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. 2018 May 15;8:7583. doi: 10.1038/s41598-018-25922-7

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© The Author(s) 2018

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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Chromatic shift in illumination. (a) An illustration of the CLSM setup to measure chromatic shift only for illumination. When the pinhole was opened to its maximum size, almost all light goes into the detector (PMT). Therefore, the chromatic shift of emission light is negligible and only that of the illumination light is measured. (b) Fixed cells were stained with phalloidin conjugated with Alexa 488 and 594. The green channel, obtained by exciting with 488 nm, was aligned with respect to the red channel, obtained by exciting with 488 (“488” blue bars) or 561 (“488 + 561” green bars) nm using the global method. The vector sum of the seven global alignment parameters is shown. Different immersion oils were used to examine the influence of spherical aberration and dispersion. (c) An illustration of WFM and 3D-SIM illumination around the sample. (d) The same samples from (b) were imaged with WFM or 3D-SIM using the same objective lens. WFM images were deconvolved (WFM Decon) and 3D-SIM raw images were averaged to create pseudo-WFM images. The red channel excited with 488 nm and the green channel excited with 488 nm were aligned with respect to the red channel excited with 561 nm and the green channel excited with 488 nm. The vector sum of the seven alignment parameters is shown.