This PDF file contains the Front Matter associated with SPIE Proceedings Volume 7891, including the Title page, Copyright information, Table of Contents, Conference Committee listing, and introduction.

Endoscopic investigation of the main pancreatic duct and biliary ducts is called endoscopic retrograde cholangiopancreatography (ERCP), and carries a risk of pancreatitis for the patient. During ERCP, a metal guidewire is inserted into the pancreatobiliary duct from a side-viewing large endoscope within the duodenum. To verify correct placement of the ERCP guidewire, an injection of radiopaque dye is required for fluoroscopic imaging, which exposes the patient and clinical team to x-ray radiation. A safer and more effective means to access the pancreatobiliary system can use direct optical imaging, although the endoscope diameter and stiffness will be significantly larger than a guidewire's. To quantify this invasiveness before human testing, a synthetic force-sensing pancreas was fabricated and attached to an ERCP training model. The invasiveness of a new, 1.7-mm diameter, steerable scanning fiber endoscope (SFE) was compared to the standard ERCP guidewire of 0.89-mm (0.035") diameter that is not steerable. Although twice as large and significantly stiffer than the ERCP guidewire, the SFE generated lower or significantly less average force during insertion at all 4 sensor locations (P<0.05) within the main pancreatic duct. Therefore, the addition of steering and forward visualization at the tip of the endoscope reduced the invasiveness of the in vitro ERCP procedure. Since fluoroscopy is not required, risks associated with dye injection and x-ray exposure can be eliminated when using direct optical visualization. Finally, the SFE provides wide-field high resolution imaging for image-guided interventions, laser-based fluorescence biomarker imaging, and spot spectral analysis for future optical biopsy.

Near infrared fluorescence (NIRF) optical imaging has been successfully demonstrated to offer a high specificity and sensitivity in detecting various diseases. However, the measurement sensitivity of NIRF optical imaging system is limited by strong backscattered excitation light leakage. Herein, appropriate filter combination and collimation optics was adapted to the NIRF optical imager. The sensitivity of near-infrared fluorescence imaging instrumentation can be dramatically improved upon using the appropriate filter combination and collimation optics. This validation and qualification approach to reduce the noise floor and improve sensitivity is presented a standardized metric for all fluorescence based imaging systems proposed.

A significant challenge in detecting cervical pre-cancer in low-resource settings is the lack of effective screening facilities and trained personnel to detect the disease before it is advanced. Light based technologies, particularly quantitative optical spectroscopy, have the potential to provide an effective, low cost, and portable solution for cervical pre-cancer screening in these communities. We have developed and characterized a portable USB-powered optical spectroscopic system to quantify total hemoglobin content, hemoglobin saturation, and reduced scattering coefficient of cervical tissue in vivo. The system consists of a high-power LED as light source, a bifurcated fiber optic assembly, and two USB spectrometers for sample and calibration spectra acquisitions. The system was subsequently tested in Leogane, Haiti, where diffuse reflectance spectra from 33 colposcopically normal sites in 21 patients were acquired. Two different calibration methods, i.e., a post-study diffuse reflectance standard measurement and a real time self-calibration channel were studied. Our results suggest that a self-calibration channel enabled more accurate extraction of scattering contrast through simultaneous real-time correction of intensity drifts in the system. A self-calibration system also minimizes operator bias and required training. Hence, future contact spectroscopy or imaging systems should incorporate a selfcalibration channel to reliably extract scattering contrast.

A real-time three dimensional (3D) fluorescence imaging system incorporating wavelength-coded and multiplexed holographic gratings is presented. Holographic gratings formed in thick Phenanthrenquinone- (PQ-) Doped Poly (methyl methacrylate) (PMMA) have narrowband spectral-spatial transmittance filtering properties to generate wavelengthspectrum selective multi-focal planes within a biological object. We demonstrate the imaging modality to obtain laserinduced fluorescent tissue structures from different depths at the excitation wavelength of 355nm.

