Biological Second and Third Harmonic Generation Microscopy

Abstract

This unit describes how higher harmonic generation microscopy (HHGM) is applied to detect native, nonstained cell and tissue structures that were previously only accessible after immunohistochemical or immunofluorescent labeling.Nonlinear optical microscopy by multiphoton excitation has developed into a popular, and powerful approach that combines different excitation and emission techniques for the three-dimensional (3-D) reconstruction of biological specimens (Denk et al., 1990;Konig, 2000). The specific approaches include detection of two-and three-photon excited fluorescence, two-photon excited fluorescence lifetime (FLIM), and the second and third harmonic generation. Specimens amenable to multiphoton microscopy include fixed and live samples, thin and thick slices or samples with transparent or relatively opaque properties, as well as intravital microscopy in anesthetized animals (see UNIT 4.11). The most obvious advantages of multiphoton microscopy include: (1) greater tissue penetration up to the millimeter range due to near-infrared excitation and consequently, less scatter;(2) reduced phototoxicity imposed onto cells and tissues due to the inherent confocality exciting only fluorophores in the focal plane and the lack of a detector pinhole resulting in improved optical detection efficiency; (3) the broader excitation range of fluorophores, compared to single photon excitation, allowing multicolor imaging using the same excitation wavelength; and (4) the possibility of combining fluorescence microscopy with other detection modes, including HHGM and FLIM. Therefore, nonlinear imaging is being used for nearly any application in cell biology that requires multicolor imaging, deep tissue penetration, low phototoxicity, and high resolution, similar to confocal microscopy.Two-photon or multiphoton excited HHGM has become one of the most popular and powerful techniques for the reconstruction of intrinsic structural and molecular properties of cells, extracellular matrix, and bone without the use of dyes or stains (Campagnola et al., 2002;Friedl, 2004). Two modalities of HHGM are presently being used for biomedical applications, the second (SHG) and third (THG) harmonic generation imaging (the principle and theoretical basis for these imaging modalities are detailed in the Commentary). SHG and THG imaging provides structural and molecular information that is complementary to two-photon fluorescence (TPF) imaging and can be simultaneously combined with other two-photon excited modalities, including two-photon photoactivation, twophoton correlated spectroscopy, two-photon single-particle tracking, and two-photon lifetime microscopy. HHGM is most useful in the imaging of connective tissue, collagen fibers and fascia, as well as striated muscle, inflammatory cells, blood vessels, and hair at a resolution near the diffraction limit of visible light.This unit will provide details on the experimental setup and some biological applications of HHGM, including 3-D connective tissue reconstruction and dynamic imaging of ce...

Mapping proteolytic cancer cell-extracellular matrix interfaces

“…the location and function of each proteolytic cell structure in the context of even more structural complex tissues. To visualize such proteolytic processes in vivo [22,56], the development of protease-specific quenched substrates is required that not only freely reach the site of interest but, after cleavage, adopt a non-diffusive state with high temporal and spatial fidelity by, e.g. precipitation in situ.…”

Visualizing dendritic cell migration within the skin

“…-helical proteins (e.g. collagen and elastin), can be visualized without the need for Xuorescent labeling due to second and third harmonic generation (SHG and THG) signals (Friedl et al 2007). …”

“…This can, for example, be achieved by the intravenous injection of Xuorescent dyes, such as labeled dextran or Qdots. In addition, using the right Wlter sets, it is possible to visualize sources of non-linear signals by virtue of SHG and THG (Friedl et al 2007). In the ear, connective tissue Wbers, striated muscle and cartilage can all be imaged by this means (Fig.…”

Unleashing Optics and Optoacoustics for Developmental Biology

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