This PDF file contains the front matter associated with SPIE Proceedings Volume 8577, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.

Stokes Shift Spectroscopy (S3) offers a novel way to rapidly measure spectral fingerprints of complex molecular mixtures in tissue. The changes of key fluorophores from normal state to the malignant state can be reflected by alteration of Stokes Shift Spectra (S3 spectra). S3 measurements can be used to acquire enough information of different key fluorophores from one spectrum to speed up spectral acquisition time. In this study, we demonstrate the usefulness of the S3 technique to distinguish the malignant tissue from the normal prostate and breast tissues. The optimal wavelength shift constant (Δλ_c) of S3 spectra measurements for prostate/breast cancer detection were determined to be 40 nm. The underlying physical and biological basis for S3 is discussed. For the first time, our work explicitly discloses how and why S3 is supreme in comparison with other conventional spectroscopic techniques.

The aim of this case study was to explore the relationship between porphyrins and colon adenocarcinoma, and to examine the potential of porphyrin-induced fluorescence for the diagnosis of colon cancer. Further studies were carried on 8 cases ex vivo colon adenocarcinoma samples which exceptionally exhibited 635 nm fluorescence emission under 405 nm excitation. The time-resolved fluorescence spectra at 635 nm emission under 405 nm excitation were also measured and two-exponential decay fitting was performed to determine the fluorescence lifetime at 635 nm emission. Significant difference was observed between the spectra of normal and cancer tissues, which included an emission peak at 635 nm under the excitation wavelengths of 405 nm. There was also a significant difference between the fluorescence lifetimes of 635 nm emission of the normal tissue and cancer tissue (P<0.05). These results demonstrate that the spectroscopic analysis method allows a selective detection of adenocarcinoma tissues. This spectral profile and lifetime of the red fluorescence resemble that of porphyrins, which suggests that porphyrin fluorescence may be a useful biomarker for characterizing colon cancers of certain patient populations.

Lumpectomy, in conjunction with radiation and chemotherapy drugs, together comprise breast-conserving treatment as an alternative to total mastectomy for patients with breast tumors. The tumor is removed in surgery and sent for pathology processing to assess the margins, a process that takes at minimum several hours, and generally days. If the margins are not clear of tumor, the patient must undergo a second surgery to remove residual tumor. This re-excision rate varies by institution, but can be as high as 60%. Currently, no intraoperative microscopic technique is used routinely to examine tumor margins in breast tissue. A new technique for rapidly scanning large areas of tissue has been developed, called confocal strip scanning, which provides high resolution and seamless mosaics over large areas of intact tissue, with nuclear and cellular resolution and optical sectioning of about 2 microns. Up to 3.5 x 3.5 cm² of tissue is imaged in 13 minutes at current stage speeds. This technique is demonstrated in freshly excised breast tissue, using a mobile confocal microscope stationed in our pathology laboratory. Twenty-five lumpectomy and mastectomy cases were used as a testing ground for reflectance and fluorescence contrast modes, resolution requirements and tissue fixturing configurations. It was concluded that fluorescent imaging provides the needed contrast to distinguish ducts and lobules from surrounding stromal tissue. Therefore the system was configured with 488 nm illumination, with acridine orange fluorescent dye for nuclear contrast, with the aim of building an image library of malignant and benign breast pathologies.

Urine is one of the diagnostically important bio fluids, as it has many metabolites and some of them are native fluorophores. There may be a variation in the distribution and the physiochemical properties of the fluorophores during any metabolic change and pathologic conditions. Native fluorescence spectroscopy has been considered as a promising tool to characterize the fluorophores present in the urine. In this study, we aimed at characterizing the urine of both normal and patients with confirmed cancer using steady state and time-resolved fluorescence spectroscopy at 280 nm and 350 nm excitation. It is observed that the metabolites indoxyl sulphate and neopterin and its derivatives are responsible for altered spectral signatures at 280 nm, and 350 nm excitation. The overall spectral data were subjected to Principal Component Analysis and the resultant components were used as input in the linear discriminant analysis. As a total, 84% and 81.8% of samples were correctly classified at 280 nm and 350 nm respectively.

Time-resolved fluorescence was used to investigate malignant and normal adjacent breast tissues stained with a conjugate of indocyanine green and octreotate. A marked increase in fluorescence lifetime intensity was seen in the breast cancer sample compared to the normal sample. The fluorescent lifetimes were also investigated and showed similar fluorescence decay curves in stained malignant and normal breast tissue. These results confirm that somatostatin receptors occur on human breast carcinomas, suggest that the presence of somatostatin receptors should be investigated as a marker of breast cancer aggressiveness, and suggest that this conjugate might be used to detect the presence of residual breast cancer after surgery, allowing better assessment of tumor margins and reducing the need for second or repeat biopsies in selected patients. These results may also provide clues for designing future treatment options for breast cancer patients.

