July 3, 2018
by Abhijit Marar
Goal: Develop a light microscope that performs 3-dimensional high resolution imaging.
Review of progress to date:
We have successfuly imaged 1um yellow-green fluorescent microspheres (Invitrogen microspheres 505/515) using incoherent holography. Due to limitations of the camera and the use of an LED instead of a laser, we were not able to image smaller beads because not enough signal was being emitted from the beads.
Using a better performing sCMOS (scientific CMOS) camera we have been able to image 200nm red fluorescent microscpheres (Invitrogen microspheres 580/605). The data is taken with the camera set to an exposure time of 500ms and laser power at 50mW. With this configuration, the signal to noise ratio at lower exposure times is not high enough to result in a good reconstruction.
Note: This exposure time is not ideal. Our goal is to get it down to ~20-50 ms).
(Above:) Hologram 1 (Phase: 0 degrees) Hologram 2 (Phase: 120 degrees) Hologram 3 (Phase: 240 degrees) Final Image: Reconstruction at focus
Each image shows faint roughly circular diffraction rings surrounding each bead’s position. The final reconstructed image resolves the 9 upper microbeads into sharply distinct light-emitting particles.
Currently we will continue to run a few more tests with the same parameters and the camera to better understand the system. Once we think we understand the system completely and can get it to work consistently, we plan to use an EMCCD camera that might allow us to look at the beads with lower exposure times and also perhaps move to smaller beads (100nm) and single molecules eventually.
Note: There are different configurations ( combinations in which the optics can be placed) to perform incoherent holography. The holograms have different characteristics in these different configurations and these characteristics determine the quality of the reconstruction. "Better understanding of the system" refers to trying out these configurations and seeing which one works best to achieve the goal.