This PDF file contains the front matter associated with SPIE Proceedings Volume 6431, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

Tumors derived from epithelial cells comprise the majority of human tumors and their growth results from the accumulation of multiple mutations affecting cellular processes critical for tissue homeostasis, including cell proliferation and cell death. To understand these processes and address the complexity of cancer cell function, multiple cellular responses to different experimental conditions and specific genetic mutations must be analyzed. Fundamental to this endeavor is the development of rapid cellular assays in genetically defined cells, and in particular, the development of optical imaging methods that allow dynamic observation and real-time monitoring of cellular processes. In this context, we are developing an optical scatter imaging technology that is intended to bridge the gap between light and electron microscopy by rapidly providing morphometric information about the relative size and shape of non-spherical organelles, with sub-wavelength resolution. Our goal is to complement current microscopy techniques used to study cells in-vitro, especially in long-term time-lapse studies of living cells, where exogenous labels can be toxic, and electron microscopy will destroy the sample. The optical measurements are based on Fourier spatial filtering in a standard microscope, and could ultimately be incorporated into existing high-throughput diagnostic platforms for cancer cell research and histopathology of neoplastic tissue arrays. Using an engineered epithelial cell model of tumor formation, we are currently studying how organelle structure and function are altered by defined genetic mutations affecting the propensity for cell death and oncogenic potential, and by environmental conditions promoting tumor growth. This talk will describe our optical scatter imaging technology and present results from our studies on apoptosis, and the function of BCL-2 family proteins.

Near-infrared optical tomography is an interesting technique of imaging with high blood-based contrast. Unfortunately non-invasive NIR tomographic imaging has been restricted to specific organs like breast that can be transilluminated externally. In this paper, we demonstrate that near-infrared (NIR) optical tomography can be employed at the endoscope-scale, and implemented at a rapid sampling speed that allows translation to in vivo use. A spread-spectral-encoding technique based on a broadband light source is combined with light delivery by linear-to-circular fiber bundle, to provide endoscopic probing of multiple source/detector fibers for tomographic imaging as well as parallel sampling of all source-detector pairs for rapid data acquisition. Endoscopic NIR tomography is demonstrated by use of a 12mm diameter probe housing 8 sources and 8 detectors at 8 Hz frame rate. Transrectal NIR optical tomography by use of tissue specimen is also presented. This novel approach provides the key feasibility studies to allow this blood-based contrast imaging technology to be tried in cancer detection of internal organs via endoscopic interrogation.

Complex neuronal structures and interactions make studying fast optical signals associated with brain activation difficult, especially in non-invasive measurements that are further complicated by the filtering effect of the scalp and skull. We have chosen to study fast optical signals in the peripheral nervous system to look at a more simplified biological neuronal structure and a system that is more accessible to non-invasive optical studies. In this study, we recorded spatially resolved electrical and optical responses of the human sural nerve to electrical stimulation. A 0.1 ms electrical stimulation was used to activate the sural nerve. Electrical signals were collected by an electromyogram machine and results showed an electrical response spanning a distance of 8 mm across the nerve. Optical signals were collected by a two-wavelength (690 and 830 nm) near-infrared spectrometer and displayed a characteristic decrease in intensity at both wavelengths. Data were taken at multiple positions and then reproduced five times. The average optical data over the five trials showed an optical signal that was spatially consistent with the electrical response to sural nerve stimulation.

We present an algorithm using data acquired with a time-resolved system with the goal of reconstructing sources of fluorescence emanating from the deep interior of highly scattering biological tissues. A novelty in our tomography algorithm is the integration of a light transport model adapted to rodent geometries. For small volumes, our analysis suggest that neglecting the index of refraction mismatch between diffusive and non-diffusive regions, as well as the curved nature of the boundary, can have a profound impact on fluorescent images and spectroscopic applications relying on diffusion curve fitting. Moreover, we introduce a new least-squares solver with bound constraints adapted for optical problems where a physical non-negative constraint can be imposed. Finally, we find that maximizing the time-related information content of the data in the reconstruction process significantly enhances the quality of fluorescence images. Preliminary noise propagation and detector placement optimization analysis are also presented.

