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This PDF file contains the front matter associated with SPIE Proceedings Volume 7560, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.
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Optical spectroscopic methods are being contemplated as adjunct/ alternative to existing 'Gold standard' of cancer
diagnosis, histopathological examination. Several groups are actively pursuing diagnostic applications of Ramanspectroscopy
in cancers. We have developed Raman spectroscopic models for diagnosis of breast, oral, stomach, colon
and larynx cancers. So far, specificity and applicability of spectral- models has been limited to particular tissue origin. In
this study we have evaluated explicitly of spectroscopic-models by analyzing spectra from already developed spectralmodels
representing normal and malignant tissues of breast (46), cervix (52), colon (25), larynx (53), and oral (47).
Spectral data was analyzed by Principal Component Analysis (PCA) using scores of factor, Mahalanobis distance and
Spectral residuals as discriminating parameters. Multiparametric limit test approach was also explored. The preliminary
unsupervised PCA of pooled data indicates that normal tissue types were always exclusive from their malignant
counterparts. But when we consider tissue of different origin, large overlap among clusters was found. Supervised
analysis by Mahalanobis distance and spectral residuals gave similar results. The 'limit test' approach where
classification is based on match / mis-match of the given spectrum against all the available spectra has revealed that
spectral models are very exclusive and specific. For example breast normal spectral model show matches only with
breast normal spectra and mismatch to rest of the spectra. Same pattern was seen for most of spectral models. Therefore,
results of the study indicate the exclusiveness and efficacy of Raman spectroscopic-models. Prospectively, these findings
might open new application of Raman spectroscopic models in identifying a tumor as primary or metastatic.
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A near-infrared Raman spectroscopy system integrated with multimodal endoscopic imaging has been developed for the early noninvasive in vivo diagnosis and detection of gastric malignancies. High-quality in vivo Raman spectra in the range 800-1800 cm-1 can be acquired from gastric normal and premalignant (dysplastic) mucosal tissue within 1 second under the guidance of white-light and narrow-band gastroscopic imaging during clinical gastroscopy. Prominent differences in Raman spectral shapes and intensities are observed between normal and dysplastic gastric mucosal tissue, particularly in the spectral ranges 800-900, 1250-1450 and 1600-1800 cm-1, which primarily contain signals related to proteins, nucleic acids and lipids. The empirical intensity ratio algorithm I875/I1450 classifies in vivo Raman spectra of dysplasia with a sensitivity of 100% and specificity of 100%. Our initial investigations show that in vivo Raman spectroscopy in conjunction with multimodal endoscopic imaging modalities holds a great promise for improving the early diagnosis of gastric malignancies.
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Breast cancer is the most frequent cancer type in women Worldwide. Sensitivity and specificity of clinical breast
examinations have been estimated from clinical trials to be approximately 54 % and 94 %, respectively. Further,
approximately 95 % of all positive breast cancer screenings turn out to be false-positive. The optimal method for early
detection should be both highly sensitive to ensure that all cancers are detected, and also highly specific to avoid the
humanistic and economic costs associated with false-positive results. In vivo optical spectroscopy techniques, Raman in
particular, have been pointed out as promising tools to improve the accuracy of screening mammography. The aim of
the present study was to apply FT-Raman spectroscopy to discriminate normal and adenocarcinoma breast tissues of
Sprague-Dawley female rats. The study was performed on 32 rats divided in the control (N=5) and experimental (N=27)
groups. Histological analysis indicated that mammary hyperplasia, cribriform, papillary and solid adenocarcinomas were
found in the experimental group subjects. The spectral collection was made using a commercial FT-Raman
Spectrometer (Bruker RFS100) equipped with fiber-optic probe (RamProbe) and the spectral region between 900 and
1800 cm-1 was analyzed. Principal Components Analysis, Cluster Analysis, and Linear Discriminant Analysis with
cross-validation were applied as spectral classification algorithm. As concluding remarks it is show that normal and
adenocarcinoma tissues discriminations was possible (correct proportion for Transcutaneous collection mode was
80.80% and for "Open Sky" mode was 91.70%); however, a conclusive diagnosis among the four lesion subtypes was
not possible.
