The ability to detect biological events at single molecule level provides unique insights in the field of biophysics. Back-focal-plane laser interferometry is a promising technique for single-molecule-scale, 3D position measurements at rates far beyond the capability of video. I present an in-situ calibration method for the back-focal-plane, low-power (non-trapping) laser interferometry. The software-based technique does not rely on any a priori model or calibration knowledge; hence the name Agnostic. The technique is sufficiently fast and non-invasive that the calibration can be performed on the fly, without interrupting or compromising the on-going experiment. The technique can be applied to track 3D, long range motion (up to 100 um) of a broad variety of microscopic biological objects. The spatiotemporal resolution achieved is of the order of a few nanometers and tens of microseconds. Three biological applications enabled by the technique are presented: firstly, a prototype of an oscillating-bead high-bandwidth frequency-response analyzer for biology, based on Agnostic Tracking as implemented in our custom-built 3D Magnetic Force Microscope (3DFM); secondly, a magnetic-force study that revealed a previously-unknown anchoring-dependent nonlinear response of a cellular membrane; last, a rheological study that revealed a novel grouping of motion characteristics of individual vesicles diffusing inside live cytoplasm
