STED Imaging of Silica Beads
To try to calibrate the STED for imaging on our membranes, Bill and I have been trying to use 50 nm silica beads that have been captured on the membrane. However, we discovered initially that the beads were actually not too easy to work with and clumped like crazy.
On STED, we weren’t really able to find individual particles, though we did have some regions that gave us relatively nice imaging of particles on the surface.
However, these particles were likely clumped and on the surface of the sample. So to try to remove the clumps, I sonicated the particles for 30 min. This still resulted in clumps, but we were able to sort of make out regions that had what appeared to be individual particles.
While this looked decent, we suspected that the particles we were imaging were still on the surface and not actually in the pores of the membrane. So we decided to adjust the focus of the STED by 150 nm to try to image particles that would be in the pores.
We started to resolve particles that were likely in the pores of the membrane. However, there was still a problem of clumping which I think can be attributed to improper rehydration of the nanoparticles. I will add ethanol to hopefully break apart the particles (from a protocol given to me by Elena Lomakina) and I will then retry the experiment.