Che-Hang Yu

In vivo and ex vivo imaging of intra-tissue elastic fibers using third-harmonic-generation microscopy

Elastin is an essential and widespread structural protein in charge of the integrity on tissues and organs. In this study, we demonstrate that elastin is a major origin of the third-harmonic-generation (THG) contrast under Cr:forsterite laser excitation operating at 1230nm, with selective visualization inside many tissues such as lung tissues and arteries. In vivo imaging of the nude mouse elastic cartilage beneath the hypodermis by epi- THG microscopy keeps the high resolution and contrast in all three dimensions. Combined with second-harmonic-generation microscopy, THG microscopy exhibits the ability to show the extraordinary proliferation of elastic fibers for the ophthalmic disease of pterygium and the capability of distinguishable visualization from collagen.

Aberrant expression of microRNAs in T cells from patients with ankylosing spondylitis contributes to the immunopathogenesis

Ankylosing spondylitis (AS) is a chronic inflammatory disorder characterized by dysregulated T cells. We hypothesized that the aberrant expression of microRNAs (miRNAs) in AS T cells involved in the pathogenesis of AS. The expression profile of 270 miRNAs in T cells from five AS patients and five healthy controls were analysed by real‐time polymerase chain reaction (PCR). Thirteen miRNAs were found potentially differential expression. After validation, we confirmed that miR‐16, miR‐221 and let‐7i were over‐expressed in AS T cells and the expression of miR‐221 and let‐7i were correlated positively with the Bath Ankylosing Spondylitis Radiology Index (BASRI) of lumbar spine in AS patients. The protein molecules regulated by miR‐16, miR‐221 and let‐7i were measured by Western blotting. We found that the protein levels of Toll‐like receptor‐4 (TLR‐4), a target of let‐7i, in T cells from AS patients were decreased. In addition, the mRNA expression of interferon (IFN)‐γ was elevated in AS T cells. Lipopolysaccharide (LPS), a TLR‐4 agonist, inhibited IFN‐γ secretion by anti‐CD3+anti‐CD28 antibodies‐stimulated normal T cells but not AS T cells. In the transfection studies, we found the increased expression of let‐7i enhanced IFN‐γ production by anti‐CD3+anti‐CD28+ lipopolysaccharide (LPS)‐stimulated normal T cells. In contrast, the decreased expression of let‐7i suppressed IFN‐γ production by anti‐CD3+anti‐CD28+ LPS‐stimulated AS T cells. In conclusion, we found that miR‐16, miR‐221 and let‐7i were over‐expressed in AS T cells, but only miR‐221 and let‐7i were associated with BASRI of lumbar spine. In the functional studies, the increased let‐7i expression facilitated the T helper type 1 (IFN‐γ) immune response in T cells.

Cell tracking and detection of molecular expression in live cells using lipid-enclosed CdSe quantum dots as contrast agents for epi-third harmonic generation microscopy.

We demonstrated that lipid-enclosed CdSe quantum dots (LEQDs) can function as versatile contrast agents in epi-detection third harmonic generation (THG) microscopy for biological applications in vivo. With epi-THG intensities 20 times stronger than corresponding fluorescence intensities from the same LEQDs under the same conditions of energy absorption, such high brightness LEQDs were proved for the abilities of cell tracking and detection of specific molecular expression in live cancer cells. Using nude mice as an animal model, the distribution of LEQD-loaded tumor cells deep in subcutaneous tissues were imaged with high THG contrast. This is the first demonstration that THG contrast can be manipulated in vivo with nanoparticles. By linking LEQDs with anti-Her2 antibodies, the expression of Her2/neu receptors in live breast cancer cells could also be easily detected through THG. Compared with fluorescence modalities, the THG modality also provides the advantage of no photobleaching and photoblinkin g effects. Combined with a high penetration 1230 nm laser, these novel features make LEQDs excellent THG contrast agents for in vivo deep-tissue imaging in the future.

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.

