KLAUS, Angela V.; SCHAWAROCH, Valerie; American Museum of Natural History; Baruch College: A Novel Methology Utilizing Confocal Laser Scanning Microscopy for Systematic Analysis in Arthropods

The use of confocal laser scanning microscopy (CLSM) for imaging arthropod structures has the potential to profoundly impact systematics of this group. Three-dimensional (3D) reconstruction of CLSM data provides high-fidelity, detailed images of miniscule structures unobtainable by traditional methods (e.g. hand-illustration, bright-field light microscopy, scanning electron microscopy). A CLSM dataset consists of a stack of 2D images (�optical slices�). The �optical sectioning� capability of the confocal microscope is a result of the placement of a pinhole aperture in front of the signal detector. The pinhole excludes out-of-focus light from each optical slice so that only the in-focus light from the specimen is captured at each imaging depth. CLSM is particularly well-suited for arthropod specimens due to the autofluorescent nature of their tissues. In the current work, we document a methodology for obtaining image stacks via CLSM, and for visualizing the data via three different rendering techniques. We also explore the distortion of image stacks due spherical aberration, and describe strategies for minimizing such distortion. Using this methodology, we have examined several diagnostic structures, including the genitalia of flies.