July 16, 2019
by Abhijit Marar
STORM imaging of the myopodia (postsynaptic filopodia in Drosophila muscles):
Our collaborators here at UGA (Dr. Daichi Kamiyama and Melissa Inal) have been trying to solve the issue of excessive background when trying to perform STORM imaging of the myopodia. From the optics side, we have tried to implement HiLo illumination ( Tokunaga, Makio, Naoko Imamoto, and Kumiko Sakata-Sogawa. "Highly inclined thin illumination enables clear single-molecule imaging in cells." Nature methods 5.2 (2008): 159. ) to reduce the background. Currently, we have had some success imaging the myopodia using the HiLo illumination configuration by bleaching out the area around the myopodia before acquiring STORM data of the myopodia. Below is a STORM image of the myopodia labelled with CF 568 dye reconstructed from 40000 frames.
Two-color STORM imaging of the neuro-muscular junction in Drosophila:
We have successfully implemented the capability to perform simulataneous two-color imaging on the current microscope without sacrificing field of view. Using the image splitter we have been able to acquire wide-field images of the neuro-muscular junction. Once the imaging conditions are worked out for the individual channels, we plan to perform simultaneous two-color STORM imaging of the neuro-musuclar junction. Below is an image of the two channels (top: 647/670 (myopodia) and bottom: 568/583 (filopodia)) being simulatenously imaged.