Suppression of auto-fluorescence from high-resolution 3D polymeric architectures fabricated via two-photon polymerization for cell biology applications

• DOI: 10.1016/j.mne.2023.100188
• Publication Date: 2023-04-11T06:50:13Z
• Authors: A. Sharaf; J.P. Frimat; G.J. Kremers; A. Accardo
• Keywords: Fluorescence-lifetime imaging microscopy

Two-photon polymerization (2PP) has provided the field of cell biology with opportunity to fabricate precisely designed microscaffolds for a wide range studies, from mechanobiology in vitro disease modelling. However, multitude commercial and in-house developed photosensitive materials employed 2PP suffers high auto-fluorescence multiple regions spectrum. In context biological this is major problem since one main methods characterization fluorescence microscopy immuno-stained cells. This undesired affects efficiency such an analysis as it often overlaps fluorescent signals stained cells rendering them indistinguishable scaffolds. Here, we propose two effective solutions suppress compare determine superiority over other: photo-bleaching powerful UV point source quenching via Sudan Black B (SBB). The used study were all commercially available, namely IP-L, IP-Dip, IP-S, IP-PDMS. Bleaching was shown be 61.7–92.5% reducing depending on material. On other hand, SBB 33–95.4% effective. worst result presence (33%) combination IP-PDMS adsorption material not sufficient fully quench auto-fluorescence. reduction significantly enhanced when activating structures oxygen plasma 30 s. Moreover, performed culture assay using human neuroblastoma line (SH-SY5Y) prove effectiveness both immunofluorescence characterization. presented lower performance especially 2PP-fabricated microchannels microcages, within which differentiated SH-SY5Y migrated extended their axon-like processes, obstructed Therefore, concluded that optimal way suppression. summary, provides systematic comparison answer most pressing issues applied paves more efficient cultured engineered microenvironments.