This PDF file contains the front matter associated with SPIE Proceedings Volume 8216, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing.

We developed a heat-sensitive microbubble (HSM) agent for intraoperative assessment of thermal ablation margins in cancer ablation therapies. The HSM agent, comprising a core of liquid perfluorocarbon (PFC) compound and a shell of biodegradable poly lactic-coglycolic acid (PLGA), was fabricated using an emulsion evaporation method. In our previous study, significant increase of ultrasound contrast was observed after heat activation of HSMs. In this study, intraoperative ultrasonic assessment of thermal ablation margins by HSMs was demonstrated in vivo in a pig model. HSMs were delivered to the pig liver by portal vein injection. Liver ablation was done using a RF ablation probe. Intraoperative ultrasound imaging with HSMs clearly delineated the ablation margin. Fluorescence images of liver tissue samples confirmed the existence and activation of HSMs. This result demonstrated that the HSM agent can be potentially utilized as a multimodal contrast agent for intraoperative ultrasonic and fluorescence assessment of thermal ablation margins in cancer ablation therapies.

The importance of cellular pH has been shown clearly in the study of cell activity, pathological feature, drug metabolism, etc. Monitoring pH changes of living cells and imaging the regions with abnormal pH values in vivo could provide the physiologic and pathologic information for the research of the cell biology, pharmacokinetics, diagnostics and therapeutics of certain diseases such as cancer. Thus, pH-sensitive fluorescence imaging of bulk tissues has been attracting great attention in the regime of near-infrared diffuse fluorescence tomography (DFT), an efficient small-animal imaging tool. In this paper, the feasibility of quantifying pH-sensitive fluorescence targets in turbid medium is investigated using both time-domain and steady-state DFT methods. By use of the specifically designed time-domain and continuous-wave systems and the previously proposed image reconstruction scheme, we validate the method through 2-dimensional imaging experiments on a small-animal-sized phantom with multiply targets of distinct pH values. The results show that the approach can localize the targets with reasonable accuracy and achieve quantitative reconstruction of the pH-sensitive fluorescent yield.

The study aims at developing an optical measurement module incorporated with an X-ray mammographic system to obtain diffuse optical images (DOI) for the detection of breast tumors. Two goals steer the study: (1) to enhance sensitivity and specificity of tumor detection through the use of functional DOI; and (2) to reduce radiation exposure by using only one mammogram, instead of two, as structure information to compute optical-coefficient images. A dual-direction (downward and upward) scanning device to project illuminated near infrared light with multiple-channel switching for both sources and detectors was designed and constructed to obtain double information. The designed and constructed NIR scanning module incorporates with GE Senographe 2000D to assist breast tumor detection.

There are visible changes during skin aging. In the extracellular matrix these changes referred to as intrinsic aging (skin areas not exposed to sunlight) and extrinsic aging can be measured using various methods, such as subjective clinical evaluation, histology and molecular analysis. In this study we developed a new parameter for the non-invasive quantitative determination of dermal skin aging utilizing a five-dimensional intravital tomography (5D-IVT). This device, also known as 5D - multi-photon laser scanning microscopy, is a powerful tool to investigate (photo)aging-associated alterations in vivo. Structural alterations in the dermis of extrinsically aged (chronically sun-exposed) and intrinsically aged (sun-protected) human skin were recorded utilizing the collagen-specific second harmonic generation (SHG) signal and the elastin-specific autofluorescence (AF) signal. Recording took place in young and elderly volunteers. The resulting images were processed in order to gain the elastin percentage and the collagen percentage per image. Then, the elastin - to - collagen ratio (ELCOR) was calculated. With respect to volar forearm skin, the ELCOR significantly increased with age. In elderly volunteers, the ELCOR value calculated for the chronically sun-exposed temple area was significantly augmented compared with the sun-protected upper arm area. Based on 5D-IVT we introduce the ELCOR as a new means to quantify age-associated alterations in the extracellular matrix of in vivo human skin. This novel parameter is compared to the currently used "SHG to AF aging index" of the dermis (SAAID).