Full field laser Doppler perfusion imaging offers advantages over scanning laser Doppler imaging as the effects of movement artifacts are reduced. The increased frame rate allows rapid changes in blood flow to be imaged. A custom made CMOS sensor offers several advantages over commercial cameras as the design can be optimized to the detected signals. For example, laser Doppler signals are known to have a bandwidth from DC up to ~20KHz and be of a low modulation depth. Therefore a design that can amplify the AC component and have a sampling rate and an antialiasing filter appropriate to the signal bandwidth would be beneficial. An additional advantage of custom made sensors is that on-chip processing of blood flow allows the data bottleneck that exists between the photo-detector array and processing electronics to be overcome, as the processed data can be read out from the image sensor to a PC or display at a low data rate. A fully integrated 64x64 pixel array for imaging blood flow is presented. On-chip analog signal processing is used to amplify the AC component, normalize the AC signal by the DC light intensity and provide anti-aliasing. On-chip digital signal processing is used to implement the filters required to calculate blood flow. The imaging array has been incorporated into a device that has been used in a clinical setting. Results are presented demonstrating changes in blood flow in occlusion and release tests.

A study of blood perfusion mapping was performed with a remote opto-physiological imaging (OPI) system coupling a sensitive CMOS camera and a custom-built resonant cavity light emitting diode (RCLED) ringlight. The setup is suitable for the remote assessment of blood perfusion in tissue over a wide range of anatomical locations. The purpose of this study is to evaluate the reliability and stability of the OPI system when measuring a cardiovascular variable of clinical interest, in this case, heart rate. To this end, the non-contact and contact photoplethysmographic (PPG) signals obtained from the OPI system and conventional PPG sensor were recorded simultaneously from each of 12 subjects before and after 5-min of cycling exercise. The time-frequency representation (TFR) method was used to visualize the time-dependent behavior of the signal frequency. The physiological parameters derived from the images captured by the OPI system exhibit comparable functional characteristics to those taken from conventional contact PPG pulse waveform measurements in both the time and frequency domains. Finally and more importantly, a previously developed opto-physiological model was employed to provide a 3-D representation of blood perfusion in human tissue which could provide a new insight into clinical assessment and diagnosis of circulatory pathology in various tissue segments.

Quantitative data on the fundamental optical properties (OPs) of biological tissue, including absorption and reduced scattering coefficients are important for elucidating light propagation during optical spectroscopy and facilitating diagnostic device design and optimization, and may enable rapid detection of early neoplasia. However, systems for in situ broadband measurement of mucosal tissue OPs in the ultraviolet-visible range have not been realized. In this study, we evaluated a fiberoptic-based reflectance system, coupled with neural network inverse models (trained with Monte Carlo simulation data), for measuring OPs in highly attenuating, two-layer turbid media. The experimental system incorporated a broadband light source, a fiberoptic probe and a CCD camera. The calibration method involved a set of standard nigrosin-microsphere phantoms as well as a more permanent spectralon phantom for quality assurance testing and recalibration. The system was experimentally evaluated using two-layer hydrogel phantoms with hemoglobin and polystyrene microspheres. The effects of tissue top-layer thickness and fitting approaches based on known absorption and scattering distributions were discussed. With our method, measurements with error less than 28% were obtained in the wavelength range of 350-630 nm.

As a new time-resolved method which combines the spread spectrum time-resolved method with single photon counting, pseudo-random single photon counting (PRSPC) has been proved to have the potential for high speed data acquisition due to high count rate achievable. A continuous wave laser modulated by a pseudo-random bit sequence is used to illuminate the sample, while single photon counting is used to build up the optical signal in response to the excitation. Periodic cross-correlation is performed to retrieve the temporal profile. Besides the high count rate, PRSPC also offers low system cost and portability which are not with the conventional time-correlated single photon counting (TCSPC). In this paper, we report a high speed PRSPC system that can be used for real time acquisition of the temporal spread function (TPSF) of diffuse photons. We also present preliminary experimental work of human blood glucose testing studies by utilizing the PRSPC system.

In this paper, we present our efforts in improving the penetration depth of MPM and the development of a multiphoton endoscope for imaging intrinsic tissue fluorescence and harmonic generation in vivo, with a main focus on instrument design and optimization.

