How thick is your light sheet?" is a question that has been asked frequently after talks showing impressive renderings of 3D data acquired by a light-sheet microscope. This question is motivated by the fact that most of the time the thickness of the light-sheet is uniquely associated to the axial resolution of the microscope. However, the link between light-sheet thickness and axial resolution has never been systematically assessed and it is still unclear how both are connected. The question is not trivial because commonly employed measures cannot readily be applied or do not lead to easily interpretable results for the many different types of light sheet. Here, we introduce a set of intuitive measures that helps to define the relationship between light sheet thickness and axial resolution by using simulation data. Unexpectedly, our analysis revealed a trade-off between better axial resolution and thinner light-sheet thickness. Our results are surprising because thicker light-sheets that provide lower image contrast have previously not been associated with better axial resolution. We conclude that classical Gaussian illumination beams should be used when image contrast is most important, and more advanced types of illumination represent a way to optimize axial resolution at the expense of image contrast.

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High-Speed tracking of several particles allows measuring dynamic long-range interactions relevant to biotechnology and colloidal physics. In this paper we extend the successful technique of 3D back-focal plane interferometry to oscillating laser beams and show that two or more particles can be trapped and tracked with a precision of a few nanometers in all three dimensions. The tracking rate of several kHz is only limited by the scan speed of the beam steering device. Several tests proof the linearity and orthogonality of our detection scheme, which is of interest to optical tweezing applications and various metrologies. As an example we show the position cross-correlations of three diffusing particles in a scanning line optical trap.

Optical traps play an increasing role in the bionanosciences because of their ability to apply forces flexibly on tiny structures in fluid environments. Combined with particle-tracking techniques, they allow the sensing of miniscule forces exerted on these structures. Similar to atomic force microscopy (AFM), but much more sensitive, an optically trapped probe can be scanned across a structured surface to measure the height profile from the displacements of the probe. Here we demonstrate that, by the combination of a time-shared twin-optical trap and nanometre-precise three-dimensional interferometric particle tracking, both reliable height profiling and surface imaging are possible with a spatial resolution below the diffraction limit. The technique exploits the high-energy thermal position fluctuations of the trapped probe, and leads to a sampling of the surface 5,000 times softer than in AFM. The measured height and force profiles from test structures and Helicobacter cells illustrate the potential to uncover specific properties of hard and soft surfaces.