direct beam of parallel rays (2). Light-dependent chloroplast movements are less obviously tied to light directionality, but they too are altered when light is diffuse. Chloroplasts normally align along walls that are parallel to an intense beam of light, in a position where they can shade each other and reduce potentially damaging light exposure. When light is bright but diffuse, chloroplasts assume a more random distribution around the periphery of the cell (3). Even photosynthesis depends on the directionality of light. Diffuse light can penetrate into the canopy better than direct light, relieving light limitation of leaves that would be shaded under direct light, and increasing canopy-wide photosynthesis (4–9). In contrast, at the individual leaf level, diffuse light is not as effective as direct light at driving photosynthesis (10). Despite the importance of the directional quality of light for plant photoresponses, few leaf optical measurements are available for leaf samples irradiated with diffuse light, primar- ily because it is difficult to measure the total amount of reflected light under these conditions. In order to explain why this is so, we will describe how measurements of reflected, transmitted and absorbed light are normally made. Such measurements use an integrating sphere, a hollow sphere coated inside with highly reflecting material and fitted with ports for admitting light and for placing samples or measure- ment devices. For visible light, the coating contains metal oxides and typically has a reflectivity of >99%. To measure transmitted light, a leaf sample is mounted on a port of an integrating sphere and irradiated with collimated light (see Appendix S1, Fig. S1a). Light that travels through the leaf is scattered and exits the leaf in many directions, so an integrating sphere is necessary to capture all this light. Transmittance (T) is the ratio of light in the sphere when the leaf is present to the light when the leaf is absent. To measure reflected light, a leaf is mounted similarly except that direct beam light is sent though the sphere from an open port opposite the leaf sample (see Appendix S1, Fig. S1b). The light reflected from the leaf is scattered and hence must be collected by the sphere. Reflectance (R) is calculated as the ratio of the amount of light in the sphere in the presence of a *Corresponding author email: hlgorton@smcm.edu (Holly L. Gorton) †Current address: Craig R. Brodersen, Department of Viticulture and Enology, University of California, One Shields Avenue, Davis, CA 95616-5270 Ó 2010 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/10 Photochemistry and Photobiology, 2010, 86: 1076–1083 1076