| 摘要: | 雙光子顯微鏡因為非線性的激發過程，會有良好的光學切片能力，其中二倍頻顯微鏡在觀察肌腱組織時，可以利用肌腱組織的非中心對稱結構，無須染色就可直接觀察樣本，再加上二倍頻訊號激發的過程中無實際能階躍遷，滿足動量守恆，不會對樣本產生光破壞，有益於長時間觀察樣本。
不同於螢光，二倍頻訊號因為是屬於無能階躍遷的機制，所以不適用於以螢光訊號為基礎的超解析度顯微鏡，但可與不受限於螢光訊號的結構照明顯微術進行結合。傳統的結構照明顯微術以廣域照明架構為基礎，但對於雙光子顯微系統而言，需要極高的激發光強度才能使激發樣本上的雙光子螢光，所以須選用點掃描式的系統來增加雙光子的激發效率。本論文將結合二倍頻顯微鏡以及點掃描式結構照明以提升二倍頻顯微術的解析度。
二倍頻訊號是同調訊號，成像理論與螢光的成像理論不同，本論文基於同調訊號的成像理論建立了掃描式二倍頻結構照明顯微術的成像原理，並證實了在重建後的影像中具有較高頻的資訊，透過模擬，在1047 nm、873 nm、748.6 nm和655 nm這四種不同週期的條紋下提升的解析度分別為1.28、1.35、1.44、1.52倍；在實驗上則利用雞翅的肌腱組織成功的在1047 nm、873 nm條紋週期下於X方向取得1.29和1.38倍的解析度提升，Y方向則取得1.3和1.4倍的解析度提升。;Two-photon microscopy has an outstanding optical sectioning capability due to its nonlinear excitation process. While observing tendon tissues with its second harmonic generation microscopy, because of the non-centrosymmetric structure, tendons can be observed directly. In addition, the excited process has no energy level transitions, meeting the conservation of momentum, therefore the sample will not be damaged by the light source, making it well suited for long time observations.
Unlike fluorescents, second harmonic signals are under the conditions of no energy level transitions, thus it does not apply to the fluorescent signal-based super-resolution microscopies, however, it could be combined with structured illumination microscopy. The original structured illumination microscopy was based on a wide field setup, yet two-photon microscopy systems require extremely high excitation intensities to produce the two-photon excitation on its samples, consequently, scanning systems were chosen to overcome such issue. This paper will combine the scanning second harmonic generation microscope and structured illumination microscopy to improve the resolution.
Second harmonic signals are coherent signals, its imaging theory is different to the incoherent signal’s. In this paper, it is proven that the reconstruction image has a higher frequency information based on the Second Harmonic Generation Scanning Structured Illumination Microscopy theory. In simulation, under four different periods: 1047 nm, 873 nm, 748.6 nm and 655 nm, the resolution is enhanced by 1.28, 1.35, 1.44, and 1.52 times respectively; in experiments, by using chicken wings tendon as samples, resolution were improved by 1.29 and 1.38 times; and in the Y direction, 1.3 and 1.4 times the resolution improvement were achieved. |
