Converting lateral scanning into axial focusing to speed up three-dimensional microscopy.
Converting lateral scanning into axial focusing to speed up three-dimensional microscopy.
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DOI：
10.1038/s41377-020-00401-9
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Fiolka R
中科院分区：
文献类型：
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作者：
Chakraborty T;Chen B;Daetwyler S;Chang BJ;Vanderpoorten O;Sapoznik E;Kaminski CF;Knowles TPJ;Dean KM;Fiolka R
在光学显微镜中，物镜或样品的缓慢轴向扫描速率传统上限制了体积成像的速度。最近，通过将可移动反射镜与远距离聚焦几何结构中的像平面共轭或将电可调透镜（ETL）与后焦平面共轭，已经实现了快速轴向扫描。然而，反射镜的机械致动限制了轴向扫描速率（对于基于压电或音圈的致动器，通常仅为10-100 Hz），而ETL引入了阻止高分辨率成像的球面和高阶像差。为了克服这些局限性，我们引入了一种新的光学设计，将横向扫描运动转换为球面无像差轴向扫描，可用于高分辨率成像。使用检流计镜，我们在远程聚焦臂中横向扫描激光束，然后从图像空间中的不同高度的镜子反射回来。我们表征这种远程聚焦技术的光学性能，并使用它来加速轴向扫描光片显微镜的数量级，允许在三维空间中的快速囊泡动力学的量化。我们还展示了共振远程聚焦在12 kHz的双光子光栅扫描显微镜，它允许快速成像的脑组织和斑马鱼的心脏动力学与衍射有限的分辨率。一种基于远程聚焦概念的新光学设计可以帮助研究人员捕捉生物过程的高速图像。虽然大多数光学显微镜通过机械调整样品和物镜之间的距离来重新聚焦，但远程聚焦设置使用可移动的反射镜来快速扫描激光沿着光轴的焦点。位于美国达拉斯的德克萨斯大学西南医学中心的Reto Fiolka博士和他的同事们现在证明，对这一概念进行调整-由移动激光扫描的固定镜子-可以在毫秒时间尺度上提供无像差的图像。实验表明，阶梯形和倾斜镜的组合使跳动的斑马鱼心脏等样本能够以比之前的远程聚焦方法高出一个数量级的帧率进行三维成像。
In optical microscopy, the slow axial scanning rate of the objective or the sample has traditionally limited the speed of volumetric imaging. Recently, by conjugating either a movable mirror to the image plane in a remote-focusing geometry or an electrically tuneable lens (ETL) to the back focal plane, rapid axial scanning has been achieved. However, mechanical actuation of a mirror limits the axial scanning rate (usually only 10–100 Hz for piezoelectric or voice coil-based actuators), while ETLs introduce spherical and higher-order aberrations that prevent high-resolution imaging. In an effort to overcome these limitations, we introduce a novel optical design that transforms a lateral-scan motion into a spherical aberration-free axial scan that can be used for high-resolution imaging. Using a galvanometric mirror, we scan a laser beam laterally in a remote-focusing arm, which is then back-reflected from different heights of a mirror in the image space. We characterize the optical performance of this remote-focusing technique and use it to accelerate axially swept light-sheet microscopy by an order of magnitude, allowing the quantification of rapid vesicular dynamics in three dimensions. We also demonstrate resonant remote focusing at 12 kHz with a two-photon raster-scanning microscope, which allows rapid imaging of brain tissues and zebrafish cardiac dynamics with diffraction-limited resolution. A new optical design based on the concept of remote focusing can benefit researchers looking to capture high-speed images of biological processes in action. While most optical microscopes refocus by mechanically adjusting the distance between the sample and the objective, remote focusing setups use movable mirrors to quickly scan the focal spot of a laser along the optical axis. Dr. Reto Fiolka at the University of Texas Southwestern Medical Center in Dallas, United States, and colleagues now demonstrate that a tweak to this concept—fixed mirrors that are scanned by a moving laser—can provide aberration-free images at millisecond time scales. Experiments revealed that combinations of step-shaped and sloped mirrors enabled samples such as beating zebrafish hearts to be imaged three-dimensionally with frame rates up to an order of magnitude higher than previous remote-focusing approaches.