Ultramicroscopy of Transparent Flies

Ultramicroscopy: Detailed image of Drosophila’s head with fluorescent molecules. (Source: M. Pende, TU Vienna)

The nervous system of an animal can be studied by cutting it up into thin layers, however this inevi­tably leads to the destruction of the cellular structures in the tissue. Analyzing complex nerve connec­tions is then hardly possible. The far more elegant method is the optical clearing of the various tissues using chemical processes that make the animal transparent. Interes­ting structures in the tissue can be selec­tively marked and analyzed. At the Vienna Uni­versity of Tech­nology, a clearing method has now been developed that can be applied to insects, which is a parti­cularly difficult task. With an ultra­microscope, it is now possible to image large nerve tissue samples and take high-resolution pictures of complex neural networks that have been labeled with fluores­cent molecules.

“We can learn a lot about the nervous systems of animals by using genetic engi­neering to insert special molecules into the nerve tissues, which can then be made to fluoresce,” says Marko Pende, a PhD student at TU Wien. The big question is how these fluores­cent molecules can be imaged without damaging the tissue. One method that has been used with great success is ultra­microscopy. Trans­parent tissue is illu­minated with a laser beam, which is widened by special optical elements, creating a two-dimensional flat surface of light. This surface pene­trates the tissue and illu­minates those fluores­cent molecules that lie exactly in that plane. Layer by layer, the tissue can be analyzed with this light-sheet, creating a three-dimen­sional model from the two-dimen­sional frames on the computer.

“We focused on the fruit fly droso­phila melano­gaster because it is of particular interest for research into the nervous system. Unfor­tunately, it is parti­cularly difficult to develop a suitable clearing method for insects,” explains Marko Pende. “For the tissue to become trans­parent, it has to be treated with special chemicals, and in insect tissues these chemicals have always destroyed the fluores­cent molecules until now.” In addition, insect tissue contains chitin, which can hardly be made transparent. Also, drosophila has parti­cularly robust pigments in it its eyes.

The team succeded together with the University of Vienna and the Medical Uni­versity in finding a way to make droso­phila flies completely transparent without destroying the fluores­cent marker molecules. This was achieved with the help of improved chemical mixtures. “It is an important step forward for the droso­phila research community,” says Thomas Hummel from the Depart­ment of Neuro­biology of the Uni­versity of Vienna.

The pictures were made possible by pio­neering optical research by Saiedeh Saghafi. She was able to signi­ficantly improve the ultra­microscope: The light-sheet, with which the plane is illu­minated layer by layer, used to be about 10 microns thick. The improved ultra­microscope now produces uniform light-sheets of only 3 μm thickness over a large area. In addition, the micro­scope was equipped with an addi­tional lens, which changes the focal point, much like to a pair of glasses: “So far, we could only focus on the outer area of the tissue, now we can take a centimeter-deep look into the tissue and still get sharp images,” says Hans Ulrich Dodt, Head of the Depart­ment of Bioelec­tronics at TU Wien. “It will enable impressive, high-reso­lution images that will give us important insights into the way the Drosophila nervous system works.”

The new technique should now help to study the connectome of drosophila. The connectome is the arrange­ment of inter­connections throughout the nervous system, the neuronal circuit diagram of the animal. This circuit diagram can then be related to behavioral patterns of drosophila. In addition, drosophila is ideal for analyzing genes that lead to neuro­degenera­tive diseases in humans, such as Alzheimer’s and Parkinson’s disease. Transparent flies now provide a unique oppor­tunity to under­stand the complex changes in various areas of the nervous system during neuro­degenera­tion. (Source: TU Wien)

Reference: M. Pende et al.: High-resolution ultramicroscopy of the developing and adult nervous system in optically cleared Drosophila melanogaster, Nat. Commun. 9, 4732 82018); DOI: 10.1038/s41467-018-07192-z

Link: Dept. of Bioelectronics, Technical University Wien, Vienna, Austria

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