Confocal theta microscope with three objective lenses

Abstract

A microscope using three water immersion objective lenses which realizes confocal, 4Pi-confocal and various confocal theta microscopies in fluorescence, transmission, scattered, and reflection mode is described. An argon-ion laser is the primary light source. A pulsed titanium-sapphire laser allows two-photon absorption fluorescence microscopy. The instrument has a predicted resolution of 100 nm along the illumination axis and a three-dimensional resolution of 5×106 nm3 for lenses each with a numerical aperture of 0.75. This is an improvement of an order of magnitude over a confocal fluorescence microscope using the same lens. Applications of the microscope range from observation of a sample at three different angles, to confocal theta fluorescence microscopy with multiphoton absorption. Since mounting and immersion media are identical, aberrations become negligible. The large working distance of 2 mm makes the instrument ideal for the observation of biological samples of up to 1.5 mm in diameter such as drosophila embryos.