Gold nanorods as probes in two-photon fluorescence correlation spectroscopy: emerging applications and potential artifacts.

Owing to the highly efficient two-photon fluorescence of gold nanorods and very short fluorescence lifetime compared with the rotational correlation time, the rotation and diffusion of a single gold nanorod can be easily observed by two-photon fluorescence correlation spectroscopy (TP-FCS). This property, along with the previous successful use as a contrast agent in two-photon fluorescence imaging, suggests a potential application in TP-FCS as well. Although the FCS measurement becomes highly efficient with gold nanorods as probes, the amplitude and temporal decay of the measured correlation functions depend critically on excitation power. Here, we investigate various photophysical processes of gold nanorods to determine the cause of such a sensitive power dependency. This understanding provides a basis for choosing appropriate FCS models to recover reasonable physical parameters. Although the correlation function amplitude G(0) is 32 times lower when the excitation power increases from 20 µW to 1.12 mW, the application of a saturation-modified FCS model yields very good fit to each data set and the fitted concentration of 0.64 nM is comparable to the 0.7 nM given by the inductively coupled plasma mass spectrometry measurement. The FCS assay appears to be an efficient method for the quantification of gold nanorods when correctly interpreted. However, even with the saturation considered in the fitting model, the fitted rotational and translational diffusion rates are getting faster as the power increases. This indicates that other effects such as photothermal effects may raise the local temperature, and thus increasing the rotational and translational diffusion rate.