This PDF file contains the front matter associated with SPIE Proceedings Volume 6848, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing.

Venipunctures to draw blood for diagnostics can be cumbersome. Multiple puncture attempts are distressing, painful and traumatic, especially for small children. Drawing blood from babies, in particular, is a problem, due to the cutaneous baby fat, tiny veins and, worst case, a pigmented skin. We developed a practical vein viewing system based on IR translumination that, contrary to commercial systems available, has the advantage of: a) low cost, b) easily implemented in routine practice, c) normal and IR image simultaneously available, d) small add-on, e) child friendly IR illuminator and f) efficient IR light coupling. Before introducing the vessel viewer for clinical application in the children's department, parameters were measured in 194 patients (age 0-17 yrs): time to draw blood, number of attempts, skin characteristics, discomfort of patient, and experience of nurse. In this control group, time to draw blood increases significantly with decreasing age of the children. The instant feedback from the nurses has been valuable for the improvements of especially the illumination sources. A clinical trial has been performed in 125 patients (age 0-6 yrs) to prove effectivity of the system in the blood withdrawal procedure. There was a significant decrease from 13% to 2% in failure rate. Also time needed to search for a vein was significantly decreased. A practical and accessible vein viewing system has been developed and is being introduced for clinical application. Although the concept of patient friendliness is already accepted, measurements need to show the effectiveness for particular groups of patients.

Near-infrared (NIR) fluorescence has the potential to provide surgeons with real-time intraoperative image-guidance. Increasing the signal-to-background ratio of fluorescent agents involves delivering a controllable excitation fluence rate of proper wavelength and/or using complementary imaging techniques such as FLIM. In this study we describe a low-cost linear driver circuit capable of driving Light Emitting Diodes (LEDs) from DC to 35 MHz, at high power, and which permit fluorescence CW and lifetime measurements. The electronic circuit Gerber files described in this article and the list of components are available online at www.frangionilab.org.

Near-infrared (NIR) optical imaging is an emerging noninvasive modality for breast cancer diagnosis. The currently available optical imaging systems towards tomography studies are limited either by instrument portability, patient comfort, or flexibility to image any given tissue volume. Hence, a novel hand-held probe based gain modulated intensified CCD camera imaging system is developed such that it can possibly overcome some of the above limitations. The unique features of this hand-held probe based optical imaging system are: (i) to perform simultaneous multiple point illumination and detection, thus decreasing the total imaging time and improving overall signal strength; (ii) to adapt to the tissue contours, thus decreasing the light leakage at contact surface; and (iii) to obtain trans-illumination measurements apart from reflectance measurements, thus improving the depth information. Phantom studies are performed to demonstrate the feasibility of performing fluorescence optical imaging under different target depths using cubical phantoms (10×6.5×10 cc). The effect of simultaneous multiple point illumination over sequential single point illumination is demonstrated from experimental phantom studies.

Continuous wave Near Infrared Spectroscopy is a well known non invasive technique for measuring changes in tissue oxygenation. Absorption changes (&Dgr;O2Hb and &Dgr;HHb) are calculated from the light attenuations using the modified Lambert Beer equation. Generally, the concentration changes are calculated relative to the concentration at a starting point in time (delta time method). It is also possible, under certain assumptions, to calculate the concentrations by subtracting the equations at different wavelengths (delta wavelength method). We derived a new algorithm and will show the possibilities and limitations. In the delta wavelength method, the assumption is that the oxygen independent attenuation term will be eliminated from the formula even if its value changes in time, we verified the results with the classical delta time method using extinction coefficients from different literature sources for the wavelengths 767nm, 850nm and 905nm. The different methods of calculating concentration changes were applied to the data collected from animal experiments. The animals (lambs) were in a stable normoxic condition; stepwise they were made hypoxic and thereafter they returned to normoxic condition. The two algorithms were also applied for measuring two dimensional blood oxygen saturation changes in human skin tissue. The different oxygen saturation levels were induced by alterations in the respiration and by temporary arm clamping. The new delta wavelength method yielded in a steady state measurement the same changes in oxy and deoxy hemoglobin as the classical delta time method. The advantage of the new method is the independence of eventual variation of the oxygen independent attenuations in time.

