Paul Campagnola

Second harmonic generation imaging of endogenous structural proteins

We find that several key endogenous structural proteins including collagen, acto-myosin, and tubulin give rise to intense second harmonic generation (SHG) and that these structures can be imaged in intact tissues on a laser-scanning microscope. Because SHG is a non-resonant process, this modality suffers little inherent photobleaching or toxicity. In this study we demonstrate the clarity of SHG optical sectioning within unfixed, unstained thick specimens, including fish scales, C. elegans, and mouse muscle, where penetration into tissue upwards of 600 microns was achieved. The simultaneous use of SHG and two-photon excited GFP fluorescence allows for the inference of the molecular isoform that gives rise to SHG from the myofilament lattice in C. elegans. The physical origin of SHG within these tissues is addressed and is attributed to the laser interaction with dipolar protein structures. SHG polarization anisotropy is also used to determine the extent of dipolar order and radial symmetry in the helical structures. Comparisons are drawn between SHG and other forms of microscopy including polarization and fluorescence microscopy, highlighting the advantages and disadvantages.

Second harmonic generation imaging of skeletal muscle tissue and myofibrils

Second Harmonic Generation (SHG) imaging microscopy is used to examine the morphology and structural properties of intact muscle tissue. Using biochemical and optical analysis, we characterize the molecular structure underlying SHG from the complex muscle sarcomere. We find that SHG from isolated myofibrils is abolished by extraction of myosin, but is unaffected by removal or addition of actin filaments. We thus determined that the SHG emission arises from domains of the sarcomere containing thick filaments. By fitting the SHG polarization anisotropy to theoretical response curves, we find an orientation for the harmonophore that corresponds well to the pitch angle of the myosin rod α-helix with respect to the thick filament axis. Taken together, these data indicate that myosin rod domains are the key structures giving rise to SHG from striated muscle. Using SHG imaging microscopy, we have also examined the effect of optical clearing with glycerol to achieve greater penetration into specimens of skeletal muscle tissue. We find that treatment with 50% glycerol results in a 2.5 fold increase in achievable SHG imaging depth. Fast Fourier Transform (FFT) analysis shows quantitatively that the periodicity of the sarcomere structure is unaltered by the clearing process. Also, comparison of the SHG angular polarization dependence shows no change in the supramolecular organization of acto-myosin complexes. We suggest that the primary mechanism of optical clearing in muscle with glycerol treatment results from the reduction of cytoplasmic protein concentration and concomitant decrease in the secondary inner filter effect on the SHG signal. The pronounced lack of dependence of glycerol concentration on the imaging depth indicates that refractive index matching plays only a minor role in the optical clearing of muscle.