Superresolution microscopy of large specimens

The objective is to utilise the full potential of the new supercontinuum lasers currently available for imaging neuronal networks. These "white light lasers" enable polychromatic imaging. This is of paramount importance for biological applications, as often the co-localisation of fluorescent labels of different colours is necessary. The proposed approach is based on our novel microscope (ultramicroscope) which allows the recording of 3D image stacks of macroscopic specimens with microscopic resolution. Preparations are made transparent by a chemical clearing which preserves fluorescent labelling of e.g. neurons. Furthermore it should be possible to obtain increased resolution with the supercontinuum lasers by using the principles of STED microscopy which provides up to a tenfold increase in resolution over confocal microscopy. As the axial resolution in the ultramicroscope does not match the lateral resolution, an increase in axial resolution by the principles of STED microscopy would greatly improve the method. To this end special optics for generating a light sheet quenched in thickness beyond diffraction limit will be developed. Ultimately, superresolution imaging of large biological specimens like mouse brains should become posssible.

have worked on developing a 3D microscopy with especially high resolution for large preparations like whole mouse brains. This is possible by clearing the mouse brains with a special procedure making them completely transparent. If they are illuminated from the side with a thin laser beam forming a light sheet, thin optical sections are generated which can be recorded from above with a microscope. This way one obtains thousands of optical sections allowing one to reconstruct the 3 dimensional structure with a computer. For the resolution it is now important that the optical sections are as thin as possible. To achieve this we wanted to apply the STED procedure on light sheet microscopy. This procedure is principally able to generate a very high resolution, however fluorescently labelled cells in the specimen are quickly bleached by the high light intensities. We have therefore finally developed new optics for the generation of light sheets which works without STED. With this optics we could record mouse brains with unprecedented resolution.