We used neural network for blood glucose level determination in this study. The data set used in this study was collected using a non-invasive blood glucose monitoring system with six laser diodes, each laser diode operating at distinct near infrared wavelength between 1500nm and 1800nm. The neural network is specifically used to determine blood glucose level of one individual who participated in an oral glucose tolerance test (OGTT) session. Partial least squares regression is also used for blood glucose level determination for the purpose of comparison with the neural network model. The neural network model performs better in the prediction of blood glucose level as compared with the partial least squares model.

Near-infrared (NIR) fluorescence imaging is a novel optical technique with an ability of probing larger volume of tissues or lesions located in deep tissue areas. An integrated fluorescence and reflectance imaging system was developed to evaluate its potential for cancer diagnosis. The results show that the NIR autofluorescence intensity of normal colon tissues is significantly higher than that of cancer, and the diagnostic accuracy of 92.8% can be achieved using NIR autofluorescence/reflectance imaging. This work demonstrates that NIR autofluorescence/NIR reflectance imaging technique has potential for colonic cancer diagnosis and detection.

Autofluorescence imaging has shown a high sensitivity for early diagnosis and detection of cancer and precancer in humans, however, this diagnostic technique has a limitation with high false positive rates resulting in a low diagnostic specificity. In this study, we develop an endoscope-based autofluorescence imaging system in combination with spectroscopy measurement system for tissue diagnostics and characterization in the head and neck. The results show that combining the spectroscopy and imaging techniques can improve both the diagnostic sensitivity and specificity for discriminating laryngeal carcinoma from normal tissue.

Diabetes mellitus is a metabolic disease that currently affects about 7% of the US population, or roughly about 20 million people. Effectively controlling diabetes requires regular measurements of the blood sugar levels to ensure the one time insulin injection when the concentration of glucose reaches a critical level. In this report, nonlinear Raman microspectroscopy is demonstrated to be a promising new way of continuous and noninvasive way of measuring the glucose concentration.

Currently, precise non-invasive diagnostics systems for the real-time multi detection and monitoring of physiological parameters and chemical analytes in the human body are urgently required by clinicians, physiologists and bio-medical researchers. We have developed a novel cost effective smart 'vanishing tattoo' (similar to temporary child's tattoos) consisting of environmental-sensitive dyes. Painlessly impregnated into the skin the smart tattoo is capable of generating optical/fluorescence changes (absorbance, transmission, reflectance, emission and/or luminescence within UV, VIS or NIR regions) in response to physical or chemical changes. These changes allow the identification of colour pattern changes similar to bar-code scanning. Such a system allows an easy, cheap and robust comprehensive detection of various parameters and analytes in a small volume of sample (e.g. variations in pH, temperature, ionic strength, solvent polarity, presence of redox species, surfactants, oxygen). These smart tattoos have possible applications in monitoring the progress of disease and transcutaneous drug delivery. The potential of this highly innovative diagnostic tool is wide and diverse and can impact on routine clinical diagnostics, general therapeutic management, skin care and cosmetic products testing as well as fundamental physiological investigations.

Neurosurgical procedures involve brain compression created by retractors. Although it is clear that retractors are causing damage to the brain tissue, the pathophysiology of the retraction was not investigated in details. In the present study we used the multiparametric monitoring approach for real time evaluation of mitochondrial function, hemodynamic, ionic and electrical activities monitored contralaterally to the retractor placement on the brain. The aims of the study were to test the effects of retractor size and severity of the compression on the degree of damage to the cerebral tissue. A special probe was lowered towards the cerebral cortex, (2mm and 4mm in depth) using a micromanipulator. Compression lasted for 30 minutes, than the retractor was elevated back to its initial position and monitoring continued for two hours. Additionally, two sizes of retractors were used 6mm and 3mm in diameter, the 3mm retractor included an intracranial pressure (ICP) probe. The results show that the combination of a large retractor with the depth of 4mm yielded high mortality rate (62%) of the rats while the use of a smaller retractor decreased significantly the percentage of mortality. Also, compression to the depth of 4mm increased tissue injury as compared to 2mm depth. In conclusion, the present study raises the importance and significance of multiparametric monitoring, and not only ICP and cerebral blood flow of the areas nearby the retractor position and not only the retraction site, as well as the effect of the retractor size on the damage induced to the cerebral tissue.

