Two color imaging of the NMJ and Light sheet plans for the STORM setup

August 12, 2019

by Abhijit Marar

Two color imaging of the Neuro-muscular junction (NMJ):

We have been able to acquire one good image of the NMJ. This image was acquired using sequential imaging (ie. 30,000 frames were first acquired to image the myopodia and then 30,000 frames of the axonal filopodia were acquired). We are currently trying to work with the splitter, but that has not yet provided successful images. Below is the STORM image of the NMJ.

STORM imaging of the NMJ: (A) Reconstructed STORM image of the axonal filopodia labelled with AF 647. (B) Reconstructed STORM image of the myopodia labelled with CF 568. (C) Composite image with (A)
and (B) overlayed on each other after drift correction.

Light-sheet design for the STORM setup:

The lab is interested in having a light-sheet illumination design for the STORM setup. This will aid in high-resolution imaging of thick-samples (the fly-embryo would fall in this category). We think this will also help with the problem of excess background noise we have been facing in incoherent holography. There are multiple ways to design such a system and currently the lab has two ideas (Thanks to another member of the Kner lab: Yang Liu). We will make a decision soon to decide on the design to be implemented. The two design's are not different in principle but may vary in difficulty in implementation, ease of use and stability. The principle being followed is discussed in detail in the following paper: ( Gustavsson, A. K., Petrov, P. N., Lee, M. Y., Shechtman, Y., & Moerner, W. E. (2018). 3D single-molecule super-resolution microscopy with a tilted light sheet. Nature communications, 9(1), 123.)

Figure 2: Light-sheet design for STORM setup: (A) Complete view of the Light sheet (LS) design  to give the viewer an idea of how the design will be implemented over
the current setup. (B) Close-up of the LS illumination scheme using a prism. (C) Close up of LS illumination scheme using a 1" mirror mount.