| 摘要: | 結構照明顯微鏡是使影像產生週期性的條紋，再藉由數學運算獲得更高頻率的空間資訊，提升光學系統的橫向解析度，達成超過繞射極限的限制，然而在過去的文獻中，結構照明顯微鏡較少被應用在雙光子螢光影像上。
雙光子螢光顯微鏡相較於單光子螢光顯微鏡，激發光對樣本有較好的穿透深度，對樣本產生較少的破壞，並且光學切片的能力較高，擁有較好的縱向解析度，然而根據繞射極限，雙光子螢光顯微鏡在橫向解析度遜色於單光子螢光顯微鏡。
想要將結構照明顯微鏡應用在雙光子螢光顯微鏡上，需要對雙光子螢光影像進行空間調制，形成條紋，若是基態耗損原 理可以讓螢光分子進入暗態而不發出雙光子螢光，就可以透過調制耗損光的方式，在雙光子螢光影像上形成條紋。
基態耗損原理尚未被用來抑制雙光子螢光訊號，本論文選擇了4種樣本進行實驗，樣本分別為R6G-PVA、Eosin Y-PVA、螢光小球F8800和F8801。首先，進行抑制單光子螢光實驗，並以過去的文獻做參考，證實樣本擁有暗態而可以抑制單光子螢光訊號。接著進行抑制雙光子螢光實驗，觀察雙光子螢光是否可以被耗損光(綠光雷射，波長為532 nm)抑制，並比較4種樣本 的抑制程度。為了應用在雙光子結構照明，以實驗量測了雙光子螢光訊號的訊雜比、積分時間對抑制雙光子螢光的影響、耗損光以及激發光(紅外光雷射，波長為1064 nm)之間的間隔對抑制雙光子螢光的影響。;Structured illumination microscopy produces periodic fringes in the image, and then obtains higher-frequency spatial information through mathematical operations, improves the lateral resolution of the optical system, and exceeds the limit of diffraction. However, in the past literature, structured illumination microscopy is rarely used for two-photon fluorescence imaging.
Compared with the single-photon fluorescence microscope, the excitation laser has a better penetration depth to the sample, causing less damage to the sample, and has a higher optical sectioning ability and better longitudinal resolution. However, according to the diffraction limit, two-photon fluorescence microscopy is inferior to single-photon fluorescence microscopy in lateral resolution.
To apply the structured illumination microscope to the two-photon fluorescence microscope, the two-photon fluorescence image needs to be spatially modulated to form stripes. If the ground-state depletion principle can make the fluorescent molecules enter the dark state without emitting two-photon fluorescence, then Stripes can be formed on two-photon fluorescent images by modulating the depletion laser.
The ground-state depletion principle has not been used to suppress the two-photon fluorescent signal. This paper selected four samples for experiments, namely R6G-PVA, Eosin Y-PVA, fluorescent beads F8800 and F8801. First, the experiment of suppressing single-photon fluorescence was carried out, and the past literature was used as a reference to confirm that the sample has a dark state and can suppress the single-photon fluorescence signal. Then, the experiment of suppressing single-photon fluorescence was carried out to observe whether the two-photon fluorescence could be suppressed by depletion laser (Green laser, wavelength of 532 nm), and compare the degree of suppression of the four samples. In order to apply to two-photon structure illumination, the signal-to-noise ratio of the two-photon fluorescent signal, influence of integration time on two-photon fluorescence, the effect of the interval time between the depletion laser and the excitation laser (Infrared laser, wavelength of 1064 nm) on the suppression of two-photon fluorescence. |
