Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence

A multiphoton microscope employing second-harmonic generation (SHG) and two-photon excited fluorescence (TPF) is used for high-resolution ex vivo imaging of rabbit cornea in a backscattering geometry. Endogenous TPF and SHG signals from corneal cells and extracellular matrix, respectively, are clearly visible without exogenous dyes. Spectral characterization of these upconverted signals provides confirmation of the structural origin of both TPF and SHG, and spectral imaging facilitates the separation of keratocyte and epithelial cells from the collagen-rich corneal stroma. The polarization dependence of collagen SHG is used to highlight fiber orientation, and three-dimensional SHG tomography reveals that approximately 88% of the stromal volume is occupied by collagen lamellae.     

Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy

The photoaging process of facial skin is investigated by use of multiphoton fluorescence and second-harmonic generation (SHG) microscopy. We obtain the autofluorescence (AF) and SHG images of the superficial dermis from the facial skin of three patients aged 20, 40, and 70 years. The results show that areas of AF increase with age, whereas areas of SHG decrease with age. The results are consistent with the histological findings in which collagen is progressively replaced by elastic fibers. The AF and SHG changes in photoaging are quantified by a SHG to autofluorescence aging index of dermis (SAAID). Our results suggest that SAAID can be a good indicator of the severity of photoaging.     

In vivo assessment of human skin aging by multiphoton laser scanning tomography

Changes of dermal collagen and elastin content are characteristic for skin aging as well as for pathological skin conditions. To evaluate these changes, we used in vivo multiphoton laser tomography to measure two-photon excited autofluorescence (AF) and second harmonic generation (SHG). We tested 18 patients of all ages and calculated the SHG-to-AF aging index of dermis (SAAID). We observed a negative relationship between the SAAID and age, which was accelerated for the female (n=7) subgroup. The current findings are the first in vivo demonstration of this relationship, and they show that specific characteristics of aged skin such as the ratio of extracellular matrix components collagen and elastin can be evaluated by in vivo AF and SHG measurements using near-IR femtosecond laser pulses.     

Nonlinear spectral imaging of human normal skin,basal cell carcinoma and squamous cell carcinoma based on two-photon excited fluorescence and second-harmonic generation

In this work, we use nonlinear spectral imaging based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) for analyzing the morphology of collagen and elastin and their biochemical variations in basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and normal skin tissue. It was found in this work that there existed apparent differences among BCC, SCC and normal skin in terms of their thickness of the keratin and epithelial layers, their size of elastic fibers, as well as their distribution and spectral characteristics of collagen. These differences can potentially be used to distinguish BCC and SCC from normal skin, and to discriminate between BCC and SCC, as well as to evaluate treatment responses.     

Diagnostic value of nonlinear optical signals from collagen matrix in the detection of epithelial precancer

During precancer development in epithelium, neoplastic cells remodel the underlying stroma, for example, the basement membrane, capillaries, fibroblasts, and extracellular matrix. The purpose of this study is to investigate the relationship between the nonlinear optical signals from the collagen matrix in stroma and the progression of early epithelial carcinogenesis. Two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) signals were measured from the stroma of hamster oral cheek pouch in vivo. We found that three features, including the intensity ratio of TPEF over SHG, the spatial frequency distribution, and the texture feature of SHG images, provide the quantitative identification of epithelial precancer at different pathologic stages. We demonstrated that the combination of all three features by using a support vector machine algorithm can significantly improve the accuracy in the detection of epithelial precancer.     

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.     

Dorsal Skin Fold Chamber for High Resolution Multiphoton Imaging

The advantages offered by multiphoton microscopy enabled this technique to be applied for in vivo understanding of physiological processes. However, multiphoton intravital microscopy also requires associated technologies to be developed. In this work, we detailed the design of a dorsal skin fold chamber made of titanium alloy that allow high resolution multiphoton fluorescence and second harmonic generation (SHG) microscopy to be achieved. We demonstrate that our apparatus is capable of obtaining high resolution, images of blood vessels and collagen matrix in nude mice. Such an imaging chamber will allow physiological processes to be investigated at high resolution     

Nonlinear optical microscopy for drug delivery monitoring and cancer tissue imaging

A multimodal nonlinear optical microscope that combines coherent anti‐Stokes Raman scattering (CARS), two‐photon excitation fluorescence (TPEF), second‐harmonic generation (SHG) and sum‐frequency generation (SFG) was developed and applied to image breast cancer tissue and MCF‐7 cells as well as monitoring anticancer drug delivery in live cells. TPEF imaging showed that drugs are preferentially localized in the cytoplasm and the nuclear envelope in resistant cells. Moreover, the extracellular matrix was observed by TPEF signals arising from elastin's autofluorescence and SHG signals from collagen fibrils in breast tissue sections. Additionally, CARS signals arising from proteins and (PO₂)⁻ allowed identification of tumors. Label‐free imaging with chemical contrast of significant components of cancer cells and tissue suggests the potential of multimodal nonlinear optical microscopy for early detection and diagnosis of cancer. Copyright © 2010 John Wiley & Sons, Ltd.     

