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Understanding and visualizing the uptake and biodistribution of pesticides inside living cells are great importance for enhancing targeting and changing application methods of pesticides.Here we reported for the first time that gold nanorods (Au NRs) with size of 39.4 nm × 11.3 nm could be used as a fluorescent tracer to examine the biodistribution of a typical herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), in tobacco bright yellow 2 (BY-2)cells, by covalently bonding 2,4-D molecules with the Au NRs.The conjugations (2,4-D-MP-Au NRs) between2,4-D and Au NRs was confirmed by using Raman spectra and X-ray photoelectron spectroscopy (XPS).Additionally, two-photon luminescence (TPL) spectra were recorded in the time-course experiments after the switching-on of the laser light with an excitation wavelength of 800 nm in Fig.1.TPL images from at least three randomly selected sites of BY-2 cells, including extracellular environment, cell wall, cytoplasm and nucleus, were acquired under identical experimental conditions.The detector positions were highlighted by red circles.In a control experiment, no TPL signal was observed in any site of the cell that contained no Au NRs.In addition, there is also no TPL signal in the extracellular environment in Fig.l(b) because the cells were rinsed by centfifugation.However, in Fig.l(c)-(h), the bright dots of different sites inside the cells can be detected.It states that Au NRs can emit strong TPL when the fs laser waver wavelength is in resonance with their longitudinal surface plasmon resonance.Moreover, TPL can be distinguished from tissue autofluorescence based on the specific spectral patterns,which agrees with what was reported in previous publication [1].The preliminary results show that 2,4-D-MP-Au NRs have entered into the BY-2 cells after 12 h.Additionally, TPL can be used as a reliable tool for quantitatively imaging of Au NRs [2].Therefore, we also investigated the TPL intensity from 2,4-D-MP-Au NRs in the insets of Fig.l(c)-(h).Then the TPL intensity from different sites inside the cells was plotted in Fig.l(i), indicating that the TPL intensity weakens gradually from cell wall to nucleus for 12 h.However, the TPL intensity is the highest value at the cytoplasm for 24 h.Besides, the strongest TPL intensity at 12 h was emitted at the cell wall, but almost no Au NRs could be detected anymore at 24 h.Taken together, the TPL intensity of different sites inside BY-2 cells decreases from 12 to 24 h, which would suggest that 2,4-D-MP-Au NRs have be internalized and excluded by the BY-2 cells with time.Therefore, the imaging of 2,4-D-MP-Au NRs inside BY-2 cells can be monitored by two-photon microscopy.Results revealed that the hybrid material could enter into the BY-2 cells, and was localized in cell wall,cytoplasm and nucleus of BY-2 cells.Their strong signal, ease of synthesis, and biocompatibility indicate that covalently functionalized Au NRs can be used as a potential tracer of pesticides for live plant cells, even plants.