How to define and optimize axial resolution in light-sheet microscopy: a simulation-based approach

“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, by using simulation data we introduce a set of intuitive measures that helps to define the relationship between light sheet thickness and axial resolution. 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.