In this work, we discuss the incorporation of a priori information into the inverse problem formulation for fluorescence optical tomography. In this respect, we first formulate the inverse problem in the optimization framework which allows the incorporation of a priori information about the solution and its gradient. Then, we consider the variational problem, which is equivalent to the optimization problem and prove the existence and uniqueness of the solution. Finally, we discuss the design of the functions that incorporate the a priori information into the inverse problem formulation and present a model problem to illustrate the design procedure.

Diffuse optical tomography is a non-invasive method aiming at the detection of breast cancer. The sensitivity and specificity of the method can be increased if a fluorescent contrast agent is used that accumulates in malignant lesions. Recently, Philips developed an optical scanner, where the patient is lying on a bed, with one breast hanging freely in a cup containing an optical matching fluid. 507 optical fibers are mounted in the surface of the measurement cup. The breast is illuminated sequentially by half of these fibers while the other half is used to collect the light that is emanating from the breast. The system uses near-infrared light of continuous wave solid-state lasers to illuminate the breast at four different wavelengths. A complete measurement takes less than ten minutes and involves five breast scans: transmission data are collected for four wavelengths, and fluorescence data for excitation at one wavelength. Here, we present the image reconstruction scheme and a novel method to assess the system performance in terms of lesion detectability. This method uses a statistical significance test on simulated data with and without a lesion. It allows the quantification of the detectability of lesions for different size, position, or contrast of the lesion. It also allows to analyze the potential impact of system improvements or to judge the performance of an image reconstruction algorithm.

The automated estimation of cell division rate plays an important role in the evaluation of a gene function in high throughput biomedical research. Using Computerized Video Time-Lapse (CVTL) microcopy , it is possible to follow a large number of cells in their physiological conditions for several generations. However analysis of this large volume data is complicated due to cell to cell contacts in a high density population. We approach this problem by segmenting out cells or cell clusters through a learning method. The feature of a pixel is represented by the intensity and gradient information in a small surrounding sub-window. Curve evolution techniques are used to accurately find the cell or cell cluster boundary. With the assumption that the average cell size is the same in each frame, we can use the cell area to estimate the cell division rate. Our segmentation results are compared to manually-defined ground truth. Both recall and precision measures for segmentation accuracy are above 95%.

Recent interest in the use of dual modality imaging in the field of optical Near Infrared (NIR) Tomography has increased, specifically with use of structural information, from for example, MRI. Although MRI images provide high resolution structural information about tissue, they lack the contrast and functional information needed to investigate physiology, whereas NIR data has been established as a high contrast imaging modality, but one which suffers from low resolution. To this effect, the use of dual modality data has been shown to increase the qualitative and quantitative accuracy of clinical information that can be obtained from tissue. Results so far have indicated that providing accurate apriori structural information is available, such dual modality imaging techniques can be used for the detection and characterization of breast cancer in-vivo, as well as the investigation of brain function and physiology in both human and small animal studies. Although there has been much interest and research into the best suitable and robust use of a-priori structural information within the reconstruction of optical properties of tissue, little work has been done into the investigation of how much accuracy is needed from the structural MRI images in order to obtain the most clinically reliable information. In this paper, we will present and demonstrate the two most common application of a-priori information into image reconstruction, namely soft and hard priori. The effect of inaccuracies of the a-priori structural information within the reconstructed NIR images are presented showing that providing that the error of the a-priori information is within 20% in terms of size and location, adequate NIR images can be reconstructed.

