Fluorescence microscopy can provide microscopy imaging with subcellular resolution and is playing an increasingly important role in preclinical study and clinical diagnosis. Graphene quantum dots (GQDs) are the latest successor to traditional fluorescent probes. We developed a new bottom-up method for the synthesis of single-crystal GQDs in solution by hydrothermal molecular hydrolysis and fusion using pyrene as a molecular precursor. This simple fusion method enables green production, low cost and large-scale functionalized high-quality graphene nanostructures. Single-crystal GQD as a fluorescent probe provides higher quality cell images, including single-photon fluorescence (Fig. 1, a-d) and double-photon fluorescence (Fig. 1, e). GQD is readily internalized into the cell and ultimately localized in the cytoplasm. In addition, single crystal GQDs provide good performance and high quality images in vivo (Fig. 1, f, g).These results indicate that GQDS is a promising and bright fluorescent probe. This study provides new evidence for the superiority of GQDS for in vitro and in vivo biomedical applications.

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