Tomographic Imaging of Collagen Fiber Orientation in Human Tissue Using Depth-Resolved Polarimetry of Second-Harmonic-Generation Light

We propose a nonlinear optical probe method to image the distribution of collagen fiber orientation in human tissue by measuring the polarization of collagen-induced second-harmonic-generation (SHG) light (SHG polarimetry). Depth-resolved SHG polarimetry, with a depth resolution of 14 μm, was used to evaluate the cross-sectional profile of collagen fiber orientation in Achilles tendon and dentin, revealing a characteristic distribution of collagen orientation along the depth direction. We evaluated the two-dimensional (2D) lateral distribution of collagen fiber orientation in back reticular dermis and anklebone by polarization-resolved SHG imaging, and confirmed an appreciable difference in the distribution profiles of the two samples. We further extended the method to a depth-resolved measurement of the three-dimensional (3D) distribution of collagen orientation in anklebone. The proposed system promises to be a powerful tool for in vivo measurement of collagen fiber orientation in human tissue.     

Two-photon autofluorescence spectroscopy and second-harmonic generation of epithelial tissue

A spectroscopy system is developed for studying the two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) of epithelial tissue in backscattering geometry. Our findings show that TPEF signals from epithelial and underlying stromal layers exhibit different spectral characteristics, providing information on the biomorphology and biochemistry of tissue. The SHG signal serves as a sensitive indicator of collagen to separate the epithelial layer from underlying stroma. The polarization dependence of the SHG signal reveals a well-ordered orientation of collagen fibers in the stromal layer. The results demonstrate the potential of depth-resolved TPEF and SHG in determining the pathology of epithelial tissue.     

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 the thermally induced structural transitions of collagen by use of second-harmonic generation microscopy

The thermal disruption of collagen I in rat tail tendon is investigated with second-harmonic generation (SHG) microscopy. We investigate its effects on SHG images and intensity in the temperature range 25 degrees-60 degrees C. We find that the SHG signal decreases rapidly starting at 45 degrees C. However, SHG imaging reveals that breakage of collagen fibers is not evident until 57 degrees C and worsens with increasing temperature. At 57 degrees C, structures of both molten and fibrous collagen exist, and the disruption of collagen appears to be complete at 60 degrees C. Our results suggest that, in addition to intensity measurement, SHG imaging is necessary for monitoring details of thermally induced changes in collagen structures in biomedical applications.     

Implementation and evaluation of a detector for forward propagated second harmonic signals

Second harmonic generation (SHG) is emerging as an alternative non-linear imaging method. The fact that most commercial multi-photon microscopes can be easily adapted to image SHG makes it appealing to explore the kind of sample information given by SHG. Here we describe an SHG detection implementation designed to optimize the collection of forward propagating light. A Hamamatsu H957-08 PMT is inserted at the back-focal plane of the condenser on an inverted Nikon TE300, and controlled by the existing electronics of a BioRad 1024MP system. Evaluation of the performance was done on common SHG generating preparations, KH₂PO₄ crystals and collagen. We concluded that positioning a detector at the back focal plane of the condenser provides a highly efficient detection system for second harmonic signals, with many advantages over a detector sited at the lamp housing.     

Two-photon excited spectroscopies of ex vivo human skin endogenous species irradiated by femtosecond laser pulses

Two-photon excited spectroscopies from ex vivo human skin are investigated by using a femtosecond laser and a confocal microscope (Zeiss LSM 510 META). In the dermis, collagen is responsible for second harmonic generation (SHG); elastin, nicotinamide adenine dinucleotide (NADH), melanin and porphyrin are the primary endogenous sources of two-photon excited autofluorescence. In the epidermis, keratin,NADH, melanin and porphyrins contribute to autofluorescence signals. The results also show that the SHG spectra have the ability to shift with the excitation wavelength and the autofluorescence spectra display a red shift of the spectral peaks when increasing the excitation wavelength. These results may have practical implications for diagnosis of skin diseases.     

