SIGNIFICANCE The large background, narrow dynamic range, and detector saturation have been the common limiting factors in stimulated emission (SE)-based pump-probe microscopy, attributed to the very small signal overriding the very intense laser probe beam. To better differentiate the signal of interest from the background, lock-in detection is used to measure the fluorescence quenching, which is termed spontaneous loss (SL). The advantages are manifold. The spontaneous fluorescence signal can be well separated from both the pump and the probe beams with filters, thus eliminating the background, enlarging the dynamic range, and avoiding the saturation of the detector. AIM We propose and demonstrate an integrated pump-probe microscopy technique based on lock-in detection for background removal and dynamic range enhancement through SL detection. APPROACH The experimental setup is configured with a pulsed diode laser at a wavelength λpu  =  635  nm, acting as a pump (excitation) and a mode-locked Ti:sapphire laser at a central wavelength λpr  =  780  nm, serving as the probe beam (stimulation). Both pulse trains are temporally synchronized through high precision delay control by adjusting the length of the triggering cables. The pump and probe beams are alternatively modulated at different frequencies f1 and f2 to extract the stimulated gain (SG) and SL signal. RESULTS SG signal shows saturation due to the irradiation of the intense probe beam onto the photodetector. However, the detector saturation does not occur at high probe beam power for SL detection. The fluorescence lifetime images are acquired with reduced background. The theoretical signal-to-noise ratios for SG and SL are also estimated by photon statistics. CONCLUSION We have confirmed that the detection of SL allows the elimination of the large background without photodetector saturation, which commonly exists in SG configuration. This modality would allow unprecedented manipulation and investigation of fluorophores in fluorescence imaging.

中文翻译：

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泵探针显微镜中的自发损失与刺激增益：概念演示的证明。

意义 大背景、窄动态范围和探测器饱和度一直是基于受激发射 (SE) 的泵浦探针显微镜的常见限制因素，这归因于覆盖非常强的激光探测光束的非常小的信号。为了更好地区分感兴趣的信号与背景，使用锁定检测来测量荧光猝灭，这称为自发损失 (SL)。优点是多方面的。自发荧光信号可以很好地从泵浦和带滤光器的探测光束中分离出来，从而消除背景，扩大动态范围，避免检测器饱和。目的 我们提出并展示了一种基于锁定检测的集成泵浦探针显微镜技术，用于通过 SL 检测去除背景和增强动态范围。方法实验装置配置有波长 λpu = 635 nm 的脉冲二极管激光器，用作泵（激发）和中心波长 λpr = 780 nm 的锁模钛：蓝宝石激光器，用作探测光束（刺激）。通过调整触发电缆的长度，通过高精度延迟控制，两个脉冲序列在时间上同步。泵浦光束和探测光束以不同的频率 f1 和 f2 交替调制，以提取受激增益 (SG) 和 SL 信号。结果 由于强探测光束照射到光电探测器上，SG 信号显示饱和。然而，对于 SL 检测，在高探测光束功率下不会发生检测器饱和。荧光寿命图像是在减少背景的情况下获得的。SG 和 SL 的理论信噪比也是通过光子统计来估计的。结论我们已经证实，SL 的检测可以消除大背景而没有光电探测器饱和，这通常存在于 SG 配置中。这种模式将允许在荧光成像中对荧光团进行前所未有的操作和研究。