Rafael Storz

Advances in Multispectral Confocal Imaging

Due to the tendencies in biological research towards multiple stainings and increased use of newly developed dyes like fluorescent proteins, the application areas of multispectral confocal imaging increase rapidly. We present here the most recent advances in this technology particularily concerning but not limited to the excitation module, the detection module, and the beamsplitter optics of multispectral confocal microscopes.

Programmable beam-splitter for confocal laser scanning microscopy

In confocal microscopy manifold combinations of dyes are used. The entire wavelength range from near-ultraviolet to near-infrared is used to excite these dyes. Their emission is then detected primarily in the visible range. In addition to the number of dyes, the number of possible excitation laser lines is already large and constantly increasing. Due to the almost unlimited number of possible combinations, programmable devices are required. This is true for excitation-modules, beam-splitters and detection modules. Systems using filters with fixed spectral properties can practically fulfill the requirements for only a very small subset of applications. Programmable devices have been realized for excitation using Acousto Optical Tunable Filters (AOTFs). Freely definable spectral detectors are available for a perfect adaptation to the desired emission bands. The missing link for a completely filter-free design is now introduced using an Acousto Optical Beam Splitter (AOBS).

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.