Jianling Chen

Visualization of collagen regeneration in mouse dorsal skin using second harmonic generation microscopy

The purpose of this study is to highlight a clearer understanding of the process of collagen regeneration during wound healing. By means of second harmonic generation (SHG) microscopy, the changes of collagen arrangement at the wound margin were analyzed at 0, 3, 5, 7, 11 and 13 days post injury. The degree of collagen disorders associated with the healing process was quantitatively obtained using the aspect ratio of polar plot image of collagen azimuthal angles and the healing status of collagen could be estimated by arithmetical mean deviation (Ra) of the collagen SHG images. Our results suggest that SHG microscopy has potential advances in the collagen studies during wound healing and the arrangement of collagen fibers gradually transformed from disorder to order so as to contract the wound. It is capable of promoting clinical application of the noninvasive imaging tool and the analysis methods of collagen disorder as an effective scar management for prevention and treatment about aberrant healing.

Monitoring wound healing of elastic cartilage using multiphoton microscopy

OBJECTIVE To demonstrate the ability of multiphoton microscopy (MPM) for monitoring wound healing of elastic cartilage. METHOD In a rabbit ear model, four cartilage specimen groups at 1-day, 1-, 4-, 20-week healing time points as well as a normal elastic cartilage were examined with MPM without using labeling agents. MPM images at wound margins were obtained from specimens at different healing stages, compared with the Hematoxylin and Eosin (H&E) stained images. Image analysis was performed to characterize the collagen morphology for quantifying the wound healing progression of elastic cartilage. RESULTS MPM provided high-resolution images of elastic cartilage at varying depths. Comparisons of the images of specimens at different healing stages show obvious cell growth and matrix deposition. The results are consistent with the histological results. Moreover, quantitative analysis results show significant alteration in the collagen cavity size or collagen orientation index during wound healing of elastic cartilage, indicating the possibility to act as indicators for monitoring wound healing. CONCLUSION Our results suggested that MPM has the ability to monitor the wound healing progression of elastic cartilage, based on the visualization of cell growth and proliferation and quantitative characterization of collagen morphology during wound healing.

Differentiating keloids from normal and hypertrophic scar based on multiophoton microscopy

We use a novel nonlinear optical technique, multiphoton microscopy, to differentiate keloids from hypertrophic and normal scars through the morphological and quantitative analysis of collagen and elastin in superficial dermis of scar tissues and boundary between normal skin and scar tissue.

Monitoring process of human keloid formation based on second harmonic generation imaging

In this paper, the morphological variation of collagen among the whole dermis from keloid tissue was investigated using second harmonic generation (SHG) microscopy. In the deep dermis of keloids, collagen bundles show apparently regular gap. In the middle dermis, the collagen bundles are randomly oriented and loosely arranged in the pattern of fine mesh while the collagen bundles are organized in a parallel manner in the superficial dermis near the epidermis. The developed parameters COI and BD can be used to further quantitatively describe these changes. Our results demonstrate the potential of SHG microscopy to understand the formation process of human keloid scar at the cellular level through imaging collagen variations in different depth of dermis.

Label-free monitoring of colorectal adenoma–carcinoma sequence based on multiphoton microscopy

The monitoring and evaluation of colorectal adenoma–carcinoma sequence during endoscopy are important for endoscopic resection of precursor lesions to disrupt the adenoma–carcinoma sequence and halt progression to invasive neoplastic disease. In this study, multiphoton microscopy (MPM) was used to identify different stages during the development of colorectal adenocarcinoma including adenoma with low-grade and high-grade dysplasia, and adenocarcinoma invading the submucosa. It was found that by combining two-photon excited fluorescence (TPEF) imaging and second harmonic generation (SHG) imaging, MPM can reveal the morphological changes of the epithelial cells and glands, identify the invasive position and depth of atypical glands and quantitatively describe the change of the cellular nucleus and the nuclear-to-cytoplasmic ratio during the stepwise progression of colorectal adenocarcinoma. These are important pathological findings for pathologists when diagnosing colorectal lesions. With the advancement of a compact and flexible multiphoton endoscope for in vivo imaging and clinical applications, MPM has the potential to provide immediate histological diagnosis for the monitoring and evaluation of the colorectal adenoma–carcinoma sequence during endoscopy.

