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. 2022 Nov 24;10:983872. doi: 10.3389/fbioe.2022.983872

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Copyright © 2022 Spedicati, Ruocco, Zoso, Mortati, Lapini, Delledonne, Divieto, Romano, Castaldo, Di Meglio, Nurzynska, Carmagnola and Chiono.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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TPEF acquisitions. (A) 3D-cellularized scaffolds reconstruction. Blue refers to DAPI staining of cell nuclei, red indicates α-SMA—Alexa Fluor 555 immunostaining. (B) z-Stack measurement on the 3D cellularized scaffold stained for α-SMA. Excitation wavelength was 950 nm. Signal acquired in the red channel (641 +/− 37.5 nm) integrates the tail of the two-photon excited fluorescence response of Alexa Fluor 555. (C) z-Stack measurement on the 3D decellularized scaffolds stained for collagen I and IV. Excitation wavelength was 950 nm. Signal acquired in the green channel (550 +/− 44 nm) integrates the two-photon excited fluorescence response of both Alexa Fluor 488 and Alexa Fluor 555.