Extended field-of-view light-sheet microscopy

Abstract

Light-sheet fluorescence microscopy enables rapid 3D imaging of biological samples. Unlike confocal and two-photon microscopes, a light-sheet microscope illuminates the focal plane with an objective orthogonal to the detection axis and images it in a single snapshot. Its combination of high contrast and minimal sample exposure make it ideal to image thick samples with sub-cellular resolution. To uniformly illuminate a wide field-of-view without compromising axial resolution, propagation-invariant light-fields such as Bessel and Airy beams have been put forward. These beams do however irradiate the sample with a relatively broad transversal structure. The fluorescence excited by the side lobes of Bessel beams can be blocked physically during recording; though at the cost of increased sample exposure. In contrast, the Airy beam has a fine transversal structure that is both curved and asymmetric. Its fine structure captures all the high-frequency components that enable high axial resolution without the need to discard useful fluorescence. This advantage does not carry over naturally to two-photon excitation where the fine transversal structure is suppressed. We demonstrate a symmetric and planar Airy light-sheet that can be used with two-photon excitation and that does not rely on deconvolution.