This PDF file contains the front matter associated with SPIE Proceedings Volume 9315, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and Conference Committee listing.

The construction of photoacoustic tomography (PAT) systems that combine tunable laser sources and ultrasound systems for bimodal imaging and spectroscopic applications such as oximetry presents novel challenges to the biophotonics researcher. We address some of the design issues, including system synchronization, cross-platform integration, and image reconstruction algorithms, and present techniques for device performance validation. Our system comprises a pulsed Nd:YAG laser-pumped optical parametric oscillator for near-infrared tunability and a research-grade ultrasound acquisition system compatible with multiple clinical transducers to enable wide variation in operating parameters. Considerations such as pulse energy variability, ultrasound transducer properties, and spectral energy compensation, and their impact on measurements are presented. Spectral imaging was performed on tissue-simulating phantoms made of a custom polyvinyl chloride (PVC) plastisol gel designed to mimic both the optical properties (absorption, scattering) and acoustic properties (sound velocity, attenuation) of human breast tissue. Phantoms contained fluid channels at various depths which were injected with either oxyhemoglobin or organic dye solutions as absorptive targets. Spectral analysis of these solutions was performed for channel depths from 0.5 to 3 cm and at radiant exposures up to the ANSI maximum permissible exposure. Recovered photoacoustic spectra are compared with absorption spectra measured using spectrophotometry. Results provide insight into the influence of factors that impact the quality of spectroscopic measurements and reconstructed images in ultrasound-PAT systems.

Standardized approaches for performance assessment of biophotonic devices have the potential to facilitate system development and intercomparison, clinical trial standardization, recalibration, manufacturing quality control and quality assurance during clinical use. Evaluation of devices based on near-infrared spectroscopy (NIRS) for detection of hemoglobin (Hb) content and oxygenation have often involved tissue-simulating phantoms incorporating artificial dyes or flow systems. Towards the development of simple, effective techniques for objective, quantitative evaluation of basic NIRS system performance, we have developed and evaluated two test methods. These methods are based on cuvette inserts in solid turbid phantoms for measuring commercially-available Hb oximetry standards and custom-formulated oxy/deoxy-Hb solutions. Both approaches incorporate solid acetal, or polyoxymethylene (POM), as a tissue-simulating matrix material. First, inverse-adding-doubling (IAD) based on measurements with a spectrophotometer and an integrating sphere was used to measure POM optical properties and their stability over time. Second, two fiberopticprobe- based NIRS systems were used to measure concentration change of oxy- and deoxy-Hb in standard Hb solutions and customized Hb solutions by adding yeast. Differences in system performance were likely due to differences in light source outputs and fiberoptic probe design. Our preliminary results indicate that simple phantom-based approaches based on commercially available polymers and inclusions containing Hb standards, or controlled oxygenation levels may be useful for benchtop assessment of NIRS device quality for a variety of biophotonic devices.

Whole Slide Imaging (WSI) systems are used in the emerging field of digital pathology for capturing high-resolution images of tissue slides at high throughput. We present a technique to measure the optical aberrations of WSI systems using a Shack-Hartmann wavefront sensor as a function of field position. The resulting full-field aberration maps for the lowest order astigmatism and coma are analyzed using nodal aberration theory. According to this theory two coefficients describe the astigmatism and coma inherent to the optical design and another six coefficients are needed to describe the cumulative effects of all possible misalignments on astigmatism and coma. The nodal aberration theory appears to fit well to the experimental data. We have measured and analyzed the full-field aberration maps for two different objective lens-tube lens assemblies and found that only the optical design related astigmatism coefficient differed substantially between the two cases, but in agreement with expectations. We have also studied full-field aberration maps for intentional decenter and tilt and found that these affect the misalignment coefficient for constant coma (decenter) and the misalignment coefficient for linear astigmatism (tilt), while keeping all other nodal aberration theory coefficients constant.

