雙光子螢光顯微術擁有良好縱向解析度(longitudinal resolution)是觀察切片樣本的有力工具，為追求影像的橫向解析度(lateral resolution)的提升，過去的研究曾結合掃描式結構照明顯微術的原理成功使橫向解析度提升為原來的1.39倍，但他們發現照明條紋的對比度受樣本散射的離焦雜訊影響變差，使得解析度難以進一步提升，對此本研究提出改善方法，減少離焦雜訊對影像的影響以提高條紋對比度。 本研究改良過去研究使用的系統，將偵測器改為光電倍增管(photomultiplier tube, PMT)，藉由同時把樣本訊號及離焦雜訊記錄為PMT影像的單一像素，可以減少離焦雜訊對PMT影像的影響，並將接收訊號由螢光改為樣本的三倍頻(third-harmonic generation)以觀察樣本界面(interface)的結構，為了對應這些變動，必須耗乏(deplete)樣本的三階非線性係數才能得到帶有弦波條紋的PMT影像，並用PMT影像重建橫向解析度有提升的影像。 模擬上述改良方法取得的重建影像，並經過參數掃描分析後得知，若要提升橫向解析度1.39倍，則選擇樣本的三倍頻要能被衰減原強度的0.278，且PMT影像之SNR值要在3以上；若樣本的三倍頻能被衰減原強度的0.586且PMT影像SNR在4以上，則解析度可提升2倍，此為結構照明顯微術的理論極限。;Two-photon fluorescence microscopy is a powerful system to observe details of sections with good longitudinal resolution. In order to get better lateral resolution, some researches combined scanning structured illumination microscopy and two-photon fluorescence microscopy to improved lateral resolution. One of them improved lateral resolution by a factor of 1.39 times, but it found that the enhancement of lateral resolution is restricted by bad contrast of illumination pattern due to defocus noise from scattering of sample. We reformed system of the research to improve contrast of illumination pattern by changing detector, and the system apply third-harmonic generation of sample as signal to observe structure of interface. The new system applies photomultiplier tube (PMT) as detector to record signal and defocus noise in one pixel of PMT image so as to reduce the effect from defocus noise. According to this architecture, it has to get PMT images with sinusoidal pattern via depleting third-order nonlinear coefficient of sample. Finally reconstruct image with improved lateral resolution by PMT images. In this study, we simulated images reconstructed by our system and analyzed the improvement of lateral resolution of reconstructed images. With parameter scanning, 1.39 times improvement of lateral resolution can be achieved if third-harmonic generation of sample could be attenuated by 0.278 of origin with SNR of PMT image better than 3; theoretical limitation of improvement of lateral resolution can be achieved if third-harmonic generation of sample could be attenuated by 0.586 of origin with SNR of PMT image better than 4.
