線掃描時域聚焦多光子顯微術(line-scanning temporal focusing multiphoton microscopy，LS-TFMPM)可在維持軸向激發侷限能力的同時提升成像效率，適合高速與大範圍螢光成像。然而，超快雷射經多個光學元件後會因光學色散造成脈衝展寬，使焦平面瞬時峰值功率下降，進而降低多光子激發效率、影像亮度與軸向解析度；此外，線形激發光束強度近似高斯分佈，易造成影像亮度不均。因此，本研究結合光學色散補償、多線激發與光束整形技術，以改善系統成像品質。
首先建立干涉式自相關(fringe-resolved autocorrelation，FRAC)系統，評估補償前後之脈衝寬度變化。結果顯示，透過液態透鏡進行群延遲色散(group delay dispersion， GDD)補償後，脈衝寬度可由約 1151.71 fs 縮短至約100.31 fs。 此外，用用間光光變變與與雷射強度分佈回饋最佳化演算法(laser intensity profile feedback-based optimization，LIPFO)，將高斯光束轉換為較均勻之平頂光場，使平頂光補償RMSE由0.0459降低至0.0075，並可用於外加強度干擾補償與正弦強度調變。
接著，本研究導入線性可變變聚焦鏡(linear deformable mirror，LDM)，可以多光子螢光強度作為回饋，搭配改良式爬山演算法補償殘餘高階色散。同時，透過光學繞射元件(diffractive optical element，DOE)產生六條線形光束，以降低振鏡掃描角度可提升成像效率。藉由整合多線激發與光學色散補償，本文提出多線激發線性可變變聚焦鏡光學色散主動補償方法(multi-line excitation-based active compensation for optical dispersion with a linear deformable mirror， MACOL)。 在加入相位干擾後，系統軸向解析度由2.8 μm惡化至4.2 μm，經MACOL補償後改善至2.6 μm。可於紙抹布中纖維分佈影像與小鼠皮膚組織成像中，補償後影像亮度與對比度皆明顯提升，顯示本文方法具備高速與大範圍生物組織成像之應用潛力。

Line-scanning temporal focusing multiphoton microscopy (LS-TFMPM) improves imaging efficiency while maintaining axial excitation confinement, making it suitable for high-speed and large-area fluorescence imaging. However, optical dispersion from multiple optical components broadens ultrafast laser pulses, reducing the instantaneous peak power at the focal plane and degrading multiphoton excitation efficiency, image brightness, and axial resolution. In addition, the Gaussian-like intensity distribution of the line-shaped excitation beam causes nonuniform image brightness. Therefore, this study integrates optical dispersion compensation, multi-line excitation, and beam shaping to improve system imaging quality.
A fringe-resolved autocorrelation (FRAC) system was first established to evaluate pulse width variation before and after dispersion compensation. With group delay dispersion (GDD) compensation using a liquid lens, the pulse width was reduced from approximately 1151.71 fs to 100.31 fs. In addition, a spatial light modulator (SLM) combined with a laser intensity profile feedback-based optimization (LIPFO) algorithm was used to transform the Gaussian beam into a more uniform flat-top beam, reducing the RMSE from 0.0459 to 0.0075. The method was also applied to external disturbance compensation and sinusoidal intensity modulation.
Furthermore, a linear deformable mirror was introduced with multiphoton fluorescence intensity as feedback, and a modified hill-climbing algorithm was used to compensate for residual higher-order dispersion. A diffractive optical element (DOE) was also used to split a single line-shaped excitation beam into six beams, reducing the required galvo scanning angle and improving imaging efficiency. By integrating multi-line excitation with dispersion compensation, this study proposes a multi-line-excitation-based active compensation for optical dispersion with a linear deformable mirror (MACOL) method. After phase disturbance, the axial resolution degraded from 2.8 μm to 4.2 μm, but improved to 2.6 μm after MACOL compensation. fluorescence-stained paper towel specimen and mouse skin tissue imaging further confirmed that MACOL effectively enhances image brightness and contrast, demonstrating its potential for high-speed, large-area biological tissue imaging.

1. A. Laskin, P. Kaiser, V. Laskin, et al., “Laser beam shaping for biomedical microscopy techniques,” Proc. SPIE 9887, 98872E (2016).
2. C. Maibohm, O. F. Silvestre, Borme, et al., “Multi-beam two-photon polymerization for fast large area 3D periodic structure fabrication for bioapplications,” Sci. Rep. 10(1), 8740 (2020).
3. D. Flamm, D. G. Grossmann, M. Sailer, et al., “Structured light for ultrafast laser micro- and nanoprocessing,” Opt. Eng. 60(2), 025105 (2021).
