This PDF file contains the front matter associated with SPIE-OSA Biomedical Optics Proceedings Volume 6628, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing.

We report on novel hyper-spectral imaging filter-modules based on acousto-optic tuneable filters (AOTF). The AOTF functions as a full-field tuneable bandpass filter which offers fast continuous or random access tuning with high filtering efficiency. Due to the diffractive nature of the device, the unfiltered zero-order and the filtered first-order images are geometrically separated. The modules developed exploit this feature to simultaneously route both the transmitted white-light image and the filtered fluorescence image to two separate cameras. Incorporation of prisms in the optical paths and careful design of the relay optics in the filter module have overcome a number of aberrations inherent to imaging through AOTFs, leading to excellent spatial resolution. A number of practical uses of this technique, both for in vivo auto-fluorescence endoscopy and in vitro fluorescence microscopy were demonstrated. We describe the operational principle and design of recently improved prototype instruments for fluorescence-based diagnostics and demonstrate their performance by presenting challenging hyper-spectral fluorescence imaging applications.

We demonstrate a novel non-intrusive technique based on tunable diode laser absorption spectroscopy to investigate human maxillary sinuses in vivo. The technique relies on the fact that free gases have much sharper absorption features (typical a few GHz) than the surrounding tissue. Molecular oxygen was detected at 760 nm. Volunteers have been investigated by injecting near-infrared light fibre-optically in contact with the palate inside the mouth. The multiply scattered light was detected externally by a handheld probe on and around the cheek bone. A significant signal difference in oxygen imprint was observed when comparing volunteers with widely different anamnesis regarding maxillary sinus status. Control measurements through the hand and through the cheek below the cheekbone were also performed to investigate any possible oxygen offset in the setup. These provided a consistently non-detectable signal level. The passages between the nasal cavity and the maxillary sinuses were also non-intrusively optically studied, to the best of our knowledge for the first time. These measurements provide information on the channel conductivity which may prove useful in facial sinus diagnostics. The results suggest that a clinical trial together with an ear-nose-throat (ENT) clinic should be carried out to investigate the clinical use of the new technique.

For non-invasive estimation of optical properties (i.e. determination of the absorption and the reduced scattering coefficients) of turbid media such as tissue, spatially resolved diffuse reflectance spectroscopy is one of most used technique. So far this has only been done for wavelengths covered by CCD-detectors (about 350-1050nm). The NIR region beyond 1050nm i.e. the second and first overtone regions, has absorption peaks of interest e.g. for tissue the glucose peak at around 1250nm and 1600nm. Thus for non-invasive medical diagnostics applications, a spatially resolved measurement system capable of estimating optical properties in this region will be very useful. Until now optical properties of tissue in this region have only been estimated using in vitro methods e.g. using an integrating sphere set-up. In this paper we describe a spatially resolved system that will extend the region up to 1700nm by using a TE cooled 320×256 pixel InGaAS detector, a white light source and a probe that consists of 9×200micron fibres spanning 0.3 to 2.7mm from the source. Across the 320 pixels 680nm will be dispersed giving a resolution of 2.125nm/pixel and a resolving power of about 14nm. The system is validated using tissue-like phantoms. Since tissue has a high concentration of water which leads to high absorption after 950nm, the diffusion approximation cannot be used to extract the optical properties from the spatially resolved measurements. Instead, two techniques based around Monte Carlo simulations of diffuse reflectance profiles, to build a lookup table coupled with interpolation using splines or a third order polynomial, have been used to calculate the optical properties for different wavelengths. The performances of these techniques are compared. It was found that the spline fitting produced lower error for the wavelength region considered.

Autofluorescence spectroscopy has been a widely explored noninvasive technique to detect the precancerous development in epithelial tissue, where NADH and FAD fluorescence are metabolism related. In this study, we investigated the methods to monitor cellular metabolism based on the ratio of NADH over FAD fluorescence and the ratio of free NADH and protein-bound NADH fluorescence, respectively. The signals of free NADH, protein-bound NADH and FAD were isolated from the intracellular autofluorescence using wavelength- and time-resolved fluorescence spectroscopy. We demonstrated that the wavelength- and time-resolved intracellular autofluorescence can be used to monitor the cellular metabolic pathways and differentiate the normal cells from the cancer cells.

Measurements of endogeous fluorophores open the possibility for evaluation of metabolic state at the eye. For interpretation of 2-dimensional measurements of time-resolved auto fluorescence in 2 separate spectral ranges at the human eye, comparing measurements were performed on porcine eyes. Determining excitation and emission spectra, attention was drawn of proof of coenzymes NADH and FAD in isolated anatomical structures cornea, aqueous humor, lens, vitreous, neuronal retina, retinal pigment epithelium (RPE), choroid, and sclera. All these structures exhibit auto fluorescence, highest in lens. Excitation at 350 nm results in local fluorescence maxima at 460 nm, corresponding to NADH, in all structures. This short-wave excitation allows metabolic studies only at the anterior eye, because of the limited transmission of the ocular media. During excitation at 446 nm the existence of FAD is expressed by local fluorescence maxima at 530 nm. The composition fluorescence spectra allow no discrimination between single ocular structures. Approximating the dynamic fluorescence by a double exponential function, the shortest lifetimes were detected in RPE and neuronal retina. The histograms of mean lifetime t_M cover each other on lens with cornea and also on sclera with choroid. Despite the lifetimes are close between RPE and neuronal retina, the relative contributions Q₁ are wide different. The gradient of trend lines in cluster diagrams of amplitudes α₂ vs. α₁ allows a discrimination of ocular structures.

Monitoring the functional status of cryo-preserved cells and tissue in-situ, i.e. in the frozen state, might allow for optimal adjustment of preservation conditions and might provide the information necessary to predict a functionality recovery rate. Here, an imaging approach with compositional sensitivity seems favourable. In our approach we use multiphoton microscopy in combination with fluorescence lifetime imaging to investigate cells, human and plant tissue at cryogenic conditions. By the non-linearity of multiphoton excitation we largely suppress image distortions attributed to scattering of incoming light. Only where the intensity of the pulsed near-infrared laser beam is sufficiently large, significant fluorescence is excited. This allows reaching penetration depth in ice comparable to the liquid state. As additional information we use the fluorescence decay to assign compositional entities. Results obtained on cells and tissues are discussed with respect to temperature dependencies and the related use for applications.

