Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases of photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive-index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.

中文翻译：

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调制超分辨率显微镜中的光漂白减少

自受激发射损耗 (STED) 显微镜首次展示以来，远场成像技术取得了重大突破。迄今为止，STED 显微镜最直接和最广泛的部署使用连续波 (CW) 激光束来激发和消耗荧光发射。CW STED 成像技术的一个主要缺点是光漂白效应，因为耗尽光束需要高光功率才能达到亚衍射分辨率。为了克服这个障碍，我们应用了一种基于调制激发激光束的同步检测方法，同时将耗尽光束保持在 CW 操作，并使用锁定放大器对收集到的信号进行频率滤波，以单独记录超分辨率荧光发射. 我们在这里证明，这种方法可以显着降低两束激光束的光功率，从而显着降低 STED 显微镜中的光漂白效果。我们报告了激发光束和耗尽光束的功率相对较低的超分辨率图像。此外，耗尽光束产生的典型的不需要的散射效应和背景信号，总是由组成样品的材料中的折射率不匹配引起的，通过使用调制 STED 方法大大减少。以相对较低的功率获取超分辨率图像的能力与研究各种样本非常相关，但对于本工作中举例说明的生物物种尤为重要。