The demand of non-invasive ocular screening is rapidly growing due to an increase of age related eye diseases worldwide. An indeed in-depth understanding of optical properties is required to elucidate nature of retinal tissue. The research aims to investigate an effective biomedical engineering approach to allow process region of interests (ROIs) in eyes to reveal physiological status. A dynamic opto-physiological model (DOPM) representing retinal microvascular circulation underlying a diffusion approximation to solve radiative transport theorem (RTT) has being developed to interpret patho-physiological phenomena. DOPM is being applied in imaging photoplethysmography (iPPG) to extract PPG signals from a series of 2D matrix images to access blood perfusion and oxygen saturation distributions. A variation of microvascular circulation could be mapped for an effectively diagnostic screening. The work presents mathematical modelling based ten layers of ocular tissue tested with four set of controlled parameters demontrated detection ratio between normal tissue damage or abnormal tissue and significant change of AC signal amplitude in these tissues. The result shows signicant change of AC signal amplitude in abnormal tissue. The preliminary results show extractable PPG signals from eye fundus video; experimented at five ROIs: whole fundus, optical disk, main vein vessel, lesion area and affected area. The outcome shows optical disk region gave a better performance compared to whole fundus region and main vein vessel. The robustness, miniaturization and artefact reduction capability of DOPM to discriminate oxygenation levels in retina could offer a new insight to access retinal patho-physiological status.

The application of midIR spectroscopy towards human cell and tissue samples is impaired by the need for technical solutions and lacking sample standards for technology development. We here present the standardization of stable test samples for the continuous development and testing of novel optical system components. We have selected cell lines representing the major cellular skin constituents keratinocytes and fibroblasts (NIH-3T3, HaCaT). In addition, two skin cancer cell types (A-375 and SK-MEL-28 cells) were analyzed. Cells were seeded on CaF₂ substrates and measured dried and under aqueous medium as well as fixated and unfixated. Several independent cell preparations were analyzed with an FTIR spectrometer in the wave number range from 1000 - 4000 cm^-1. The obtained data demonstrate that fixed and dehydrated cells on CaF₂ can be stored in pure ethanol for several weeks without significant losses in quality of the spectral properties. The established protocol of cell seeding on CaF₂ substrates, chemical fixation, dehydration, storage under ethanol and air-drying is suitable for the production of reliable midIR standards. The retrieved spectra demonstrate that fixed cells on CaF₂ can be prepared reproducibly; with stable midIR spectral properties over several weeks and properties mimicking reliable unfixed cells. Moreover, the fixated samples on CaF₂ show clear differences in the cell type specific spectra that can be identified by principle component analysis. In summary, the standardized cell culture samples on CaF₂ substrates are suitable for the development of a midIR device and the optimization of the specific midIR spectra.

Near-infrared spectroscopy (NIRS) has been extensively developed for in-vivo measurements of tissue vascular oxygenation, breast tumor detection, and functional brain imaging, by groups of physicists, biomedical engineers, and mathematicians. To quantify concentrations of oxyhemoglobin, deoxyhemoglobin, and total hemoglobin (hemodynamics), extinction coefficients of hemoglobin (ε) have to be employed. However, it is still controversial what ε values should be used and relatively what calibration should be done in NIRS quantification to achieve the highest precision, although that the differences in ε values among published data resulted in ~20% variation in quantification of hemoglobin concentration is reported based a single human blood test. We collected 12 blood samples from 12 healthy people, and with each blood sample performed blood tissue model experiments. 4 teams of published extinction value widely used in NIRS fields were employed respectively in our quantification. Calibrations based least square analysis and regression between real and estimated hemodynamics for 12 subjects were performed with each team of ε values respectively. We found that: Moaveni’s ε values contributed to highest accuracy; Regression method produced quite effective calibration, and when it combined with Moaveni’s ε values, the calibration reduced the std/mean of estimation by two orders of magnitude. Thus Moaveni’s ε values are most recommended to use in NIRS quantification, especially with our calibration matrix based on regression analysis with a group of subjects’ blood sample.

Colonoscopy is the preferred procedure for the detection, biopsy and removal of neoplastic lesions of the colon. It is estimated that about 14 million colonoscopies will have been performed in the US in 2014. The number of patients undergoing colonoscopy worldwide is also increasing, however, the procedure is far from perfect and recent studies have questioned its impact on colon cancer prevention, particularly in the proximal colon. Whereas standard endoscopes are designed to provide a view of a cylindrical lumen, the colon is not a simple hollow tube, but a tortuous organ with many folds that can prevent polyps from being seen. Poor color contrast of some flat lesions also make detection more difficult. A number of techniques have been developed to increase the surface area of the colon viewed, from accessories that can be used with existing colonoscopes to new endoscopy systems. Methods to improve lesion contrast are also being developed. The ideal device should not only maximize the surface of the colon viewed and improve lesion contrast to aid detection, but should do so inexpensively and without increasing the complexity and duration of the procedure. Healthcare reform will soon require endoscopists to report the quality of their procedures, as measured by individual rates of adenoma detection. Therefor the need to develop new devices that improve lesion detection is profound, but for any product to be clinically assimilated, it needs to be easy to use and affordable.

