Browsing by Author "Sie, Yong Da"

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Fast and improved bioimaging via temporal focusing multiphoton excitation microscopy with binary digital-micromirror-device holography
(2018-11-01)
Sie, Yong Da
;
Chang, Chia-Yuan
;
Lin, Chun-Yu
;
Chang, Nan-Shan
;
Campagnola, Paul J.
Conventional temporal focusing-based multiphoton excitation microscopy (TFMPEM) can offer wide-field optical sectioning with an axial excitation confinement of a few microns. To improve the axial confinement of TFMPEM, a binary computer-generated Fourier hologram (CGFH) via a digital-micromirror-device (DMD) was implemented to intrinsically improve the axial confinement by filling the back-focal aperture of the objective lens. Experimental results show that the excitation focal volume can be condensed and the axial confinement improved about 24% according to the DMD holography. In addition, pseudouniform MPE can be achieved using two complementary CGFHs with rapid pulse-width modulation switching via the DMD. Furthermore, bioimaging of CV-1 in origin with SV40 genes-7 cells demonstrates that the TFMPEM with binary DMD holography can improve image quality by enhancing axial excitation confinement and rejecting out-of-focus excitation. (C) 2018 Society of Photo-Optical Instrumentation Engineers (SPIE)
Volumetric bioimaging based on light field microscopy with temporal focusing illumination
(2018-01-01)
Hsu, Feng-Chun
;
Sie, Yong Da
;
Lai, Feng-Jie
;
Chen, Shean-Jen
;
光電學院
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.