National Cheng Kung University, Department of Mechanical Engineering, Tainan, Taiwan.
National Chiao Tung University, College of Photonics, Tainan, Taiwan.
J Biomed Opt. 2021 Jan;26(1). doi: 10.1117/1.JBO.26.1.016501.
Line scanning-based temporal focusing multiphoton microscopy (TFMPM) has superior axial excitation confinement (AEC) compared to conventional widefield TFMPM, but the frame rate is limited due to the limitation of the single line-to-line scanning mechanism. The development of the multiline scanning-based TFMPM requires only eight multiline patterns for full-field uniform multiphoton excitation and it still maintains superior AEC.
The optimized parallel multiline scanning TFMPM is developed, and the performance is verified with theoretical simulation. The system provides a sharp AEC equivalent to the line scanning-based TFMPM, but fewer scans are required.
A digital micromirror device is integrated in the TFMPM system and generates the multiline pattern for excitation. Based on the result of single-line pattern with sharp AEC, we can further model the multiline pattern to find the best structure that has the highest duty cycle together with the best AEC performance.
The AEC is experimentally improved to 1.7 μm from the 3.5 μm of conventional TFMPM. The adopted multiline pattern is akin to a pulse-width-modulation pattern with a spatial period of four times the diffraction-limited line width. In other words, ideally only four π / 2 spatial phase-shift scans are required to form a full two-dimensional image with superior AEC instead of image-size-dependent line-to-line scanning.
We have demonstrated the developed parallel multiline scanning-based TFMPM has the multiline pattern for sharp AEC and the least scans required for full-field uniform excitation. In the experimental results, the temporal focusing-based multiphoton images of disordered biotissue of mouse skin with improved axial resolution due to the near-theoretical limit AEC are shown to clearly reduce background scattering.
基于线扫描的时频聚焦多光子显微镜（TFMPM）与传统宽场 TFMPM 相比具有优越的轴向激发限制（AEC），但由于单线到线扫描机制的限制，帧率受到限制。基于多线扫描的 TFMPM 的发展仅需要 8 个多线图案即可实现全场均匀多光子激发，并且仍然保持优越的 AEC。
开发优化的并行多线扫描 TFMPM，并通过理论模拟验证其性能。该系统提供了相当于基于线扫描的 TFMPM 的锐利 AEC，但所需的扫描次数更少。
在 TFMPM 系统中集成了数字微镜器件，用于生成多线图案进行激发。基于具有锐利 AEC 的单线图案的结果，我们可以进一步对多线图案进行建模，以找到具有最高占空比和最佳 AEC 性能的最佳结构。
AEC 从传统 TFMPM 的 3.5μm 实验改善到 1.7μm。所采用的多线图案类似于具有空间周期为四倍衍射受限线宽的脉冲宽度调制图案。换句话说，理想情况下，只需进行四次π/2 空间相移扫描即可形成具有优越 AEC 的全二维图像，而无需依赖于图像尺寸的线到线扫描。
我们已经证明了所开发的基于并行多线扫描的 TFMPM 具有锐利 AEC 的多线图案和实现全场均匀激发所需的最少扫描次数。在实验结果中，显示了由于接近理论极限 AEC，改善了轴向分辨率的无序生物组织的基于时间聚焦的多光子图像，明显减少了背景散射。