4. Y. Kozawa, T. Nakamura, Y. Uesugi, et al., “Wavefront engineered light needle microscopy for axially resolved rapid volumetric imaging,” Biomed. Opt. Express 13(3), 1702–1717 (2022).
5. K. Wen, X. Fang, Y. Ma, et al., “Large-field structured illumination microscopy based on 2D grating and a spatial light modulator,” Opt. Lett. 47(11), 2666–2669 (2022).
6. K. C. Phillips, H. H. Gandhi, E. Mazur, et al., “Ultrafast laser processing of materials: a review,” Adv. Opt. Photonics 7(4), 684–712 (2015).
7. D. Tan, B. Zhang, and J. Qiu, “Ultrafast laser direct writing in glass: Thermal accumulation engineering and applications,” Laser Photonics Rev. 15(9), 2000455 (2021).
8. M. Göppert‐Mayer, “Elementary processes with two quantum transitions,” Ann. Phys. 18(7–8), 466–479 (2009).
9. P. T. C. So, C. Y. Dong, B. R. Masters, et al., “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 2(1), 399–429 (2000).
10. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
11. P. J T. Wang and C. Xu, “Three-Photon neuronal imaging in deep mouse brain,” Optica 7(8), 947–960 (2020).
12. L. Perrin, B. Bayarmagnai, and B. Gligorijevic, “Frontiers in intravital multiphoton microscopy of cancer,” Cancer Rep. 3(1), e1192 (2020).
13. S. V. Plotnikov, A. M. Kenny, S. J. Walsh, et al., “Measurement of muscle disease by quantitative second-harmonic generation imaging,” J. Biomed. Opt. 13(4), 044018 (2008).
14. J. A. Armstrong, “Measurement of picosecond laser pulse widths,” Appl. Phys. Lett. 10, 16–18 (1967).
15. H. P. Weber, “Method for Pulsewidth Measurement of Ultrashort Light Pulses Generated by Phase-Locked Lasers using Nonlinear Optics,” J. Appl. Phys. 38(5), 2231–2234 (1967).
16. J-C. M. Diels, J. J. Fontaine, I. C. McMichael, et al., “Control and measurement of ultrashort pulse shapes (in amplitude and phase) with femtosecond accuracy,” Appl. Opt. 24(9), 1270–1282 (1985).
17. O. Bryngdahl, “Geometrical transformations in optics*,” J. Opt. Soc. Am. 64, 1092–1099 (1974).
18. O. Bryngdahl, “Optical map transformations,” Opt. Commun. 10, 164–168 (1974).
19. H. Aagedal, M. Schmid, S. Egner, et al., “Analytical beam shaping with application to laser-diode arrays,” J. Opt. Soc. Am. A 14, 1549–1553 (1997).
20. D. Nodop, J. Ruecker, S. Waechter, et al., “Hyperbolic phase function used in a spatial light modulator for flat top focus generation,” Opt. Lett. 44, 2169–2172 (2019).
21. W. Denk, J. H. Strickler, and W. W. Webb, “Two-Photon Laser Scanning Fluorescence Microscopy,” Science 248(4951), 73–76 (1990).
22. G. H. Zhu, J. van Howe, M. Durst, et al., “Simultaneous spatial and temporal focusing of femtosecond pulses,” Opt. Express 13(6), 2153–2159 (2005).
23. B. Sun, P. S. Salter, and M. J. Booth, “Effects of sample dispersion on ultrafast laser focusing,” J. Opt. Soc. Am. B 32(7), 1272–1280 (2015).
24. R. L. Fork, O. E. Martinez, and J. P. Gordon, “Negative dispersion using pairs of prisms,” Opt. Lett. 9(5), 150–152 (1984).
25. S. Akturk, X. Gu, M. Kimmel, et al., “Extremely simple single-prism ultrashort-pulse compressor,” Opt. Express 14(21), 10101–10108 (2006).
26. A. Weiner, “Femtosecond pulse shaping using spatial light modulators,” Rev. Sci. Instrum. 71(5), 1929–1960 (2000).
27. K. Seno, K. Suzuki, N. Ooba, et al., “Demonstration of channelized tunable optical dispersion compensator based on arrayed-waveguide grating and liquid crystal on silicon,” Opt. Express 18(18), 18565–18579 (2010).
28. A. M. Weiner, D. E. Leaird, J. S. Patel, et al., “Programmable femtosecond pulse shaping by use of a multielement liquid-crystal phase modulator,” Opt. Lett. 15(6), 326–328 (1990).