We introduce a method based on optical reflectivity changes to segment the retinal nerve fiber layer (RNFL) in images recorded using swept source spectral domain optical coherence tomography (OCT). The segmented image is used to determine the RNFL thickness. Simple filtering followed by edge detecting techniques can successfully be applied to segment the RNFL from recorded images and estimate RNFL thickness. The method is computationally more efficient than previously reported approaches. Higher computational efficiency allows faster segmentation and provides the ophthalmologist segmented retinal images that better utilize advantages of spectral domain OCT instrumentation. OCT B-scan and fundus images of the retina are recorded for 5 patients. The segmentation method is applied on B-scan images recorded from all patients. An expert ophthalmologist separately demarcates the RNFL layer in the OCT images from the same patients in each B-scan image. Results from automated image processing software are compared to the boundary demarcated by the expert ophthalmologist. The absolute error between the boundaries demarcated by the expert and the algorithm is expressed in terms of area and is used as an error metric. Ability of the algorithm to accurately segment the RNFL in comparison with an expert ophthalmologist is reported.

We have developed a photonic technology that allows for precise immobilisation of proteins to sensor surfaces. The technology secures spatially controlled molecular immobilisation since the coupling of each molecule to a support surface can be limited to the focal point of the UV laser beam, with dimensions as small as a few micrometers. The ultimate size of the immobilized spots is dependent on the focal area of the UV beam. The technology involves light induced formation of free, reactive thiol groups in molecules containing aromatic residues nearby disulphide bridges. It is not only limited to immobilizing molecules according to conventional patterns like microarrays, as any bitmap motif can virtually be used a template for patterning. We now show that molecules (proteins) can be immobilized on a surface with any arbitrary pattern according to diffraction patterns of light. The pattern of photo-immobilized proteins reproduces the diffraction pattern of light expected with the optical setup. Immobilising biomolecules according to diffraction patterns of light will allow achievement of smaller patterns with higher resolution. The flexibility of this new technology leads to any patterns of photo-imprinted molecules, with micrometer resolution, thus being of relevance for present and future applications in nanotechnologies.

We present a novel probe-based portable clinical system for early detection and margin demarcation of melanoma and non-melanoma skin cancers. The system collects both white light reflectance and fluorescence from tissue in real time. We use gated detection to eliminate effects of room lights and make the system clinically compatible. Instrument control and spectral calibration is automated using a personal computer. The total acquisition time for data collection is less than a second. We use a spectrally-constrained inverse model for our probe geometry to fit the diffuse reflectance and extract hemoglobin content, oxygen saturation, tissue micro-architecture and melanin content. We demonstrate system performance and present results from tissue simulating phantoms. The mean rms errors in estimating scattering and absorption coefficients in tissue phantoms over a physiologically relevant range were 9.8% and 11.8% respectively. Using a photon migration model and least-squares regression we were able to extract the intrinsic fluorescence line shapes and estimate fluorophore concentrations from measured fluorescence spectra with an rms error of less than 10%.

Specific protein concentrations in human body fluid can serve as diagnostic markers for some diseases, and a quantitative and high-throughput technique for multiplexed protein detection would speed up diagnosis and facilitate medical research. For this purpose, our group developed the BioCD, a spinning-disc interferometric biosensor on which antibody is immobilized. The detection system adopts a common-path scheme making it ultra stable. The scaling mass sensitivity is below 10 pg/mm for protein surface density. A 25000-spot antibody BioCD was fabricated to measure the concentration of prostate specific antigen (PSA), a protein indicating prostate cancer if its level is high. Statistical analysis of our immunoassay results projects that the detection limit of PSA would reach 20 pg/ml in a 2 mg/ml background solution. For future prospects, a multiplexed BioCD can be produced for simultaneous diagnosis of diverse diseases. For instance, 100 markers above 200 pg/ml could be measured on a single disc given that the detection limit is inversely proportional to square root of the number of spots.

