在本研究中，透過文獻回顧得知PMT之不穩定行為在實驗量測下，無論是短期可逆的漂移效應(Drift effect)，抑或是長期不可逆的疲乏效應(Fatigue effect)，都會對訊號產生一定程度的影響，導致使用基態耗損顯微鏡(Ground state depletion microscopy, GSD)對樣本進行基態耗損效應的量測時，無法確認雙光子螢光訊號的抑制趨勢是否來自於基態電子的耗盡所產生的螢光關閉現象。為此，本研究將利用基態耗損顯微系統進行實驗，透過不同實驗條件下所量測之雙光子螢光訊號行進行分析，探討疲乏效應的產生原因及特徵表現，藉由實驗結果可以得知在訊號強度過高的狀況下，PMT放大訊號的過程中容易使得增益值產生變化，並且需要在多道光源於激發的過程中，加入適當的無光激發時長才能逐漸恢復至穩定狀態。本研究結果於未來使用此系統量測時，可提升整體系統的準確性與穩定性，避免因PMT導致的訊號失真情形。;In super-resolution fluorescence microscopy, the fluorescence signal is generally weaker compared to signals obtained using conventional optical microscopy. To enhance signal intensity and improve image quality, photomultiplier tubes (PMTs) are widely employed for signal amplification and detection. However, under prolonged exposure and high-intensity excitation light, PMTs are prone to fatigue effects, leading to reduced gain and sensitivity, which in turn results in signal distortion.
In this study, a literature review reveals that PMT instability—whether reversible short-term drift effects or irreversible long-term fatigue effects—can significantly affect signal integrity. This issue becomes particularly critical when using ground state depletion microscopy (GSD) to investigate the depletion of ground-state electrons in samples. It becomes challenging to determine whether the observed suppression of the two-photon fluorescence signal genuinely originates from fluorescence quenching due to ground-state depletion.
To address this issue, this study conducts experiments using a GSD microscopy system. By analyzing two-photon fluorescence signals acquired under various experimental conditions, we investigate the causes and characteristics of PMT fatigue effects. The results indicate that when the signal intensity is excessively high, the gain of the PMT tends to fluctuate during the signal amplification process. It is also observed that introducing an appropriate period of non-illumination between multi-source excitation cycles can help restore the system to a stable state.
The findings of this research contribute to improving the accuracy and stability of future measurements conducted with this system, by reducing signal distortion caused by PMT-induced effects.
