Guan-Yu Zhuo

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.

Three‐dimensional structural imaging of starch granules by second‐harmonic generation circular dichroism

Chirality is one of the most fundamental and essential structural properties of biological molecules. Many important biological molecules including amino acids and polysaccharides are intrinsically chiral. Conventionally, chiral species can be distinguished by interaction with circularly polarized light, and circular dichroism is one of the best‐known approaches for chirality detection. As a linear optical process, circular dichroism suffers from very low signal contrast and lack of spatial resolution in the axial direction. It has been demonstrated that by incorporating nonlinear interaction with circularly polarized excitation, second‐harmonic generation circular dichroism can provide much higher signal contrast. However, previous circular dichroism and second‐harmonic generation circular dichroism studies are mostly limited to probe chiralities at surfaces and interfaces. It is known that second‐harmonic generation, as a second‐order nonlinear optical effect, provides excellent optical sectioning capability when combined with a laser‐scanning microscope. In this work, we combine the axial resolving power of second‐harmonic generation and chiral sensitivity of second‐harmonic generation circular dichroism to realize three‐dimensional chiral detection in biological tissues. Within the point spread function of a tight focus, second‐harmonic generation circular dichroism could arise from the macroscopic supramolecular packing as well as the microscopic intramolecular chirality, so our aim is to clarify the origins of second‐harmonic generation circular dichroism response in complicated three‐dimensional biological systems.

Effect of Lorentz local field for optical second order nonlinear susceptibility in ZnO nanorod

Nonlinear optical properties of ZnOnanorods (NRs) are strongly influenced by its dimension and aspect ratio. Size-dependent second harmonic generation(SHG) in ZnO NRs has been investigated with polarized excitations recently. However, detailed description to the SHG dependency with NR dimensions has not yet been given. In this paper, the relationship between rod diameter/length and corresponding χ(2) values based on Lorentz local field is established, both theoretically and experimentally, for the first time. Theoretically,Lorentz local field induced spectral red shift and the consequent dielectric constant modification is used to elucidate the size effect for χ(2) under the condition that both excitation and SHG wavelengths are far from the band gap. Experimentally, χ(2) of ZnOnanorods with various sizes is measured via Maker fringe technique, and the results fit well to our theory.

Multiphoton imaging to identify grana, stroma thylakoid, and starch inside an intact leaf

BackgroundGrana and starch are major functional structures for photosynthesis and energy storage of plant, respectively. Both exhibit highly ordered molecular structures and appear as micrometer-sized granules inside chloroplasts. In order to distinguish grana and starch, we used multiphoton microscopy, with simultaneous acquisition of two-photon fluorescence (2PF) and second harmonic generation (SHG) signals. SHG is sensitive to crystallized structures while 2PF selectively reveals the distribution of chlorophyll.ResultThree distinct microstructures with different contrasts were observed, i.e. “SHG dominates”, “2PF dominates”, and “SHG collocated with 2PF”. It is known that starch and grana both emit SHG due to their highly crystallized structures, and no autofluorescence is emitted from starch, so the “SHG dominates” contrast should correspond to starch. The contrast of “SHG collocated with 2PF” is assigned to be grana, which exhibit crystallized structure with autofluorescent chlorophyll. The “2PF dominates” contrast should correspond to stroma thylakoid, which is a non-packed membrane structure with chrolophyll. The contrast assignment is further supported by fluorescence lifetime measurement.ConclusionWe have demonstrated a straightforward and noninvasive method to identify the distribution of grana and starch within an intact leaf. By merging the 2PF and SHG images, grana, starch and stroma thylakoid can be visually distinguished. This approach can be extended to the observation of 3D grana distribution and their dynamics in living plants.

Reversible suppression of second harmonic generation in dye-doped liquid crystal by light-induced thermal phase transition on sub-micrometer scale

Optically controllable signals are fundamental to various applications from communication to super-resolution imaging. However, literature on non-fluorescent, nonlinear optical signals that can be reversibly turned on/off on a sub-micrometer scale is scant. In this work, we experimentally demonstrate a scheme for the reversible suppression of second harmonic generation (SHG) based on dye-doped nematic liquid crystal molecules. Under a pump (suppressing SHG) and probe (generating SHG) setup with a tightly focusing microscope and a time-gated detection, outstanding modulation depth (>80%) has been realized. Surprisingly, the mechanism of liquid crystal SHG switch on a sub-micrometer scale was found to be light-induced thermal phase transition as against optical Fredericks transition. Quantitative analysis of the optical nonlinearity χ ( 2 ) versus local heating shows an excellent agreement of SHG signal suppression as well as its dependence on the liquid crystal molecular order and phase change. Our work provides an innovative example of applying nonlinear optical properties of soft materials, and can be further optimized for all-optical modulation applications.

