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. 2017 Jun 13;7:3393. doi: 10.1038/s41598-017-03414-4

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© The Author(s) 2017

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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3D cell culture conditions have a strong impact on intranuclear lamin distribution and overall morphology of MEF cells. (A) 3D cell culture system for single molecule microscopy and irradiation experiments: Cells embedded in the collagen-hydrogel are placed on a coverslip. A thin, highly inclined laminar optical sheet (HILO) is used to illuminate a subfraction of fluorescently labeled primary integrin antibodies of cells cultured in 3D in a distance of of 20–50 µm to the coverslip. By combining STORM and HILO a sufficient imaging contrast is obtained to detect single molecule signals in 3D cultured cells. (B) Heat map of a Lamin A/C immunostaining of 2D versus (C) 3D cultured cells. Scale bar is 5 µm, respectively. (D) A β-Tubulin (red) and H2A (green) immunostaining visualize the cytoskeleton and the nucleus of 2D cultured MEF cells. Scale bar is 25 µm. (E) CMO (green) staining of the PM of a 3D cultured MEF cell cultured in a collagen hydrogel (red). Scale bar is 10 µm. (F) MEF cells after a week-long 3D culture (maximum projection of a 3D confocal stack). (G) 3D volume rendering of (F).