Yujie Sun, Yale E. Goldman
A new concept for z-tracking of particles or single fluorphores is to split the light from the microscope objective to form two images based on rays from two halves of the objective’s field of view. This is similar to looking at the sample from the side.
The parallax method to obtain z-FIONA is shown left. Green and red groups of rays view the object from two opposite sides. The splitting mirror edge is located conjugate to the back focal plane of the objective. When the sample moves z-ward, its two images move closer together.
Calibration of Parallax View (right). A 200 nm bead was imaged using the optics at the left. The nano-stage was moved in 20 nm x-increments at the red bars and 20 nm z-wards in between the red bars. Tracking the positions with Gaussian fitting and averaging their horizontal positions gives x. Subtracting their vertical positions gives z.