Second-harmonic generation as a DNA malignancy indicator of prostate glandular epithelial cells

This paper first demonstrates second-harmonic generation (SHG) in the intact cell nucleus, which acts as an optical indicator of DNA malignancy in prostate glandular epithelial cells. Within a scanning region of $2.7~\mu$m$\times 2.7~\mu$m in cell nuclei, SHG signals produced from benign prostatic hyperplasia (BPH) and prostate carcinoma (PC) tissues (mouse model C57BL/6) have been investigated. Statistical analyses ($t$ test) of a total of 405 measurements (204 nuclei from BPH and 201 nuclei from PC) show that SHG signals from BPH and PC have a distinct difference ($p < 0.05)$, suggesting a potential optical method of revealing very early malignancy in prostate glandular epithelial cells based upon induced biochemical and/or biophysical modifications in DNA.     

肿瘤细胞DNA样品基于二次谐波发生(SHG)的非线性光谱学成像

二次谐波的产生是一个二阶非线性光学过程,这个过程只发生在中心对称系数不为零的非中心对称区域。利用二次谐波显微技术可以对各种具有内源性信号的生物组织进行无损伤实时成像,如结缔组织的胶原纤维或肌细胞的肌动球蛋白。在生物化学和结构生物学中,虽然DNA是由脱氧核糖核苷酸构成而蛋白质是由氨基酸残基组成,但是DNA和蛋白质大分子高级结构的形成机制是相似的。利用光谱学成像技术对不同DNA样品进行检测,获取DNA样品的SHG信号并进行高解析度成像。这些DNA样品包括基因组DNA溶液、细胞核提取物以及培养细胞的细胞核。实验结果表明在常规条件下可以获得基因组DNA溶液和细胞核提取物的SHG信号,但几乎观测不到来自培养细胞核区的SHG信号。通过在培养基中添加少量无水乙醇(体积比小于5%),可以在培养细胞的细胞核区域检测到SHG信号。推测在培养细胞中乙醇和DNA相互作用引起DNA分子构象发生变化,这些变化可能导致了DNA分子非线性光学性质的改变。     

Macromolecular structure of cellulose studied by second-harmonic generation imaging microscopy

The macromolecular structure of purified cellulose samples is studied by second-harmonic generation (SHG) imaging microscopy. We show that the SHG contrast in both Valonia and Acetobacter cellulose strongly resembles that of collagen from animal tissues, both in terms of morphology and polarization anisotropy. Polarization analysis shows that microfibrils in each lamella are highly aligned and ordered and change directions by 90 degrees in adjacent lamellae. The angular dependence of the SHG intensity fits well to a cos2 theta distribution, which is characteristic of the electric dipole interaction. Enzymatic degradation of Valonia fibers by cellulase is followed in real time by SHG imaging and results in exponential decay kinetics, showing that SHG imaging microscopy is ideal for monitoring dynamics in biological systems.     

Study of van Gieson''s picrofuchsin staining on second-harmonic generation in type I collagen

Second-harmonic generation （SHG） microscopy is a recently developed nonlinear optical imaging modality for imaging tissue structures with submicron resolution and is a potent tool for visualizing pathological effects of diseases. In this letter, we present our investigation on the influence of van Gieson＇s （VG） alcoholic pierofuchsin staining on SHG in type I collagen （from tendon-rich C57BL/6）. Multi-channel imaging and spectra analysis show that the strong SHG signal produced in fresh collagen type I fiber has been greatly suppressed after VG staining, which indicates that staining may induce the structural or characteristic changes of SHG-dependent crystal formed by collagen constituents, such as glycine, proline, and hydroxyproline.     

Refractive index dispersion properties of collagen for microscopic second-harmonic generation

We theoretically simulate second-harmonic generation (SHG) in collagen under linearly polarized focused laser beam. With this model, the effects of numerical aperture (NA) and refractive index dispersion on SHG emission have been investigated. Dispersion properties of collagen are significant under incident wavelength in the visible range. Our results show that the efficient SHG is obtained by controlling the NA, and the higher NA is a necessity when the dispersion effect is considered. Our theoretical simulation results provide useful clues for experimental study of microscopic SHG emission in collagen excited by focused beam.     

Spectral-domain optical coherence phase and multiphoton microscopy

We describe simultaneous quantitative phase contrast and multiphoton fluorescence imaging by combined spectral-domain optical coherence phase and multiphoton microscopy. The instrument employs two light sources for efficient optical coherence microscopic and multiphoton imaging and can generate structural and functional images of transparent specimens in the epidirection. Phase contrast imaging exhibits spatial and temporal phase stability in the subnanometer range. We also demonstrate the visualization of actin filaments in a fixed cell specimen, which is confirmed by simultaneous multiphoton fluorescence imaging.