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The contribution demonstrates how the molecular contrast of Fourier transform infrared
(FTIR), Raman and coherent anti-Stokes Raman scattering (CARS) microscopic imaging can be applied
for the histopathological assessment of brain tumors. Human brain tissue specimens were obtained from
patients undergoing neurosurgery. Thin sections of control brain tissue from an epilepsy patient and
tumor tissue from a meningioma patient were prepared on calciumfluoride slides which were appropriate
substrates for data acquisition in transmission and reflection mode. All CARS images correlate well with
the FTIR and Raman images. Whereas CARS images were collected within seconds, exposure times
were minutes for FTIR imaging and hours for Raman imaging. CARS images in the interval 2750-3000
cm-1 mainly probed spectral contributions of lipids which are important diagnostic markers of brain
tumors. It was demonstrated that the CARS profile in the interval 2750-3000 cm-1 differed between the
control sample and meningioma. Full spectral information could be extracted from Raman and FTIR
images that enabled to distinguish different tissue types in brain tumors. Based on the current results we
suggest a complementary application of FTIR, Raman and CARS imaging. FTIR and Raman imaging
defines spectral regions and spectral markers that are essential for tissue classification. CARS images at
different Stokes shifts or in the multiplex mode probe these spectral descriptors at video-time frame rates.
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Raman spectroscopy is a powerful tool for analysis of the chemical composition in living tissue and cells without
destructive processes such as fixation, immunostaining, and fluorescence labeling. Raman microspectroscopic technique
enables us to obtain a high quality spectrum from a single living cell. We demonstrated in situ cell cycle analysis with
Raman microspectroscopy with the excitation wavelength of 532 nm. Cell cycle phases, G0/G1 and G2/M were able to
be identified in the present study. The result of in situ Raman analysis was evaluated with flow cytometry analysis.
Although the Raman spectra of living cells showed complex patterns during cell cycle, several Raman bands could be
useful as markers for the cell cycle identification. A single cell analysis using Raman microspectroscopy predicted a
possibility to observe directly molecular dynamics intracellular molecules of proteins, lipids and nucleic acids. Our
current study focused on cytoplasm region and resonant Raman signals of cytochrome c in mitochondrion, and discussed
how the Raman signals from cellular components contribute to the Raman spectral changes in cell cycle change in the
human living cell (lung cancer cell).
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Monitoring the sterility of cell or tissue cultures is of major concern, particularly in the fields of regenerative medicine
and tissue engineering when implanting cells into the human body. Our sterility-control system is based on a Raman
micro-spectrometer and is able to perform fast sterility testing on microliters of liquid samples. In conventional sterility
control, samples are incubated for weeks to proliferate the contaminants to concentrations above the detection limit of
conventional analysis. By contrast, our system filters particles from the liquid sample. The filter chip fabricated in
microsystem technology comprises a silicon nitride membrane with millions of sub-micrometer holes to retain particles
of critical sizes and is embedded in a microfluidic cell specially suited for concomitant microscopic observation. After
filtration, identification is carried out on the single particle level: image processing detects possible contaminants and
prepares them for Raman spectroscopic analysis. A custom-built
Raman-spectrometer-attachment coupled to the
commercial microscope uses 532nm or 785nm Raman excitation and records spectra up to 3400cm-1. In the final step,
the recorded spectrum of a single particle is compared to an extensive library of GMP-relevant organisms, and
classification is carried out based on a support vector machine.
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Urinary tract infection diagnosis and antibiogram require a minimum of 48 hours using standard laboratory practice. This
long waiting period contributes to an increase in recurrent infections, rising health care costs, and a growing number of
bacterial strains developing resistance to antibiotics. In this work, Surface Enhanced Raman Spectroscopy (SERS) was
used as a novel method for classifying bacteria and determining their antibiogram. Five species of bacteria were
classified with > 90% accuracy using their SERS spectra and a classification algorithm involving novel feature extraction
and discriminant analysis. Antibiotic resistance or sensitivity was determined after just a two-hour exposure of bacteria
to ciprofloxacin (sensitive) and amoxicillin (resistant) and analysis of their SERS spectra. These results can become the
basis for the development of a novel method that would provide same day diagnosis and selection of the most
appropriate antibiotic for most effective treatment of a urinary tract infection.
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Here we present our latest results concerning the application of Raman microspectroscopy in combination with
innovative chemometrics to characterize biological cells. The first part of this manuscript deals with the application of
micro-Raman spectroscopy to identify microbial contaminations while the main focus within the second part of this
presentation is concerned with Raman studies on eukaryotic cells where we will report about the development of an
algorithm to differentiate between breast cancer cells and normal epithelial cells.