Noninvasive, real-time monitoring of brain tissue viability is crucial for the patients with stroke, traumatic brain injury, etc. For this purpose, measurement of intrinsic optical signal (IOS) is attractive because it can provide direct information about the viability of brain tissue noninvasively. We performed simultaneous measurements of IOSs that are related to morphological characteristics, i.e., light scattering, and energy metabolism for rat brains during saline infusion as a model with temporal loss of brain tissue viability. The results showed that the scattering signal was steady in an initial phase but showed a drastic, triphasic change in a certain range of infusion time, during which the reduction of CuA in cytochrome c oxidase started and proceeded rapidly. The start time of triphasic scattering change was delayed for about 100 s by lowering brain temperature from 29°C to 24°C, demonstrating the optical detection of cerebroprotection effect by brain cooling. Electron microscopic observation showed morphological changes of dendrite and mitochondria in the cortical surface tissue after the triphasic scattering change, which was thought to be associated with the change in light scattering we observed. These findings suggest that the simultaneous measurement of the intrinsic optical signals related to morphological characteristics and energy metabolism is useful for monitoring tissue viability in brain.

Tissue engineered constructs can be employed to graft wounds or replace diseased tissue. Non-invasive methods are required to assess cellular viability in these constructs both pre- and post-implantation into patients. In this study, Monte Carlo simulations and fluorescence experiments were executed on ex vivo produced oral mucosa equivalent (EVPOME) constructs to investigate the fluorescence emitted at 355 nm excitation from these constructs. Both simulations and experiments indicated the need to investigate alternative excitation wavelengths in order to increase the cellular fluorescence from these constructs, while decreasing contributions from extra-cellular fluorophores.

Head and neck (H&N) cancer patients have a high incidence of second primary tumours in the tracheobronchial tree. Diagnostic autofluorescence bronchoscopy (DAFE) has shown promising results in the detection of early neoplastic and pre-neoplastic changes in the bronchi. We have investigated the medical impact of DAFE in a population of H&N cancer patients. The bronchoscopies were performed using a modified commercially available DAFE system. Endoscopic imaging of the tissue autofluorescence (AF) was combined with an online image analysis procedure allowing to discriminate between true and false positive results. White light (WL) bronchoscopy was performed as a control. Twenty-one patients with high lung cancer risk factors underwent WL and AF bronchoscopy with this improved system. Forty-one biopsies were taken on macroscopicall suspicious (WL or AF positive) sites. Seven patients were found to have second primary tumours in the bronchi. The sensitivity for the detection of these early lesions with the DAFE was 1.6 times larger than the sensitivity of WL bronchoscopy only. The positive predictive value (PPV) for AF is 79% (33% for WL alone). The PPV of both methods together is 100%. DAFE proved to be efficient for the detection of second primary lesions in H&N cancer patients and can be used as a simple addition to pre-operative work-up or follow-up in this patient population.

Early diagnosis of head and neck tumors is usually achieved via surgical tissue biopsy. By measuring the specific autofluorescence of endogenous fluorophores with tumor-specific distributions, it might be possible to non-invasively judge tissue dignities ("optical biopsy"). A total of 22 patients with suspicious lesions of the oral cavity and 7 healthy volunteers were included into the study. Using a mercury vapour lamp as a light source, excitation and detection of endogenous fluorophores (tryptophan, NADH, FAD) was achieved using corresponding filter sets in an automated system. By including simultaneously recorded remission spectra into the analysis, it was possible to calculate "intrinsic" fluorescence spectra. Subsequently, the histopathological results of the lesions were compared to the spectroscopic findings. In a quantitative analysis, the intrinsic fluorescence spectra from (pre)malignant mucosal lesions regularly differed in fluorescence intensities when compared to healthy tissue. Whereas NADH and FAD yielded tumor specific intensity profiles with statistically significant differences in Student's t-test (p≤0.05), no definite spectral differences were found for tryptophan (p=0.22). The mucosa of the healthy volunteers showed a similar spectral pattern as the non-cancerous control areas in tumor patients. With regards to the results in this pilot study, MFA might serve as a helpful tool in early diagnosis of malignant lesions of head and neck.

In gastrointestinal surgery, leakage of anastomoses in general is a challenging problem because of the related mortality and morbidity1,2. The highest incidence of anastomotic leakage is found at the most proximal and most distal parts of the digestive tract, i.e. esophageal and colorectal anastomoses. Increased strain and limited vascular supply at the anastomoses are the two main reasons of leakage, especially in the absence of a serosal layer at these sites2,3,4. Apart from these local risk factors, several general risk factors attributed to the occurrence of anastomotic failure, of which smoking, cardiovascular disease, gender, age and malnutrition are the most important2,5-8. Most of these factors suggest local ischemia as an important cause of anastomotic dehiscence. In esophageal resection the blood supply to the remaining esophageal end is compromised due to ligation of arteries and resection of surrounding mediastinal tissue. Furthermore, the gastric conduit, usually only based on the right gastroepiploic artery, is transposed from its anatomical abdominal position into the thoracic cavity and cervical region. Apart from co-existing morbidities such as sepsis, cardiovascular and several systemic diseases, the altered vascular supply frequently compromises the microcirculation at both ends of the anastomosis, and is as such responsible for the higher rate of leakage compared to small and other large bowel anastomoses9,10.

Prostate cancer is now the most commonly diagnosed male cancer in Europe. Histopathology, the current gold standard for diagnosing prostate pathology is subjective and limited by intra and inter-observer variation. Fourier transform infrared spectroscopy (FTIR) enables objective tissue analysis on the basis of biochemical and structural tissue components. This study examined FTIR's ability to discriminate between benign, premalignant and malignant prostate pathologies. Biochemical fitting using non negative least squares was performed on the spectral datasets of individual prostate pathologies. This novel technique was applied to estimate the relative concentrations of the dominant components in prostate tissue from different prostate pathologies. Preliminary results of the biochemical fitting were promising and gave an early insight into possible biomarkers which could be used in the future to classify stages of the carcinogenesis process. The Principal Component Analysis fed Linear Discriminant Analysis enabled good separation of the pathologies with sensitivities and specificities comparable with the gold standard histopathology. Further work will develop the biochemical fitting technique and increase sample size.