Optical Coherence Tomography (OCT) is an efficient technique for in-depth optical biopsy of biological tissues, relying on interferometric selection of ballistic photons. Full-Field Optical Coherence Tomography (FF-OCT) is an alternative approach to Fourier-domain OCT (spectral or swept-source), allowing parallel acquisition of en-face optical sections. Using medium numerical aperture objective, it is possible to reach an isotropic resolution of about 1x1x1 ìm. After stitching a grid of acquired images, FF-OCT gives access to the architecture of the tissue, for both macroscopic and microscopic structures, in a non-invasive process, which makes the technique particularly suitable for applications in pathology. Here we report a multimodal approach to FF-OCT, combining two Full-Field techniques for collecting a backscattered endogeneous OCT image and a fluorescence exogeneous image in parallel. Considering pathological diagnosis of cancer, visualization of cell nuclei is of paramount importance. OCT images, even for the highest resolution, usually fail to identify individual nuclei due to the nature of the optical contrast used. We have built a multimodal optical microscope based on the combination of FF-OCT and Structured Illumination Microscopy (SIM). We used x30 immersion objectives, with a numerical aperture of 1.05, allowing for sub-micron transverse resolution. Fluorescent staining of nuclei was obtained using specific fluorescent dyes such as acridine orange. We present multimodal images of healthy and pathological skin tissue at various scales. This instrumental development paves the way for improvements of standard pathology procedures, as a faster, non sacrificial, operator independent digital optical method compared to frozen sections.

Current methods for detection of oral cancer lack the ability to delineate between normal and precancerous tissue with adequate sensitivity and specificity. The usual diagnostic mechanism involves visual inspection and palpation followed by tissue biopsy and histopathology, a process both invasive and time-intensive. A more sensitive and objective screening method can greatly facilitate the overall process of detection of early cancer. To this end, we present a multimodal imaging system with fluorescence lifetime imaging (FLIM) for wide field of view guidance and reflectance confocal microscopy for sub-cellular resolution imaging of epithelial tissue. Moving from a 12 x 12 mm² field of view with 157 ìm lateral resolution using FLIM to 275 x 200 μm² with lateral resolution of 2.2 μm using confocal microscopy, hamster cheek pouch model is imaged both in vivo and ex vivo. The results indicate that our dual modality imaging system can identify and distinguish between different tissue features, and, therefore, can potentially serve as a guide in early oral cancer detection..

Cutaneous aging is a complicated biological process affecting different constituents of skin, which can be divided into two types: the chronological aging and the photo-aging. The two cutaneous aging processes often co-exist accompanying with each other. The effects are often overlapped including changes in epithelium and dermis. The degeneration of collagen is a major factor in dermal alteration with aging. In this study, multiphoton microscopy (MPM) with its high resolution imaging and optical coherence tomography (OCT) with its depth resolved imaging were used to study the anti-aging dermatology in vivo. It was attempted to make the optical parameter and texture feature to evaluate the process of aging skin using mathematical image processing. The links among optical parameter, spectrum and texture feature in collagen with aging process were established to uncover mechanism of aging skin.

Near-Infrared Spectroscopy (NIRS) measures the functional hemodynamic response occuring at the surface of the cortex. Large pial veins are located above the surface of the cerebral cortex. Following activation, these veins exhibit oxygenation changes but their volume likely stays constant. The back-reflection geometry of the NIRS measurement renders the signal very sensitive to these superficial pial veins. As such, the measured NIRS signal contains contributions from both the cortical region as well as the pial vasculature. In this work, the cortical contribution to the NIRS signal was investigated using (1) Monte Carlo simulations over a realistic geometry constructed from anatomical and vascular MRI and (2) multimodal NIRS-BOLD recordings during motor stimulation. A good agreement was found between the simulations and the modeling analysis of in vivo measurements. Our results suggest that the cortical contribution to the deoxyhemoglobin signal change (ΔHbR) is equal to 16-22% of the cortical contribution to the total hemoglobin signal change (ΔHbT). Similarly, the cortical contribution of the oxyhemoglobin signal change (ΔHbO) is equal to 73-79% of the cortical contribution to the ΔHbT signal. These results suggest that ΔHbT is far less sensitive to pial vein contamination and therefore, it is likely that the ΔHbT signal provides better spatial specificity and should be used instead of ΔHbO or ΔHbR to map cerebral activity with NIRS. While different stimuli will result in different pial vein contributions, our finger tapping results do reveal the importance of considering the pial contribution.

