Volumetric bioimaging based on light field microscopy with temporal focusing illumination

Abstract

Light field technique at a single shot can get the whole volume image of observed sample. Therefore, the original frame rate of the optical system can be taken as the volumetric image rate. For dynamically imaging whole micron-scale biosample, a light field microscope with temporal focusing illumination has been developed. In the light field microscope, the f-number of the microlens array (MLA) is adopted to match that of the objective; hence, the subimages via adjacent lenslets do not overlay each other. A three-dimensional (3D) deconvolution algorithm is utilized to deblur the out-of-focusing part. Conventional light field microscopy (LFM) illuminates whole volume sample even non interesting parts; nevertheless, whole volume excitation causes even more damage on bio-sample and also increase the background noise from the out of range. Therefore, temporal focusing is integrated into the light field microscope for selecting the illumination volume. Herein, a slit on the back focal plane of the objective is utilized to control the axial excitation confinement for selecting the illumination volume. As a result, the developed light field microscope with the temporal focusing multiphoton illumination (TFMPI) can reconstruct 3D images within the selected volume, and the lateral resolution approaches to the theoretical value. Furthermore, the 3D Brownian motion of two-micron fluorescent beads is observed as the criterion of dynamic sample. With superior signal-to-noise ratio and less damage to tissue, the microscope is potential to provide volumetric imaging for vivo sample.