Graduate Field of Biophysics, Cornell University, Ithaca, NY, USA.
Applied and Engineering Physics, Cornell University, Ithaca, NY, USA.
Methods Mol Biol. 2022;2393:127-152. doi: 10.1007/978-1-0716-1803-5_7.
Azimuthal beam scanning, also referred to as circle scanning, is an effective way of eliminating coherence artifacts with laser illumination in widefield microscopy. With a static excitation spot, dirt on the optics and internal reflections can produce an uneven excitation field due to interference fringes. These artifacts become more pronounced in TIRF microscopy, where the excitation is confined to an evanescent field that extends a few hundred nanometers above the coverslip. Unwanted intensity patterns that arise from these imperfections vary with path of the excitation beam through the microscope optical train, so by rapidly rotating the beam through its azimuth the uneven illumination is eliminated by averaging over the camera exposure time. In addition to being useful from TIRF microscopy, it is also critical for scanning angle interference microscopy (SAIM), an axial localization technique with nanometer-scale precision that requires similar instrumentation to TIRF microscopy. For robust SAIM localization, laser excitation with a homogeneous profile over a range of polar angles is required. We have applied the circle scanning principle to SAIM, constructing an optimized instrument configuration and open-source hardware, enabling high-precision localization and significantly higher temporal resolution than previous implementations. In this chapter, we detail the design and construction of the SAIM instrument, including the optical configuration, required peripheral devices, and system calibration.
方位角光束扫描，也称为圆扫描，是消除宽场显微镜中激光照明相干伪影的有效方法。在静态激发光斑的情况下，光学器件上的污垢和内部反射会由于干涉条纹而产生不均匀的激发场。这些伪影在 TIRF 显微镜中更为明显，因为激发被限制在一个衰减场中，该衰减场在盖玻片上方延伸几百纳米。由于这些不完美，从这些不完美中产生的不需要的强度模式随激发光束通过显微镜光学器件的路径而变化，因此通过快速旋转光束使其方位角旋转，可以通过在相机曝光时间内进行平均来消除不均匀的照明。除了在 TIRF 显微镜中有用之外，它对于扫描角干涉显微镜（SAIM）也至关重要，SAIM 是一种具有纳米级精度的轴向定位技术，需要类似于 TIRF 显微镜的仪器。为了实现稳健的 SAIM 定位，需要在一系列极角上具有均匀轮廓的激光激发。我们已经将圆扫描原理应用于 SAIM，构建了优化的仪器配置和开源硬件，实现了比以前实现更高的定位精度和更高的时间分辨率。在本章中，我们详细介绍了 SAIM 仪器的设计和构建，包括光学配置、所需的外围设备和系统校准。