The concept and design for a multifocal confocal spectral microscope is discussed. The multifocal confocal spectral microscope utilizes an array of focal beams to capture to the spectrum of multiple points simultaneously. Experimental results from a 3x3 multifocal confocal spectral microscope with spectral resolution less than 2 nm are shown.

In biophotonics imaging, one important and quantitative task is layer-thickness estimation. In this study, we investigate the approach of combining optical coherence tomography and a maximum-likelihood (ML) estimator for layer thickness estimation in the context of tear film imaging. The motivation of this study is to extend our understanding of tear film dynamics, which is the prerequisite to advance the management of Dry Eye Disease, through the simultaneous estimation of the thickness of the tear film lipid and aqueous layers. The estimator takes into account the different statistical processes associated with the imaging chain. We theoretically investigated the impact of key system parameters, such as the axial point spread functions (PSF) and various sources of noise on measurement uncertainty. Simulations show that an OCT system with a 1 μm axial PSF (FWHM) allows unbiased estimates down to nanometers with nanometer precision. In implementation, we built a customized Fourier domain OCT system that operates in the 600 to 1000 nm spectral window and achieves 0.93 micron axial PSF in corneal epithelium. We then validated the theoretical framework with physical phantoms made of custom optical coatings, with layer thicknesses from tens of nanometers to microns. Results demonstrate unbiased nanometer-class thickness estimates in three different physical phantoms.

Quantification of leukocyte subpopulations and characterization of antigen-expression pattern on the cellular surface can play an important role in diagnostics. The state of cellular immunology on the single-cell level was analyzed by polychromatic flow cytometry in a recent comparative study within the average Leipzig population (LIFE - Leipzig Research Centre for Civilization Diseases). Data of 1699 subjects were recorded over a long-time period of three years (in a total of 1126 days). To ensure compatibility of such huge data sets, quality-controls on many levels (stability of instrumentation, low intra-laboratory variance and reader independent data analysis) are essential. The LIFE study aims to analyze various cytometric pattern to reveal the relationship between the life-style, the environmental effects and the individual health. We therefore present here a multi-step quality control procedure for long-term comparative studies.

We study experimentally the scanning functions of galvanometer-based scanners (GSs) in order to optimize them for biomedical imaging in general, and for Optical Coherence Tomography (OCT) in particular. The main scanning parameters of the scanning process are taken into account: theoretical duty cycle (of the input signal of the GS), scan frequency (f_s), and scan amplitude (θ_m). Triangular to sawtooth scanning regimes are thus considered. We demonstrate that when increasing the scan frequency and amplitude, the scanning function (i.e., the angular position of the galvomirror) is not able to follow anymore the input signal. Furthermore, as the theoretical duty cycle is increased, the result is unexpected: the effective duty cycle actually decreases – for high f_s and θ_m. A saturation of the device therefore occurs. The practical limits of this phenomenon are discussed. GS users are thus provided with a multi-parameter analysis that allows them for optimizing their scanning regimes and to avoid pushing the devices to their limit – when that actually results in decreasing the quality of the images obtained, by example in OCT. Gabor Domain Optical Coherence Microscopy (GD-OCM) images are made to show effects of this phenomenon.

Whispering gallery modes (WGM) within microsphere cavities have demonstrated the ability to provide label-free, highly sensitive and selective detection down to a single molecule level, emerging as a promising technology for future biosensing applications. Currently however, the majority of biosensing work utilizing WGMs has been conducted in resonators made from either silica or polystyrene while other materials have been largely uninvestigated. This work looks to predict the optimal combinations of material, resonator size and excitation/coupling scheme to provide guidelines to assist in decision making when undertaking refractive index biosensing in a range of situations.

Soliton self-frequency shift (SSFS) is a versatile technique for generating ultrashort optical pulses with customized wavelengths suitable for nonlinear optical microscopy. However, spectral overlapping of the soliton with the residual sometimes causes extra power deposition onto the biological samples, which may further induce optical damage. Consequently, how to choose the optimal optical filter is of vital importance for increasing signal level and minimizing damage. Here we propose maximizing the ratio between multi-photon signal and the nth power of the excitation pulse energy as a criterion for optimal spectral filtering in SSFS. This optimization is based on most efficient signal generation and entirely depends on physical quantities that can be easily measured experimentally.