Near-infrared (NIR) fluorescence is an alternative modality for molecular imaging that has been demonstrated in animals and recently in humans. Fluorescence-enhanced optical tomography (FEOT) using continuous wave or frequency domain photon migration techniques could be used to provide quantitative molecular imaging in vivo if it could be validated against "gold-standard," nuclear imaging modalities, using dual-labeled imaging agents. Unfortunately, developed FEOT systems are not suitable for incorporation with CT/PET/SPECT scanners because they utilize benchtop devices and require a large footprint. In this work, we developed a miniaturized fluorescence imaging system installed in the gantry of the Siemens Inveon PET/CT scanner to enable NIR transillumination measurements. The system consists of a CCD camera equipped with NIR sensitive intensifier, a diode laser controlled by a single board compact controller, a 2-axis galvanometer, and RF circuit modules for homodyne detection of the phase and amplitude of fluorescence signals. The performance of the FEOT system was tested and characterized. A mouse-shaped solid phantom of uniform optical properties with a fluorescent inclusion was scanned using CT, and NIR fluorescence images at several projections were collected. The method of high-order approximation to the radioactive transfer equation was then used to reconstruct the optical images. Dual-labeled agents were also used on a tumor bearing mouse to validate the results of the FEOT against PET/CT image. The results showed that the location of the fluorophore obtained from the FEOT matches the location of tumor obtained from the PET/CT images. Besides validation of FEOT, this hybrid system could allow multimodal molecular imaging (FEOT/PET/CT) for small animal imaging.

Time Reversal Optical Tomography (TROT) is developed to locate extended target(s) in a highly scattering turbid medium, and estimate their optical strength and size. The approach uses Diffusion Approximation of Radiative Transfer Equation for light propagation along with Time Reversal (TR) Multiple Signal Classification (MUSIC) scheme for signal and noise subspaces for assessment of target location. A MUSIC pseudo spectrum is calculated using the eigenvectors of the TR matrix T, whose poles provide target locations. Based on the pseudo spectrum contours, retrieval of target size is modeled as an optimization problem, using a "local contour" method. The eigenvalues of T are related to optical strengths of targets. The efficacy of TROT to obtain location, size, and optical strength of one absorptive target, one scattering target, and two absorptive targets, all for different noise levels was tested using simulated data. Target locations were always accurately determined. Error in optical strength estimates was small even at 20% noise level. Target size and shape were more sensitive to noise. Results from simulated data demonstrate high potential for application of TROT in practical biomedical imaging applications.

Skin assessment technology based on comparative analysis of single-pixel RGB signal values at poly-chromatic illumination has been proposed. Multi-spectral imaging information from a single snapshot RGB image data set with the inter-channel crosstalk correction can be extracted this way. Proof-of-concept evaluations and measurement results are presented and discussed. Potential of bi-chromatic illumination for skin hemoglobin mapping during arterial occlusion test has been demonstrated.

Recent development in multimodal imaging and image-guided therapy requires multifunctional microparticles that encapsulate several imaging and therapeutic agents in the same carrier for simultaneous detection and treatment of the diseases. However, commonly used microfabrication processes for these microparticles have multiple limitations such as the low encapsulation efficiency and the loss of bioactivity for the encapsulated biological cargos. To overcome these limitations, we have carried out both the experimental and the theoretical studies on coaxial electrospray of poly(lactide-co-glycolide) PLGA microparticles. On the experimental side, a coaxial electrospray setup has been developed and tested. The setup consists of a customized coaxial needle assembly, two ring electrodes, two high-voltage power supplies, two syringe infusion pumps, a particle collection reservoir, and a process monitoring system. On the theoretical side, a classical normal mode method has been used for instability analysis of the coaxial electrified jet based on the experimental parameters. The effects of different dimensionless process parameters on the formation of different unstable modes have also been studied. The reported research represents the first step toward the quantitative control and optimization of the coaxial electrospray process for the fabrication of multifunctional microparticles in multimodal imaging and image-guided therapy.

