Reiner Rygiel

High-resolution imaging of biological samples

Confocal laser scanning microscopy is used in many different fields of research nowadays. Therefore, high spatial resolution is required as well as high temporal resolution. Further, the quality of the resulting images has to be considered. Doing imaging with laser scanning microscopes, the balance between spatial resolution, speed and signal-to-noise ratio has to be defined for every specimen and experiment individually. Special adaptations to standard laser scanning microscopes improve the efficiency of the Leica TCS SP2 and Leica TCS SP2 RS for high-resolution low-noise imaging. Here, we want to report from the acousto-optical beam splitter, the resonant K-scanner and new objectives.

Compact 4pi beam scanning microscope for imaging cells with a sevenfold increase in axial resolution

Confocal microscopy is the method of choice in biological 3D-imaging, however, the axial resolution is limited to ~500 nm. During the last decade it has been successfully demonstrated that the axial resolution can be substantially improved with 4Pi microscopy. We report a 4Pi microscope realized as a fast beam scanning system consisting of a 4Pi-module linked to a state-of-the-art confocal microscope. As a result, the advantages of the confocal system such as scanning, sensitive multicolor detection and imaging speed are combined with the superior resolution of 4Pi microscopy. This novel microscope is eminently suited for biological applications. It is designed both for single-photon and for two-photon picosecond excitation, and also enables joint coherent illumination and detection, i.e. 4Pi type C. The superior PSF and OTF of the system enable 80 nm axial resolution in cells mounted in aqueous media. We present the optical design of the system and demonstrate an up to 7-fold improved optical sectioning in live cells.