PMID- 34837177 OWN - NLM STAT- MEDLINE DCOM- 20220118 LR - 20230510 IS - 1940-6029 (Electronic) IS - 1064-3745 (Linking) VI - 2393 DP - 2022 TI - Azimuthal Beam Scanning Microscope Design and Implementation for Axial Localization with Scanning Angle Interference Microscopy. PG - 127-152 LID - 10.1007/978-1-0716-1803-5_7 [doi] AB - 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. CI - (c) 2022. Springer Science+Business Media, LLC, part of Springer Nature. FAU - Colville, Marshall AU - Colville M AD - Graduate Field of Biophysics, Cornell University, Ithaca, NY, USA. FAU - Park, Sangwoo AU - Park S AD - Graduate Field of Biophysics, Cornell University, Ithaca, NY, USA. FAU - Singh, Avtar AU - Singh A AD - Applied and Engineering Physics, Cornell University, Ithaca, NY, USA. AD - Broad Institute, Cambridge, MA, USA. FAU - Paszek, Matthew AU - Paszek M AD - Graduate Field of Biophysics, Cornell University, Ithaca, NY, USA. AD - Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA. AD - Field of Biomedical Engineering, Cornell University, Ithaca, NY, USA. AD - Kavli Institute for Nanoscale Science, Cornell University, Ithaca, NY, USA. FAU - Zipfel, Warren R AU - Zipfel WR AD - Graduate Field of Biophysics, Cornell University, Ithaca, NY, USA. wrz2@cornell.edu. AD - Applied and Engineering Physics, Cornell University, Ithaca, NY, USA. wrz2@cornell.edu. AD - Kavli Institute for Nanoscale Science, Cornell University, Ithaca, NY, USA. wrz2@cornell.edu. AD - Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA. wrz2@cornell.edu. LA - eng GR - R33 CA193043/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PL - United States TA - Methods Mol Biol JT - Methods in molecular biology (Clifton, N.J.) JID - 9214969 SB - IM MH - Artifacts MH - Lasers MH - Microscopy, Fluorescence MH - *Microscopy, Interference MH - Optics and Photonics OTO - NOTNLM OT - Azimuthal beam scanning TIRFM OT - Circle-scanned TIRFM OT - Fluorescence microscopy OT - Interference microscopy OT - Live-cell imaging OT - Localization microscopy OT - Scanning-angle interference microscopy EDAT- 2021/11/28 06:00 MHDA- 2022/01/19 06:00 CRDT- 2021/11/27 06:05 PHST- 2021/11/27 06:05 [entrez] PHST- 2021/11/28 06:00 [pubmed] PHST- 2022/01/19 06:00 [medline] AID - 10.1007/978-1-0716-1803-5_7 [doi] PST - ppublish SO - Methods Mol Biol. 2022;2393:127-152. doi: 10.1007/978-1-0716-1803-5_7.