Nanoscale Imaging of Synaptic Connectivity in the Drosophila Larva
The availability of a number of genetic tools for drosophila melanogaster has made it a suitable model system for studying brain processes, structure and connectivity. In particular, it is possible to attach fluorescent markers or drive the expression of fluorescent proteins in specific brain regions or subsets of neurons. With the rise of powerful microscopy techniques, such as laser scanning confocal microscopy, it is now possible to accurately visualize those fluorescent structures. There is, however, a limitation on the minimum size of the object to be studied, the so called diffraction limit. Optical imaging techniques can only resolve objects larger than about λ/2, where λ is the wavelength of the light involved in the imaging process.
This project focuses in the imaging of chemical synapses whose sizes range between 100 and 200 nm, rendering it impossible to use traditional fluorescence microscopy techniques to observe specific populations (pre- or post-synaptic sites) of proteins expressed in sub-micrometer structures, often encountered in the system of interest. A more elaborate imaging method is therefore needed.
We’ve implemented the Stochastic Optical Reconstruction Microscopy (STORM) (Rust, Bates and Zhuang, Nature Methods 2006) technique with which shall be able to overcome the diffraction limit of resolution. STORM’s principle is that of locating single fluorescent centers by the use of photoswitchable molecules or optical switches. There are several possibilities for optical switches (Mark Bates, et al., Science 2007) by bringing together two fluorescent dye species, namely one reporter dye and one activator dye, simultaneously conjugated to an antibody. It is then possible to perform superresolution microscopy on one or more protein populations, making the technique suitable for the study of multicomponent structures in biological specimens, such as our system of interest.
Schlagworte: STORM, Superresolution, Synaptic Connectivityzurück