Microalcifications can be found in both benign and malignant breast lesions and their composition can indicate the disease state. Type I microcalcifications are composed of calcium oxalate dihydrate (COD) and are associated mainly with benign tissue, whereas hydroxyapatite (HAP) can be present in both tissue types. As current practices such as mammography and histopathology examine the morphology of the specimen, they can not reliably distinguish between the two types of calcification, which frequently are the only mammographic features that indicate the presence of a cancerous lesion. Analysis of tissue by Fourier transform infrared microspectroscopy (FTIR) allows biochemical information to be achieved from the sample. Spectral maps have been carried out on paraffinized sections of breast tissue from 9 patients of different pathology types containing calcification. The chemical composition of the calcifications and surrounding tissue has been analysed and correlated with tissue pathology. This preliminary study has demonstrated the ability to conduct FTIR in paraffinized sections of breast tissue, and initial observations show a correlation between HAP carbonate substitution and tissue pathology. It is hoped that this and further studies will give insight into how the calcifications are linked to the disease process and will give an increased understanding of the significance of calcifications in breast tissue. If type II microcalcifications can be differentiated in benign and malignant tissue by spectroscopic techniques, this may have positive implications in early diagnosis if the techniques can be applied in vivo and spectroscopy of paraffin sections enables biochemical information to accompany histopathology of the sample.

Most women with early breast cancer have the option of breast conserving therapy, which involves the complete removal of the primary breast lesion (a lumpectomy) with tumor-free margins, followed by radiotherapy. Since the presence of tumor at or near the margin is strongly correlated with the risk of local tumor recurrence, there is a need to develop a non-invasive, real-time tool that can differentiate normal breast tissue from tumor at the margins to assure complete removal. Our previous studies have demonstrated the ability of combined autofluorescence and diffuse reflectance spectroscopy to differentiate normal from non-normal breast tissue ex vivo. Using a portable, combined fluorescence and reflectance spectroscopy system, measurements were taken from each of the six surfaces of the tissue mass immediately following removal during lumpectomies for 24 patients. After correlation with histopathology, a multivariate statistical algorithm classified the 27 tumor spectra and 102 normal spectra with 78% sensitivity and 99% specificity, or 94% negative and 95% positive predictive value. Since point spectroscopy is ultimately insufficient for this application due to its small sampling area, fluorescence and reflectance spectral images from a lumpectomy specimen were gathered and used to show the feasibility of this approach.

We have used elastic light scattering spectroscopy to differentiate between malign and benign skin lesions. The system consists of a UV spectrometer, a single optical fiber probe and a laptop. The single optical fiber probe was used for both delivery and detection of white light to tissue and from the tissue. The single optical fiber probe received singly scattered photons rather than diffused photons in tissue. Therefore, the spectra are correlated with morphological differences of the cells. It has been shown that spectra of malign skin lesions are different than spectra of benign skin lesions. While slopes of the spectra taken on benign lesions or normal skin tissues were positive, slopes of the spectra taken on malign skin lesions tissues were negative. In vivo experiments were conducted on 20 lesions from 18 patients (11 men with mean age of 68 ± 9 years and 7 women with mean age of 52 ± 20 years) applied to the Department of Dermatology and Venerology. Before the biopsy, spectra were taken on the lesion and adjacent (approximately 1 cm distant) normal-appearing skin. Spectra of the normal skin were used as a control group. The spectra were correlated to the pathology results with sensitivity and specificity of 82% and 89%, respectively. Due to small diameter of fiber probe and limited number of sampling (15), some positive cases are missed, which is lowered the sensitivity of the system. The results are promising and could suggest that the system may be able to detect malignant skin lesion non-invasively and in real time.

Caries, a common and widespread infectious disease, has to be detected as early as possible. Based on the need for an easy and handy tool for preventing invasive treatment a new fluorescence camera system has been developed. Using this camera the so-called porphyrins, metabolic products of oral pathogenic bacteria can be visualized. Thereby fluorophores are excited at a wavelength of 405nm by the built-in GaN-LEDs. Healthy and diseased dental hard tissues fluoresce in the green and in the red spectral range, respectively, thus allowing differentiation by coulor. To prove the reliability of this fluorescence camera system, freshly extracted teeth were examined. Three different methods of analysis were verified and compared to give information about the lesions (sensitivity & selectivity): The extent of the fluorescence area, the integral of the red/green ratio of the lesion and the maximum red/green ratio in the area of interest. Histological sections of the teeth served as reference. In addition, the camera was compared to a tip probe sensor already available on the market. In total, our results show that regarding the three different algorithms of analysis, the maximum of the red/green ratio is a preferential method to evaluate carious lesions. Sound tissue, enamel caries and dentin caries can be clearly distinguished. The new fluorescence camera is a handy, efficient and fast device in order to detect lesions and seems to be superior to the tip probe sensor regarding the positioning. Further studies are required.

A method of noninvasive monitoring of human tissue and blood components based on optical diffuse scattering spectroscopy combined with metabolic heat measurements has been developed. Developed compact fiber optical and thermal sensor measures thermal generation, heat balance, blood flow rate, hemoglobin and it's derivative concentrations and environment conditions. It contains thermal and optical detectors, halogen lamp and LEDs, multi-leg fiber optical bundle to measure diffuse light scattering inside and through the patient body including vascular system, which contributes to the control of the body temperature. Measurements of surface cutaneous tissue thermal radiation, ambient room temperature and background radiation temperature are used to measure conduction, convection, and radiation of heat from the human body. Blood flow rate in the body is estimated from the change in temperature for the contact and adjacent thermal detectors. Multi wavelength spectroscopy provides a reflectance spectra which are converted to absorbance values. The calibration and measurement processes are performed independently. Methods of multivariate statistical analysis involving the variables from sensor signals, polynomials from various variables, regression analysis of individual patients, and cluster analysis of patients group were applied to convert various signals from the sensor pickup into physicochemical variables. Obtained data show that the method provides a foundation for noninvasive measuring several biochemical parameters of blood. Further developments of the technology which is under progress now are the following: clinical studies to further characterize the performance of this technology and development of compact and low cost sensor device for home diagnostics.

