The ability to image at high speeds is necessary in biological imaging to capture fast-moving or transient events or to efficiently image large samples. However, due to the lack of rigidity of biological specimens, carrying out fast, high-resolution volumetric imaging without moving and agitating the sample has been a challenging problem. Pupil-matched remote focusing has been promising for high NA imaging systems with their low aberrations and wavelength independence, making it suitable for multicolor imaging. However, owing to the incoherent and unpolarized nature of the fluorescence signal, manipulating this emission light through remote focusing is challenging. Therefore, remote focusing has been primarily limited to the illumination arm, using polarized laser light for facilitating coupling in and out of the remote focusing optics. Here we introduce a novel optical design that can de-scan the axial focus movement in the detection arm of a microscope. Our method splits the fluorescence signal into S and P-polarized light and lets them pass through the remote focusing module separately and combines them with the camera. This allows us to use only one focusing element to perform aberration-free, multi-color, volumetric imaging without (a) compromising the fluorescent signal and (b) needing to perform sample/detection-objective translation. We demonstrate the capabilities of this scheme by acquiring fast dual-color 4D (3D space + time) image stacks, with an axial range of 70 μm and camera limited acquisition speed. Owing to its general nature, we believe this technique will find its application to many other microscopy techniques that currently use an adjustable z-stage to carry out volumetric imaging such as confocal, 2-photon, and light sheet variants.

中文翻译：

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在光片显微镜检测臂中使用远程聚焦进行轴向去扫描

在生物成像中，高速成像的能力对于捕获快速移动或瞬态事件或有效地对大样本进行成像是必要的。然而，由于生物样本缺乏刚性，在不移动和搅拌样本的情况下进行快速、高分辨率的体积成像一直是一个具有挑战性的问题。光瞳匹配远程聚焦因其低像差和波长独立性而有望用于高数值孔径成像系统，使其适用于多色成像。然而，由于荧光信号的不相干和非偏振性质，通过远程聚焦来操纵这种发射光具有挑战性。因此，远程聚焦主要限于照明臂，使用偏振激光来促进远程聚焦光学器件的耦合进出。在这里，我们介绍一种新颖的光学设计，可以对显微镜检测臂中的轴向焦点移动进行反扫描。我们的方法将荧光信号分成S偏振光和P偏振光，让它们分别通过远程聚焦模块并与相机结合。这使我们能够仅使用一个聚焦元件来执行无像差、多色、体积成像，而不会 (a) 损害荧光信号和 (b) 需要执行样本/检测目标转换。我们通过采集快速双色 4D（3D 空间 + 时间）图像堆栈来展示该方案的功能，轴向范围为 70 μm，相机采集速度有限。由于其一般性质，我们相信该技术将应用于许多其他显微镜技术，这些技术目前使用可调节的 z 载物台来进行体积成像，例如共焦、2 光子和光片变体。