The use of parallax for accurate three-dimensional single particle tracking has been widely used to study the three-dimensional dynamic processes of cells with high spatial and temporal resolution. This imaging method routinely either uses a series of strategically positioned mirrors or a wedge prism to create a duplicate-split image of one object on a portion of the same imaging sensor (e.g., EMCCD and sCMOS cameras). The difference in the relative lateral positions of these split images is then used to calculate the depth (z-position) of the object. Furthermore, by introducing an active feedback control, this method allows for high-resolution in-focus three-dimensional tracking, however, its accuracy could be improved. To restore y-position differences and keep the object in focus, the sample stage must be moved (commonly 5-50 nm) per snapshot. This stage migration risks movement of the subject and exposes the technique to inaccuracy of z-position, rotation, and other three-dimensional data. Here, we employ an electronic focus tunable lens (ETL) for automatic in-focus three-dimensional single particle tracking using parallax PSF engineering. This methodology would eliminate the need to move the sample stage. The tunable lens works to modify the optical power of the objective and therefore modify the point-spread function (PSF) by means of variable working distance of effective tube lens with equivalent resolution. This variable working distance replaces the need to move a sample stage in the z-direction while maintaining resolution and depth of field. Our methodology allows for improved accuracy of rotational information and more precise three-dimensional localization of particles in biological systems.

Electronically tunable lens in three-dimensional single particle tracking by means of parallax

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