Quantifying the refractive index dispersion of a pigmented biological tissue using Jamin–Lebedeff interference microscopy

Jamin–Lebedeff polarizing interference microscopy is a classical method for determining the refractive index and thickness of transparent tissues. Here, we extend the application of this method to pigmented, absorbing biological tissues, based on a theoretical derivation using Jones calculus. This novel method is applied to the wings of the American Rubyspot damselfly, Hetaerina americana. The membranes in the red-colored parts of the damselfly’s wings, with a thickness of ∼2.5 µm, contain a pigment with maximal absorption at ∼490 nm and a peak absorbance coefficient of ∼0.7 µm−1. The high pigment density causes a considerable and anomalous dispersion of the refractive index. This result can be quantitatively understood from the pigment absorbance spectrum by applying the Kramers–Kronig dispersion relations. Measurements of the spectral dependence of the refractive index and the absorption are valuable for gaining quantitative insight into how the material properties of animal tissues influence coloration. Scientists have used Jamin–Lebedeff interference microscopy to measure the refractive index of strongly pigmented biological tissue. Doekele Stavenga and co-workers from the University of Groningen in the Netherlands used the technique to analyze the refractive index, and its variation with wavelength, of the red wings of the American Rubyspot Damselfly. Until now, this microscopy scheme has been applied only to transparent, unpigmented tissue. The researchers report that the high density of pigment in the damselfly wing results in a significant anomalous dispersion of its refractive index, with the index increasing for longer wavelengths. This is believed to enhance the reflectivity in the red spectral region and thus improve the colour contrast of the wing decorations. Similar contrast shown effects are thought to be present in the eyes of some crustaceans.