We used two fluorescence techniques based on the Indocyanine Green contrast agent to study the effectiveness of antiangionenic drugs in mice. To this purpose, the volume of the active vasculature in different tumor models implanted in mice was assessed by means of a low noise fluorescence imaging setup and by a photon counting system working in transmittance geometry. Using a first tumor model (carcinoma MDA-MB-435) we observed that mice treated with a Vascular Disrupting Agent (ZD6126) showed a reduction in fluorescence emission of the contrast agent with respect to control mice. This was a clear indication of the vascular shutdown that took place in tumors. The effectiveness of the treatment was also confirmed by histological sections. Then, in a second experiment we considered a second tumor model (carcinoma 1A9-VS1) overexpressing the Vascular Endotelial Growth Factor (VEGF121), which is used by tumor cells to promote angiogenesis. We measured the Indocyanine Green fluorescence in mice treated with an antioangiogenic drug (Avastin^TM) and in control mice. In tumors of treated mice we observed an ICG emission lower than the one detected in control mice. This demonstrated that VEGF activity was effectively blocked by the treatment with Avastin. In conclusion, ICG fluorescence provides a simple and reliable way to assess the effectiveness of vascular targeting therapies. Measurements of the fluorescence signal can be repeated every 24 hours, thus allowing oncologists to perform longitudinal studies on the same animals.

VTT Technical Research Centre of Finland has developed two reader prototypes for immunodiagnostic tests. VTT has also developed a one-step, homogeneous noncompetitive immunoassay for small analytes using recombinant antibodies and morphine as the model analyte. VTT developed reader for lateral flow test. Lateral flow test is a strip, which has a sample area and a detection area. In the sample area there are antibodies attached to gold or fluorescence particles, which are captured into the detection area, if a sample has a desired analyte. The concentration of the measured sample is then calculated from the fluorescence detection or color change. The second developed prototype reader is based on Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET). In this reader samples are put on microwell array. There are two fluorophores in each of the wells and emission of both fluorophores is measured. The sample concentration is calculated from these emission signals. The optimization of homogenous FRET assays for morphine was included to this project. The first results obtained with the TR-FRET reader prototype show that the sensitivity of the current morphine test is clearly adequate.

This paper presents a novel method to characterise spectral differences that distinguish leukaemia and lymphoma cell lines. This is based on objective spectral measurements of major cellular biochemical constituents and multivariate spectral processing. Fourier transform infrared (FT-IR) maps of the lymphoma, lymphoid and myeloid leukaemia cell samples were obtained using a Perkin-Elmer Spotlight 300 FT-IR imaging spectrometer. Multivariate statistical techniques incorporating principal component analysis (PCA) and linear discriminant analysis (LDA) were used to construct a mathematical model. This model was validated for reproducibility. Multivariate statistical analysis of FTIR spectra collected for each cell sample permit a combination of unsupervised and supervised methods of distinguishing cell line types. This resulted in the clustering of cell line populations, indicating distinct bio-molecular differences. Major spectral differences were observed in the 4000 to 800 cm^-1 spectral region. Bands in the averaged spectra for the cell line were assigned to the major biochemical constituents including; proteins, fatty acids, carbohydrates and nucleic acids. The combination of FT-IR spectroscopy and multivariate statistical analysis provides an important insight into the fundamental spectral differences between the cell lines, which differ according to the cellular biochemical composition. These spectral differences can serve as potential biomarkers for the differentiation of leukaemia and lymphoma cells. Consequently these differences could be used as the basis for developing a spectral method for the detection and identification of haematological malignancies.

Tissue specific progenitor cells and its differentiations have got a lot of attentions in regenerative medicine. The process of differentiations, the formation of tissues, has become better understood by the study using a lot of cell types progressively. These studies of cells and tissue dynamics at molecular levels are carried out through various approaches like histochemical methods, application of molecular biology and immunology. However, in case of using regenerative sources (cells, tissues and biomaterials etc.) clinically, they are measured and quality-controlled by non-contact and non-destructive methods from the view point of safety. Or the analysis with small quantities of materials could be possible if the quantities of materials are acceptable. A non-contact and non-destructive quality control method has been required. Recently, the use of Fourier Transform Infrared spectroscopy (FT-IR) has been used to monitor biochemical changes in cells, and has gained considerable importance. The changes in the cells and tissues, which are subtle and often not obvious in the histpathological studies, are shown to be well resolved using FT-IR. Moreover, although most techniques designed to detect one or a few changes, FT-IR is possible to identify the changes in the levels of various cellular biochemicals simultaneously under in vivo and in vitro conditions. The objective of this study is to establish the infrared spectroscopy of tissue specific progenitor cell differentiations as a quality control of cell sources for regenerative medicine. In the present study, as a basic study, we examine the adipose differentiation kinetics of preadipose cells (3T3-L1) and the osteoblast differentiation kinetics of mesenchymal stem cells (Kusa-A1) to analyze the infrared absorption spectra.

We report on first successful alignment of a surfactant lyotropic nematic between rubbed glass substrates either clean or spin-coated with a polymer layer. Worsening of the alignment either at small number of rubbings (less than 60), at long time delay before filling the cell or when washing the rubbed substrates in deionized water, suggest that the rubbinginduced electrostatic charging of the substrate surface is the main alignment mechanism. Prepared surfonematic cells have good optical quality allowing us to perform optical characterization of the studied surfonematic. The surfonematic is optically negative with the weak light wavelengths dispersion. The absolute value of the birefringence is low: -(6x10^-4). For the first time we report that hemoglobin molecules can be dissolved in the surfonematic matrix. Hemoglobin dissolved in well-aligned surfonematic matrix display polarization spectra which differ from its spectrum in water: 1) wide absorption band in the spectral range 540-640nm for hemoglobin in the surfonematic is different from that for water solutions of hemoglobin; 2) polarization spectra of hemoglobin in aligned surfonematic display weak dichroism leading to the estimation of the orientation order parameter for hemoglobin molecules to be about 10^-2. These spectral features indicate that hemoglobin residues form anisotropic complexes with positively charged surfactant residues. At the studied concentration these complexes are weakly aligned by the surfonematic matrix but are not birefringent and do not affect the orientational order of the surfonematic matrix.

