Dibaji Hassan, Kazemi Nasaban Shotorban Ali, Grattan Rachel M, Lucero Shayna, Schodt David J, Lidke Keith A, Petruccelli Jonathan, Lidke Diane S, Liu Sheng, Chakraborty Tonmoy
Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM, USA.
Comprehensive Cancer Center, University of New Mexico Health Sciences Center, Albuquerque, NM, USA.
Nat Commun. 2024 Jun 12;15(1):5019. doi: 10.1038/s41467-024-49291-0.
Rapid, high-resolution volumetric imaging without moving heavy objectives or disturbing delicate samples remains challenging. Pupil-matched remote focusing offers a promising solution for high NA systems, but the fluorescence signal's incoherent and unpolarized nature complicates its application. Thus, remote focusing is mainly used in the illumination arm with polarized laser light to improve optical coupling. 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, 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 in 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.
在不移动重型物镜或不干扰脆弱样本的情况下进行快速、高分辨率的体积成像仍然具有挑战性。瞳孔匹配的远程聚焦为高数值孔径系统提供了一个有前景的解决方案，但荧光信号的非相干和非偏振特性使其应用变得复杂。因此，远程聚焦主要用于带有偏振激光的照明光路中以改善光学耦合。在此，我们介绍一种新颖的光学设计，它可以消除显微镜检测光路中轴向焦点的移动。我们的方法将荧光信号分离为S偏振光和P偏振光，让它们分别通过远程聚焦模块，然后与相机组合。这使我们仅使用一个聚焦元件就能进行无像差、多色的体积成像，且（a）不影响荧光信号，（b）无需进行样本/检测物镜的平移。我们通过采集轴向范围为70μm且相机限制采集速度的快速双色4D（3D空间 + 时间）图像堆栈来展示该方案的能力。由于其通用性，我们相信这项技术将在许多目前使用可调节Z轴载物台进行体积成像的其他显微镜技术中得到应用，如共聚焦、双光子和光片变体技术。
