Chang Chia-Yuan, Lin Cheng-Han, Lin Chun-Yu, Sie Yong-Da, Hu Yvonne Yuling, Tsai Sheng-Feng, Chen Shean-Jen
Center for Micro/Nano Science and Technology, National Cheng Kung University, 701, Tainan, Taiwan.
Department of Engineering Science, National Cheng Kung University, 701, Tainan, Taiwan.
J Biophotonics. 2018 Jan;11(1). doi: 10.1002/jbio.201600287. Epub 2017 May 2.
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 spatially modulated illumination at 90° (center) and 0° (right) orientations.
一种改进的基于时间聚焦的多光子激发显微镜（TFMPEM）具有数字微镜器件（DMD），该器件不仅用作闪耀光栅进行光的空间色散，还同时用于图案化照明。在此，TFMPEM已得到扩展，以实现结构化频率和方向的空间调制照明，从而增加物镜后焦孔径处的光束覆盖范围。TFMPEM的轴向激发限制（AEC）可以从3.0μm压缩到1.5μm，提高了50%。通过将TFMPEM与HiLo技术结合使用，作为相同空间频率但不同方向的两种结构化照明，根据压缩后的AEC结构化照明重建的生物组织图像在对比度和散射抑制方面明显更优。图：常规TFMPEM（左）、以HiLo技术作为90°（中）和0°（右）方向的1.09μm空间调制照明的TFMPEM拍摄的伊红染色小鼠小脑皮质的双光子激发荧光（TPEF）图像。
