Pantazis, P., Maloney, J., Wu, D., and Fraser, S.E. (2010) Second harmonic generating (SHG) nanoprobes for in vivo imaging. Proceedings of the National Academy of Sciences of the United States of America. 107(33):14535-14540.
Fluorescence microscopy has profoundly changed cell and molecular biology studies by permitting tagged gene products to be
followed as they function and interact. The ability of a fluorescent dye to absorb and emit light of different wavelengths
allows it to generate startling contrast that, in the best cases, can permit single molecule detection and tracking. However,
in many experimental settings, fluorescent probes fall short of their potential due to dye bleaching, dye signal saturation,
and tissue autofluorescence. Here, we demonstrate that second harmonic generating (SHG) nanoprobes can be used for in vivo
imaging, circumventing many of the limitations of classical fluorescence probes. Under intense illumination, such as at the
focus of a laser-scanning microscope, these SHG nanocrystals convert two photons into one photon of half the wavelength; thus,
when imaged by conventional two-photon microscopy, SHG nanoprobes appear to generate a signal with an inverse Stokes shift
like a fluorescent dye, but with a narrower emission. Unlike commonly used fluorescent probes, SHG nanoprobes neither bleach
nor blink, and the signal they generate does not saturate with increasing illumination intensity. The resulting contrast and
detectability of SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues.