We want to end this chapter by briefly introducing three high-resolution imaging techniques from the aspects of super-resolution visualization, direct biomechanical characterization, and deep molecular imaging. We expect increased application of these techniques in the exciting field of tissue engineering due to their unique and powerful individual properties.

Spatial resolution is one of the most critical parameters of microscopy, and improvement in spatial resolution has always expanded the limit of what people can see and therefore understand at new spatial levels. Resolutions of traditional optical microscopy techniques, as discussed above, are all limited by diffraction, which is on the order of a half-wavelength of the light (Born et al., 1999). Relying on fluorescence imaging, super-resolution microscopy techniques, such as stimulated emission depletion microscopy (Hell and Wichmann, 1994), stochastic optical reconstruction microscopy (Huang et al., 2008), and photo-activated localization microscopy (Hess et al., 2006), have been developed to break the diffraction limit and can feature spatial resolutions as good as ~20 nm (Huang et al., 2009). This spatial imaging scale allows time-resolved localization of individual molecules in live cells (Godin et al., 2014). This technology can prove useful in providing detailed molecular analysis of the cellular response to scaffolds and external stimuli.

Brillouin scattering is the inelastic light scattering between photons and acoustic phonons, which allows viscoelastic characterization of the material (Dil, 1982). Scarcelli et al. have developed confocal Brillouin microscopy that employs Brillouin scattering in tissue to probe biomechanics (Scarcelli and Yun, 2007). With a highly sensitive spectrometer, the Brillouin shift from tissue can be measured noninva- sively with a 3-D spatial resolution as high as that seen in confocal microscopy. The Brillouin shift can be utilized as a quantitative measure of tissue elasticity and is well correlated with the traditionally used elastic modulus (Scarcelli et al., 2011). Confocal Brillouin microscopy has been extensively employed to study ocular tissue biomechanics (Scarcelli et al., 2011, 2014, 2015a), including the acrylic intraocular lens (Scarcelli and Yun, 2007). Noncontact 3-D mapping of intracellular hydromechanical properties using Brillouin microscopy has been demonstrated (Scarcelli et al., 2015b), which can be utilized as a new and efficient approach to assess the cellular biomechanical changes in tissue engineering.

For a long time, molecular imaging has suffered from a limited depth of view in highly scattering tissues. Photothermal OCT, which employs the OCT phase signal to probe the externally induced photothermal expansion inside tissue (Tucker-Schwartz et al., 2012; 2015), can achieve 3-D molecular contrast with millimeter-level imaging depth in scattering samples (Zhou et al., 2010). With photon-absorption nanostructures targeting specific molecules, upon modulated light excitation, molecular specificity can be obtained, which has been used for cell identification (Skala et al., 2008). Since the unique imaging scale of OCT is highly useful in a number of applications in tissue engineering, the addition of molecular contrast holds the promise for robust structural, functional, and molecular characterization of tissue constructs within a single system.

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