Resonance Raman (RR) has the potential to reveal the differences between cancerous and normal breast and brain tissues in vitro. This differences caused by the changes of specific biomolecules in the tissues were displayed in resonance enhanced of vibrational fingerprints. It observed that the changes of reduced collagen contents and the number of methyl may show the sub-methylation of DNA in cancer cells. Statistical theoretical models of Bayesian, principal component analysis (PCA) and support vector machine (SVM) were used for distinguishing cancer from normal based on the RR spectral data of breast and meninges tissues yielding the diagnostic sensitivity of 80% and 90.9%, and specificity of 100% and 100%, respectively. The results demonstrated that the RR spectroscopic technique could be applied as clinical optical pathology tool with a high accuracy and reliability.

Confocal resonance Raman (RR) spectra were collected from single proliferating cells and analyzed to detect spectral patterns that are cell-cycle dependent, as a consequence of cellular proliferation — normal or abnormal. The cells’ biochemical age at each time point was confirmed by immunohistochemical staining to identify the presence or absence of cellular components that appear and/or disappear as the cells proceed through the cell-cycle. The RR spectra were collected and compared for each time point as the cells proceeded through the cell cycle to determine what spectral vibrational patterns are cell-cycle dependent. In this study, the question is whether the cell-cycle dependent RR spectral patterns of the vibrational modes observed in proliferating normal and neoplastic single cells are due to a state of cancer or are simply the consequences of the cells’ changing internal biochemistry due to the process of cellular proliferation --- normal or abnormal.

The study of bone composition is very important for the development of new technologies, for instance, the improvement of biocompatible implants and artificial bone matrix. Some kinds of bone matrix as tibia might be regenerated by two different routes, the endochondral and the intramembranous. In this work We analyze the composition of regenerated tibia by the two mentioned routes. They are compared with the composition of the healthy portion of a normal tissue. Our results show mainly differences in the quantity of lipids, water and in the vibrations of Amide I and Amide II.

The liver is responsible for several basic functions in human body how the syntheses of the most main proteins and degradation process of toxins, drugs and alcohols. In present days, the viral hepatitis C is one of the highest causes of chronic hepatic illness worldwide, affecting around 3% of the world population. The liver biopsy is considered the gold standard for diagnosing hepatic fibrosis; however, the biopsies may be questioned because of potential sampling error, morbidity, possible mortality and relatively high costs. Spectroscopy techniques such as Raman spectroscopy have been used for diagnosis of human tissues, with favorable results. Raman spectroscopy has been employed to distinguish normal from hepatic lesions through spectral features mainly of proteins, nucleic acids and lipids. In this study, eleven patients with diagnoses of chronic hepatitis C underwent hepatic biopsies having two hepatic fragments collected: one was scored through METAVIR system and the other one was submitted to near-infrared Raman spectroscopy using a dispersive spectrometer (830 nm wavelength, 300 mW laser power and 20 s exposure time). Five spectra were collected in each fragment and submitted to Principal Components Analysis (PCA). Results showed a good correlation between the Raman spectroscopy features and the stage of hepatic fibrosis and inflammation. PCA showed that samples with higher degree of fibrosis presented higher amount of protein features (collagen), whereas samples of higher degree of inflammation presented higher features of hemoglobin, in accordance to the expected evolution of the chronic hepatitis. It has been found an important biomarker for the beginning of hepatic lesion (quinone) with a spectral feature at 1595 cm^-1.

A rapid image-guided Raman endoscopy system integrated with on-line diagnostic scheme is developed for in vivo Raman tissue diagnosis (optical biopsy) in the upper GI during clinical gastrointestinal endoscopy under multimodal wide-field imaging guidance. The real-time Raman endoscopy technique was tested prospectively on new gastric patients (n=4) and could identify dysplasia in vivo with sensitivity of 81.5% (22/27) and specificity of 87.9% (29/33). This study realizes for the first time the novel image-guided Raman endoscopy as a screening tool for real-time, online diagnosis of gastric cancer and precancer in vivo at endoscopy.

The combination of Raman spectroscopy with fiber optic probes enables analyzing the biochemical composition of tissues without markers in a non-destructive way. A small diameter (1 mm) fiber optic probe with one excitation fiber, 11 detection fibers and integrated filters (Emvision, USA) was recently coupled to a Raman spectrometer (Kaiser Optical Systems) to study excised arteries ex vivo and rabbit arteries in vivo. The current contribution introduces a novel fiber optic Raman probe with in-line fiber Bragg gratings (FBGs) as notch filter in the collection path. Multi-core single-mode fibers (MCSMF) were drawn integrating 19 and 61 single-mode cores to improve collection efficiency. Raman probes were assembled with one fiber for excitation and six MCSMF with inscribed FBGs for collection. The diameter of the 6 around 1 geometry can be reduced down to 0.375 mm. Background suppression, collection efficiency and distance dependence of the probes were characterized and first Raman measurements are presented. The advantages of the novel probes are discussed and further applications to Raman-on-chip detection schemes are described.