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The overexpression of HER2 (human epidermal growth factor receptor 2) in breast cancer is associated with
increased disease recurrence and worse prognosis. Current diagnosis of HER2 positive breast cancer is time
consuming with an estimated 20% inaccuracy. Raman spectroscopy is a proven method for pathological diagnosis
based on the molecular composition of tissues. This study aimed to determine the feasibility of Raman spectroscopy
to differentially identify the amplification of HER2 in cells. Three cell lines including BT474 (HER2 overexpressing
breast cancer cell), MCF-10A (human breast epithelial cell), and MCF-10A with overexpressing HER2, were
investigated using a bench top confocal Raman system. A diagnostic algorithm based on generalized linear model
(GLM) with elastic-net penalties was established to discriminate 318 spectra collected from the cells, and to identify
the spectra regions that differentiate the cell lines. The algorithm was able to differentially identify BT474 breast
cancer cells with an overall sensitivity of 100% and specificity of 99%. The results demonstrate the capability of
Raman spectroscopy to determine HER2 status in cells. Raman spectroscopy shows promise for application in the
diagnosis of HER2 positive breast cancer in clinical practice.
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Birds are traditionally classified as male or female based on their anatomy and plumage color as judged by the human
eye. Knowledge of a bird's gender is important for the veterinary practitioner, the owner and the breeder. The accurate
gender determination is essential for proper pairing of birds, and knowing the gender of a bird will allow the veterinarian
to rule in or out gender-specific diseases. Several biochemical methods of gender determination have been developed for
avian species where otherwise the gender of the birds cannot be determined by their physical appearances or
characteristics. In this contribution, we demonstrate that FT-IR spectroscopy is a suitable tool for a quick and objective
determination of the bird's gender. The method is based on differences in chromosome size. Male birds have two Z
chromosomes and female birds have a W-chromosome and a Z-chromosome. Each Z-chromosome has approx.
75.000.000 bps whereas the W-chromosome has approx. 260.00 bps. This difference can be detected by FT-IR
spectroscopy. Spectra were recorded from germ cells obtained from the feather pulp of chicks as well as from the
germinal disk of fertilized but non-bred eggs. Significant changes between cells of male and female birds occur in the
region of phosphate vibrations around 1080 and 1120 cm-1.
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The continuous monitoring of the concentration of glucose provides an essential tool for the improved glycemic
control for people with diabetes. Most of the present approaches of transcutaneous, continuous glucose monitors
are based on chemical detection and require the insertion of reagents into the body. In contrast, we aim at the
reagent-free monitoring of glucose by means of mid-infrared spectroscopy.
A quantum cascade laser provides narrow band radiation at wavelengths around the absorption bands of glucose
(≈ 10 μm). At the same time it yields sufficient energy to allow for a good signal-to-noise ratio in transmission
measurements despite the strong background absorption of water.
We investigated various concepts for the sensor head based on the light-guiding properties and handling of
materials such as AgCl/AgBr or silicon. In-vitro experiments were performed using a custom-made, temperaturestabilized
measurement flow chamber. In preparation for future in-vivo applications first results of biotoxicity
tests of the fiber sensors are given.
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We report on ultrafast visible pump/mid-infrared probe spectroscopy of the carboxy form of heme proteins by
employing the recently developed chirped-pulse upconversion technique, which allows both high resolution and
sensitivity over an extremely broad spectral range. Commonly, the bleach signal due to ligand dissociation and
the incipient docking-site absorption signal, being about 200 cm-1 apart and differing by more than an order of
magnitude in absorptivity, are studied in separate experiments. We here monitor them simultaneously, allowing
a direct observation and a concurrent analysis of the initial processes after photoinduced ligand dissociation, for
instance, the formation of hot vibrational bands.
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Thyroid gland is a small gland in the neck consisting of two lobes connected by an isthmus. Thyroid's main function is to
produce the hormones thyroxine (T4), triiodothyronine (T3) and calcitonin. Thyroid disorders can disturb the production of
these hormones, which will affect numerous processes within the body such as: regulating metabolism and increasing
utilization of cholesterol, fats, proteins, and carbohydrates. The gland itself can also be injured; for example, neoplasias,
which have been considered the most important, causing damage of to the gland and are difficult to diagnose. There are
several types of thyroid cancer: Papillary, Follicular, Medullary, and Anaplastic. The occurrence rate, in general is between 4
and 7%; which is on the increase (30%), probably due to new technology that is able to find small thyroid cancers that may
not have been found previously. The most common method used for thyroid diagnoses are: anamnesis, ultrasonography, and
laboratory exams (Fine Needle Aspiration Biopsy- FNAB). However, the sensitivity of those test are rather poor, with a high
rate of false-negative results, therefore there is an urgent need to develop new diagnostic techniques. Raman spectroscopy
has been presented as a valuable tool for cancer diagnosis in many different tissues. In this work, 27 fragments of the thyroid
were collected from 18 patients, comprising the following histologic groups: goitre adjacent tissue, goitre nodular tissue,
follicular adenoma, follicular carcinoma, and papillary carcinoma. Spectral collection was done with a commercial FTRaman
Spectrometer (Bruker RFS100/S) using a 1064 nm laser excitation and Ge detector. Principal Component Analysis,
Cluster Analysis, and Linear Discriminant Analysis with cross-validation were applied as spectral classification algorithm.
Comparing the goitre adjacent tissue with the goitre nodular region, an index of 58.3% of correct classification was obtained.
Between goitre (nodular region and adjacent tissue) and papillary carcinoma, the index of correct classification was 64.9%,
and the classification between benign tissues (goitre and follicular adenoma) and malignant tissues (papillary and follicular
carcinomas), the index was 72.5%.
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Preterm labor is the second leading cause of neonatal mortality and leads to a myriad of complications like delayed
development and cerebral palsy. Currently, there is no way to accurately predict preterm labor, making its prevention
and treatment virtually impossible. While there are some at-risk patients, over half of all preterm births do not fall into
any high-risk category. This study seeks to predict and prevent preterm labor by using Raman spectroscopy to detect
changes in the cervix during pregnancy. Since Raman spectroscopy has been used to detect cancers in vivo in organs like
the cervix and skin, it follows that spectra will change over the course of pregnancy. Previous studies have shown that
fluorescence decreased during pregnancy and increased during post-partum exams to pre-pregnancy levels. We believe
significant changes will occur in the Raman spectra obtained during the course of pregnancy. In this study, Raman
spectra from the cervix of pregnant mice and women will be acquired. Specific changes that occur due to cervical
softening or changes in hormonal levels will be observed to understand the likelihood that a female mouse or a woman
will enter labor.
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Comparative Raman spectra of ex vivo, saline-perfused, injured and healthy rat spinal cord as well as experiments using
enzymatic digestion suggest that proteoglycan over expression may be observable in injured tissue. Comparison with
authentic materials in vitro suggest the occurrence of side reactions between products of cord digestion with
chondroitinase (cABC) that produce lactones and similar species with distinct Raman features that are often not
overlapped with Raman features from other chemical species. Since the glial scar is thought to be a biochemical and
physical barrier to nerve regeneration, this observation suggests the possibility of using near infrared Raman
spectroscopy to study disease progression and explore potential treatments ex vivo and if potential treatments can be
designed, perhaps to monitor potential remedial treatments within the spinal cord in vivo.
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In this contribution we present results of tip-enhanced Raman spectroscopy (TERS) measurements on a single crystal of
cystine. The observed spectral features lead to the conclusion that the S-S bond was cleaved due to interactions with the
silver tip. The spectra differ strongly depending on the site of the molecule interacting with the silver tip. Additionally,
first TERS spectra bovine serum albumin (BSA) indicate, that the disulfide bridges of cystine in the protein remain
unchanged. This fact can be used for a structural discussion of the secondary structure of the protein. A thorough band
assignment was feasible based on an extensive collection of previously obtained TERS spectra of selected amino acids.
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Ultra-low spatial resolution Raman (ULSRR) mapping using fibre probes has been performed on mammalian and human
tissues. This will provide an understanding of the potential for in vivo surveillance of the lining of organs using such a
technique and for identifying abnormal tissues such as residual tumours within a surgical field.
The aim of the study was to create Raman probe map images of excised oesophageal specimens following radical and
palliative oesophagectomy procedures. A reproducible mapping grid was placed over the excised tissue surface and
Raman mapping at 830nm performed at regular intervals to provide images of 200 pixels over the region of interest.
Principal component analysis was used to create pseudocolour score images of both porcine phantoms and a human
resected oesophagus.
A principal component fed linear discriminant (LD) classification model of 72 biopsy samples from 35 patients was
created using a novel single fibre Raman probe. A subset of the training dataset was used to populate a matrix of 200
pixels to simulate a Raman probe map. Spectra from the simulated map were then projected onto the LD model and a
pseudocolour LD pathology map created.
Delineation of clinically significant pathology groups was demonstrated therefore this study has shown the feasibility of
in vivo ULSRR for margin assessment using a Raman probe.
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Light can be coupled into imaging spectrographs through the use of fiber-optic bundles. Ideally, the collected
spectra from adjacent optical fibers should be resolved and independent. However, this assumption breaks down if a
partial overlap of adjacent fibers on the detector CCD results from either diffraction or uncorrected monochromatic
aberrations. In addition, spectral mixing can be caused by optical cross-talk among tightly packed fibers, particularly if
the fiber-optic buffer has been removed in order to use the CCD area more efficiently and increase the linear fiber
packing density. These coupling effects can become sources of systematic error, especially when fiber bundles are used
for imaging or when branches of a multi-leg fiber array are interrogating different samples. Coupling errors can mix
spectra from different spatial regions of the sample. In this paper, the mixing in fiber bundles will be assessed, and
mathematical methods to resolve overlapped signals and correct for signal mixing will be discussed. Using data
processing to correct for optical coupling errors, instead of physically changing the spacing between fibers in the bundle,
allows for the efficient use of the limited spectrograph detector area by having a larger number of fibers.
