Multimodal Imaging System Research Articles

Development and Investigation of a Long-Range Time-of-Flight and Color Imaging System

Time-of-flight (Tof) imaging based on the photonic mixer device (PMD) or similar ToF imaging solutions has been limited to short distances in the past, due to limited lighting devices and low sensitivity of ToF imaging chips. Long-range distance measurements are typically the domain of laser scanning systems. In this paper, PMD based medium- and long-range lighting devices working together with a 2-D/3-D camera are presented and several measurement results are discussed. The proposed imaging systems suffer from two systematic limitations in addition to problems due to wind and insufficient lighting: a low lateral resolution of the depth imaging chip and ambiguities in the distance measurements. In order to provide a robust and flexible system, we introduce algorithms to obtain unambiguous depth values (phase unwrapping) and to perform a joint motion compensation and super-resolution. Several experiments were conducted in order to evaluate the components of the multimodal imaging system.

Multi-modal molecular diffuse optical tomography system for small animal imaging

A multi-modal optical imaging system for quantitative 3D bioluminescence and functional diffuse imaging is presented, which has no moving parts and uses mirrors to provide multi-view tomographic data for image reconstruction. It is demonstrated that through the use of trans-illuminated spectral near-infrared measurements and spectrally constrained tomographic reconstruction, recovered concentrations of absorbing agents can be used as prior knowledge for bioluminescence imaging within the visible spectrum. Additionally, the first use of a recently developed multi-view optical surface capture technique is shown and its application to model-based image reconstruction and free-space light modelling is demonstrated. The benefits of model-based tomographic image recovery as compared to two-dimensional (2D) planar imaging are highlighted in a number of scenarios where the internal luminescence source is not visible or is confounding in 2D images. The results presented show that the luminescence tomographic imaging method produces 3D reconstructions of individual light sources within a mouse-sized solid phantom that are accurately localized to within 1.5 mm for a range of target locations and depths, indicating sensitivity and accurate imaging throughout the phantom volume. Additionally the total reconstructed luminescence source intensity is consistent to within 15%, which is a dramatic improvement upon standard bioluminescence imaging. Finally, results from a heterogeneous phantom with an absorbing anomaly are presented, demonstrating the use and benefits of a multi-view, spectrally constrained coupled imaging system that provides accurate 3D luminescence images.

Hybrid PET/MR imaging of tumors using an oleanolic acid-conjugated nanoparticle

Research into multifunctional nanoparticles is focused on creating an agent for use in an all-in-one multimodal imaging system that includes diagnostic imaging, drug delivery, and therapeutic monitoring. We designed a new dual-modality tumor-targeting agent with a new tumor-targeting molecule, oleanolic acid (OA), which is derived from a natural compound and coupled with a macrocyclic chelating agent such as 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), iron oxide nanoparticles (IONP), and radiolabeling components such as 68Ga for dual-modality positron emission tomography (PET)/magnetic resonance imaging (MRI). We attempted to obtain fusion PET/MR images with the 68Ga–NOTA–OA–IONP hybrid tumor-targeting imaging agent using colon cancer (HT-29) xenograft mice models. The HT-29 cancer cells showed high uptake of 68Ga–NOTA–OA–IONP, which also had an inhibitory effect on the cells. Moreover, we obtained PET and MRI tumor images as well as fusion PET/MRI images of the tumors using 68Ga–NOTA–OA–IONP. Therefore, the dual-modality cancer-targeting radiolabeled nanoparticle reported here is a potent imaging agent that is suitable for PET, MRI, and PET/MRI-based diagnosis of tumors; it also has the advantage of not only detecting tumor functionality, but also simultaneously aiding in tumor resolution.

Fluorescence Tomography Reconstruction With Simultaneous Positron Emission Tomography Priors

In this paper, fluorescence molecular tomography (FMT) imaging guided by priors from simultaneous positron emission tomography (PET) was performed on a multi-modality imaging system combining PET and FMT. The target prior information from PET images was employed to the FMT reconstruction procedure using the iteratively reweighted least-squares method. Numerical simulations and phantom experiments were performed to validate the proposed method. The results indicate that incorporating the PET prior information into the FMT reconstruction can potentially improve the spatial resolution of FMT.