In the last few years, the propagation of diffuse photons in scattering media has become an important field of interest. This is mainly due to the possibility offered by the low absorption of light in the range 700 to 900nm. Indeed, this property leads to a potential deep penetration. But a non negligible limitation appears: the scattering processes strongly reduce both the contrast and the resolution. In this paper, the time-dependent light propagation in highly scattering media containing an inclusion is solved by means of a finite element method, tacking into account Robin type air-tissue boundary conditions. This study is devoted to the depth localization of a tumor enclosed into a breast-like slab. The tissue is modeled by a rectangular meshed domain that mimics a breast compressed between two transparent plates. Cartesian coordinates are used in order to solve the time-dependent diffusion approximation. A short laser pulse of 1ps is considered. The transillumination technique is able to laterally detect the object when the source and detector are moved together on the same axis. In order to perform the localization of the inclusion in this study, the optical properties of the object were varied. Knowing the lateral position of the inclusion, we derive interesting temporal contrast functions based on the mean time of flight of photons. These functions allow to localize in depth the inclusion under the assumption that the object is very diffusing. To conclude, our study demonstrates the possibility to detect laterally and axially a tumor-like inclusion enclosed in breast-like tissues.

Photon transport in complex biological tissues is most accurately predicted via Monte Carlo (MC) modeling methods, which often require lengthy computation times. In this report, a semi-analytical technique (henceforth referred to as PI-scaling) was derived that combines MC simulation, absorption scaling, and path integrals (PI) to rapidly reconstruct time-resolved reflectance from the surface of bilayered epithelial tissue models. Comparisons to forward MC simulations indicated that the PI−scaling method was accurate to better than 10% for tissue models where the optical properties of the top layer did not greatly influence the time-resolved reflectance. Employing such a method should provide a novel solution to the first step of the problem of rapid simulation of time-resolved reflectance of photons in layered tissues.

Neoplastic progression in epithelial tissues is accompanied by structural and morphological changes in the stromal collagen matrix. We used the Finite-Difference Time-Domain (FDTD) method, a popular computational technique for full-vector solution of complex problems in electromagnetics, to establish a relationship between structural properties of collagen fiber networks and light scattering, and to analyze how neoplastic changes alter stromal scattering properties. To create realistic collagen network models, we acquired optical sections from the stroma of fresh normal and neoplastic oral cavity biopsies using fluorescence confocal microscopy. These optical sections were then processed to construct three-dimensional collagen networks of different sizes as FDTD model input. Image analysis revealed that volume fraction of collagen fibers in the stroma decreases with neoplastic progression, and statistical texture features computed suggest that fibers tend to be more disconnected in neoplastic stroma. The FDTD modeling results showed that neoplastic fiber networks have smaller scattering cross-sections compared to normal networks of the same size, whereas high-angle scattering probabilities tend to be higher for neoplastic networks. Characterization of stromal scattering is expected to provide a basis to better interpret spectroscopic optical signals and to develop more reliable computational models to describe photon propagation in epithelial tissues.

Skin cancer full resection implies an evaluation of safety margins around the visible tumour. For melanomas such margins are proportional to tumour's thickness also known as "Breslow Index". In order to see if Diffuse Reflectance Spectroscopy (DRS) could be used to non-invasively evaluate Breslow Index, an in vitro study as well as numerical simulations were performed. Bilayered phantoms were made : a lower layer mimicking dermis underneath an absorbing layer mimicking a melanoma. Five groups of phantoms each having a specific top layer's thickness were made : 2, 3, 4, 5 or 6 mm. For wavelengths longer than 600 nm, Diffuse Reflectance spectra were significantly different (p<0.05) for each thickness at every Collecting to Excitation Fibre Separations (CEFS) : 271, 536, 834, 1076 and 1341 &mgr;m. Monte Carlo simulations were performed to check if DRS could detect smaller (i.e. 0.5 mm) thickness variations. Both experimental and numerical results showed the DR signal intensity linearly (R²>0.9) decreases as CEFS increases. The thicker the melanic layer was the smaller the slope (absolute value) was. These in vitro results will help setting up a clinical trial to non invasively evaluate Breslow Index : the bandwidth should be the NIR one (wavelengths longer than 600 nm) and CEFS should be shorter than 1 mm. Calibration will have to be made in order to relate slope to Breslow Index.

The significant achievements of medical science over the last century are evident in the increasing age of the global population, however this now brings new problems, the most prominent being the growth in the number of people suffering from dementia. Over half the people with dementia in the UK are sufferers of Alzheimer's disease, a condition in which intraneuronal neurofibrillary tangles and extraneuronal senile tangles take over neurons prompting their death. A definitive diagnosis is still only currently available post-mortem, whilst current symptom based processes of elimination are far from perfect, especially when the only treatments available are symptom inhibiting drugs. Principal component analysis (PCA) of the Raman spectra taken from brain tissue has proved to be a potential tool in the diagnosis. However, this work now has to be refined in order to progress to tissue less associated with the symptoms of Alzheimer's disease. The first step of this has already been taken in progressing from frontal tissue to occipital tissue point spectra taken at random positions from bulk tissue. Now we present initial work into acquiring Raman spectral maps from across a tissue area, in pursuit of identifying unique plaque and tangle spectra. These spectra are presented alongside synthetic β-Amyloid spectra, in a study of the role that the peptide plays in the biomarker spectra, and how this information can aid the PCA of bulk tissue, and point towards a Raman spectroscopic test on less sensitive tissue, such as blood.

We present a novel field of view (FOV) zoom scanning multifocal multiphoton microscopy (MMM) system that is based on a microlens array for producing discrete excitation spot array, and the combining of a pair of galvo mirrors and a sample stage for changing the resolution and FOV with fixed optical system and objective. The system can be operated not only in low resolution and large FOV, but also in high resolution and small FOV applications, without compromising the performances of the optical elements in the system. By implementing dedicated system control protocol and image reconstruction algorithm, fluorescence images in different FOV and resolution can be obtained according to user's definition. The performance of the system is demonstrated with two-photon excitation fluorescence imaging of a fluorescence resolution test target, prepared plant stem slide and fluorescence microspheres suspension. Preliminary results indicate that the system has high spatial resolution and holds potential for providing high image acquisition rate. This FOV zoom scanning protocol can be used in our simultaneous time- and spectrum-resolved MMM system, which can provide spectral and lifetime information simultaneously in fluorescence microscopy for biomedical imaging.