We demonstrate that the sidelobes of a Bessel function generated by an axicon were suppressed by more than 20 dB over 5 mm depth of focus in a Fourier domain optical coherence tomography using a single mode fiber. We also show a rotational image in biological tubular in vivo samples with 2.5 μm axial resolution, 8 μm lateral resolution, and 5 mm depth of focus using a custom-designed axicon.

We demonstrate micron-class-resolution frequency-domain OCT at high acquisition-speeds using a commercial supercontinuum source (i.e. 300nm bandwidth centered at 800nm) and a broadband custom astigmatism-corrected spectrometer (i.e. < 0.1 nm spectral resolution over 400nm bandwidth). We achieved 1.3 μm axial resolution in, in vivo skin tissue, with an acquisition speed of 23 A-scans per sec using the implemented FD-OCT.

We have reported a technique of full-range imaging so-called a Dual-Detection Frequency Domain Optical Coherence Tomography (DD-FD-OCT), in which two spectra, representing real and imaginary components of a complex spectral interference, are simultaneously acquired by two detection channels. In this paper, we will discuss in detail a mirror rejection performance of DD-FD-OCT. Furthermore, we present an implementation of DD-FD-OCT for phase-resolved Doppler imaging. The DD-FD-OCT signal is achieved without manipulation of the phase relation between consecutive axial lines and hence its phase information is almost identical to that acquired by the conventional FD-OCT. As a result, the full-range DD-FD-OCT is fully applicable to phase-resolved Doppler imaging without either degradation in the fullrange performance or reduction in the velocity dynamic range in flow measurement. An in vivo flow imaging within biological samples using the DD-FD-OCT is demonstrated.

Here we present a novel coherent optical receiver that can be easily adapted to biomedical imaging systems. The proposed receiver provides amplitude, phase and polarization information. The principle of operation is discussed and the design and characterization of the receiver is presented.

The photon-scattering imaging data of Liposyn II intravenous emulsion solution samples of different concentrations and different thicknesses is reported and analyzed. The scattering Mueller matrix element m₁₁ data shows that the maximum number of multi-photon scatterings is an increasing function of concentration and sample thickness.

In hyper-spectral imaging systems with a wide spectral range, axial optical aberrations may lead to a significant blurring of image intensities in certain parts of the spectral range. Axial optical aberrations arise from the indexof- refraction variations that is dependent on the wavelength of incident light. To correct axial optical aberrations the point-spread function (PSF) of the image acquisition system needs to be identified. We proposed a multiframe joint blur identification and image restoration method that maximizes the likelihood of local image energy distributions between spectral images. Gaussian mixture model based density estimate provides a link between corresponding spatial information shared among spectral images so as to find and restore the image edges via a PSF update. Model of the PSF was assumed to be a linear combination of Gaussian functions, therefore the blur identification process had to find only the corresponding scalar weights of each Gaussian function. Using the identified PSF, image restoration was performed by the iterative Richardson-Lucy algorithm. Experiments were conducted on four different biological samples using a hyper-spectral imaging system based on acousto-optic tunable filter in the visible spectral range (0.55 - 1.0 μm). By running the proposed method, the quality of raw spectral images was substantially improved. Image quality improvements were quantified by a measure of contrast and demonstrate the potential of the proposed method for the correction of axial optical aberrations.

Near-infrared hyperspectral imaging is becoming a popular tool in the biomedical field, especially for detection and analysis of different types of cancers, analysis of skin burns and bruises, imaging of blood vessels and for many other applications. As in all imaging systems, proper illumination is crucial to attain optimal image quality that is needed for best performance of image analysis algorithms. In hyperspectral imaging based on filters (AOTF, LCTF and filter wheel) the acquired spectral signature has to be representative in all parts of the imaged object. Therefore, the whole object must be equally well illuminated - without shadows and specular reflections. As there are no restrictions imposed on the material and geometry of the object, the desired object illumination can only be achieved with completely diffuse illumination. In order to minimize shadows and specular reflections in diffuse illumination the light illuminating the object must be spatially, angularly and spectrally uniform. We present and test two diffuse illumination system designs that try to achieve optimal uniformity of the above mentioned properties. The illumination uniformity properties were measured with an AOTF based hyperspectral imaging system utilizing a standard white diffuse reflectance target and a specially designed calibration target for estimating the spatial and angular illumination uniformity.