In vivophotoacoustic lifetime imaging of tumor hypoxia in small animals

Tumor hypoxia is an important factor in assessment of both cancer progression and cancer treatment efficacy. This has driven a substantial effort toward development of imaging modalities that can directly measure oxygen distribution and therefore hypoxia in tissue. Although several approaches to measure hypoxia exist, direct measurement of tissue oxygen through an imaging approach is still an unmet need. To address this, we present a new approach based on in vivo application of photoacoustic lifetime imaging (PALI) to map the distribution of oxygen partial pressure (pO2) in tissue. This method utilizes methylene blue, a dye widely used in clinical applications, as an oxygen-sensitive imaging agent. PALI measurement of oxygen relies upon pO2-dependent excitation lifetime of the dye. A multimodal imaging system was designed and built to achieve ultrasound (US), photoacoustic, and PALI imaging within the same system. Nude mice bearing LNCaP xenograft hindlimb tumors were used as the target tissue. Hypoxic regions were identified within the tumor in a combined US/PALI image. Finally, the statistical distributions of pO2 in tumor, normal, and control tissues were compared with measurements by a needle-mounted oxygen probe. A statistically significant drop in mean pO2 was consistently detected by both methods in tumors.

Luminescent Magnetic Quantum Dots for In Vitro/In Vivo Imaging and Applications in Therapeutics

The quest for design of newer/advanced methods for medical diagnosis and targeted therapeutics are of utmost interest and challenging too, because of its importance in clinical diagnosis. Currently available diagnosis methodologies have their own disadvantages. These shortcomings can be overcome by using multimodal imaging systems where two or more imaging modalities may be coupled. Nanoparticles being widely studied for targeted drug delivery and as biological contrasting agents, might play a decisive role in such findings. This review is focused towards the ongoing research in the area of hybrid nanocomposites which can be used for both as MRI contrasting agent (magnetic nanoparticles) and molecular imaging studies (using fluorescent quantum dots) at in vitro and in vivo level. Though several reports are available in literature for such bimodal imaging systems, their clinical trials are very restricted, possibly because of the lack of communication between the in vitro and in vivo studies. This review is expected to bridge the gap between such studies.

Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer.

Optical imaging techniques using a variety of contrast mechanisms are under evaluation for early detection of epithelial precancer; however, tradeoffs in field of view (FOV) and resolution may limit their application. Therefore, we present a multiscale multimodal optical imaging system combining macroscopic biochemical imaging of fluorescence lifetime imaging (FLIM) with subcellular morphologic imaging of reflectance confocal microscopy (RCM). The FLIM module images a 16×16 mm² tissue area with 62.5 μm lateral and 320 ps temporal resolution to guide cellular imaging of suspicious regions. Subsequently, coregistered RCM images are acquired at 7 Hz with 400 μm diameter FOV, <1 μm lateral and 3.5 μm axial resolution. FLIM-RCM imaging was performed on a tissue phantom, normal porcine buccal mucosa, and a hamster cheek pouch model of oral carcinogenesis. While FLIM is sensitive to biochemical and macroscopic architectural changes in tissue, RCM provides images of cell nuclear morphology, all key indicators of precancer progression.

Abstract 2864: Molecular imaging of early inflammatory response during hyperplasia and/or colitis in response to bacterial infection.

Abstract Inflammatory diseases such as ulcerative colitis recruit myeloid cells and result in inflammatory responses such as generation of reactive oxygen species (ROS). Development of non-invasive luminescence imaging approaches is needed to evaluate these inflammatory responses. We developed methodologies to show that vibration induced gut inflammation can be determined using luminescence imaging approach in a non-invasive fashion (Papineni and Vizard, World Molecular Imaging Congress 2012). Citrobacter rodentium (CR) is a natural bacterial pathogen that infects the distal colon of mice and induces transmissible murine colonic hyperplasia (TMCH). Here we determined the early inflammatory response (ROS activity) to intestinal infection by CR in a longitudinal study. We further assessed the effects of allelic variation on murine chromosome 11 (B6.CAST. 11) on the inflammatory response. The ROS activity was monitored in real-time in vivo using L-012 (8-amino-5-chloro-7-phenylpyridol [3,4-d]pyridazine-1,4(2H,3H) dione), a chemiluminescence reporter, using planar multimodal imaging system. 0.1 ml of 1 mg/ml L-012 probe was injected (i.p.) in control and the experimental mice that were subjected to CR infection. Necrosis was imaged (post-infection day-19) using a fluorescent probe containing a bis (zinc2+dipicolylamine, Zn- DPA) motif which binds with high affinity to apoptotic, necrotic, and bacterial surfaces. Significant difference in the basal gut ROS activity was found in mice with allelic variation on chromosome 11 compared to the wild type littermates. Robust elevation in ROS activity was observed in both the wild type and B6.CAST.11mice that were infected with CR (post infection 9, 12, 19 days). The variation in the levels of ROS activity however was very distinct between the wild type and B6.CAST.11 mice indicating the contribution of molecular determinants at the chromosome 11. The potential advantages in these real-time ROS monitoring methodologies by in vivo imaging, in understanding mechanisms of infection, inflammation, and development of better intervention strategies will be elaborated. Citation Format: Rao Papineni, Ishfaq Ahmed, Steven LeVine, Kenneth Bradley, William Mclaughlin, John Pizzonia, Shahid Umar. Molecular imaging of early inflammatory response during hyperplasia and/or colitis in response to bacterial infection. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2864. doi:10.1158/1538-7445.AM2013-2864

