We’ve implemented the Stochastic Optical Reconstruction Microscopy (STORM) technique with which the resolution limit imposed by diffraction has been overcome. Our goal is to image nanostructures in the Drosophila brain with superresolution in order to have an accurate view of the connectivity in the central nervous system that would allow for a better understanding of the flow of information, in specific, from the visual system to the different neurons.

With the aid of photoswitchable dyes, we obtain images with a sparse subset of fluorescent molecules imaged in a frame so that the point spread function (PSF) of two adjacent emitters do not overlap and their centroids can be located with high accuracy, on the order of 10 times better than optical resolution. One can bring fluorophores from dark to bright states in a stochastic manner and after repeating this process a number of times a final diffraction-unlimited image is reconstructed.

The technique has been developed in recent years and the Multicolor and 3D versions have been realized with success in model systems as well as in tissue. Below is a picture of our Multicolor-STORM setup, with laser lines 407, 459, 532 and 647nm. The different lines are combined into a single beam and the stimulation is controlled with an acousto-optical tunable filter (AOTF). The AOTF allows for fast and efficient diffraction of light, so that the wavelength and the intensity of light is selected with it. The diffracted beams are then coupled into an optic fiber and this feeds the TIRF arm of a Nikon Ti microscope.


The next is an example of a staining of the 3p3-GFP staining in the adult head of Drosophila. Left: TIRF images. Right: STORM images.


Ricardo Alberto Armenta Calderón
University of Fribourg
Physics Department - Soft Matter and Photonics
Biology Department - Drosophila Group

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