CONFOCAL MICROSCOPY APPLIED TO PALEONTOLOGICAL SPECIMENS

AbstractConfocal laser scanning microscopy is a well-established optical technique allowing for three-dimensional (3-D) visualization of fluorescent specimens with a resolution close to the diffraction limit of light. Thanks to the availability of a wide range of fluorescent dyes and selective staining using antibodies, the technique is commonly used in life sciences as a powerful tool for studying different biological processes, often at the level of single molecules. However, this type of approach is often not applicable for specimens that are preserved in historical slide collections, embedded in amber, or are fossilized, and cannot be exposed to any form of selective staining or other form of destructive treatment. This usually narrows the number of microscopic techniques that can be used to study such specimens to traditional light microscopy or scanning electron microscopy. However, these techniques have their own limitations and cannot fully reveal 3-D structures within such barely accessible samples. Can confocal microscopy be of any help? The answer is positive, and it is due to the fact that many paleontological specimens exhibit a strong inherent autofluorescence that can serve as an excellent source of emitted photons for confocal microscopy visualizations either through reconstruction of the induced autoflourescent signal from the sample, or through reconstruction of the reflected signal from the sample surface. Here, we describe the workflow and methodology involved in acquiring confocal data from a sample and reprocessing the resulting image stack using the image-processing program imageJ before reconstructing the data using the open-source 3-D rendering program, Drishti. This approach opens new possibilities for using confocal microscopy in a nondestructive manner for visualizing complex paleontological material that has never previously been considered as suitable for this type of microscopic technique.