Laser scanning microscopy plays an essential role in the 3D volumetric imaging of biological tissue. However, conventional point-scanning techniques commonly have limited acquisition speed. Other well-developed high-speed imaging methods, e.g., using spinning disk, line scanning, light sheet illumination, etc., still require mechanical sampling scanning for imaging at different depths. To overcome this problem, the authors of this Biomedical Optics Express
publication developed a novel approach to 3D volumetric fluorescence imaging based on a multiplexed computer-generated hologram (CGH) method. A light needle excites the fluorophores, and the wavefront of the emission signals is modulated at the pupil of the objective lens. The wavefront modulation cancels the defocus component and adds wavefront tilts to the fluorescence emission within the defined depth range. As a result, the axial positions of emission signals linearly translate to lateral positions on the detector plane, thus enabling depth-resolved imaging. The authors demonstrated this technology in a two-photon microscope with Bessel beam illumination and a 1D array detector with 16 channels for imaging at different axial positions. They have shown real-time, video-rate 3D imaging on thick fixed biological specimens with about ten times faster imaging speed. This technique provides a new approach to enabling high-speed volumetric imaging without mechanical scanning.
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