Two simulation programs of a stimulated Raman scattering microscopy (SRS) imaging system with lock-in amplifier (LIA) detection were developed. SRS is an imaging technique based on the vibrational Raman cross-section as the contrast mechanism and enables fast, label-free imaging. Most SRS implementations are based on LIA detection of a modulated signal. However, building and operating such SRS set-ups still poses a challenge when selecting the LIA parameter settings for optimized acquisition speed or image quality. Moreover, the type of sample, e.g. a sparse sample vs. a densely packed sample, the required resolution as well as the Raman cross-section and the laser powers affect the parameter choice. A simulation program was used to find these optimal parameters. The focal spot diameters of the individual lasers (pump and Stokes) were used to estimate the effective SRS signal focal spot and the (optical) spatial resolution. By calibrating the signal and noise propagation through an SRS system for a known molecule, we estimated the signal and noise input to the LIA. We used a low pass filter model to simulate the LIA behavior in order to find the optimal parameters (i.e. filter order and time constant). Optimization was done for either image quality (expressed as contrast to noise ratio) or acquisition time. The targeted object size was first determined as a measure for the required resolution. The simulation output consisted of the LIA parameters, pixel dwell time and contrast to noise ratio. In a second simulation we evaluated SRS imaging based on the same principles as the optimal setting simulation, i.e. the signals were propagated through an imaging system and LIA detection. The simulated images were compared to experimental SRS images of polystyrene beads. Finally, the same software was used to simulate multiplexed SRS imaging. In this study we modeled a six-channel frequency-encoded multiplexed SRS system demodulated with six LIA channels. We evaluated the inter-channel crosstalk as a function of chosen LIA parameters, which in multiplex SRS imaging also needs to be considered. These programs to optimize the contrast to noise ratio, acquisition speed, resolution and crosstalk will be useful for operating stimulated Raman scattering imaging setup, as well as for designing novel setups.

中文翻译：

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用于成像优化的受激拉曼散射模拟

开发了具有锁定放大器 (LIA) 检测的受激拉曼散射显微镜 (SRS) 成像系统的两个模拟程序。SRS 是一种基于振动拉曼截面作为对比机制的成像技术，可实现快速、无标记的成像。大多数 SRS 实现基于调制信号的 LIA 检测。然而，在选择 LIA 参数设置以优化采集速度或图像质量时，构建和操作此类 SRS 设置仍然是一个挑战。此外，样本的类型，例如稀疏样本与密集样本、所需的分辨率以及拉曼横截面和激光功率都会影响参数选择。使用模拟程序来寻找这些最佳参数。单个激光器（泵浦和斯托克斯）的焦斑直径用于估计有效的 SRS 信号焦斑和（光学）空间分辨率。通过为已知分子校准通过 SRS 系统的信号和噪声传播，我们估计了输入到 LIA 的信号和噪声。我们使用低通滤波器模型来模拟 LIA 行为，以找到最佳参数（即滤波器阶数和时间常数）。针对图像质量（表示为对比度与噪声比）或采集时间进行了优化。目标对象大小首先被确定为所需分辨率的度量。模拟输出包括 LIA 参数、像素驻留时间和对比度噪声比。在第二次模拟中，我们根据与最佳设置模拟相同的原则评估 SRS 成像，即。e. 信号通过成像系统和 LIA 检测传播。将模拟图像与聚苯乙烯珠的实验 SRS 图像进行比较。最后，使用相同的软件来模拟多路 SRS 成像。在这项研究中，我们模拟了一个用六个 LIA 通道解调的六通道频率编码多路复用 SRS 系统。我们将通道间串扰作为所选 LIA 参数的函数进行评估，这在多路 SRS 成像中也需要考虑。这些优化对比度噪声比、采集速度、分辨率和串扰的程序将有助于操作受激拉曼散射成像装置，以及设计新颖的装置。最后，使用相同的软件来模拟多路 SRS 成像。在这项研究中，我们模拟了一个用六个 LIA 通道解调的六通道频率编码多路复用 SRS 系统。我们将通道间串扰作为所选 LIA 参数的函数进行评估，这在多路 SRS 成像中也需要考虑。这些优化对比度噪声比、采集速度、分辨率和串扰的程序将有助于操作受激拉曼散射成像装置，以及设计新颖的装置。最后，使用相同的软件来模拟多路 SRS 成像。在这项研究中，我们模拟了一个用六个 LIA 通道解调的六通道频率编码多路复用 SRS 系统。我们将通道间串扰作为所选 LIA 参数的函数进行评估，这在多路 SRS 成像中也需要考虑。这些优化对比度噪声比、采集速度、分辨率和串扰的程序将有助于操作受激拉曼散射成像装置，以及设计新颖的装置。这在多重 SRS 成像中也需要考虑。这些优化对比度噪声比、采集速度、分辨率和串扰的程序将有助于操作受激拉曼散射成像装置，以及设计新颖的装置。这在多重 SRS 成像中也需要考虑。这些优化对比度噪声比、采集速度、分辨率和串扰的程序将有助于操作受激拉曼散射成像装置，以及设计新颖的装置。