December 13, 2018
by Abhijit Marar
We have moved to our new lab space at the Miller plant sciences building and the microscopes have been setup. The holographic microscope has been setup and we have run some initial tests to look at the point spread function (PSF) (one can say, this is the “signature” of the microscope, it tells us how are system will perform optically). With the move, we have made certain changes to the setup on both the excitation arm and the emission arm. The changes to the excitation arm will not affect the excitation beam in any way but they make the microscope more user-friendly (easier for the user to align the system). In the emission arm, the spatial light modulator (SLM) has been replaced with a Michelson interferometer. The Michelson interferometer is more light efficient in comparison to the SLM. The direction of the alignment of the liquid crystals in the SLM force the SLM to only reflect light that is vertically polarized, thus discarding 50% of the fluorescence emitted from the sample. The Michelson interferometer consists of two mirrors and a beam splitter and thus uses all the light coming from the sample. One arm of the Michelson interferometer has a plane mirror and the other arm has a concave mirror with a focal length of 300 mm.
The two arms of the Michelson interferometer have been aligned and we have been able to acquire holograms of 200 nm dark-red fluorescent microspheres (660/680). The resulting final image has been reconstructed from a single hologram (only 1 phase). Due to this, we see what we think is the real image (bright sharp spot in the center) overlapped with the virtual image (weak large circle surrounding). This is normal in holography and is called the twin image problem. To overcome this usually we take three images with different phases (as we have been doing with the SLM). We have purchased a linear translational stage with a piezo controller on which we are going to place one of the mirrors from the Michelson interferometer to be able to take the three images with the three phases ( its called phase-shifting holography in the article). We think this will solve the problem of the twin image.
Below is an image that compares the hologram from the SLM with the hologram from the Michelson interferometer. Both images were taken under similar light conditions ( 10ms exposure time, 10mW laser power). In the image from the SLM, you can see that there is a signal that rises above the background, but the concentric circles are not very clear ( The circles should get clearer with greater signal). The image from the Michelson interferometer the rings appear to be clearer thus showing that it is more light efficient than the SLM.We still have to get curtains around the table and also make an enclosure to make the system more light tight. That would reduce the noise in the system including the Michelson interferometer.