Distinguishing a multitude of optical labels is crucial to improving the spatial and temporal resolution of bioimaging. However, current multicolor imaging approaches are limited by the spectral overlap of employed fluorophores. We here discern different instances of a single optical label type through their emission intensity. Such multilevel optical labels are enabled by an optical writing process that permanently modifies their spectral response in a predictable manner and by a separate spectral feature that serves as normalization in the presence of sample variability. The proposed approach was realized by independently controlling the emission properties of highly functionalized fluorescent nanodiamond. Upon laser irradiation, the contribution of the spectral region associated with the N3 color center decreases in a predictable and permanent fashion, while the nitrogen vacancy (NV) emission remains stable. This selective photobleaching of N3 centers was found to originate from a two-photon-assisted dissociation process that results in a 105 higher mobility of photoexcited carriers in N3 centers compared to NV. The resulting write once read many (WORM) memory exhibits multiple distinct memory levels that can be stored and read out with high robustness and reproducibility. The potential of our approach was demonstrated by characterizing markers in HeLa cells with high fidelity, despite the complex emission background. Finally, direct manipulation of label information inside of cells was demonstrated, opening up new routes in advanced bioimaging.
Keywords: NV center; bioimaging; biolabeling; memory; nanodiamond.