This paper presents an optical system for performing excitation resolved imaging of fluorescent dyes, tissue phantoms and ex vivo tissues. The excitation source was a supercontinuum generated in a highly nonlinear fibre, spectrally filtered using dispersive optics and a movable slit or digital micromirror device. This allowed excitation with multiple spectra, which may be chosen to optimize the fluorescence yield from a particular fluorophore, or to maximize discrimination between multiple labels. As an initial validation, the analysis of fluorescent dye solutions embedded in collagen gels in the presence of scatterers and absorbers by using fixed reference isomap (FR-IsoMap) is presented, followed by initial evaluation with human lung bronchial tissue. The proposed system has potential applications for in vivo fluorescence endoscopy of cancerous tissues through excitation spectral selection and analysis with FR-IsoMap.

Living skin for basic and clinical research can be evaluated by Confocal Laser Scanning Microscope (CLSM) non-invasively. CLSM imaging system can achieve skin image its native state either "in vivo" or "fresh biopsy (ex vivo)" without fixation, sectioning and staining that is necessary for routine histology. This study examines the potential fluorescent CLSM with a various exogenous fluorescent contrast agent, to provide with more resolution images in skin. In addition, in vivo fluorescent CLSM researchers will be extended a range of potential clinical application. The prototype of our CLSM system has been developed by Prof. Gweon's group. The operating parameters are composed of some units, such as illuminated wavelength 488 nm, argon illumination power up to 20mW on the skin, objective lens, 0.9NA oil immersion, axial resolution 1.0μm, field of view 200μm x 100μm (lateral resolution , 0.3μm). In human volunteer, fluorescein sodium was administrated topically and intradermally. Animal studies were done in GFP transgenic mouse, IRC mouse and pig skin. For imaging of animal skin, fluorescein sodium, acridine orange, and curcumine were used for fluorescein contrast agent. We also used the GFP transgenic mouse for fluorescein CLSM imaging. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. Curcumin is a yellow food dye that has similar fluorescent properties to fluorescein sodium. Acridin Orange can be highlight nuclei in viable keratinocyte. In vivo CLSM of transgenic GFP mouse enable on in vivo, high resolution view of GFP expressing skin tissue. GFP signals are brightest in corneocyte, kertinocyte, hair and eccrine gland. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. In papillary dermis, fluorescein distribution is more homogeneous. Curcumin is a yellow food dye that has similar fluorescent properties to fluorescein sodium. In vivo CLSM of transgenic GFP mouse enable on in vivo, high resolution view of GFP expressing skin tissue. GFP signals are brightest in corneocyte, kertinocyte, skin appendage and blood vessels. In conclusion, this study demonstrates the usefulness of CLSM as technique for imaging skin in vivo. In addition, CLSM is non-invasive, the same tissue site may be imaged over a period of time to monitor the various change such as wound healing, severity of skin diseases and effect of therapeutic management.

The recent development of electro-optical instrumentation allowed constructing 4D (3D + time) structure-light scanners which may be used to measure the surface of human body in motion. The main advantage of structure-light scanners is the possibility of capturing data from the whole measured body surface, while traditional marker-based systems acquire data only form markers attached to skin of the examined patient. The paper describes new parameters describing the local shape of measured surface. The distribution maps of these parameters allow discrimination of various surface types and in effect localization and tracing of under-skin anatomical structures in time. The presented parameters give similar results to well-known curvatures but are easier and quicker to calculate. Moreover the calculation process of the new parameters is more numerically stable itself. The developed path of processing and analysis of 4D measurement data has been presented. It contains the following stages: data acquisition, volumetric model creation, calculations of shape parameters, selecting areas of interest, locating and tracing of anatomical landmarks. Exemplary results of application of developed parameters and methods to real measurement and computer generated data are also presented.