Spontaneous expression is associated with physiological states, i.e., heart rate, respiration, oxygen saturation (SpO2%), and heart rate variability (HRV). There have yet not sufficient efforts to explore correlation of physiological change and spontaneous expression. This study aims to study how spontaneous expression is associated with physiological changes with an approved protocol or through the videos provided from Denver Intensity of Spontaneous Facial Action Database. Not like a posed expression, motion artefact in spontaneous expression is one of evitable challenges to be overcome in the study. To obtain a physiological signs from a region of interest (ROI), a new engineering approach is being developed with an artefact-reduction method consolidated 3D active appearance model (AAM) based track, affine transformation based alignment with opto-physiological mode based imaging photoplethysmography. Also, a statistical association spaces is being used to interpret correlation of spontaneous expressions and physiological states including their probability densities by means of Gaussian Mixture Model. The present work is revealing a new avenue of study associations of spontaneous expressions and physiological states with its prospect of applications on physiological and psychological assessment.

Photonics is an inherently interdisciplinary endeavor, as technologies and techniques invented or developed in one scientific field are often found to be applicable to other fields or disciplines. We present two case studies in which optical spectroscopy technologies originating from stellar astrophysics and optical telecommunications multiplexing have been successfully adapted for biomedical applications. The first case involves a design concept called the High Throughput Virtual Slit, or HTVS, which provides high spectral resolution without the throughput inefficiency typically associated with a narrow spectrometer slit. HTVS-enhanced spectrometers have been found to significantly improve the sensitivity and speed of fiber-fed Raman analysis systems, and the method is now being adapted for hyperspectral imaging for medical and biological sensing. The second example of technology transfer into biomedicine centers on integrated optics, in which optical waveguides are fabricated on to silicon substrates in a substantially similar fashion as integrated circuits in computer chips. We describe an architecture referred to as OCTANE which implements a small and robust "spectrometer-on-a-chip” which is optimized for optical coherence tomography (OCT). OCTANE-based OCT systems deliver three-dimensional imaging resolution at the micron scale with greater stability and lower cost than equivalent conventional OCT approaches. Both HTVS and OCTANE enable higher precision and improved reliability under environmental conditions that are typically found in a clinical or laboratory setting.

This study presents an effective engineering approach for human vital signs monitoring as increasingly demanded by personal healthcare. The aim of this work is to study how to capture critical physiological parameters efficiently through a well-constructed electronic system and a robust multi-channel opto-electronic patch sensor (OEPS), together with a wireless communication. A unique design comprising multi-wavelength illumination sources and a rapid response photo sensor with a 3-axis accelerometer enables to recover pulsatile features, compensate motion and increase signal-to-noise ratio. An approved protocol with designated tests was implemented at Loughborough University a UK leader in sport and exercise assessment. The results of sport physiological effects were extracted from the datasets of physical movements, i.e. sitting, standing, waking, running and cycling. t-test, Bland-Altman and correlation analysis were applied to evaluate the performance of the OEPS system against Acti-Graph and Mio-Alpha.There was no difference in heart rate measured using OEPS and both Acti-Graph and Mio-Alpha (both p<0.05). Strong correlations were observed between HR measured from the OEPS and both the Acti-graph and Mio-Alpha (r = 0.96, p<0.001). Bland-Altman analysis for the Acti-Graph and OEPS found the bias 0.85 bpm, the standard deviation 9.20 bpm, and the limits of agreement (LOA) -17.18 bpm to +18.88 bpm for lower and upper limits of agreement respectively, for the Mio-Alpha and OEPS the bias is 1.63 bpm, standard deviation SD8.62 bpm, lower and upper limits of agreement, - 15.27 bpm and +18.58 bpm respectively. The OEPS demonstrates a real time, robust and remote monitoring of cardiovascular function.

We studied Čerenkov radiation generated in irradiated optical fibers and demonstrated a generic spectroscopic method for separation of Čerenkov radiation from the transmitted signal in fiber optic dosimetry based on the assumption that the recorded signal is a linear superposition of two basis spectra: Čerenkov radiation and characteristic luminescence of the phosphor medium. Experimentally, we evaluated this technique by using fiber optic probes irradiated by electron beams generated by a clinical linear accelerator. This method can be used for optical characterization of various scintillating materials, such as phosphor nanoparticles, in ionizing radiation fields of high energy. We performed Monte Carlo simulations of the Čerenkov radiation generated in the fiber and found a strong dependence of the recorded Čerenkov radiation on the numerical aperture of the fiber at shallow phantom depths; however, beyond the depth of maximum dose that dependency is minimal. Our simulation results agree well with the experimental results for Čerenkov radiation generated in fibers irradiated with 6 MeV electrons.