Fluorescence techniques are known for their high sensitivity and are widely used as analytical tools and detection methods for product and process control, material sciences, environmental and bio-technical analysis, molecular genetics, cell biology, medical diagnostics, and drug screening. According to DIN/ISO 17025 certified standards are used for fluorescence diagnostics having the drawback of giving relative values for fluorescence intensities only. Therefore reference materials for a quantitative characterization have to be related directly to the materials under investigation. In order to evaluate these figures it is necessary to calculate absolute numbers like absorption/excitation cross sections and quantum yield. This can be done for different types of dopands in different materials like glass, glass ceramics, crystals or nano crystalline material embedded in polymer matrices. Based on the optical spectroscopy data we will discuss options for characteristic doped glasses and glass ceramics with respect to scattering and absorption regime. It has shown recently for YAG:Ce glass ceramics that for a proper determination of the quantum efficiency in these highly scattering media a reference material with similar scattering and fluorescent properties is required. This may be performed using the emission decay measurement diagnostics, where the decay time is below 100 ns. In this paper we present first results of these aspects using well performing LUMOGEN RED organic pigments for a comparison of mainly transparent glass with glass ceramics doped with various amounts of dopands e.g. ions of raw earth elements and transition metals. The LUMOGEN red is embedded in silica and polyurethane matrices. Characterisations on wavelength accuracy and lifetime for different environmental conditions (temperature, UV irradiation) have been performed. Moreover intensity patterns and results for homogeneity, isotropy, photo and thermal stability will be discussed. In a next step we will show the transfer of the characterisation methods to inorganic fluophores (YAG:Ce) in silicon. Fluorescence (steady state, decay time) and absorption (remission, absorption) spectroscopy working in different temperature regimes (10 − 350 K) are employed diagnostic methods in order to get a microscopic view of the relevant physical processes and to prove the correctness of the obtained data. The work is funded by BMBF under project number 13N8849.

In gastrointestinal surgery, leakage of anastomoses in general is a challenging problem because of the related mortality and morbidity1,2. The highest incidence of anastomotic leakage is found at the most proximal and most distal parts of the digestive tract, i.e. esophageal and colorectal anastomoses. Increased strain and limited vascular supply at the anastomoses are the two main reasons of leakage, especially in the absence of a serosal layer at these sites2,3,4. Apart from these local risk factors, several general risk factors attributed to the occurrence of anastomotic failure, of which smoking, cardiovascular disease, gender, age and malnutrition are the most important2,5-8. Most of these factors suggest local ischemia as an important cause of anastomotic dehiscence. In colorectal anastomosis the vascular supply is compromised due to resection of the diseased bowel segment. The vascular supply of the rectal stump is compromised by resection of the proximal feeding sigmoidal vessels. Apart from co-existing morbidities such as sepsis, cardiovascular and several systemic diseases, the altered vascular supply frequently compromises the microcirculation at both ends of the anastomosis, and is as such responsible for the higher rate of leakage compared to small and other large bowel anastomoses9,10.

Carotenoid is an important antioxidant in human body, which can eliminate the free radicals and other harmful reactive oxygen species. The standard technique for measuring carotenoid is high-pressure liquid chromatography which involves using chemicals and is invasive. In this paper, we present a portable resonance Raman spectroscopy system for measuring carotenoid in vivo, which is noninvasive, highly sensitive and compact. A small diode-pumped all solid-state 473nm laser instead of a 488nm Argon ion laser is used to excite in vivo the carotenoid in the thumb, and the resonance Raman scattering light intensity is measured to assess the carotenoid level. Basically, it is difficult to detect the very weak resonance Raman scattering light because it is overlapping with the strong fluorescence. Our investigation shows that matching glycerol can help to reduce tissue scattering and increase light collecting efficiency. We demonstrate that the employment of optical matching technology for measuring carotenoid resonance Raman spectra in tissue can improve the signal-to-noise ratio by 3.9dB.

In dermatology, biophotonic methods offer high sensitivity and non-invasive measurements of skin tissue optical properties, in various physiological and pathological conditions. There are numerous skin processes, which can be examined and characterized using diagnostic optical spectroscopy, as the monitoring of skin aging, diagnosis of benign and malignant cutaneous lesions, dosimetry in photodynamic therapy (PDT), etc. Several mathematical models have been used to calculate the tissue optical properties from experimental measurements and to predict the light propagation in soft tissues, like skin, based on transport theory or Monte Carlo modeling. This work analyses the phenomena which are observed experimentally during the irradiation of skin, such as the absorption, reflectance, scattering, fluorescence and transmission of laser light. The study was carried out on animal skin samples, extracted post-mortem. In this work we also tried to evaluate the utility of diffusion approximation modeling for measuring the light intensity distribution in the skin samples with cw visible laser beam (&lgr;=632.8 nm). The diffusion theory model was tested for the simulation results of the spatial light distribution within a five-layer model of animal skin tissue. We have studied the dependence towards the depth and the radial distance of the photon density of the incident radiation.

Large-scale analysis of proteins, which can be regarded as functional biomolecule, assumes an important role in the life science. A MALDI using an ultraviolet laser (UV-MALDI) is one of ionization methods without fragmentation and has achieved conformation analysis of proteins. Recently, protein analysis has shifted from conformation analysis to functional and direct one that reserves posttranslational modifications such as the sugar chain addition and phosphorylation. We have proposed a MALDI using a mid-infrared tunable laser (IR-MALDI) as a new ionization method. IR-MALDI is promising because most biomolecules have a specific absorption in mid-infrared range, and IR-MALDI is expected to offer; (1) use of various matrices, (2) use of biomolecules such as water and lipid as the matrix, and (3) super-soft ionization. First, we evaluated the wavelength dependence of ionization of different matrices using a difference frequency generation (DFG) laser, which can tune the wavelength within a range from 5.5 to 10.0 &mgr;m. As results, ionization was specifically occurred at 5.8 &mgr;m which the C=O vibration stretching bond in matrix material and mass spectrum was observed. Next, protein mass spectrum was observed in the culture cells, MIN6, which secrete insulin, without the conventional cell-preparation processes. We demonstrate that the IR-MALDI has an advantage over the conventional method (UV-MALDI) in direct analysis of intracellular proteins.