In biomedical application study using phase contrast X-ray, both sample thickness or density and absorption difference are very important factors in aspects of contrast enhancement. We present experimental evidence that synchrotron hard X-ray are suitable for radiological imaging of biological samples down to the cellular level. We investigated the potential of refractive index radiology using un-monochromatized synchrotron hard X-rays for the imaging of cell and tissue in various diseases. Material had been adopted various medical field, such as apoE knockout mouse in cardiologic field, specimen from renal and prostatic carcinoma patient in urology, basal cell epithelioma in dermatology, brain tissue from autosy sample of pakinson's disease, artificially induced artilrtis tissue from rabbits and extracted tooth from patients of crack tooth syndrome. Formalin and paraffin fixed tissue blocks were cut in 3 mm thickness for the X-ray radiographic imaging. From adjacent areas, 4 μm thickness sections were also prepared for hematoxylin-eosin staining. Radiographic images of dissected tissues were obtained using the hard X-rays from the 7B2 beamline of the Pohang Light Source (PLS). The technique used for the study was the phase contrast images were compared with the optical microscopic images of corresponding histological slides. Radiographic images of various diseased tissues showed clear histological details of organelles in normal tissues. Most of cancerous lesions were well differentiated from adjacent normal tissues and detailed histological features of each tumor were clearly identified. Also normal microstructures were identifiable by the phase contrast imaging. Tissue in cancer or other disease showed clearly different findings from those of surrounding normal tissue. For the first time we successfully demonstrated that synchrotron hard X-rays can be used for radiological imaging of relatively thick tissue samples with great histological details.

Photodynamic diagnosis (PDD) is a method to diagnose the possibility of cancer, both by the principle that if a photosensitizer is injected into an organic tissue, it is accumulated in the tissue of a malignant tumor selectively after a specific period, and by a comparison of the intensity of the fluorescence of normal tissue with abnormal tissue after investigating the excitation light of a tissue with accumulated photosensitizer. Currently, there are two methods of PDD: The first is a way to acquire incitement fluorescence by using a photosensitizer, and the second is a way to use auto-fluorescence by green fluorescence protein (GFP) and red fluorescence protein (RFP) such as NADH+ active factors within the organic body. Since the selection of the wavelength band of excitation light has an interrelation with fluorescence generation according to the selection of a photosensitizer, it plays an important role in PDD. This study aims at designing and evaluating light source devices that can stably generate light with various kinds of wavelengths in order to make possible PDD using a photosensitizer and diagnosis using auto-fluorescence. The light source was a Xenon lamp and filter wheel, composed of an optical output control through Iris and filters with several wavelength bands. It also makes the inducement of auto-fluorescence possible because it is designed to generate a wavelength band of 380-420nm, 430-480nm, 480-560nm. The transmission part of the light source was developed to enhance the efficiency of light transmission. To evaluate this light source, the characteristics of light output and wavelength band were verified. To validate the capability of this device as PDD, the detection of auto-fluorescence using mouse models was performed.

We describe a new system for automated temperature control during laser hyperthermia. The system includes a PC with special software, in which a regulator is realized, infra red camera as a detector of temperature changes and laser with the 810 nm irradiation wavelength, which operates under PC control. The regulation was realized as a negative feedback adaptive control system, which tunes it self during the operating and do not need any prior information about the parameters of the irradiated tissue. The system was tested on different experimental models, the parameters of which varied in a wide range. The system showed a stable work with times of the transients about 60 seconds. The error of temperature stabilization was about ± 0.8 °C.

We present our preliminary results on two-dimensional (2-D) optical tomographic imaging of hemodynamic changes of two preterm infant brains in different ventilation settings conditions. The investigations use the established two-wavelength, 16-channel time-correlated single photon counting system for the detection, and the generalized pulse spectrum technique based algorithm for the image reconstruction. The experiments demonstrate that two-dimensional diffuse optical tomography may be a potent and relatively simple way of investigating the functions and neural development of infant brains in the perinatal period.

Image-assisted diagnosis and therapy is becoming more commonplace in medicine. However, most imaging techniques suffer from voluntary or involuntary motion artifacts, especially cardiac and respiratory motions, which degrade image quality. Current software solutions either induce computational overhead or reject out-of-focus images after acquisition. In this study we demonstrate a hardware-only gating circuit that accepts multiple, pseudo-periodic signals and produces a single TTL (0-5 V) imaging window of accurate phase and period. The electronic circuit Gerber files described in this article and the list of components are available online at www.frangionilab.org.