Conventional three-dimensional (3D) images of biological samples are typically assembled from a stack of twodimensional images acquired sequentially at different focal planes. This time-consuming manner hinders the application of 3D imaging techniques to the investigation of fast biochemical dynamics and light-sensitive biological events. The concept of multifocus imaging, which enables simultaneous acquisition of images from multiple focal planes, was introduced to achieve rapid 3D imaging. In the present study, we achieved multifocus imaging through polarization wavefront shaping via a micro-retarder array which splits the incident linearly polarized light into three beamlets that are focused to three axially-offset focal planes with ~100 μm separation. Append to an existing beam-scanning microscope, this multifocus system enables rapid 3D imaging compatible with a variety of optical microscopic approaches including laser transmittance, two-photon excited fluorescence, and second harmonic generation microscopy.

Date Published: 17 February 2020
PDF: 8 pages
Proc. SPIE 11245, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVII, 112450T (17 February 2020); doi: 10.1117/12.2546543

Published in SPIE Proceedings Vol. 11245:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVII
Thomas G. Brown; Tony Wilson; Laura Waller, Editor(s)