Noninvasive in vitro and in vivo assessment of epidermal hyperkeratosis and dermal fibrosis in atopic dermatitis

Atopic dermatitis (AD) is characterized by hyperkeratosis of epidermis and fibrosis within dermis in chronic skin lesions. Thus far, the histology of skin lesions has been evaluated only by examination of excised specimens. A noninvasive in vivo tool is essential to evaluate the histopathological changes during the clinical course of AD. We used Cr:forsterite laser-based multimodality nonlinear microscopy to analyze the endogenous molecular signals, including third-harmonic generation (THG), second-harmonic generation (SHG), and two-photon fluorescence (TPF) from skin lesions in AD. Significant differences in thickness of epidermis and stratum corneum (SC), and modified degrees of fibrosis in dermis (measured by THG signals and SHG signals, respectively), are clearly demonstrated in in vitro studies. Increased TPF levels are positively associated with the levels of the THG signals from the SC. Our in vitro observations of histological changes are replicated in the in vivo studies. These findings were reproducible in skin lesions from human AD. For the first time, we demonstrate the feasibility of preclinical applications of Cr:forsterite laser-based nonlinear microscopy. Our findings suggest that the optical signatures of THG, TPF, and SHG can be used as molecular markers to assess the pathophysiological process of AD and the effects of local treatment.

Multi-photon resonance enhancement of third harmonic generation in human oxyhemoglobin and deoxyhemoglobin

Cancer cells require excessive oxygen and nutrition to support their rapid growth, so angiogenesis and decrease of blood oxygen are often associated with areas of cancer development. Current technologies for blood oxygen measurement, however, do not possess high spatial resolution and therefore cannot be used to detect small tumors in their early stage. In this paper, we studied the third harmonic generation (THG) spectra of oxy- and deoxyhemoglobin in the 1170-1365 nm region, which is strongly influenced by the multi-photon resonance effect, especially around the Soret transition band. Our spectroscopic results thus indicate the high potential of THG spectroscopic microscopy for oxygen depletion level measurement of a single red blood cell in vivo.

Miniaturized video-rate epi-third-harmonic-generation fiber-microscope

With a micro-electro-mechanical system (MEMS) mirror, we successfully developed a miniaturized epi-third-harmonic-generation (epi-THG) fiber-microscope with a video frame rate (31 Hz), which was designed for in vivo optical biopsy of human skin. With a large-mode-area (LMA) photonic crystal fiber (PCF) and a regular microscopic objective, the nonlinear distortion of the ultrafast pulses delivery could be much reduced while still achieving a 0.4 microm lateral resolution for epi-THG signals. In vivo real time virtual biopsy of the Asian skin with a video rate (31 Hz) and a sub-micron resolution was obtained. The result indicates that this miniaturized system was compact enough for the least invasive hand-held clinical use.

Direct backward third-harmonic generation in nanostructures

We theoretically and experimentally demonstrated that direct backward third harmonic generation (THG) waves can be comparable in magnitude with forward THG waves in nanostructures, such as ZnO thin films and nanoparticles of CdSe and Fe3O4.

A sub-100fs self-starting Cr:forsterite laser generating 1.4W output power

Without cavity dumping or external amplification, we report a femtosecond Cr:forsterite laser with a 1.4 W output power and 2 W in continuous wave (CW) operated with a crystal temperature of 267 K. In the femtosecond regime, the oscillator generates Kerr-lens-mode-locked 84 fs pulses with a repetition rate of 85 MHz, corresponding to a high 16.5 nJ pulse energy directly from a single Cr:forsterite resonator. This intense femtosecond Cr:forsterite laser is ideal to pump varieties of high power fiber light sources and could be thus ideal for many biological and spectroscopy applications.

In vivo Molecular-Resonant Third Harmonic Generation Microscopy of Hemoglobin

Through molecular-resonant third-harmonic-generation (THG) in hemoglobin, in vivo molecular THG microscopy of erythrocytes can be realized without using fluorescence and exogenous contrast agents. Studies in live hamster oral cavity indicate its superiority to image angiogenesis.