Specific Binding and Internalization: Fluorescence Lifetime Imaging Microscopy of Gold Nanoclusters and Cells

Fluorescent gold nanoclusters (AuNCs) are novel promising nanomaterials for biomedical applications because of their unique physiochemical properties. AuNCs stabilized with mercaptoundecanoic acid (MUA-AuNCs) were used, which show a fluorescence emission that peaks at a wavelength around 525 nm, which is in the same wavelength region as the autofluorescence of a biological cell. However, the fluorescence decay time of the MUA-AuNCs (100 ns) is much longer in comparison to the autofluorescence (3 ns). Fluorescence lifetime imaging microscopy (FLIM) is a powerful method to discriminate between emitters of different fluorescence lifetimes. These MUA-AuNCs were biofunctionalized with biomolecules that specifically bind to the receptor expressed on the cell's membrane. To get an image of the whole cell and the bound and internalized AuNCs we use cross-sectional FLIM scans in axial direction at different heights through the cell. To distinguish between specifically bound and internalized MUA-AuNCs on and in cells different FLIM methods were applied and supported by electron microscopic and analytical methods.