High-resolution ultramicroscopy of the developing and adult nervous system in optically cleared Drosophila melanogaster.

The fruit fly, Drosophila melanogaster, is an important experimental model to address central questions in neuroscience at an organismic level. However, imaging of neural circuits in intact fruit flies is limited due to structural properties of the cuticle. Here we present a novel approach combining tissue clearing, ultramicroscopy, and data analysis that enables the visualisation of neuronal networks with single-cell resolution from the larval stage up to the adult Drosophila.

Reshaping a multimode laser beam into a constructed Gaussian beam for generating a thin light sheet.

Based on the modal analysis method, we developed a model that describes the output beam of a diode-pumped solid state (DPSS) laser emitting a multimode beam. Measuring the output beam profile in the near field and at the constructed far field the individual modes, their respective contributions, and their optical parameters are determined. Using this information, the beam is optically reshaped into a quasi-Gaussian beam by the interference and superposition of the various modes.

Outlook on optimizing ultramicroscopy imaging technique through optical characterization.

Here, we present an optically optimized system for static ultramicroscopy imaging technique. The unit for generating an ultra-thin light sheet employs aspheric and meso-optical elements (meso-aspheric system). An analytical as well as an experimental comparison between the light sheet produced by the standard system (using a rectangular slit aperture and one cylindrical lens) and the one produced by our latest optimized system, which converts a symmetrical Gaussian beam into an ultra-thin light sheet is presented.

Ultramicroscopy: development and outlook.

We present an overview of the ultramicroscopy technique we developed. Starting from developments 100 years ago, we designed a light sheet microscope and a chemical clearing to image complete mouse brains. Fluorescence of green fluorescent protein (GFP)-labeled neurons in mouse brains could be preserved with our 3DISCO clearing and high-resolution three-dimensional (3-D) recordings were obtained.

Reduction of photo bleaching and long term archiving of chemically cleared GFP-expressing mouse brains.

Tissue clearing allows microscopy of large specimens as whole mouse brains or embryos. However, lipophilic tissue clearing agents as dibenzyl ether limit storage time of GFP-expressing samples to several days and do not prevent them from photobleaching during microscopy. To preserve GFP fluorescence, we developed a transparent solid resin formulation, which maintains the specimens' transparency and provides a constant signal to noise ratio even after hours of continuous laser irradiation.