The scope is back, up and running

May 22, 2018

by Abhijit Marar

SMARACT STAGE/ MICROSCOPE UPDATE:

The SmarAct stage was returned to the Kner lab with the final conclusion that the stage is functioning consistently with its specifications. The clicking noise we were concerned about previously has been attributed to the stick-slip behavior of the piezo-electric material (https://www.youtube.com/watch?v=zVNYxsudul0).The reason as to why such a behavior should cause a shift in the position of a bead is still unknown. The aluminum plate on which the stage was resting has been upgraded. The current version of the plate is smaller and is made from steel due to its increased stiffness.

We have decided to go ahead and start taking data under these conditions. The microscope has been set up again and we have collected some initial data with 100 nm fluorescent microspheres (Invitrogen microspheres 580/605). To benchmark the system, 2D and 3D PSF (Point Spread Function) measurements have been taken to assess the abberations in the system. A 10 minute time-lapse dataset has also been acquired to measure the stability of the system. 60 images were collected over 10 minutes with an image taken every 10 seconds.

2D/ 3D PSF:

The PSF's show that there is spherical aberration in the system. The cause of it can be attributed to a refractive index mismatch between the lens immersion medium and the mouting medium. It could also be due to the lenses being placed at the wrong distances but this has been checked for without a significant improvement in the PSF quality. The FWHM (Full- Width Half Maximum) (https://en.wikipedia.org/wiki/Full_width_at_half_maximum) of the PSF was measured to be ~305.06 nm using a 60x1.4 NA oil objective.

      

STABILITY TEST:

The fluorescent bead drifted ~ 140 nm over the course of 10 minutes. We are currently attributing this to air currents in the room. We hope this issue will be solved by enclosing the microscope stage, thus protecting it from air-currents and temperature deviations in the room. Shown below are the localizations of the 60 frames where the drift can be seen and a plot charectersing the drift.

 

ALPHA TEST OF INCOHERENT HOLOGRAPHY:

An initial test of incoherent holography was successfully performed on one micrometer yellow-green fluorescent microspheres (Invitrogen microspheres 505/515). The test was performed on the existing wide-field microscope in the Kner lab. A Blackfly sCMOS camera was used to capture the data and a phase only SLM from meadowlark optics was used to write the patterns on. To benchmark the system we would have to successfully perform this on the 100 nm beads. This was not possible this time due to the limitations of the camera and also because the fluorescent beads were imaged with blue LED instead of a laser which meant reduced intensities leading to low number photons thus leading to less information about the object of interest (100 nm fluorescent microsphere in this case). We believe that the camera being used for the microcscope being built and the use of a laser will help us overcome this problem.