Saturable scattering and its application to superresolution microscopy

Recently, several superresolution methods have been demonstrated to revolutionize the way we access the nano-world. However, these techniques are based on switching or saturation of fluorescence, and consequently are limited by switching reversibility and photobleaching. Here we show first superresolution far-field imaging based on scattering, which is a universal phenomenon without bleaching. Our principal finding is that scattering from plasmonics particles is saturable. By extracting the saturated part, sub-80-nm resolution is achieved. This work not only expands the horizon of superresolution imaging from fluorescence to scattering, but also points out the possibility of resolution enhancement for all imaging modalities that utilize plasmon properties.

Multiphoton imaging to distinguish grana and starch inside an intact leaf

We have demonstrated a straightforward and noninvasive method to identify the distribution of grana and starch within an intact leaf. Grana and starch are the major functional structures for photosynthesis and energy storage of plant, respectively. Both exhibit highly ordered molecular structures and appear as micrometer-sized granules inside chloroplasts. In order to distinguish grana and starch, we used multiphoton microscopy, with simultaneous acquisition of two photon fluorescence (2PF) and second harmonic generation (SHG) signals. Consequently, SHG is found on both grana and starch while 2PF from chlorophyll indicates the identity of grana.

Optically switchable second harmonic generation in a liquid crystal thin film within femtoliter volume

Nematic liquid crystal (NLC) is one of most useful soft matters. Because the molecular orientation can be controlled electrically, NLC is widely applied to display devices. It is known that NLC exhibits strong second-harmonic-generation (SHG) due to its orderly arranged molecules. The strength of SHG is strongly dependent on the angle between the incident beam polarization and the NLC molecular orientation, so the SHG in NLC can be switched on/off by rotating the NLC director. However, it is very difficult to control the orientation of NLC director electrically within a micrometer spatial domain. In this report, we demonstrated the orientation control of NLC with sub-micrometer spatial resolution based on optical Freedericksz transition (OFT) combined with a high-numerical-aperture objective. We used azo-dye doped NLC to reduce the intensity threshold of OFT with 473-nm excitation. Interestingly, we found that the threshold of OFT increases with tighter focuses. This effect can be explained by the intermolecular forces from the NLC molecules around the focal spot. By incorporating both the blue laser and a femtosecond near-infrared laser into an optical scanning microscope, we have successfully demonstrated switch of SHG inside a NLC thin film. Note that SHG is confined within femtoliter focal volume due to its intrinsic nonlinearity. That is, we have achieved an ultrasmall switch of nonlinear optical signal in NLC. This work will find applications in optical communication as well as optical-base storage system.

Chirality study inside biological tissue by second harmonic generation circular dichroism

Many biological systems are composed of chiral molecules and their functions depend strongly on their chirality. For example, most amino acids are of left-handed chirality while most polysaccharides are of right-handed chirality. Both of them are vital for human life, so it is important to perform chiral detection inside bio-tissues. Here we demonstrated second harmonic generation circular dichroism (SHG-CD) as a novel chiral imaging contrast in thick biotissue. Compared with conventional chiral detection, SHG-CD provides at least three orders higher contrast. In addition, due to the nonlinear nature of SHG, this technique provides optical sectioning capability, so the axial contrast is much better. The advantages of nonlinear optical microscopy are optical sectioning and deep penetration capabilities. The SHG-CD achieved 100% signal contrast with sub-micrometer spatial resolution. This method is expected to offer a novel contrast mechanism of imaging chirality inside complex bio-tissues.

Relationship of second order susceptibility to the dimensions of ZnO nanorods based on Lorentz local field

With the aid of Maker fringe technique, we have observed two nonlinear optical (NLO) phenomena separately on diameter and length of ZnO nanorod (NR). One is second harmonic generation (SHG) saturation in rod diameter, and the other is SHG enhancement in rod length. Besides that, the model based on Lorentz local field is proposed for the first time to elucidate the above phenomena. The deduced second order susceptibility χ(2) with various sizes of ZnO NR matches well to our theory, demonstrating that the size effect on χ(2) is governed by Lorentz local field. Our theory provides a theoretical basis to explain the mechanism of light-material interaction in nano-dimensions and is readily to be extended to other kind of semiconductor nanostructures when addressing NLO properties in them.