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Optical higher harmonic generation, including second harmonic generation and third harmonic generation, leaves no energy deposition to its interacted matters due to an energy-conservation characteristic, providing the “noninvasiveness” nature desirable for biological studies. Combined with its nonlinearity, higher harmonic generation microscopy provides excellent three-dimensional (3D) sectioning capability, offering new insights into the studies of embryonic morphological changes and complex developmental processes. By choosing a laser working in the biological penetration window, here we present a noninvasive in vivo light microscopy with sub-micron 3D resolution and millimeter penetration, utilizing endogenous higher harmonic generation signals in live specimens. Noninvasive imaging was performed in live zebrafish (Danio rerio) embryos. The complex developmental processes within >1-mm-thick zebrafish embryos can be observed in vivo without any treatment. No optical damage was found even with high illumination after long-term observations and the examined embryos all developed normally at least to the larval stage. The excellent 3D resolution of the demonstrated technology allows us to capture the subtle developmental information on the cellular or sub-cellular levels occurring deep inside the live embryos and larvae. This technique can not only provide in vivo observation of the cytoarchitecture dynamics during embryogenesis with submicron resolution and millimeter penetration depth, but would also make strong impact in developmental and structural biology studies. [Copyright &y& Elsevier]

Second-harmonic-generation (SHG) has proved itself as an important contrast mechanism in microscopic applications. Its noninvasiveness, optical sectioning capability, and high-penetrability provide attractive features in observation of thick biological tissues. Fibrous proteins, such as myosin and collagen, are dominant SHG harmonophores in vertebrates. Due to their biophotonic crystal nature, SHGs from these proteins are known to exhibit specific polarization dependencies, reflecting local molecule arrangements. Here the authors demonstrate a scheme to distinguish SHG from myosin-based muscle fibers and intertwined collagenous perimysium through polarization selection, without complicated staining or sample/image processing required. [ABSTRACT FROM AUTHOR]

Confocal pump-probe backscattering was used to study phonon dynamics of single nanoparticles, which can extend to confocal pump-probe microscopy for investigating carrier dynamics of single nanomaterials. Compared to previous pump-probe microscopy studies, in which nonlinear optical generation was widely utilized, spectral filters were employed in this work to separate the spectra from the optical pulses for pump/probe beams. This not only reduces the complexity of the experimental setup but can also circumvent the aberration issue. Phonon dynamics, including acoustic vibrations and heat diffusion, of a single gold nanoparticle were measured with a temporal delay of 8 ns, and a 0.5 K rise in the temperature of the surrounding media was resolved. The relative optical changes induced by the photoexcitation of the pump pulses in confocal reflection pump-probe traces were orders of magnitude larger than those in typical transmission-type pump probe traces. [ABSTRACT FROM AUTHOR]