Three-dimensional multi-modal microscopy

Advances in lasers in recent decades have led to novel techniques of microscopy that are only possible with powerful coherent sources. Modern electronic cameras and computers have made it possible to capture, process, and display the resulting images. The continued growth in computing power now makes it possible to combine images from different microscopic imaging modalities. The W. M. Keck Three-Dimensional Fusion Microscope brings together multiple modes of microscopy on a single platform, with the ability to switch from one to another rapidly without moving the specimen. With this microscope, it is possible to examine the spatial relationships among components of the specimen each of which is visualized with a different mode, to use one mode as validation for another, and to combine multiple modes to exact information that would not be available from any single mode alone. In addition to the commonly used modes of brightfield, differential interference, epi-fluorescence, this instrument has confocal and multi-photon fluorescence, second-harmonic, frequency-agile confocal reflectance, fluorescence recovery after photo-bleaching, and optical quadrature microscopy. The last of these is used for full-field quantitative phase imaging. The initial applications of the microscope are in the imaging of embryos, skin, and nano-fabricated structures. Sample results will be shown, and the design issues that arise in the combination of multiple modalities will be discussed.