Time-resolved transmittance measurements were performed in the wavelength range of 610 or 700 to 1050 nm on phantom slabs and bone tissue cubes of different sizes. The data were best fitted with solutions of the diffusion equation for an infinite slab and for a parallelepiped to investigate how size and optical properties of the samples affect the results obtained with the two models. When small samples are considered, the slab model overestimates both optical coefficients, especially the absorption. The parallelepiped model largely compensates for the small sample size and performs much better also when the absorption spectra are interpreted with the Beer's law to estimate bone tissue composition.

The new developments of SINPHOS project (SINgle PHOton Spectrometer) are reported. The realised device is able to measure simultaneously with high accuracy time distribution and the wavelength spectrum of photons coming several physical and biological systems. Such device is essentially composed by a grading spectrometer and an array of SPADs (Single Photon Avalange Diodes).

Infrared and Raman spectroscopy are main methods for urine stones type identification today. At the same time they have a limited application for some types of urine stones. Moreover these methods are time-consuming, often hampered by pellet breakage and interpretation of spectra for quantifying urinary calculus constituents in mixtures is difficult, requiring expert knowledge by trained technicians. The other known methods for analyse urinary calculus constituents besides their merits have also such demerits as inexactitude in type identification and high cost of analysis. We propose a new method for urine stones type determination. It is based on luminescent spectroscopy. In this work the results of researches of spectrums of excitation and luminescence of dry tailings of urine are considered. The nature of watched luminescence of urine is explained. Dependence between urine stone type and luminescence maximum localization is investigated.

The optical parameters of tissue-equivalent phantoms are determined by matching the normalized backscattering intensity (NBI) profiles iteratively with that obtained by Monte Carlo simulation procedure. Tissue equivalent optical phantoms (control and with abnormality) were prepared by mixing measured quantities of paraffin wax with wax colors. Abnormalities to be placed in the phantoms were prepared by controlling the absorption and scattering coefficients. The NBI profiles of the phantoms are obtained by an automatic non-contact scanning multi-probe laser reflectometer and are displayed as gray level images after processing. The NBI variations from the abnormality phantoms have distinct variations based on the optical characteristics of the abnormality embedded at various locations and depths. There is a considerable decrease or increase in the NBI variations for different detector probes based on the increase or decrease in absorption and scattering coefficients of abnormalities, respectively. From the profile of subtracted image the peak corresponds to the location of the abnormality and from the full width at half maximum the size of the abnormality is obtained. By further scanning of the image of the phantom with abnormality the depth of the embedded abnormality is obtained.

A novel technique ensuring parallel recording of reflection photoplethysmography signals in broad spectral range has been tested for assessment of pressure-induced vascular changes at various depths from the skin surface. PPG signals have been simultaneously detected at three combinations of the cw laser wavelengths 405 nm, 532 nm, 645 nm, 807 nm and 1064 nm. The PPG baseline responses to the probe-skin contact pressure changes and shapes of the PPG pulses originated from the same heartbeat but recorded at different wavelengths have been detected and analyzed.

We performed reflectance measurements with a time-resolved white-light spectroscopy system to monitor concentrations changes in a two-layer liquid phantom with optical properties similar to human tissues. By varying the concentrations of three inks with different spectral features, we changed the absorption coefficient of the upper and lower layer to simulate either haemodynamics changes in the muscle covered by adipose layer, or functional brain activation with systemic response in the scalp. Data were analyzed by a time-resolved spectrally constrained fitting method based on a homogeneous model of photon diffusion. Although this approach is based on a homogeneous model and employs a single 2cm source-detector distance, the technique is able to monitor changes in the lower layer, while it is scarcely affected by variation in the upper layer. Preliminary in vivo measurements have been performed on one healthy volunteer to monitor oxy- and deoxy-haemoglobin changes in the muscle during arterial occlusion and in the brain during a motor task. Even if the overall sensitivity of the technique is reduced, in vivo results are in general agreement with the findings of dedicated system for tissue oximetry.

Near-infrared spectroscopy (NIRS) is widely applied for applications monitoring skeletal muscle oxygenation. However, this method is obstructed by the subcutaneous adipose tissue thickness (ATT) which might vary between < 1 mm to more than 12 mm. Though diffuse optical imaging can be applied to measure ATT, the objective here is to get this measure from spectroscopic data of a single source-detector distance. For the measurement of the optical lipid signal we used a broad band spatially resolved system (SRS), which is based on measurements of the wavelength dependence of the attenuation A for source detector distances &rgr; between 29 mm and 39 mm. Ultrasound images served as an anatomical reference of the lipid layer. The measurements were taken on 5 different muscle groups of 20 healthy volunteers, each for left and right limbs, e.g. vastus medialis, vastus lateralis and gastrocnemius muscle on the leg and ventral forearm muscles and biceps brachii muscle on the arm. Different analysis strategies were tested for the best calculation of ATT. There is a good non-linear correlation between optical lipid signal and ultrasound data, with an overall error in ATT prediction of about 0.5 mm. This finding is supported experimentally by additional MRI measurements as well as a multi-layer Monte Carlo (MC) model. Based on this data of the ATT thickness, a newly developed algorithm which exploits the wavelength dependence of the slope in attenuation with respect to source-detector distance and MC simulation for these parameters as a function of absorption and scattering coefficients delivers a considerably better fit of reflectance spectra when fitting haemoglobin concentrations. Implications for the monitoring of muscle oxygen saturation are discussed.

Light-induced autofluorescence spectroscopy provides many possibilities for medical diagnostics needs for differentiation of tissue pathologies including cancer. For the needs of clinical practice scientists collect spectral data from patients in vivo or they study different tumor models to obtain objective information for fluorescent properties of every kind of normal and diseased tissue. Therefore it is very important to find the most appropriate and close to the human skin samples from the point of view of laser-induced fluorescence spectroscopy, which will give the possibility for easier transfer of data obtained in animal models to spectroscopic medical diagnostics in humans. In this study are presented some results for in vitro detection of the autofluorescence signals of the animal skin (pig and chicken) with using of LEDs as excitation sources (maximum emission at 365, 375, 385 and 400 nm). The autofluorescence signals from in vivo human skin were also detected for comparison with the models' results. Specific features of the spectra measured are discussed and there are proposed some of the origins of the fluorescence signals obtained. Fluorescence maxima detected are addressed to the typical fluorophores existing in the cutaneous tissues. Influence of main skin absorbers, namely melanin and hemoglobin, is also discussed.

