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. 2020 Apr 23;10:6881. doi: 10.1038/s41598-020-63991-9

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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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Two-photon TPA excitation in living cells. HeLa cells were pre-incubated with 2-µM TPA for 2 h at 37 °C before two-photon excitation at 830 nm for TP2Bzim (A), TP3Py (B), TP3Pyo (C) or 850 nm for TP2Pyo (D) (irradiance, 1.25 W/cm²) (see Methods for emission slit settings). Plasma membrane blebs are indicated in DIC transmission images by blue arrows. The time scale corresponds to the real time (black axis) or the effective illumination time accounting for the z-stack as explained in Methods (blue axis) (number of slices: 8 for TP3Py and TP3Pyo; 9 for TP2Bzim; 10 for TP2Pyo). (E) Photobleaching properties of TPAs under continuous two-photon irradiation. TPAs were subjected to prolonged NIR irradiation and the normalized fluorescence intensity was plotted as a function of irradiation time. The two-photon fluorescence was measured in vitro as described in Methods. λ_exc,2-hν: 760 nm (TP3Bzim), 830 nm (TP2Bzim, TP3Py and TP3Pyo), 850 nm (TP2Pyo), 860 nm (TP2Py).