Chang et al. solve this problem rather elegantly by scanning the beam in Z (depth), rather than moving the sample or the objective—they do this by employing an electrically tunable lens. The tunable lens shifts the focus of the laser that excites fluorescence in the sample and also shifts the focal plane of the confocal detection slit. Much like a galvanometer mirror scans the beam laterally in a laser scanning confocal microscope (eliminating the need to raster-scan the sample relative to the objective), the electro-optical lens means the sample can stay still in Z as well. This means that force and position measurements with optical tweezers can be carried out at the same time as 3D imaging, allowing the team to measure the motion of fluorescently labelled receptors along a connection between two cells—at the same time as taking force measurements with an optical trap.
The approach of using a tunable lens, or indeed various other methods of "remote focusing" such as extra objectives combined with fast translation stages, is gaining ground—often because it means that the advantages of a conventional 3D imaging system can be more easily combined with other techniques. The single tunable element used here is an elegant and relatively simple approach that should enable many exciting experiments in the future.
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