29. Y. Xue, K. P. Berry, J. R. Boivin, et al., “Scattering reduction by structured light illumination in line-scanning temporal focusing microscopy,” Biomed. Opt. Express 9(11), 5654–5666 (2018).
30. P. Rupprecht, R. Prevedel, F. Groessl, et al., “Optimizing and extending light-sculpting microscopy for fast functional imaging in neuroscience,” Biomed. Opt. Express 6(2), 353–368 (2015).
31. X. Liang, W. Hu, and Ling Fu, “Pulse compression in two-photon excitation fluorescence microscopy,” Opt. Express 18(14), 14893–14904 (2010).
32. Edmund, Introduction of mode-locking, From:https://www.edmundoptics.com.tw/knowledge-center/application-notes/lasers/ultrafast-dispersion/
33. Thorlabs, Introduction of AMP100, From:https://www.thorlabs.com/drawings/1dbbb6a273214bea-2C890B5A-9424-FD24-FA067F1C3CCE7100/AMP100-SpecSheet.pdf
34. M. H. Eybposh, N. W. Caira, M. Atisa, P. Chakravarthula, et al., “DeepCGH: 3D computer-generated holography using deep learning,” Opt. Express 28(18), 26636–26650 (2020).
35. E. Papagiakoumou, E. Ronzitti, and V. Emiliani, “Scanless two-photon excitation with temporal focusing,” Nat. Methods 17, 571–581 (2020).
36. J. Harriman, S. Serati, and J. Stockley, “Comparison of transmissive and reflective spatial light modulators for optical manipulation applications,” Proc. SPIE 5930, 59302D (2005).
37. F. G. Micallef, P. K. Shrestha, D. Chu, et al., “Transparent conductors for Mid-infrared liquid crystal spatial light modulators,” Thin Solid Films 660, 411–420(2018).
38. HOLOEYE, PLUTO-2.1-NIR-134 Manual, from:https://holoeye.com/products/spatial-light-modulators/pluto-2-1-lcos-phase-only-refl/
39. M. Agour, E. Kolenovic, C. Falldorf, et al., “Suppression of higher diffraction orders and intensity improvement of optically reconstructed holograms from a spatial light modulator,” J. Opt. A: Pure Appl. Opt. 11(10), 105405 (2009).
40. Application of Powell lens working principle, From:Application of Powell lens working principle - Knowledge - Elite Optoelectronics Co.,Ltd
41. Coherent, Bringing Lasers in Line, From:Powell-Linsen-Laserliniengeneratoren | Coherent
42. Z. Li, J. Hou, J. Suo, et al., “Contrast and resolution enhanced optical sectioning in scattering tissue using line-scanning two-photon structured illumination microscopy,” Opt. Express 25(25), 32010-32020 (2017).
43. A. Sandmeyer, M. Lachetta, H. Sandmeyer, et al., “DMD-based super-resolution structured illumination microscopy visualizes live cell dynamics at high speed and low cost,” bioRxiv 797670 (2019).
44. Optotune, Introduction of EL-12-30-TC-VIS-16D, From:https://www.optotune.com/el-12-30-tc-lens
45. Edmund, Introduction to Liquid Lenses, From:https://www.edmundoptics.com.tw/knowledge-center/application-notes/imaging/introduction-to-liquid-lenses/
46. Optotune, Introduction of ML-20-37-VIS-36D-C, From:https://www.optotune.com/product/ml-20-37/
47. T. Bifano, “MEMS deformable mirrors,” Nat. Photonics 5(1), 21–23 (2011).
48. Active Optical Systems, Manual of MDM1-32S-4, From:https://www.aos-llc.com/wp-content/uploads/Deformable-Mirror-Manual.pdf
49. Flexible Optical B.V. OKO TECH, specification of linear deformable mirror, From:https://www.okotech.com/images/pdfs/MMDM39lin_pasport.pdf
50. KMLABS, Introduction of GriffinTM - Ultrafast Ti:sapphire oscillator series, From:https://www.kmlabs.com/product/griffin/
51. R. D. Boyd, J. A. Britten, D. E. Decker, et al., “High-efficiency metallic diffraction gratings for laser applications,” Appl. Opt. 34, 1697–1706 (1995).
52. HOLOEYE, specification of DE 812, From:https://holoeye.com/product/de-812/
53. M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing in nonlinear microscopy,” Opt. Commun. 281(7), 1796–1805 (2008).
54. LIGHT CONVERSION, FLINT-FL2, From:https://lightcon.com/products/flint-high-repetition-rate-lasers/
55. HOLOEYE, PLUTO-2.1-NIR-149 Manual, from:https://holoeye.com/product/pluto-2-1-nir-149/