A dual-wavelength pulse oximetry system combined with laser Doppler was developed for the assessment of perfusion. Red and infrared PPG and Doppler signals were recorded from a healthy volunteer in three studies at different measurement sites to investigate the interference between PPG and laser Doppler flowmetry (LDF). Good quality photoplethysmographic (PPG) and Doppler signals were detected simultaneously using this combined probe from the skin of the finger. The influence of the PPG light sources on LDF measurements was investigated; also the influence of the LDF light sources to the PPG measurements was studied. In the worst case, the apparent change in PPG amplitude when the LDF system was switched on was less than 8%, and the change in LDF flux amplitude when the PPG system was switched on was 14.7%.

The extinction spectra and optical coefficients of human cancerous and normal prostate tissues were investigated in the spectral range of 750 nm - 860 nm. The scattering coefficient (μ_s) was determined from the extinction measurements on thin prostate tissue and Beer’s law. The absorption coefficient (μ_a) and the reduced scattering coefficient (μ_s') were extracted from integrate sphere intensity measurements on prostate tissue of which the thickness is in the multiple scattering range. The anisotropy factor (g) was calculated using the extracted values of μ_s and μ_s'. A micro-optical model of soft biological tissue was introduced to simulate the numerical computation of the absolute magnitudes of its scattering coefficients from the refractive index and a particle distribution function based on the Mie theory. A key assumption of the model is that the refractive index variations caused by microscopic tissue elements can be treated as particles with sizes distributed according to a skewed log-normal distribution function. The particle distribution and mean particle size of the two types of tissues were then calculated. Results show that the mean diameter of the particle size of cancerous tissue is larger than that of the cancerous tissue, which is responsible for larger reduced scattering coefficient of normal tissue in comparison with cancerous tissue. The results can be explained the change of tissue during prostate cancer evolution defined by Gleason Grade. The difference of the particles distribution and optical coefficients of cancerous and normal prostate tissues may present a potential criterion for prostate cancer detection.

To evaluate the feasibility of measuring the myocardial blood flow using 320 row detector CT by first-pass technique. Heart was simulated with a container that was filled with pipeline of 3mm diameter; coronary artery was simulated with a pipeline of 2 cm diameter and connected with the simulated heart. The simulated coronary artery was connected with a big container with 1500 ml saline and 150ml contrast agent. One pump linking with simulated heart will withdraw with a speed of 10 ml/min, 15 ml/min, 20 ml/min, 25 ml/min and 30 ml/min. First CT scan starts after 30 s of pumpback with certain speed. The second CT scan starts 5 s after first CT scans. CT images processed as follows: The second CT scan images subtract first CT scan images, calculate the increase of CT value of simulated heart and the CT value of the unit volume of simulated coronary artery and then to calculate the total inflow of myocardial blood flow. CT myocardial blood flows were calculated as: 0.94 ml/s, 2.09 ml/s, 2.74 ml/s, 4.18 ml/s, 4.86 ml/s. The correlation coefficient is 0.994 and r² = 0.97. The method of measuring the myocardial blood flow using 320 row detector CT by 2 scans is feasible. It is possible to develop a new method for quantitatively and functional assessment of myocardial perfusion blood flow with less radiation does.

The purpose of this study is to quantitatively assess the myocardial perfusion by first-pass technique in swine model. Numerous techniques based on the analysis of Computed Tomography (CT) Hounsfield Unit (HU) density have emerged. Although these methods proposed to be able to assess haemodynamically significant coronary artery stenosis, their limitations are noticed. There are still needs to develop some new techniques. Experiments were performed upon five (5) closed-chest swine. Balloon catheters were placed into the coronary artery to simulate different degrees of luminal stenosis. Myocardial Blood Flow (MBF) was measured using color microsphere technique. Fractional Flow Reserve (FFR) was measured using pressure wire. CT examinations were performed twice during First-pass phase under adenosine-stress condition. CT HU Density (HUDCT) and CT HU Density Ratio (HUDRCT) were calculated using the acquired CT images. Our study presents that HUDRCT shows a good (y=0.07245+0.09963x, r²=0.898) correlation with MBF and FFR. In receiver operating characteristic (ROC) curve analyses, HUDR_CT provides excellent diagnostic performance for the detection of significant ischemia during adenosine-stress as defined by FFR indicated by the value of Area Under the Curve (AUC) of 0.927. HUDRCT has the potential to be developed as a useful indicator of quantitative assessment of myocardial perfusion.