英国剑桥大学Steven F. Lee课题组开发出用于生命科学的即时分子取向显微镜。相关论文于2024年10月7日发表在《自然—方法学》杂志上。

研究人员提出了POLCAM，一种基于极化检测的简化单分子取向定位显微镜（SMOLM）方法，使用极化相机，可以轻松地在任何宽场荧光显微镜上实现。为了使极化相机与单分子检测兼容，研究人员开发了理论以最小化视场误差，使用模拟优化实验设计。

研究人员开发了一种基于斯托克斯参数估计的快速算法，其操作速度比现有技术快超过1000倍，实现了近乎即时的分子各向异性确定。

为了促进POLCAM的采用，研究人员开发了开源图像分析软件和一个详细说明硬件安装和软件使用的网站。为了说明POLCAM在生命科学中的潜力，研究人员将其应用于研究α-突触核蛋白纤维、哺乳动物细胞的肌动蛋白细胞骨架、成纤维样细胞以及活人T细胞的质膜。

据悉，当前的SMOLM方法需要光学设备和算法，这可能过于缓慢和复杂，限制了其在生物应用中的广泛采用。

附：英文原文

Title: POLCAM: instant molecular orientation microscopy for the life sciences

Author: Bruggeman, Ezra, Zhang, Oumeng, Needham, Lisa-Maria, Krbel, Markus, Daly, Sam, Cheetham, Matthew, Peters, Ruby, Wu, Tingting, Klymchenko, Andrey S., Davis, Simon J., Paluch, Ewa K., Klenerman, David, Lew, Matthew D., OHolleran, Kevin, Lee, Steven F.

Issue&Volume: 2024-10-07

Abstract: Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, which can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field-of-view errors, used simulations to optimize experimental design and developed a fast algorithm based on Stokes parameter estimation that can operate over 1,000-fold faster than the state of the art, enabling near-instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study α-synuclein fibrils, the actin cytoskeleton of mammalian cells, fibroblast-like cells and the plasma membrane of live human T cells.

DOI: 10.1038/s41592-024-02382-8

Source: https://www.nature.com/articles/s41592-024-02382-8