Shuangmu Zhuo

Multimode nonlinear optical imaging of the dermis in ex vivo human skin based on the combination of multichannel mode and Lambda mode

A Multimode nonlinear optical imaging technique based on the combination of multichannel mode and Lambda mode is developed to investigate human dermis. Our findings show that this technique not only improves the image contrast of the structural proteins of extracellular matrix (ECM) but also provides an image-guided spectral analysis method to identify both cellular and ECM intrinsic components including collagen, elastin, NAD(P)H and flavin. By the combined use of multichannel mode and Lambda mode in tandem, the obtained in-depth two photon-excited fluorescence (TPEF) and second-harmonic generation (SHG) imaging and TPEF/SHG signals depth-dependence decay can offer a sensitive tool for obtaining quantitative tissue structural and biochemical information. These results suggest that the technique has the potential to provide more accurate information for determining tissue physiological and pathological states.

Quantitatively linking collagen alteration and epithelial tumor progression by second harmonic generation microscopy

Collagen alteration is critical for epithelial tumor initiation and progression. Quantitatively linking collagen alteration and epithelial tumor progression is essential for developing an optical endoscopy to evaluate epithelial tumor progression. In this work, we established a quantitative link between collagen alteration and epithelial tumor progression using second harmonic generation (SHG) microscopy. It was found that SHG microscopy can provide quantitative features to effectively evaluate epithelial tumor progression, and to locate tumor and determine the margin of tumor regions. These results suggest that SHG microscopy has the potential in offering a noninvasive in vivo imaging tool to quantify epithelial tumor progression.

Nonlinear spectral imaging of human hypertrophic scar based on two-photon excited fluorescence and second-harmonic generation.

Background  A noninvasive method using microscopy and spectroscopy for analysing the morphology of collagen and elastin and their biochemical variations in skin tissue will enable better understanding of the pathophysiology of hypertrophic scars and facilitate improved clinical management and treatment of this disease.

Extracting diagnostic stromal organization features based on intrinsic two-photon excited fluorescence and second-harmonic generation signals

Intrinsic two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) signals are shown to differentiate between normal and neoplastic human esophageal stroma. It was found that TPEF and SHG signals from normal and neoplastic stroma exhibit different organization features, providing quantitative information about the biomorphology and biochemistry of tissue. By comparing normal with neoplastic stroma, there were significant differences in collagen-related changes, elastin-related changes, and alteration in proportions of matrix molecules, giving insight into the stromal changes associated with cancer progression and providing substantial potential to be applied in vivo to the clinical diagnosis of epithelial precancers and cancers.

The layered–resolved microstructure and spectroscopy of mouse oral mucosa using multiphoton microscopy

The layered-resolved microstructure and spectroscopy of mouse oral mucosa are obtained using a combination of multiphoton imaging and spectral analysis with different excitation wavelengths. In the keratinizing layer, the keratinocytes microstructure can be characterized and the keratinizing thickness can be measured. The keratin fluorescence signal can be further characterized by emission maxima at 510 nm. In the epithelium, the cellular microstructure can be quantitatively visualized with depth and the epithelium thickness can be determined by multiphoton imaging excited at 730 nm. The study also shows that the epithelial spectra excited at 810 nm, showing a combination of NADH and FAD fluorescence, can be used for the estimation of the metabolic state in epithelium. Interestingly, a second-harmonic generation (SHG) signal from DNA was observed for the first time within the epithelial layer in backscattering geometry and provides the possibility of analyzing the chromatin structure. In the stroma, the combination of multiphoton imaging and spectral analysis excited at 850 nm in tandem can obtain quantitative information regarding the biomorphology and biochemistry of stroma. Specifically, the microstructure of collagen, minor salivary glands and elastic fibers, and the optical property of the stroma can be quantitatively displayed. Overall, these results suggest that the combination of multiphoton imaging and spectral analysis with different excitation wavelengths has the potential to provide important and comprehensive information for early diagnosis of oral cancer.

