Second harmonic generation imaging of endogenous structural proteins

We show that structural protein arrays consisting largely of collagen, myosin, and tubulin, and their associated proteins can be imaged in three dimensions with high contrast and resolution by laser-scanning second harmonic generation (SHG) microscopy. SHG is a nonlinear optical scheme and this form of microscopy shares several common advantages with multiphoton excited fluorescence, namely, intrinsic three-dimensionality and reduced out-of-plane photobleaching and phototoxicity. SHG does not arise from absorption and in-plane photodamage considerations are therefore also greatly reduced. In particular, structural protein arrays that are highly ordered and birefringent produce large SHG signals without the need for any exogenous labels. We demonstrate that thick tissues including muscle and bone can be imaged and sectioned through several hundred micrometers of depth. Combining SHG with two-photon excited green fluorescent protein (GFP) imaging allows inference of the molecular origin of the SHG contrast in Caenorhabditis elegans sarcomeres. Symmetry and organization of microtubule structures in dividing C. elegans embryos are similarly studied by comparing the endogenous tubulin contrast with that of GFP::tubulin fluorescence. It is found that SHG provides molecular level data on radial and lateral symmetries that GFP constructs cannot. The physical basis of SHG is discussed and compared with that of two-photon excitation as well as that of polarization microscopy. Due to the intrinsic sectioning, lack of photobleaching, and availability of molecular level data, SHG is a powerful tool for in vivo imaging.