Our goal is to use optical coherence tomography (OCT) and multiphoton microscopy (MPM) to detect early tumor development in a mouse model of ovarian neoplasia. We hope to use information regarding early tumor development to create a diagnostic test for high-risk patients. In this study we collect in vivo images using OCT, second harmonic generation and two-photon excited fluorescence from non-vinylcyclohexene diepoxide (VCD)-dosed and VCD-dosed mice. VCD causes follicular apoptosis (simulating menopause) and leads to tumor development. Using OCT and MPM we visualized the ovarian microstructure and were able to see differences between non-VCD-dosed and VCD-dosed animals. This leads us to believe that OCT and MPM may be useful for detecting changes due to early tumor development.

Precise information on dispersion of the nonlinear optical susceptibility of Raman active media is essential in order to get an insight into physics and chemistry of intra- and inter-molecular interactions. We propose and experimentally demonstrate a method that is capable of resolving both real and imaginary parts of third-order nonlinearity (χ⁽³⁾) in the vicinity of Raman resonances. Dispersion of χ⁽³⁾ can be obtained from a medium probed within microscopic volumes with a spectral resolution of better than 0.1 cm^-1 thus making our approach an essential tool in quantitative microscopic characterization of complex biological media. Time-domain CARS transients traced with femtosecond pulses within orders of magnitude in the signal decay can lead to resolution of fine spectral features in χ⁽³⁾ dispersion that can not be reliably detected by frequency-domain Raman based spectroscopy/microscopy techniques, including coherent methods. We will present results of the method’s application in biological cells and tissue. Namely, we accessed a protein line at 1245 cm^-1 in E-coli cell, major DNA and protein lines in red blood cells and triglyceride Raman active peaks in fat tissue.

Oral cancer is one of the most frequently diagnosed human cancers and leading causes of cancer death all over the world, but the prognosis and overall survival rate are still poor because of delay in diagnosis and lack of early intervention. The failure of early diagnosis is due to insufficiency of proper diagnostic and screening tools and most patients are reluctant to undergo biopsy. Optical virtual biopsy techniques, for imaging cells and tissues at microscopic details capable of differentiating benign from malignant lesions non-invasively, are thus highly desirable. A novel multi-harmonic generation microscope, excited by a 1260 nm Cr:forsterite laser, with second and third harmonic signals demonstrating collagen fiber distribution and cell morphology in a sub-micron resolution, was developed for clinical use. To achieve invivo observation inside the human oral cavity, a small objective probe with a suction capability was carefully designed for patients’ comfort and stability. By remotely changing its focus point, the same objective can image the mucosa surface with a low magnification, illuminated by side light-emitting diodes, with a charge-coupled device (CCD) for site location selection before the harmonic generation biopsy was applied. Furthermore, the slow galvanometer mirror and the fast resonant mirror provide a 30 fps frame rate for high-speed real-time observation and the z-motor of this system is triggered at the same rate to provide fast 3D scanning, again ensuring patients’ comfort. Focusing on the special cytological and morphological changes of the oral epithelial cells, our preliminary result disclosed excellent consistency with traditional histopathology studies.

We report here a study of confocal microscope images to classify cervical precancers by a multifractal analysis. This study is performed using an inverted confocal microscope with laser scanning fluorescence imaging. The periodic structure of collagen present in the stromal region of cervical tissue gets disordered with progress in grade of dysplasia. This disorder is investigated through the β-exponent of a Discrete Fourier Transform (DFT) of the confocal images, enabling us to discriminate between the lowest and highest grades of dysplasia in human cervical tissue sections. The Holder exponent from 2D images further classifies various grades of dysplasia from normal tissue sections though Gd3 and Gd1 are indistinguishable. DFT however, clearly distinguishes Gd3 from Gd1. In addition to stromal images, epithelial images were also investigated for better classification. The cellular density of epithelium increases with depth for various grades of dysplasia and is not uniform. The Holder exponent, which measures multifractality, is higher for dysplastic tissue sections than for normal ones because of the above morphological differences. Extraction of subtle fluctuations from optical images through multifractal studies promise to be a powerful diagnostic technique.

The alteration of native fluorophores among different types of cancer cell lines was investigated by the fluorescence spectroscopy. Different types of cancer cell lines with different risk levels, such as moderate metastatic (DU-145) and advanced metastatic (PC-3) cell lines as well as normal cell line (Fibroblast), were excited by the selective excitation wavelength of 300 nm to explore changes of the relative contents of tryptophan and NADH using principal component analysis (PCA). The higher relative content of tryptophan was observed in the advanced metastatic cancer cell lines in comparison with the moderate metastatic and non aggressive cell lines.