Depth-resolved spectral imaging of rabbit oesophageal tissue based on two-photon excited fluorescence and second-harmonic generation

A novel depth-resolved spectral imaging based on two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) is developed for simultaneously investigating images and spectra at different depths within rabbit oesophageal tissues in backscattering geometry. Our results show that this method has a capability to identify the layered structures of oesophageal tissue including the keratinizing layer, epithelial cell layer and stromal layer, which are strongly correlated to tissue pathology. By integrating several system analysing tools, morphology and spectroscopy in different layers can be quantitatively obtained. Our findings demonstrate that this technique has the potential to provide more accurate and comprehensive information for the pathological diagnosis of tissues with stratified squamous epithelia.

Multiphoton microscopy study of the morphological and quantity changes of collagen and elastic fiber components in keloid disease

Multiphoton microscopy was used to study the extracellular matrix of keloid at the molecular level without tissue fixation and staining. Direct imaging of collagen and elastin was achieved by second harmonic generation and two-photon excited fluorescence, respectively. The morphology and quantity of collagen and elastin in keloid were characterized and quantitatively analyzed in comparison to normal skin. The study demonstrated that in keloid, collagen content increased in both the upper dermis and the deep dermis, while elastin mostly showed up in the deep dermis and its quantity is higher compared to normal skin. This suggests the possibility that abnormal fibroblasts synthesized an excessive amount of collagen and elastin at the beginning of keloid formation, corresponding to the observed deep dermis, while after a certain time point, the abnormal fibroblast produced mostly collagen, corresponding to the observed upper dermis. The morphology of collagen and elastin in keloid was disrupted and presented different variations. In the deep dermis, elastic fibers showed node structure, while collagen showed obviously regular gaps between adjacent bundles. In the upper dermis, collagen bundles aligned in a preferred direction, while elastin showed as sparse irregular granules. This new molecular information provided fresh insight about the development process of keloid.

Depth-cumulated epithelial redox ratio and stromal collagen quantity as quantitative intrinsic indicators for differentiating normal, inflammatory, and dysplastic epithelial tissues

Multiphoton microscopy was used to isolate the intrinsic emission contribution of epithelial cellular origins and stromal collagen in normal, inflammatory, and dysplastic epithelial tissues, and quantify the depth-cumulated epithelial redox ratio and stromal collagen quantity. It was found that both inflammatory and dysplastic epithelial tissues display a large decrease in stromal collagen quantity but have very different epithelial redox ratio. These results suggest that probing differences in epithelial redox ratio in addition to stromal collagen quantity can serve as quantitative intrinsic indicators for differentiating normal, inflammatory, and dysplastic epithelial tissues.

Label-free monitoring of colonic cancer progression using multiphoton microscopy

Abstract: Real-time histology or virtual biopsy for the diagnosis of colonic cancer is of great medical significance. In this work, we show that label-free multiphoton imaging is feasible and effective in monitoring colonic cancer progression by providing cellular and subcellular details in fresh, unfixed, unstained colonic specimens. Our results also demonstrate the capability of using tissue quantitative analysis of the redox ratio for quantifying colonic cancer progression. These results suggest that multiphoton microscopy has potential to become an in situ histological tool, which is free from the labeling requirement of conventional methods, for the early diagnosis and detection of malignant lesions in the colon.

Establishing diagnostic features for identifying the mucosa and submucosa of normal and cancerous gastric tissues by multiphoton microscopy

BACKGROUND Establishing diagnostic features is essential and significant for developing multiphoton endoscopy to make an early diagnosis of gastric cancer at the cellular level. Until now, these diagnostic features have not been clearly described and understood. DESIGN Study of diagnostic features based on multiphoton microscopy (MPM). OBJECTIVE Establishing diagnostic features to identify the mucosa and submucosa of human normal and cancerous gastric tissues by investigating their multiphoton microscopic images. SETTING Fujian Normal University and Fujian Provincial Tumor Hospital. PATIENTS Ten pairs of normal and cancerous specimens were obtained from 10 patients (ages 51-68 years) undergoing radical gastrectomy. INTERVENTIONS MPM was performed on specimens. MAIN OUTCOME MEASUREMENTS Establishment of diagnostic features. RESULTS MPM has the ability to exhibit not only the mucosal and submucosal microstructures of normal and cancerous gastric tissues but also the distribution and content of abnormal cells in these 2 layers. More importantly, it can provide the diagnostic features to qualitatively and quantitatively differentiate between normal and cancerous gastric tissues. LIMITATIONS The selection bias and preparation of specimen. CONCLUSIONS These findings provide the groundwork for further establishing diagnostic criteria.