High-Contrast Multimodel Nonlinear Optical Imaging of Collagen and Elastin

Collagen and elastin, as the major components in the extracellular matrix (ECM), are intrinsic indicators of physiological and pathological states. Here, we have developed a high-contrast multimodel nonlinear optical imaging technique to imaging collagen and elastin by detecting simultaneously two photon-excited fluorescence (TPEF) from elastin and second-harmonic generation (SHG) from collagen. Our results show that this technique can obtain a high-contrast TPEF/SHG image in human dermis and permit direct visualization of collagen and elastin. It was shown that the technique can provide collagen and elastin structural information to determine collagen and elastin fibril orientation and distribution and acquire some morphometric properties. It was found that the in-depth TPEF/SHG imaging and 3-D reconstruction of TPEF/SHG images can extract more collagen and elastin structural and biochemical information. The study results suggest that the high-contrast multimodel nonlinear imaging provides a powerful tool to study ECM intrinsic components and has the potential to provide more important information for the diagnosis of tissue.

Effect of light polariztion on pattern illumination super-resolution imaging

Far-field fluorescence microscopy has made great progress in the spatial resolution, limited by light diffraction, since the super-resolution imaging technology appeared. And stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM) can be grouped into one class of the super-resolution imaging technology, which use pattern illumination strategy to circumvent the diffraction limit. We simulated the images of the beads of SIM imaging, the intensity distribution of STED excitation light and depletion light in order to observe effects of the polarized light on imaging quality. Compared to fixed linear polarization, circularly polarized light is more suitable for SIM on reconstructed image. And right-handed circular polarization (CP) light is more appropriate for both the excitation and depletion light in STED system. Therefore the right-handed CP light would be the best candidate when the SIM and STED are combined into one microscope. Good understanding of the polarization will provide a reference for the patterned illumination experiment to achieve better resolution and better image quality.

Structured illumination microscopy and its new developments

Optical microscopy allows us to observe the biological structures and processes within living cells. However, the spatial resolution of the optical microscopy is limited to about half of the wavelength by the light diffraction. Structured illumination microscopy (SIM), a type of new emerging super-resolution microscopy, doubles the spatial resolution by illuminating the specimen with a patterned light, and the sample and light source requirements of SIM are not as strict as the other super-resolution microscopy. In addition, SIM is easier to combine with the other imaging techniques to improve their imaging resolution, leading to the developments of diverse types of SIM. SIM has great potential to meet the various requirements of living cells imaging. Here, we review the recent developments of SIM and its combination with other imaging techniques.

Numerical Simulations of Femtosecond-Laser-Induced Dynamic Alignment of Molecules in the High-Frequency Off-Resonance Regime

The motion equation for ϑ between the molecular axis and laser polarization direction in a high-frequency off-resonance femtosecond laser field is deduced while simultaneously examining the effects of a permanent dipole moment and field-induced polarizability and hyperpolarizability to molecular rotation. Femtosecond-laser-induced dynamic alignment of CO, N2, and Br2 molecules are investigated by numerically solving the obtained rotation equation for the angle ϑ. The effects of the molecular permanent dipole moment and the field-induced polarizability and hyperpolarizability on the degree of alignment are presented at different intensities. Our computational results show that the dynamic alignment of molecules is primarily determined by field-induced polarizability and the second hyperpolarizability for the laser intensity range from 5 × 1014 to 5 × 1016 W/cm2. The contributions of higher order correction terms to molecular alignment can usually be neglected. The polarizability-field interaction makes the angular distributions of a molecule have a maximum along the polarization axis and a minimum perpendicular to it. The role of the second hyperpolarizability keeps the molecular counts maximum along the laser polarization direction but minimum at an angle of 45° between the molecular axis and the polarization direction. There is also a second maximum of molecular counts perpendicular to the polarization axis. For CO, N2, and Br2 molecules, the dependences of laser-induced dynamic alignment on laser intensity exhibit completely different characteristics.

Effect of oxidative stress stimulation on intracellular PTEN trafficking in ovarian cancer by fluorescence correlation spectroscopy

The phosphatase and tensin homolog on chromosome 10 (PTEN) is one of important tumor suppressor proteins in ovarian cancer via negatively regulating the phosphatidylinositol 3-kinase–AKT signaling pathway and controlling genomic stability. Recent studies showed the physiological function of PTEN was closely related with its subcellular compartments. But only a few technologies could quantitatively measure the concentration of PTEN at different subcellular compartments in living cells. In this study, we used fluorescence correlation spectroscopy to measure the concentrations and dynamics of EGFP-PTEN in ovarian cancer cells HO-8910. Our results showed the increasing concentration of PTEN in the cytoplasm had an opposite trends with the nucleus after the oxidative stress stimulation which was induced by H2O2. Furthermore, the altered diffusion of PTEN at different subcellular compartments also illustrated the PTEN was trafficked from the cytoplasm to nucleus.