Techniques for documenting and quantifying biofluorescence through digital photography and color quantization

Abstract

Ultraviolet (UV) induced biofluorescence is being discovered with increasing frequency across the tree of life. However, there is not yet a standardized, low-cost, photographic methodology used to document, quantify, and minimize sources of bias that often accompany reports made by researchers new to fluorescence imaging. Here, a technique is described to create accurate photographs of biofluorescent specimens as well as how to use these images to quantify fluorescence via color quantization, using open-source code that utilizes K-means clusters within the International Commission on Illumination L*a*b* (CIELAB) color space. The complexity of photographing different excitation and emission wavelengths and methods to reduce bias from illumination source and/or camera color sensitivity (e.g., white balance) without the use of modified equipment is also addressed. This technique was applied to preserved southern flying squirrel (Glaucomys volans) specimens to quantify the color shift between illumination under visible and UV light and analyze variation among specimens. This relatively simple methodology can be adapted to future studies across a range of fluorescent wavelengths and project goals, and assist future research in which unbiased photographs and analyses are key to understanding the physiological and ecological role of biofluorescence.