Observed Z-dependent lateral PSF shift (wobble).

<p>(a) A wobble-free system is denoted by the straight, light blue line (left) as compared to a system with wobble (right). The wobble effect shown in a) causes a lateral displacement of localizations as a function of axial displacement due to unwanted aberrations introduced by the microscope system. (b) Cartoon of true structures (left) and resultant warped structures (right) imaged on a system with wobble. (c) The lateral shift (or wobble), in the x and y directions as a function of the axial position (z). Dashed, green and violet lines represent data from 10 different beads over the field of view. The green and magenta solid lines represent the average value based on all 10 beads in the y and x-directions respectively. We observe a more significant shift in the y-direction, which is ~ 10 times greater than that in the x-direction. (d) Wobble curves after our correction method is applied; accuracy of correction according to error bars ≤ 6 nm. Error bars are standard deviation (S.D) values of the shift. Maximum S.D for curves in 1c: 6 nm. All data was collected on a modified Olympus IX71 inverted microscope equipped with an Olympus NA 1.40 oil immersion objective.