The MechanoBiology Institute prides itself having some of the fastest and most accurate optical tracking machinery for force sensing by deflection measurements of transparent polydimethyl­siloxane pillars. Optical tracking allows for long term in-vivo observation of dense (up to about one vector per square mm) force maps with low pN accuracy. This translates into hundreds of frames per second recording at better than 4nm localization. The convection of the medium is used to provide local cooling for the region where the pump light penetrates the biological material and the immersion medium itself must feature a low absorption of the tracking wavelengths in order to serve as a coolant.

The refractive index difference of the pillars from this medium provides both the necessary contrast mechanism as well as a noticeable distortion of the recorded optical image of those pillars. Scattering in the specimen itself, small devices brought into the specimen, or the small chamber above the specimen further harm the imaging of the pillars. We believe that – as contrast and artefact are generate by the same mechanism – that these distortions can be minimized but not entirely avoided.

We present some design steps to limit the optical distortion of the images and some image processing insights that allow for the discrimination of the pillar projection and scattering artifacts along the beam path.

As a result the useful range of these observations and the scope of where these observations are accurate are greatly expanded and allow for some simplifications of crucial cellular force sensing experiments.