August 2, 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 200nm yellow-green fluorescent microspheres (Invitrogen microspheres 505/515) using incoherent holography, however the imaging conditions were not practical with regards to STORM microscopy. The camera exposure time for the data taken was 500ms which was very slow. Typically while performing STORM imaging, the exposure time is 20-50ms. The readout noise from the sCMOS camera used earlier was too high for imaging 200nm beads at such conditions.
Given the thought that the read noise might be too high in the sCMOS camera thus affecting the performance of the microscope, we decided to go back to our simulations to see how sensitive the technique of incoherent holography was to read noise at low light levels. Our simulations showed that at low light levels (500 photons), the system is very sensitive to read noise. When the read noise was greater than 0.1 e- the technique does not work to provide a good reconstruction. After this realization we changed the camera and are currently using an EMCCD camera (iXon Life 897). In the EMCCD camera the read noise can be brought down to below 0.1 e- when using it in EM gain mode. After making the necessary changes and finding the optimal camera conditions to work with we were successfully able to image 200 nm red (580/605) fluorescent microspheres at 50 ms exposure times and reconstruct the signal.
The next goals are to image smaller beads (100 nm), and also take a 3D stack while moving the bead in a 10um range and reconstruct the holograms at the best focus of each of the z-positions.