Radiation therapy (RT) is a standard treatment after lumpectomy for breast cancer, involving a typical course of approximately 6-7 weeks of daily treatment. Many women find this cumbersome and costly, and therefore many are left with the option of mastectomy. Many groups are now investigating novel ways to deliver RT, by using different techniques and shortening the course of treatment. However, the efficacy and side effects of these strategies are not known. In this project, we wish to develop noninvasive imaging tools that would allow us to measure radiation dose effects in women with breast cancer. We hope this will lead to new ways to identify individuals who may not need radiation therapy, who may safely be treated with new accelerated techniques, or who should be treated with the standard radiation therapy approach. We propose to study the effect of radiation therapy using a combination of two imaging modalities: 1) magnetic resonance imaging (MRI) which will provide detailed information on breast structures and blood vessels and 2) near infra-red diffuse optical spectroscopy (DOS), which measures local biologic properties of breast tissue. Our hypothesis is that by using a combination of modalities we will be able to better characterize radiation effects in breast tissue, by measuring differences between the radiated and non-irradiated breast. The development of novel non-invasive tools providing information about how individuals respond to radiation therapy can lead to important improvement of radiation treatment, and ultimately help guide individualized treatment programs in the future.

Frequency domain diffuse optical tomography (DOT) is a recently emerging technique that uses arrays of sources and detectors to obtain spatially dependent optical parameters of tissue. Here, we describe the design of a hybrid MR-DOT system for dynamic imaging cancer. The combined system acquires both MR and optical data simultaneously. The performance of the system is tested with phantom and in-vivo studies. Gd-DTPA and ICG was used for this purpose and the enhancement kinetics of both agents are recorded using the hybrid system.

Recent years have seen significant efforts deployed to apply optical imaging techniques in clinical indications. Optical mammography as an adjunct to X-ray mammography is one such application. 3D optical mammography relies on the sensitivity of near-infrared light to endogenous breast chromophores in order to generate in vivo functional views of the breast. This work presents prospective tissue characterization results from a multi-site clinical study targeting optical tomography as an adjunct to conventional mammography. A 2^nd -generation multi-wavelength time-domain acquisition system was used to scan a wide population of women presenting normal or suspicious X-ray mammograms. Application specific algorithms based on a diffusive model of light transport were used to quantify the breast's optical properties and derive 3D images of physiological indices. Using histopathological findings as a gold standard, results confirm that optically derived parameters provide statistically significant discrimination between malignant and benign tissue in wide population of subjects. The methodology developed for case reviews, lesion delineation and characterization allows for better translation of the optical data to the more traditional x-ray paradigm while maintaining efficacy. They also point to the need for guidelines that facilitate correlation of optical data if those results are to be confirmed in a clinical setting.

A dynamic frequency domain tomography imaging system has been developed for pressure-enhanced breast imaging with data acquisition times less than 15 sec. The amplitude and phase data at three wavelengths, 660, 785 & 826nm, are acquired simultaneously and used for reconstructing images of total hemoglobin (Hb_T), blood oxygen saturation (S_tO₂) and water concentrations in breast. The results of a homogenous blood phantom study indicate that the percent errors of average estimated chromophore values are less than 8% compared with the true values. Crosstalk between the channels in simultaneous acquisition mode resulted in error increases of 6.3% to 7.6% and 3% to 8% over data acquired in sequential mode.

Combining 2D X-ray mammography or 3D tomosynthesis with diffuse optical tomography for breast imaging is advantageous in facilitating clinical diagnosis by fusing the structural X-ray images with functional optical images. In this study, we imaged 65 patients with a combined tomosynthesis/diffuse optical breast imaging system developed at Massachusetts General Hospital. The bulk optical properties and patient demographics were summarized in this paper. The averaged total-hemoglobin for 60 healthy breasts is 21 &mgr;M which is comparable with literature values given the applied mammographic compression in our experiments. The averaged oxygen saturation is 76%. The comparison of contra-lateral breast measurements also demonstrated correlations in total hemoglobin and oxygen saturation. Image reconstructions of the healthy breasts with moderate-sized fibroglandular regions correctly recovered the chest-wall muscle, fibro-glandular tissue as well as the surrounding fatty tissue. For dense breasts, the contrast between the chest-wall and the fibro-glandular region is small and the most pronounced feature of the image is a low-absorption region in the center of the breast. We hypothesized that this is caused by pressure induced blood-redistribution. Supportive evidence for this hypothesis had been shown with mechanical simulations of breast compression.