Nanochemistry offers stimulating opportunities for a wide variety of applications in the biosciences. Understanding of the interaction of nanoparticles with biomolecules such as proteins is very important as it can help better design and fabricate nanocomposites for applications in diagnostics, drug delivery, and cell monitoring. In this work, the interaction of Bovine Serum Albumin (BSA) and two types of metal oxide nanoparticles (titanium and tin) have been studied using the intrinsic fluorescence of tryptophan residue from the proteins measured by steady state and time resolved fluorescence techniques. The nanoparticles which were fabricated using a novel synthetic process have average sizes of ~2 nm (SnO₂) and ~6 nm (estimated for TiO₂) and have very high solubilities in a variety of solvents. The Stern-Volmer plots indicate an effective quenching process by TiO₂ nanoparticles whereas SnO₂ nanoparticles have a lower quenching efficiency for BSA fluorescence. Static quenching is the major contribution in the overall process which may indicate a high degree of association between protein and nanoparticles. The difference in BSA fluorescence quenching efficiency between the two types of nanoparticles can be explained by the non-covalent interaction differences and the thermal stability of protein-nanoparticle associated species for both materials.

Attempts were made to non-invasively detect glucose-specific spectral signals in the skin by ATR-FTIR spectroscopy. In vivo spectra were collected from the inner wrists of healthy, prediabetes and diabetes subjects in the 750-4000 cm^-1 region, with a closer assessment of the glucose-related region between 1000 and 1180 cm^-1. Spectra in vivo showed glucose-specific peaks at 1030, 1080, 1118 and 1151 cm^-1, as a variety of glucose solutions are found in vitro. Based on the differences of intensities at 1030 and 1118 cm^-1 two spectral patterns were seen: I₁₁₁₈ > I₁₀₃₀ for a diabetes and I₁₀₃₀> I₁₁₁₈ for non-diabetes subjects. The peak at 1030 cm^-1 was used to assess glucose concentrations in the skin due to its good correlation with glucose concentrations in vitro. Calculated mean values of the peak at 1030 cm^-1 showed evidence of correlation with blood glucose levels when grouped as ≤ 140, 140-200 and ≥ 200 mg/dL, though there was no constant correlation between them when compared before/after OGTT or at the fasting/postprandial states. Absorbances at 1030 cm^-1 were not only increased in a dose-dependent manner in a diabetes patient, but were also generally higher than in non-diabetes subjects at 30 min OGTT assessment. Also we could monitor absorbances at 1030 cm^-1 and determine their changes in the skin tissue at different times of OGTT. We assume that our approach to in vivo measurement and monitoring of glucose concentrations at 1030 cm^-1 may be one of the indicators to assess glucose activity level and its changes in the skin tissue, and has further implications in the study of clinical and pathophysiological aspects of hyperglycemia in diabetes and non-diabetes subjects by ATR-FTIR spectroscopy.

The spectroscopic study of dental enamel by LIBS (laser induced breakdown spectroscopy), FTIR (Fourier transform infrared) and XRD (X-ray diffraction) are represented. The changes of enamel structure and composition in process of natural (caries) and artificial demineralization and restoration were studied. In comparison of sound and carious enamel LIBS showed a decrease of the content of Ca, P and change of the content of some other macro-and trace elements (Mn, Na, Fe, Zn etc). The character of the elemental composition variation was stipulated by the concrete disease. Analysis of FTIR and XRD spectra of dental samples, subjected to artificial demineralization and restoration, showed that restoration action reveals slower, than demineralization. And in some cases the damage of crystals after restoration is more significant than after demineralization.

The present work aims a new medical probe for surgeons devoted to brain cancers, in particular glioblastoma multiforme. Within the last years, our group has started the development of a new intra-operative beta imaging probe. More recently, we took an alternative approach for the same application: a fluorescence probe. In both cases the purpose is to differentiate normal from tumor brain tissue. In a first step, we developed set-ups capable to measure autofluorescence. They are based on a dedicated epi-fluorescence design and on specific fiber optic probes. Relative signal amplitude, spectral shape and fluorescence lifetime measurements are foreseen to distinguish normal and cancer tissue by analyzing fluorophores like NADH, lipopigments and porphyrines. The autofluorescence spectra are recorded in the 460-640 nm range with a low resolution spectrometer. For lifetime measurements a fast detector (APD) is used together with a TCSPC-carte. Intrinsic wavelength- and time-resolutions are a few nm and 200 ps, respectively. Different samples have been analyzed to validate our new detection system and to allow a first configuration of our medical fluorescence probe. First results from the tissue measurements are shown.

The ability of multi-modal optical spectroscopy to detect signals from pancreatic tissue was demonstrated by studying human pancreatic cancer xenografts in mice and freshly excised human pancreatic tumor tissue. Measured optical spectra and fluorescence decays were correlated with tissue morphological and biochemical properties. The measured spectral features and decay times correlated well with expected pathological differences in normal, pancreatitis and adenocarcinoma tissue states. The observed differences between the fluorescence and reflectance properties of normal, pancreatitis and adenocarcinoma tissue indicate a possible application of multi-modal optical spectroscopy to differentiating between the three tissue classifications.

Stratum corneum (horny layer) is a superficial skin layer consisting of dead cells. To reveal in-depth penetration profiles of substances topically applied onto skin surface, a minimally invasive method called tape stripping is widely used. It introduces consecutive removal of micrometer-thick cell layers of stratum corneum from the same treated skin area using an adhesive tape. Prerequisite to the substance penetration profile is the reconstruction of the removed stratum corneum by analyzing the amount of corneocytes (cells of stratum corneum from) stuck to each tape strip. Before application in vivo on humans, porcine skin is often used for such kind of studies. In this paper, we present results of the experiments with porcine skin in vitro (ears of freshly slaughtered pigs) and compare them with those carried out on humans in vivo (flexor forearm) taken from references. As we proved experimentally, there is a linear dependence between the absorbance (equals to logarithm of inverse transmittance) and thickness of the corneocytes on tape strips for all wavelength of the investigated region (300-1050 nm). Dependence of the cumulative absorbance of removed stratum corneum on tape strip number can be satisfactory fitted by an exponential function. This relationship allows evaluation of the relative share of the removed stratum corneum without complete removal of the layer. All the obtained results correlate well with those obtained on humans.