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Human transdermal in vivo spectroscopic applications for tissue analysis involving near infrared (NIR) light often must
contend with broadband NIR fluorescence that, depending on what kind of spectroscopy is being employed, can degrade
signal to noise ratios and dynamic range. Such NIR fluorescence, i.e. "autofluorescence" is well known to originate in
blood tissues and various other endogenous materials associated with the static tissues. Results of recent experiments on
human volar side fingertips in vivo are beginning to provide a relative ordering of the contributions from various
sources. Preliminary results involving the variation in the bleaching effect across different individuals suggest that for
830 nm excitation well over half of the total fluorescence comes from the static tissues and remainder originates with the
blood tissues, i.e. the plasma and the hematocrit. Of the NIR fluorescence associated with the static tissue, over half
originates with products of well-known post-enzymatic glycation reactions, i.e. Maillard chemistry, in the skin involving
glucose and other carbohydrates and skin proteins like collagen and cytosol proteins.
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In this paper we investigated the NIR absorption spectrum of aqueous glucose by using a FTIR spectrometer after
glucose solution passing through a permanent magnetic field. When glucose solution flows through the permanent
magnetic field, some of the aqueous glucose molecules are magnetized and glucose absorption is enhanced in the NIR
range of 1000-2500nm. The experimental results show that glucose absorbance in its combination region and first
overtone region is increased when the permanent magnetic field is introduced into the experiment. The increment of
absorbance in first overtone region is greater than that in combination region.
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The skin aging process is mainly accelerated by external agents such as sunlight, air humidity and
surfactants action. Changes in protein structures and hydration during the aging process are
responsible for skin morphological variations. In this work the human skin was investigated by in vivo
Raman spectroscopy before and after the topical applications of a cosmetic on 17 healthy volunteers
(age 60 to 75). In vivo Raman spectra of the skin were obtained with a Spectrometer SpectraPro- 2500i
(Pi-Acton), CCD detector and a 785 nm laser excitation source, collected at the beginning of experiment
without cream (T0), after 30 (T30) and 60 (T60) days using the product. The primary changes occurred
in the following spectral regions: 935 cm-1 (νCC), 1060 cm-1 (lipids), 1174 to 1201 cm-1 (tryptofan,
phenylalanine and tyrosine), 1302 cm-1 (phospholipids), 1520 to 1580 cm-1 (C=C) and 1650 cm-1 (amide
I). These findings indicate that skin positive effects were enhanced by a continuous cream application.
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In recent years FT-IR microspectroscopy has been developed for microbiology analysis and applied successfully
in pure cultures of microorganisms to rapidly identify strains of bacteria, yeasts and fungi. The investigation and
characterization of microorganism mixed cultures is also of growing importance, especially in hospitals where it is common to
poly-microbial infections. In this work, the rapid identification of bacteria in pure and mixed cultures was studied. The bacteria were obtained from the Institute Oswaldo Cruz culture collection at Brazil. Escherichia
coli ATCC 10799 and Staphylococcus aureus ATCC 14456 were analyzed, 3 inoculations were examined in triplicate: Escherichia coli, Staphylococcus aureus and a mixed culture of them. The inoculations were prepared according to McFarland 0.5, incubated at 37 ° C for 6 hours, diluted in saline, placed in the CaF2 window and
store for one hour at 50°C to obtain thin film. The measurement was performed by Spectrum Spotlight 400 (Perkin-Elmer) equipment in the range of 4000-900 cm-1, with 32 scans using a transmittance technique with point
and image modes. The data were processed (baseline, normalization, calculation of first derivate followed by
smoothing with 9 point using a Savitzky-Golay algorithm) and a cluster analysis were done by Ward's algorithm and an excellent discrimination between pure and mixed culture was obtained. Our preliminary results indicate that the FT-IR microspectroscopy associated with cluster analysis can be used to discriminate between pure and
mixed culture.
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We are developing a handheld multispectral imaging device to non-invasively inspect stage I
pressure ulcers in dark pigmented skins without the need of touching the patient's skin. This
paper reports some preliminary test results of using a
proof-of-concept prototype. It also talks
about the innovation's impact to traditional multispectral imaging technologies and the fields that
will potentially benefit from it.
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