Incorporating near infrared (NIR) diffuse optical tomography into magnetic resonance imaging (MRI) increases the value of MR breast cancer imaging because it adds functional imaging of hemoglobin, oxygen saturation, water, lipid content, and scattering parameters, properties that infer tissue health. Reconstruction algorithms that incorporate MR into a diffusive modality accrue unavoidable errors from improper tissue segmentation of the MR image, which create inaccuracies in the structural prior. This paper focuses on identifying the most accurate reconstruction approach based on imperfect prior knowledge of tissue boundaries. Specifically, it focuses on how unavoidable segmentation errors of different breast densities affect edge-constraining reconstruction methods to determine the correct approach. Results show that these reconstruction methods all retain the improperly defined edges, but are quantitatively accurate even when the anatomical boundaries mismatch the optical boundaries by as much as 50%. The most accurate approach is one where the problem has been reduced to the least number of unknowns, and the edges are constrained through regularization.

Dynamic multi-wavelength fluorescence imaging was accomplished using a liquid crystal tunable filter (LCTF). Since several different emission wavelengths can be selected by tuning the LCTF, two wavelength dynamic fluorescence imaging was conducted in mice bearing human melanoma M21 and M21L after injection of a mixture of (i) RGD peptide conjugated with a near-infrared (NIR) dye that targeted integrin &agr;v&bgr;3 and (ii) non-specific dye, Cy5.5. Dynamic multi-wavelength imaging with LCTF can differentiate the uptake of the two different fluorescent contrast agents between tumor and normal tissue ROIs in the M21 and M21L xenograft models. Although the LCTF attenuated fluorescence signals by a factor of two when compared to holographic and bandpass filter sets used previously, Tumor to background ratio (TBR) from NIR fluorescence images with a bandpass and holographic filter were not statistically different from those acquired with the LCTF. Therefore, the benefit of spectral information as well as dynamic multi-wavelength may outweigh the impact of the lower transmission efficiencies, and could enable in vivo small animal imaging.

Multi-spectral images of human tissue taken in-vivo often contain image alignment problems as patients have difficulty in retaining their posture during the acquisition time of 20 seconds. Previously, it has been attempted to correct motion errors with image registration software developed for MR or CT data but these algorithms have been proven to be too slow and erroneous for practical use with multi-spectral images. A new software package has been developed which allows the user to play a decisive role in the registration process as the user can monitor the progress of the registration continuously and force it in the right direction when it starts to fail. The software efficiently exploits videocard hardware to gain speed and to provide a perfect subvoxel correspondence between registration field and display. An 8 bit graphic card was used to efficiently register and resample 12 bit images using the hardware interpolation modes present on the graphic card. To show the feasibility of this new registration process, the software was applied in clinical practice evaluating the dosimetry for psoriasis and KTP laser treatment. The microscopic differences between images of normal skin and skin exposed to UV light proved that an affine registration step including zooming and slanting is critical for a subsequent elastic match to have success. The combination of user interactive registration software with optimal addressing the potentials of PC video card hardware greatly improves the speed of multi spectral image registration.

A small animal multimodality tomographer dedicated to the co-registration of fluorescence optical signal and X-rays measurements has been developed in our laboratory. The purpose of such a system is to offer the possibility to get in vivo anatomical and functional information at once. Moreover, anatomical measurements can be used as a regularization factor in order to get the reconstructions of the biodistribution of fluorochromes more accurate and to speed up the treatment. The optical system is basically composed with a CW laser (Krypton, 752 nm) for an optimal excitation of Alexa-Fluor 750 fluorochromes, and a CCD camera coupled with a combination of filters for the fluorescence detection. The animal is placed inside a transparent tube filled with an index matching fluid. In order to perform multiple views of fluorescence data acquisitions, the cylinder is fixed to a rotating stage. The excitation beam is brought to the cylinder via two mirrors mounted on translation plates allowing a vertical scan. The optical data acquisitions are performed with a high sensitivity CCD camera. The X-ray generator and the X-ray detector have been placed perpendicularly to the optical chain. A first study on phantoms was conducted to evaluate the feasibility, to test the linearity and the reproducibility, and to fix the parameters for the co-registration. These test experiments were reproduced by considering mice in the oesophagus of which the previous tubes were inserted. Finally, the performance of the system was evaluated in vivo on mice bearing tumours in the lungs, tagged with Transferrin-AlexaFluor 750.