Most of the reported fluorescence imaging methods and systems highlight the need for three-dimensional information of the inspected region surface geometry. The scope of this manuscript is to introduce an epi-illumination fluorescence imaging system, which has been enhanced with a binocular machine vision system for the translation of the inverse problem solution to the global coordinates system. The epi-illumination fluorescence imaging system is consisted of a structured scanning excitation source, which increases the spatial differentiation of the measured data, and a telecentric lens, which increases the angular differentiation. On the other hand, the binocular system is based on the projection of a structured light pattern on the inspected area, for the solution of the correspondence problem between the stereo pair. The functionality of the system has been evaluated on tissue phantoms and calibration objects. The reconstruction accuracy of the fluorophores distribution, as resulted from the root mean square error between the actual distribution and the outcome of the forward solver, was more than 80%. On the other hand, the surface three-dimensional reconstruction of the inspected region presented 0.067±0.004 mm accuracy, as resulted from the mean Euclidean distance between the three-dimensional position of the real world points and those reconstructed.

Image formation in fluorescence diffuse optical tomography is critically dependent on construction of the Jacobian matrix. For clinical and preclinical applications, because of the highly heterogeneous characteristics of the medium, Monte Carlo methods are frequently adopted to construct the Jacobian. Conventional adjoint Monte Carlo method typically compute the Jacobian by multiplying the photon density fields radiated from the source at the excitation wavelength and from the detector at the emission wavelength. Nonetheless, this approach assumes that the source and the detector in Green's function are reciprocal, which is invalid in general. This assumption is particularly questionable in small animal imaging, where the mean free path length of photons is typically only one order of magnitude smaller than the representative dimension of the medium. We propose a new method that does not rely on the reciprocity of the source and the detector by tracing photon propagation entirely from the source to the detector. This method relies on the perturbation Monte Carlo theory to account for the differences in optical properties of the medium at the excitation and the emission wavelengths. Compared to the adjoint methods, the proposed method is more valid in reflecting the physical process of photon transport in diffusive media and is more efficient in constructing the Jacobian matrix for densely sampled configurations.

Gastric cancer is the second cause of cancer-related death in the world, and it remains difficult to cure because it has been in late-stage once that is found. Early gastric cancer detection becomes an effective approach to decrease the gastric cancer mortality. Bioluminescence tomography (BLT) has been applied to detect early liver cancer and prostate cancer metastasis. However, the gastric cancer commonly originates from the gastric mucosa and grows outwards. The bioluminescent light will pass through a non-scattering region constructed by gastric pouch when it transports in tissues. Thus, the current BLT reconstruction algorithms based on the approximation model of radiative transfer equation are not optimal to handle this problem. To address the gastric cancer specific problem, this paper presents a novel reconstruction algorithm that uses a hybrid light transport model to describe the bioluminescent light propagation in tissues. The radiosity theory integrated with the diffusion equation to form the hybrid light transport model is utilized to describe light propagation in the non-scattering region. After the finite element discretization, the hybrid light transport model is converted into a minimization problem which fuses an l1 norm based regularization term to reveal the sparsity of bioluminescent source distribution. The performance of the reconstruction algorithm is first demonstrated with a digital mouse based simulation with the reconstruction error less than 1mm. An in situ gastric cancer-bearing nude mouse based experiment is then conducted. The primary result reveals the ability of the novel BLT reconstruction algorithm in early gastric cancer detection.

As one of molecular imaging, bioluminescence tomography (BLT) aims to recover internal source from surface measurement. Being an ill-posed inverse problem, BLT source reconstruction is usually converted to an optimization problem through regularization. In this contribution, we build a bimodal hybrid imaging system consisting of BLT and micro-CT, and then propose an improved source reconstruction method based on adjoint diffusion equations (ADEs). Compared with conventional methods based on constrained minimization problem (CMP), ADEs-based method replaces expensive iterative computation with solving a group of linear ADEs. Given surface flux density, internal source power density and photon fluence rate can be efficiently determined in one step. Both numerical and physical experiments are performed to evaluate the bimodal BLT/micro-CT imaging system and this novel reconstruction method. The relevant results demonstrate the feasibility and potential of this source reconstruction method.

