September 11, 2019
by Abhijit Marar
Imaging the NMJ:
The project has taken a slightly different direction given the obstacles we have been facing in imaging the NMJ. Our collaborators at this point think that they will be able to convey their message if they can get a good STORM image of the Myopodia and an image of the Fas2 protein that is present in the filopodia. To get a good STORM image of the myopodia, our approach involves taking more single-molecule frames of the myopodia by staining it multiple times during the imaging process. After the first staining step, the subsequent staining steps will be done. on the microscope. We have tried this protocol once with satisfactory results (i.e., the drift was not significant and the sample continues to blink after secondary staining process), however we are yet to optimize the imaging protocol. We are also planning on having 50 nm gold particles as fiducial markers to track the amount of drift in the system. To stain the sample multiple times, Daichi had special slides made with two holes in the slide that allows him to change the buffer and perform the entire staining protocol on the microscope.
Incoherent holography by separating the excitation and emission paths:
Before submitting the manuscript describing incoherent holography with 100 nm beads, we decided to try imaging single molecules one more time using incoherent holography, but this time we separated the excitation and emission paths (i.e., the light from the laser is no longer going through the objective to excite the sample). This was done with the goal of removing/reducing excess background caused by the laser light when imaging at low photon counts. The picture below shows the modified setup.
Unfortunately, this did not result in successful imaging of single-molecules, and we are still limited to detecting 13000 photons. There is still a huge amount of laser light going through the collection objective. This was reduced when we switched to an objective lens with a lower NA (i.e., smaller collection angle), however this will also reduce the amount of fluorescence collected from the single molecule. Nonetheless, this an experiment worth trying and I will continue to work on it after I am done with my proposal defense.