In the developing fly wing, secreted morphogens such as Decapentaplegic (Dpp) and Wingless (Wg) form gradients of concentration providing positional information. Dpp forms a longer-range gradient than Wg. To understand how the range is controlled, we measured the four key kinetic parameters governing morphogen spreading: the production rate, the effective diffusion coefficient, the degradation rate, and the immobile fraction. The four parameters had different values for Dpp versus Wg. In addition, Dynamin-dependent endocytosis was required for spreading of Dpp, but not Wg. Thus, the cellular mechanisms of Dpp and Wingless spreading are different: Dpp spreading requires endocytic, intracellular trafficking.

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.