The new "scientific CMOS" (sCMOS) sensor technology has been tested for use in hyperspectral imaging. The sCMOS offers extremely low readout noise combined with high resolution and high speed, making it attractive for hyperspectral imaging applications. A commercial HySpex hyperspectral camera has been modified to be used in low light conditions integrating an sCMOS sensor array. Initial tests of fluorescence imaging in challenging light settings have been performed. The imaged objects are layered phantoms labelled with controlled location and concentration of fluorophore. The camera has been compared to a state of the art spectral imager based on CCD technology. The image quality of the sCMOS-based camera suffers from artifacts due to a high density of pixels with excessive noise, attributed to the high operating temperature of the array. Image processing results illustrate some of the benefits and challenges of the new sCMOS technology.

The application of hyperspectral imaging requires rigorous characterization of the spatial and spectral imaging domains of the system. We present a microarray printing methodology for the testing of absorption or reflectance microscopy measurements. This controlled system can serve as a platform for inter-system calibration and provides a common framework for the development of post-processing algorithms. Calibration of the illumination at the objective plane using a transfer standard spectroradiometer allows comparison of light levels regardless of the illumination used, different apertures, and different microscopes. The method uses standard commercial optomechanical components. Printed dyes enable multiplexed testing of the spectral capability of a hyperspectral instrument. The spectral signatures of individual or blended dyes can be analyzed and applied to the testing of spectral image processing tools. Customized programming of the microarrayer allows for arbitrary patterning of dye samples onto the substrate, allowing for the testing of image processing algorithms involving the spatial distribution of spectral features.

Optical diagnostics has the potential to provide real-time diagnosis of tissue noninvasively, and many optical diagnostic techniques are receiving extensive attention and being developed. Frequency domain (FD) near-infrared diffuse spectroscopy (NIRS) is one of the three common techniques in NIRS field. Generally, a FD system modulates the light intensity in radio frequency and measures the amplitude attenuation and phase delay of the diffused light using heterodyne detection. This article deals with the method for eliminating or calibrating both coupling factor and the intrinsic parameters of the measurement system, which include the intrinsic amplitude attenuation and intrinsic phase delay. Several calibration methods are proposed, namely, calibration with standard phantom, calibration based on multiple source-detector separations (SDS), and calibration with the combination of standard phantom and multiple SDS. Two solid tubular phantoms with known optical properties are adopted to evaluate the proposed calibration methods. Endoscopic measurements on the phantoms were carried on to obtain the amplitude attenuation and phase delay while Monte Carlo simulation was employed to calculate the "real" ones. Results show that the calibration method with the combination of standard phantom and multiple SDS gets the minimum relative error of amplitude.

We present real-time, full-field, fluorescence polarization microscopy and its calibration and validation methods to monitor the absorption dipole orientation of fluorescent molecules. A quarter-wave plate, in combination with a liquid crystal variable retarder (LCVR), provides a tunable method to rotate a linear polarized light prior to being coupled into a fluorescence microscope. A series of full-field fluorescence polarization images are obtained of fluorescent molecules interleaved into the lipid bilyaer of liposomes. With this system, the dynamic dipole orientation of the fluorescent lipid analog tetramethylindocarbocyanine (DiI)-labeled lipids inserted in liposomes are probed and found to be aligned with the liposome in a tangential manner. The dipole orientation of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)- labeled lipids are expected to be aligned perpendicularly in the liposome membrane. Spectral separation of fluorescent lipid analogs into separate images provide an internal control and the ability to quantitatively correlate the membrane structure and fluctuations, within an optical section, in real-time. Application of this technique to the identification of characteristic features of cellular processes such as adhesion, endocytosis, and apoptosis are being investigated.