In biomedical optics, the Monte Carlo (MC) simulation is widely recognized as a gold standard for its high accuracy and versatility. However, in fluorescence regime, due to the requirement for tracing a huge number of the consecutive events of an excitation photon migration, the excitation-to-emission convention and the resultant fluorescent photon migration in tissue, the MC method is prohibitively time-consuming, especially when the tissue has an optically heterogeneous structure. To overcome the difficulty, we present a parallel implementation of MC modeling for fluorescence propagation in tissue, on the basis of the Graphics Processing Units (GPU) and the Compute Unified Device Architecture (CUDA) platform. By rationalizing the distribution of blocks and threads a certain number of photon migration procedures can be processed synchronously and efficiently, with the single-instruction-multiple-thread execution mode of GPU. We have evaluated the implementation for both homogeneous and heterogeneous scenarios by comparing with the conventional CPU implementations, and shown that the GPU method can obtain significant acceleration of about 20-30 times for fluorescence modeling in tissue, indicating that the GPU-based fluorescence MC simulation can be a practically effective tool for methodological investigations of tissue fluorescence spectroscopy and imaging.

Diffuse fluorescence tomography (DFT) provides spatial distributions of fluorescence parameters by measuring fluorescence signals of probes or agents that are targeted to interior specific molecules or tissues. The potential applications of DFT can be found in drug development and early tumor diagnosis. This work proposes a CT-analogous mode of DFT, where the imaging chamber is impinged by collimated beam from a fiber-coupled laser diode and the resultant fluorescence re-emissions on the opposite side, i.e., the so-called "projections", are collected by eight detection fibers placed from 101.25º to 258.75º perspectives opposite to the incidence that are then successively filtered out into a photon-counting channel for quantification. By rotating the imaging chamber or phantom at an angular, the system acquires the "projections" of surface-emitted fluorescence under different perspectives as a CT system does. This ease of acquiring a large data-set enables realization of high-quality imaging. Pilot experiments on phantoms with Cy5.5-target embedded have validated the efficacy of the proposed method.

In vivo biomedical imaging using near-infrared light must overcome the effects of highly light scattering, which limit the spatial resolution and affect image quality. The high-resolution, sensitive and quantitative fluorescence imaging tool is an urgent need for the applications in small-animal imaging and clinical cancer research. A CT-analogous method for fluorescence molecular tomography (FMT) on small-animal-sized models is presented to improve the spatial resolution of FMT to a limit of several millimeters, depending on the size of the tissue region to be imaged. The method combines FMT physics with the filtered back-projection scheme for image reconstruction of the fan-beam computed tomography, based on the early-photon detection of time-resolved optical signals, and is suitable for two-dimensional (2D) imaging of small size biological models. By use of a normalized Born formulation for the inversion, the algorithm is validated using full time-resolved simulated data for 2D phantom that are generated from a hybrid finite-element and finite-time-difference photon diffusion modeling, and its superiority in the improvement of the spatial resolution is demonstrated by imaging different target-to-background contrast ratios.

Traditionally, volume based finite element method (FEM) or finite difference method (FDM) are applied to the forward problem of the time-domain diffuse fluorescence tomography (DFT), this paper presents a new numerical method for solving the problem: the boundary element method (BEM). Using BEM forward solver is explored as an alternative to the FEM or FDM solution methodology for the elliptic equations used to model the generation and transport of fluorescent light in highly scattering media. In contrast to the FEM or FDM, the boundary integral method requires only representation of the surface meshes, thus requires many fewer nodes and elements than the FEM and FDM. By using BEM forward solver for time-domain DFT, we can simultaneously reconstruct both fluorescent yield and lifetime images. The results have demonstrated that the BEM is suitable for solving the forward problem of time-domain DFT.

