METHODS FOR CHARACTERIZATION OF FLUORESCENCE IN FOSSILIZED PLANTS

Fluorescence microscopy, the collection and analysis of signals from molecules with differing fluorescence properties, has been frequently utilized for studying living plants but very rarely for quantifying the fluorescence of fossilized plants. Understanding what compounds produce the ubiquitous fluorescence in fossil plants and determining whether they are original to the plant or the result of fossilization may provide information about (1) fluorescence in ancient plants or (2) processes of fossilization. Here we have employed a spectrally resolved two-photon micro-spectroscope with line scan excitation on a variety of fossil plants to interpret and quantify their fluorescence emission spectra. A combination of three features in the advanced micro-spectroscope are essential for accurately characterizing the excitation and emission spectra in plant fossils: (i) pixel-level spectral resolution, (ii) two-photon excitation, and (iii) line-scan excitation. Pixel-level spectral resolution enables the distinction of molecular species possessing different emission spectrum profiles, even near the boundaries between adjacent sample features. When combined with spectral deconvolution, it enables the quantification of different types of fluorescence sources, even if they reside in the same image pixel. Two-photon excitation allows the collection of the entire emission spectrum for any excitation wavelength, which is superior to single-photon excitation instruments, whose spectral detection bands change with changing excitation wavelength and, therefore, might fail to detect critical changes in emission spectra as a function of excitation wavelength. Finally, line-scan excitation enhances the signal level, and thus sensitivity, by acquiring in parallel from sample voxels along a line-shaped laser beam illuminating the said voxels simultaneously. This increase in sensitivity allows for a significant decrease in excitation power, which is critical for imaging fossilized plant specimens, which are susceptible to laser damage. In this talk, the methods for obtaining detailed emission spectra from various common types of plant fossils – e.g., amber or leaf compression fossils – will be presented, and results obtained using these methods will be discussed.