Three-Dimensional High-Resolution Second-Harmonic Generation Imaging of Endogenous Structural Proteins in Biological Tissues

Abstract

We find that several key endogenous protein structures give rise to intense second-harmonic generation (SHG)-nonabsorptive frequency doubling of an excitation laser line. Second-harmonic imaging microscopy (SHIM) on a laser-scanning system proves, therefore, to be a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of live cell and tissue architecture. Unlike fluorescence, SHG suffers no inherent photobleaching or toxicity and does not require exogenous labels. Unlike polarization microscopy, SHIM provides intrinsic confocality and deep sectioning in complex tissues. In this study, we demonstrate the clarity of SHIM optical sectioning within unfixed, unstained thick specimens. SHIM and two-photon excited fluorescence (TPEF) were combined in a dual-mode nonlinear microscopy to elucidate the molecular sources of SHG in live cells and tissues. SHG arose not only from coiled-coil complexes within connective tissues and muscle thick filaments, but also from microtubule arrays within interphase and mitotic cells. Both polarization dependence and a local symmetry cancellation effect of SHG allowed the signal from species generating the second harmonic to be decoded, by ratiometric correlation with TPEF, to yield information on local structure below optical resolution. The physical origin of SHG within these tissues is addressed and is attributed to the laser interaction with dipolar protein structures that is enhanced by the intrinsic chirality of the protein helices.

Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation

“…1) and exhibit broad blue fluorescence. SHG in tissue arises primarily from collagen (5) and to a lesser degree from microtubules and skeletal muscle (13). Table 1 summarizes the MPE characteristics of cell and tissue components accessible in the 700-to 1000-nm wavelength region.…”

“…To date, most biological MPM has depended on labeling with conventional fluorophores or fluorescent proteins such as the GFPs; however, a few studies have used two-photon excitation (2PE) of intrinsic molecules such as NAD(P)H (6)(7)(8) and flavins (9), three-photon excitation (3PE) of serotonin (10)(11)(12), and SHG of collagen, skeletal muscle, and microtubules (2,13). The combination of intrinsic and extrinsic signals is particularly powerful.…”

In vivo multiphoton fluorescence imaging: A novel approach to oral malignancy

“…Many wavelengths can be used; visualization/analysis of deeper subsurface structures such as components of the extracellular matrix, cellular and vascular elements is possible [22][23][24][25][26][27][28].…”

In vivo imaging of skeletal muscle in mice highlights muscle defects in a model of myotubular myopathy

“…[29][30][31] Owing to its superior imaging property and its compatibility with non-linear optical processes, Two-Photon-Excitation-Microscopy (2PEM) allows simultaneous tracking of fluorescent events in vivo as well as detection of non-centrosymetric molecules by second harmonic generation (SHG) such as myosin in muscle, 32 and collagen in the extracellular matrix. 33,34 2PEM and SHG were previously used to assess the structure of the contractile apparatus and tubular system in isolated mouse muscle fibers, 35 in preserved muscles from myopathic mice, 36,37 or in muscle in place in the animal after removal of the skin. 38 Minimally invasive imaging of muscle sarcomeres was achieved in mouse and human using microendoscopes.…”