Ultra-long anti-diffracting beam volume imaging using a single-photon excitation microscope

Abstract

We studied a novel volumetric single-photon excitation microscope with an ultralong anti-diffracting (UAD) beam as illumination. Volumetric fluorescence image direct mapping showed that the axial imaging range of the UAD beam was approximately 14 times and 2 times that of conventional Gaussian and Airy beams, respectively, while maintaining a narrow lateral width. We compared the imaging capabilities of the Gaussian, Airy, and UAD modes through a strongly scattering environment mixed with fluorescent microspheres and agarose gel. Thick samples were scanned layer by layer in the Gaussian, Airy, and UAD modes, and then the three-dimensional structural information was projected onto a two-dimensional image. Benefiting from the longer focal length of the UAD beam, a deeper axial projection was provided, and the volume imaging speed was vastly increased. To demonstrate the performances of the UAD microscope, we performed dynamic volumetric imaging on the cardiovascular system of zebrafish labeled with green fluorescent proteins in the three modes and dynamically monitored substance transport in zebrafish blood vessels. In addition, the symmetrical curve trajectory of the UAD beam and the axial depth of the lateral position can be used for localization of micro-objects.