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. 2020 Mar 24;10:5312. doi: 10.1038/s41598-020-62377-1

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Optical setup and design considerations. (A) Schematic of an RCM setup with annular illumination. Excitation light from the fiber is collimated by lens L5 and directed to an annular obstruction target (A) which is placed at the focal point of lens L6. The combination of L6 and lens L7 is a magnifying telescope. The light is directed onto a scanning mirror pair (SM1) through a mirror (M) and the dichroic mirror (DM). The focal point of the scan lens L1 is placed at the telecentric plane in between the 2 scanning mirrors of SM1. The focal plane of L1 coincides with the image plane of the microscope. Fluorescence emission from the microscope is descanned by SM1 and directed to a pinhole (PH) via DM and lens L2. Lens L3 collects the emission light from the PH and directs it to a second pair of scanning mirrors (SM2) through an emission filter (F). The rescanned light is collected by rescan lens L4, which forms an image at its focal distance which is captured by a camera. (B) Plot of lateral FWHM of the main lobe of the excitation PSF relative to the annulus size. (C) Plot of lateral FWHM of the microscope PSF vs. the sweep factor.