Mohs surgery is a procedure for microscopically excising basal cell carcinomas (BCCs) while preserving maximal surrounding normal skin. Each serial excision is guided by examination of the frozen histology of the previous excision. Because several (2-20) excisions must be made and frozen histology prepared for each excision. Mohs surgery is time-consuming (15-45 minutes per excision) and tedious. Real-time confocal reflectance mosaicing enables detection of BCCs directly in fresh excisions, following contrast-enhancement by acetowhitening. A confocal mosaic allows rapid observation of 15x15 mm2 of tissue, which is equivalent to a low magnification, 2X view of the excision. Relatively large superficial nodular and micronodular BCCs are rapidly detectable in confocal reflectance mosaics, whereas detection of much smaller infiltrative and sclerosing BCCs is a challenge due to the lack of sufficient nuclear/dermis contrast in acetowhitened excisions. Multimodal contrast, combining reflectance with either fluorescence or autofluorescence may make it possible to detect infiltrative and sclerosing BCCs. A reflectance image shows both nuclei and the surrounding dermis, whereas an autofluorescence image (excitation at 488nm, detection 500-700nm) shows only the dermis. Thus, ability of a composite (i.e., reflectance-less-autofluorescence) image shows significantly darkened dermis, with stronger enhancement of nuclear/dermis contrast. Preliminary results illustrate that this may enable detection of infiltrative and sclerosing BCCs. The use of reflectance and autofluorescence parallels the use of two stains (hematoxylin and eosin) in histology, thus allowing a more complete optical detection method.

The theoretical modeling of fluorescence excitation, emission, and propagation within living tissue has been a limiting factor in the development and calibration of in vivo small animal fluorescence imagers. To date, no definitive calibration standard, or phantom, has been developed for use with small animal fluorescence imagers. Our work in the theoretical modeling of fluorescence in small animals using solid modeling software is useful in optimizing the design of small animal imaging systems, and in predicting their response to a theoretical model. In this respect, it is also valuable in the design of a fluorescence phantom for use in in vivo small animal imaging. The use of phantoms is a critical step in the testing and calibration of most diagnostic medical imaging systems. Despite this, a realistic, reproducible, and informative phantom has yet to be produced for use in small animal fluorescence imaging. By modeling the theoretical response of various types of phantoms, it is possible to determine which parameters are necessary for accurately modeling fluorescence within inhomogenous scattering media such as tissue. Here, we present the model that has been developed, the challenges and limitations associated with developing such a model, and the applicability of this model to experimental results obtained in a commercial small animal fluorescence imager.

A recently developed, freely available, application specifically designed for the visualization of multimodal data sets is presented. The application allows multiple 3D data sets such as CT (x-ray computer tomography), MRI (magnetic resonance imaging), PET (positron emission tomography), and SPECT (single photon emission tomography) of the same subject to be viewed simultaneously. This is done by maintaining synchronization of the spatial location viewed within all modalities, and by providing fused views of the data where multiple data sets are displayed as a single volume. Different options for the fused views are provided by plug-ins. Plug-ins typically used include color-overlays and interlacing, but more complex plug-ins such as those based on different color spaces, and component analysis techniques are also supported. Corrections for resolution differences and user preference of contrast and brightness are made. Pre-defined and custom color tables can be used to enhance the viewing experience. In addition to these essential capabilities, multiple options are provided for mapping 16-bit data sets onto an 8-bit display, including windowing, automatically and dynamically defined tone transfer functions, and histogram based techniques. The 3D data sets can be viewed not only as a stack of images, but also as the preferred three orthogonal cross sections through the volume. More advanced volumetric displays of both individual data sets and fused views are also provided. This includes the common MIP (maximum intensity projection) both with and without depth correction for both individual data sets and multimodal data sets created using a fusion plug-in.

