Shi-Wei Chu

Multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser.

We demonstrate a novel multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser at 1230 nm. By acquiring the whole nonlinear spectrum in the visible and near-NIR region, this novel technique allows a combination of different imaging modalities, including second-harmonic generation, third-harmonic generation, and multiple-photon fluorescence. Combined with the selected excitation wavelength, which is located in the IR transparency window, this microscopic technique can provide high penetration depth with reduced damage and is ideal for studying living cells.

In vivo developmental biology study using noninvasive multi-harmonic generation microscopy

Morphological changes and complex developmental processes inside vertebrate embryos are difficult to observe noninvasively with millimeter-penetration and sub-micrometer-resolution at the same time. By using higher harmonic generation, including second and third harmonics, as the microscopic contrast mechanism, optical noninvasiveness can be achieved due to the virtual-level-transition characteristic. The intrinsic nonlinearity of harmonic generations provides optical sectioning capability while the selected 1230-nm near-infrared light source provides the deeppenetration ability. The complicated development within a ~1.5-mm thick zebrafish (Danio rerio) embryo from initial cell proliferation, gastrulation, to tissue formation can all be observed clearly in vivo without any treatment on the live specimen.

Real-time second-harmonic-generation microscopy based on a 2-GHz repetition rate Ti:sapphire laser.

The problem of weak harmonic generation signal intensity limited by photodamage probability in optical microscopy and spectroscopy could be resolved by increasing the repetition rate of the excitation light source. Here we demonstrate the first photomultiplier-based real-time second-harmonic-generation microscopy taking advantage of the strongly enhanced nonlinear signal from a high-repetition-rate Ti:sapphire laser. We also demonstrate that the photodamage possibility in common biological tissues can be efficiently reduced with this high repetition rate laser at a much higher average power level compared to the commonly used ~80- MHz repetition rate lasers.

Nonlinear bio-photonic crystal effects revealed with multimodal nonlinear microscopy

Highly optically active nonlinear bio‐photonic crystalline and semicrystalline structures in living cells were studied by a novel multimodal nonlinear microscopy. Numerous biological structures, including stacked membranes and aligned protein structures are highly organized on a nanoscale and have been found to exhibit strong optical activities through second‐harmonic generation (SHG) interactions, behaving similarly to man‐made nonlinear photonic crystals. The microscopic technology used in this study is based on a combination of different imaging modes including SHG, third‐harmonic generation, and multiphoton‐induced fluorescence. With no energy release during harmonic generation processes, the nonlinear‐photonic‐crystal‐like SHG activity is useful for investigating the dynamics of structure–function relationships at subcellular levels and is ideal for studying living cells, as minimal or no preparation is required.

Multiharmonic-generation biopsy of skin

Because it avoids the in-focus photodamage and phototoxicity problem of two-photon-fluorescence excitation, multiharmonic-generation biopsy based on a 1200-1300-nm light source could provide a truly noninvasive and highly penetrative optical sectioning of skin. We study multiharmonic-generation biopsy of fixed mouse skin. Our preliminary study suggests that this technique could provide submicrometer-resolution deep-tissue noninvasive biopsy images in skin without the use of fluorescence and exogenous markers.

Noninvasive harmonics optical microscopy for long-term observation of embryonic nervous system development in vivo

Nervous system development is a complicated dynamic process, and many mechanisms remain unknown. By utilizing endogenous second-harmonic-generation as the contrast of polarized nerve fibers and third-harmonic-generation (THG) to reveal morphological changes, we have successfully observed the vertebrate embryonic nervous development from the very beginning based on a 1230-nm light source. The dynamic development of the nerve system within a live zebrafish embryo can be recorded continuously more than 20 hr without fluorescence markers. Since the THG process is not limited by the time of gene expression and differentiation as fluorescence-based techniques are, the observable stages can be advanced to the very beginning of the development process. The complete three-dimensional brain development from a neural plate to a neural tube can be uncovered with a submicron lateral resolution. We have, for the first time, also reported the generation of SHG from myelinated nerve fibers and the outer segment of the photoreceptors with a stacked membrane structure. Our study clearly indicates the fact that higher-harmonics-based optical microscopy has the strong potential to long-term in vivo study of the nervous system, including genetic disorders of the nervous system, axon pathfinding, neural regeneration, neural repair, and neural stem cell development.

Second harmonic generation imaging - a new method for unraveling molecular information of starch.

We present a new method, second harmonic generation (SHG) imaging for the study of starch structure. SHG imaging can provide the structural organization and molecular orientation information of bio-tissues without centrosymmetry. In recent years, SHG has proven its capability in the study of crystallized bio-molecules such as collagen and myosin. Starch, the most important food source and a promising future energy candidate, has, for a decade, been shown to exhibit strong SHG response. By comparing SHG intensity from different starch species, we first identified that the SHG-active molecule is amylopectin, which accounts for the crystallinity in starch granules. With the aid of SHG polarization anisotropy, we extracted the complete χ((2)) tensor of amylopectin, which reflects the underlying molecular details. Through χ((2)) tensor analysis, three-dimensional orientation and packing symmetry of amylopectin are determined. The helical angle of the double-helix in amylopectin is also deduced from the tensor, and the value corresponds well to previous X-ray studies, further verifying amylopectin as SHG source. It is noteworthy that the nm-sized structure of amylopectin inside a starch granule can be determined by this far-field optical method with 1-μm excitation wavelength. Since SHG is a relatively new tool for plant research, a detailed understanding of SHG in starch structure will be useful for future high-resolution imaging and quantitative analyses for food/energy applications.

Measuring plasmon-resonance enhanced third-harmonic χ(3) of Ag nanoparticles

By coinciding the plasmon frequency with the third-harmonic frequency of the excitation light, the authors determined the plasmon-resonance enhanced optical third-harmonic-generation (THG) susceptibility of a polyvinylpyrrolidone-coated Ag nanoparticle with a 5–7nm diameter. With dispersed Ag nanoparticles on a quartz surface and through measuring the frequency dependent THG intensities, interface THG showed evident enhancement when the third harmonic of excitation matched the Ag-nanoparticle’s plasmon-resonant frequency. According to the effective medium theory and by analyzing the interface THG under focused Gaussian beams, the ensemble-averaged χ(3)(3ω:ω,ω,ω) of a Ag nanoparticle can be estimated to be on the order of 2×10−11esu.

Chiral imaging of collagen by second-harmonic generation circular dichroism

We provide evidence that the chirality of collagen can give rise to strong second-harmonic generation circular dichroism (SHG-CD) responses in nonlinear microscopy. Although chirality is an intrinsic structural property of collagen, most of the previous studies ignore that property. We demonstrate chiral imaging of individual collagen fibers by using a laser scanning microscope and type-I collagen from pig ligaments. 100% contrast level of SHG-CD is achieved with sub-micrometer spatial resolution. As a new contrast mechanism for imaging chiral structures in bio-tissues, this technique provides information about collagen morphology and three-dimensional orientation of collagen molecules.

Thickness dependence of optical second harmonic generation in collagen fibrils

Simultaneous backward and forward second harmonic generations from isolated type-I collagen matrix are observed. Optical interference behaviors of these nonlinear optical signals are studied with accurately determined fibril thickness by an atomic force microscope. The nonlinear emission directions are strongly dependent on the coherent interaction within and between collagen fibrils. A linear relationship is obtained to estimate collagen fibril thickness with nanometer precision noninvasively by evaluating the forward/backward second harmonic generation ratio.