泵浦探测显微镜中的自发损耗与激发增益：概念验证演示。

Spontaneous loss versus stimulation gain in pump-probe microscopy: a proof of concept demonstration.

机构信息

National Yang-Ming University, Institute of Biophotonics, Taipei, Taiwan.

China Medical University, Institute of New Drug Development, Taichung, Taiwan.

出版信息

J Biomed Opt. 2020 Mar;25(3):1-11. doi: 10.1117/1.JBO.25.3.036501.

DOI:10.1117/1.JBO.25.3.036501

PMID:32170858

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7068216/

Abstract

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.

摘要

意义

在基于受激辐射（SE）的泵浦-探测显微镜中，大的背景、窄的动态范围和探测器饱和一直是常见的限制因素，这归因于非常小的信号会覆盖非常强的激光探测光束。为了更好地区分感兴趣的信号与背景，采用锁相检测来测量荧光猝灭，这被称为自发损耗（SL）。其优势是多方面的。自发荧光信号可以通过滤波器与泵浦和探测光束很好地分离，从而消除背景、扩大动态范围并避免探测器饱和。

目的

我们提出并演示了一种基于锁相检测的集成泵浦-探测显微镜技术，通过 SL 检测实现背景去除和动态范围增强。

方法

实验装置配置了一个波长为 λpu = 635 nm 的脉冲二极管激光器，用作泵浦（激发），以及一个中心波长为 λpr = 780 nm 的锁模钛宝石激光器，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对两个脉冲序列进行时间同步。泵浦和探测光束以不同频率 f1 和 f2 交替调制，以提取受激增益（SG）和 SL 信号。