Volumetric bioimaging based on light field microscopy with temporal focusing illumination

Feng Chun Hsu, Yong Da Sie, Feng Jie Lai, Shean-Jen Chen

Research output: Chapter in Book/Report/Conference proceedingConference contributionpeer-review

1 Scopus citations


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 noninteresting 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.

Original languageEnglish
Title of host publicationThree-Dimensional and Multidimensional Microscopy
Subtitle of host publicationImage Acquisition and Processing XXV
EditorsTony Wilson, Carol J. Cogswell, Thomas G. Brown
ISBN (Electronic)9781510614833
StatePublished - 1 Jan 2018
EventThree-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXV 2018 - San Francisco, United States
Duration: 29 Jan 201831 Jan 2018

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
ISSN (Print)1605-7422


ConferenceThree-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXV 2018
CountryUnited States
CitySan Francisco


  • light field
  • temporal focusing

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