Spectroscopy and Kubelka-Munk Theory to Examine Flower Color

Researchers from the University of Groningen, Netherlands are merging the disciplines of physics and biology in the quantitative analysis of the reflectance spectra of flowers.

Image Credit: Eduardo Ramos Castaneda/Shutterstock.com

The latest work on the Coloration of the Chilean Bellflower has significant implications in the relationship between pollinators and plants and the study of evolutionary biology. Additionally, this work enhances our understanding of light scattering and pigmentation, as well as adding valuable reflectance spectra data for the Nolana paradoxa to the world’s catalog of reference spectra data.

Applying Biology and Physics

The application of the Kubelka-Munk layer stack theory to plant colorization analysis also has industrial and commercial implications related to materials, paints, dyes, and coatings; especially in the world of automobile and auto parts manufacturing and semiconductor fabrication.

Flower Petal Anatomy and the Stack Model

Considerable effort has been made for studying the physical structure of plants, especially the colorization of flowers, in large part because of the relationship in signaling pollinators. Dr. Casper J. van der Kooi, professor of plant physiology at the Groningen Institute for Evolutionary Life, has done a large amount of work to further quantitative analysis of light interaction in order to explain flower coloration.

Outer epidermal layers and inner pigmented and light scattering layers of the mesophyll together make up the petals of a flower. Incident light is selectively absorbed by pigment layers within a particular wavelength, while vacuoles and light scattering structures backscatter the incident light in all directions. A consistent visual effect is created by this diffuse reflectance when viewed from multiple angles.

In his latest work, partnered with Dr. Doekle G. Stavenga, professor of computational physics at the Zernike Institute for Advanced Materials, University of Groningen, Dr. van der Kooi studied the Chilean Bellflower, Nolana paradoxa, which has a distinct color differentiation between the unpigmented abaxial (lower) surface and the saturated, vivid purple of its adaxial (upper) surface.

Earlier efforts to explain the interaction of light within a plant to produce flower color depended on geometrical optics, however plant structures are not homogenous, making direct optical analysis inconvenient. Additionally, these methods need knowledge of vital optical parameters such as a refractive index and absorption coefficients of the component structures; however, optical constants of botanical samples such as these are not available in the existing body of knowledge. Alternatively, the Kubelka-Munk theory for absorbing and diffusely scattering media enables deriving the absorption and scattering coefficients from measured transmission and reflectance spectra.

Stavenga and van der Kooi have successfully used this method in earlier work on a non-invasive method for estimating chlorophyll content using the Kubelka-Munk theory and treating a plant leaf as a stack of absorbing and reflective plates. In this related spectra, analysis work sets the basis for comparison of the coloration strategies of flowers and future quantitative analysis.

Methods and Results

Drs. van der Kooi and Stavenga described flower coloration by developing an optical model treating a flower petal as a stack of layers, and then used the Kubelka-Munk theory for diffuse scattering and absorbing media to the layers. This method uses the combined stack reflectance and transmission spectra. It is capable of estimating the reflectance and transmission spectra for each layer based on the awareness of the number and relative thickness of the layers.

These researchers measured the distribution and physical structure of pigments by examining cross sections of bellflower petal under magnification and discovered that the Nolana paradoxa petals have a pigmented, strongly scattering adaxial mesophyll layer and an unpigmented moderately reflective abaxial layer.

Researchers captured spectrophotometric measurements by employing a deuterium-halogen lamp (AvaLight-D(H)-S) to deliver light via optical fiber to an integrating sphere (AvaSphere-50-Refl). By positioning the corolla (the sum group of petals of a flower) in the sphere and illuminated directionally, the reflected light was then captured by a second optical fiber and collected with the AvaSpec-ULS2048XL-USB2 spectrometer with a 2048 pixel back-thinned CCD image sensor.

This measured transmission and reflectance spectra for the corolla was then analyzed by Stavenga and van der Kooi using the Kubelka-Munk absorbing stack layer model in order to estimate the transmission and reflectance spectra of the observed layers of the petal interior.

Subsequent measurements sought to confirm the results of applying the layer stack theory by separately measuring the absorption spectra for the pigmented adaxial layer and the reflective properties unpigmented abaxial layer. These subsequent experiments carried out on the isolated layers appeared to prove the spectral analysis derived from using the stack layer modeling.

Conclusion and Continuing Research

Casper van der Kooi and Doekele Stavenga have considerably improved our understanding of how plants use light. The team's growing body of work together with other frequent contributors has examined the physical interaction of light that provides flowers their colorful appearance and how the spectral display of flowers is affected by the competition for pollinators.

This research into the efficacy of the Kubelka-Munk method can have potentially wide-reaching impact on both the potential relevance to other applications which concern stack metrology, and the study of plants with potential agricultural applications.

References and Further Reading

• van der Kooi, CJ; ElzengaJTM; Staal M; Stavenga DG. (2016) How to Colour a Flower: on the Optical Principles of Flower Coloration. Proceedings of the Royal Society B283:20160429
• van der Kooi, CJ; Pen, I.; Stall, M. (2016) Competition for Pollinators and Intra-communal Spectral Dissimilarities of Flowers. Plant Biology Journal 18:1 10.111/plb12328
• Ozawa, A.; Uehara, T.; Sekiguchi, F.; et al. (2009) Spectral Analysis of Scattered Light from Flowers' Petals Optical Review 16:458. doi:10.1007/s10043-009-0088-2
• van der Kooi, C.; Wilts, B.; Leertouwer, H.; Staal, M.; Elzenga, T.; Stavenga, DG. (2014) Iridescent Flowers? Contribution of Surface Structures to Optical Signaling. New Phytologist 203:2 10.1111/nph.12808
• The Anatomy of Flower Color (2016) Phy.org May 10, 2016 Accessed February 24, 2017

This information has been sourced, reviewed and adapted from materials provided by Avantes BV.

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