Quad stereo-microscopy

Stereo-microscopy is a technique that enables a sample to be imaged from two directions simultaneously, allowing the tracking of microscopic objects in three dimensions. This is achieved by illuminating the sample from different directions, each illumination direction producing an individual image. These images are superimposed in the image plane but can be easily separated using a diffractive optical element in the Fourier plane of the imaging arm. Therefore this enables 3-dimensional coordinates to be reconstructed using simple 2-dimensional image tracking and parallax. This is a powerful technique when combined with holographic optical tweezers (HOT), where multiple objects can be trapped and tracked simultaneously in three dimensions. In this work, we extend this concept to four different illumination directions: quad stereo-microscopy. This allows us to measure the accuracy of tracking in three dimensions, and to optimise the system.