Manipulation of Interstitial Fluid Pressure (IFP) has clinical potential when used in conjunction with near infrared spectroscopy for detection and characterization of breast cancer. IFP is a function of blood chemistry, vessel microanatomy, mechanical properties of the tissue, tissue geometry, and external force. IFP has been demonstrated higher in tumors than normal tissue, and it has been suggested that increased IFP can lead to changes in near infrared absorbing and scattering coefficients. While it is known that external forces can increase IFP, the relationship of force to IFP in a viscoelastic, hyperelastic solid such as tissue is complex. Fluid pressure measurements were taken in gelatin phantoms of equivalent elastic modulus to adipose and glandular tissues of the breast using a WaveMap pressure transducer. 3D pressure maps were obtained for the volumes of the phantoms with an externally applied force of 10mmHg, demonstrating the contribution of shear stress, non-linear mechanical properties, and tissue geometry. Linear elastic computational models were formulated for breast tissue with and without an inclusion of tumor-like mechanical properties. Comparison of experimental and computational model data indicates that light external pressure can lead to heterogeneous IFP distribution within tissues and increased IFP gradients around tumor-like inclusions.

Multimodality molecular imaging that combines anatomical and functional information has shown promise in development of tumor-targeted pharmaceuticals for cancer detection or therapy. Most multimodality imaging techniques are based on nuclear imaging modalities and MRI or CT. Fluorescence molecular tomography (FMT) is an emerging optical modality for non-invasive functional imaging and early diagnosis of carcinoma. Three-dimensional FMT can differentiate tissue physiological changes in vivo to provide functional information when used in conjunction with cancer cell selectively targeted probes. In this study, we present the design of such a system for multimodality molecular imaging. A frequency domain radio frequency technique based on commercial amateur radio equipment has been developed. A heterodyne method is used to transfer a low frequency oscillation into a single-side-band at radio frequency. The difference in phase, caused by fluorescence photon density wave, is detected between a transmitting fiber and a receiving fiber bundle, and then measured at lower frequency after demodulation. To achieve multimodality molecular imaging, a new fluorescent labeled tumor-targeting probe, fluorescent bombesin conjugates, has been developed with high affinity and specificity for targeting breast cancer cells. The developed multimodality fusion strategy will provide increased sensitivity/specificity for cancer cells, with respect to any single imaging modality.

Osteoarthritis (OA), characterized by the damage of the articular cartilage, is the most common joint problem worldwide. In the effort of developing new clinical tools with the potential to alter the natural history of OA, near-infrared diffuse optical tomography (DOT) has received much attention due to its unique advantages. For optical imaging in highly heterogeneous media such as the finger joints, prior information could improve the quality of optical imaging. We report a hybrid imaging system for early detection of OA in the finger joints by imposing the geometry information obtained by X-ray on three-dimensional near-infrared DOT. X-ray tomosynthesis was employed to recover the three-dimensional structure of the two bones based on 16 X-ray projections generated with a mini C-arm system at different directions within a range of 180 degrees. The interface was carefully designed to guarantee an accurate co-registration of the optical and x-ray modalities. The prior structural information of bones was incorporated into our multi-modality imaging reconstruction algorithm to enhance the recovery of the optical properties of joint tissues. Several healthy and OA finger joints were examined. The initial clinical results showed that this hybrid imaging system had the ability to provide much enhanced image resolution and contrast than DOT alone for OA detection.

We present a design for frequency domain instrument that allows for simultaneous gathering of magnetic resonance and diffuse optical tomographic imaging data. This small animal imaging system combines the high anatomical resolution of magnetic resonance imaging (MRI) with the high temporal resolution and physiological information provided by diffuse optical tomography (DOT). The DOT hardware comprises laser diodes and an intensified CCD camera, which are modulated up to 1 GHz by radio frequency (RF) signal generators. An optical imaging head is designed to fit inside the 4 cm inner diameter of a 9.4 T MRI system. Graded index fibers are used to transfer light between the optical hardware and the imaging head within the RF coil. Fiducial markers are integrated into the imaging head to allow the determination of the positions of the source and detector fibers on the MR images and to permit co-registration of MR and optical tomographic images. Detector fibers are arranged compactly and focused through a camera lens onto the photocathode of the intensified CCD camera.