Temporal focusing‐based widefield multiphoton microscopy with spatially modulated illumination for biotissue imaging

• Published in: Journal of biophotonics Vol. 11; no. 1
• Main Authors: Chang, Chia‐Yuan, Lin, Cheng‐Han, Lin, Chun‐Yu, Sie, Yong‐Da, Hu, Yvonne Yuling, Tsai, Sheng‐Feng, Chen, Shean‐Jen
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 01.01.2018
• Subjects: Calibration; Cerebellum; Cortex (temporal); Digital imaging; Equipment Design; Excitation; Frequency dependence; Illumination; Image contrast; Image reconstruction; Light; Microscopy; Microscopy, Fluorescence, Multiphoton - instrumentation; Microscopy, Fluorescence, Multiphoton - methods; multiphoton processes; Neuroimaging; nonlinear microscopy; three-dimensional microscopy; multiphoton processes; nonlinear microscopy; three-dimensional microscopy
• ISSN: 1864-063X; 1864-0648
• DOI: 10.1002/jbio.201600287

A developed temporal focusing‐based multiphoton excitation microscope (TFMPEM) has a digital micromirror device (DMD) which is adopted not only as a blazed grating for light spatial dispersion but also for patterned illumination simultaneously. Herein, the TFMPEM has been extended to implement spatially modulated illumination at structured frequency and orientation to increase the beam coverage at the back‐focal aperture of the objective lens. The axial excitation confinement (AEC) of TFMPEM can be condensed from 3.0 μm to 1.5 μm for a 50 % improvement. By using the TFMPEM with HiLo technique as two structured illuminations at the same spatial frequency but different orientation, reconstructed biotissue images according to the condensed AEC structured illumination are shown obviously superior in contrast and better scattering suppression. Picture: TPEF images of the eosin‐stained mouse cerebellar cortex by conventional TFMPEM (left), and the TFMPEM with HiLo technique as 1.09 μm−1 spatially modulated illumination at 90° (center) and 0° (right) orientations. TPEF images of the eosin stained mouse cerebellar cortex by conventional TFMPEm (left), and the TFMPEM with HiLo technique as 1.09 μm−1 spatially modulated illumination at 90° (center) and 0° (right) orientations.