A fiber-based non-contact scheme of the time-domain diffuse fluorescence yield and lifetime tomography is described that combines the time-correlated single photon counting technique for high-sensitive, time-resolved detection and CT-analogous configuration for high throughput data collection. A pilot validation of the methodology is performed for two-dimensional scenarios using simulated and experimental data. The results demonstrated the potential of the proposed scheme in improving the image quality.

A time domain noncontact fluorescence tomography system and the corresponding reconstruction algorithm towards the early diagnosis of breast cancer are developed. The time domain system based on the time-correlated single photon counting technique is adopted to provide both the high sensitivity in detection and good capability in multi-parameter reconstruction. Comparing to the conventional contact measurement mode, the noncontact system with light scanning can provide more measurement data for improving the spatial resolution of the images. The performance and efficacy of the system is evaluated with measurements on solid phantoms. For the phantom with single fluorescent target, the fluorescence yield and lifetime were simultaneously reconstructed with good quality. For the phantom with two fluorescent targets, the targets with the center-to-center separation of 20mm and the edge separation of 15mm can be distinguished. Measurements also show that the reconstructed yields are linear to the concentration of the fluorescence dye. The results demonstrated the potential of the system in the in vivo diagnosis of the early breast cancer.

A novel method for optical breast imaging was presented based on fluorescence guided diffusion optical tomography (DOT). In this paper, the time-domain fluorescence parameters (yield and lifetime) were reconstructed based on discrete wavelet transform at first, then the fluorescence images were used to guide and constrain the diffusion optical tomography reconstruction, and the image segmentation strategy based on wavelet coefficient was applied to improve the image quality in DOT. To validate the proposed method, the numerical simulation was performed to demonstrate its computational efficacy. The results showed the feasibility of this method, and the spatial resolution, quantification and computational efficiency in fluorescence diffusion optical tomography and DOT were enhanced evidently.

Multimodal microscope which obtains various images for the same interest area simultaneously plays an important role in the process of comprehensive analysis for biological information. Confocal imaging for reflection and fluorescence emission light and two-photon imaging dealing with nonlinear optical effect are operated in the unified platform by sharing system configuration commonly used for those imaging. Reflection light, fluorescence light and nonlinear optical signal from the same focal point are detected through separate channels and converted to respective images. Common optical system is especially customized to satisfy their requirements considering wavelength band of light used for imaging. Multimodal microscope is implemented and verified through multichannel images for bioexperiments. Confocal reflection imaging for label-free specimen, confocal fluorescence imaging and two-photon fluorescence imaging for specifically stained target are realized and make complementary analysis. The used light signals, continuous wave light from NUV to NIR and pulsed light, is verified through imaging results of designed multimodal microscope.

Axonal demyelination is a common phenomenon in the nervous system in human. Conventional measured approaches such as surface recording electrode and diffusion tensor imaging, are hard to fast and accurately determine the demyelinated status of a fiber. In this study, we first presented a mathematical model of nerve fiber demyelination, and it was combined with second harmonic generation(SHG) technique to study the characteristics of action-potential-encoded SHG and analyze the sensitivity of SHG signals responded to membrane potential. And then, we used this approach to fast examine the injured myelin sheaths resulted from demyelination. Each myelin sheath of a fiber was examined simultaneously by this approach. The results showed that fiber demyelination led to observable attenuation of action potential amplitude. The delay of action potential conduction would be markedly observed when the fiber demyelination was more than 80%. Furthermore, the normal and injured myelin sheaths of a myelinated fiber could be distinguished via the changes of SHG signals, which revealed the possibility of SHG technique in the examination of a demyelinated fiber. Our study shows that this approach may have potential application values in clinic.

Intravascular ultrasound (IVUS) is mature imaging modality to diagnose blood vessel disease, especially for calcification characterization. Based on the intrinsic optical absorption, intravascular photoacoustic (IVPA) works as a complementary method to IVUS. In this paper, we develop a miniature intravascular probe combined photoacoustic and ultrasound imaging. The optical components and ultrasound transducer were integrated to achieve internal illumination. Atherosclerotic human artery was imaged ex vivo, which demonstrates the imaging ability of the multi-functional probe and illustrate its clinical potential.