Intraoperative combined color and fluorescent images–based sentinel node mapping in the porcine lung: Comparison of indocyanine green with or without albumin premixing

We developed a multimodal optical imaging system for intraoperative visualization of sentinel lymph nodes (SLNs). This study is to validate our system by showing SLNs in the lung through combined optical color and fluorescent image with indocyanine green (ICG) and ICG with human serum albumin (HSA). Identical ICG concentrations of ICG only or ICG:HSA was injected into the rat footpad and porcine lung. Absolute amounts of the fluorescents were scaled on the basis of animal weights. The entire procedures were recorded using color and near-infrared (NIR) charge-coupled device (CCD) cameras simultaneously, and the 2 images were merged by real-time image processing software. All fluorescence intensity signals to background ratio (SBR) and retention rates at SLN for both fluorescents were estimated and compared. This newly developed intraoperative color and fluorescence optical imaging system successfully visualized the SLNs in animal experiments. The SLNs were identified 100% for both rat and pig under invivo conditions. Real-time image processing software overcame the low signal of NIR fluorescence images. ICG and ICG:HSA provided no significantly different SBR in the SLN images for both rat thigh and pig lung. The intraoperative optical imaging system enabled real-time image-guided surgery during SLN mapping in lung in an animal model. The ICG retention rate was similar to ICG:HSA. ICG alone can be useful for SLN imaging during lung cancer surgery.

Sensitivity of fluorescein angiography alone or with SD-OCT for the diagnosis of myopic choroidal neovascularization

Myopic choroidal neovascularization (mCNV) has certain characteristics and features that distinguish it from choroidal neovascularization secondary to age-related macular degeneration. There may be angiographic diagnostic difficulties even when using the scanning laser ophthalmoscope, which gives more contrast and better definition than traditional angiography. The aim of the study is to compare the sensitivity of fluorescein angiography (FA) alone or combined with Spectral Domain Optical Coherence Tomography (SD-OCT) for assessing the incidence of mCNV. In this retrospective study, two authors reviewed the charts and images of patients with recent (<30 days) vision deterioration, pathologic myopia, axial length >26 mm, documentation or suspicion of mCNV or macular exudative pathologies at FA and OCT. They only examined the images at first presentation obtained by the multi-modal imaging system that combines Infrared reflectance, FA, and SD-OCT, (Spectralis, Heidelberg Engineering, Germany). The images selected were then evaluated by three other investigators in blinded, independent conditions, in order to make their diagnosis, which was noted or rated as doubtful if it could not be decided on the basis of FA alone. SD-OCT images were then shown and compared to IR and FA by each of the three investigators individually to formulate a conclusive diagnosis. A total of 71 eyes of 69 patients were suitable for the study, mean age 65.97±14.57 years, spherical equivalent refraction -8.82 ± 2.51 diopters. Concordance between the three examiners' interpretations of FA features and FA-guided SD-OCT was 50/71 (70.4 %) and 67/71 (94 %) respectively. Total agreement on diagnosis between the three examiners was achieved in 55 % of cases for FA (κ = 0.53, p < 0.001), and 94 % for FA-guided SD-OCT (k = -0.01, p = 0.5). The final diagnosis with FA and FA-guided SD-OCT differed in 29 cases (40 %; 95 % C.I. 29-42 %), whereas 12 (17 %) mCNV were overlooked at FA, and in 11 (15 %) cases none of the examiners reached a diagnosis based on FA alone. On the basis of FA alone, active mCNV can be misdiagnosed. The use of SD-OCT combined with FA should therefore be strongly considered.

EndoTOFPET-US data acquisition system

The EndoTOFPET-US system is a multi-modal imaging system, combining Time of Flight PET (200 ps time resolution) and Ultra-Sound into an endoscopic probe. The PET component has an asymmetric configuration. The in-probe detector is based on 0.8 × 0.8 × 10 mm3 LYSO crystal fibers, read by a Geiger mode SIPM with digital output. The other detector is a 200 × 200 mm external plate, based on 3 × 3× 15 mm3 LYSO crystals, coupled to discrete 3 × 3 SiPMs read by a 64 channel ASIC. A compact data acquisition system based on digital reconfigurable electronics is proposed. FGPAs on the front end concentrate event data and transmit it to an external trigger. The probe electronics also transmit event data to the trigger. The trigger is a single PCIe card which sits directly in the acquisition computer. A preliminary event selection is performed directly in the trigger FPGA, while more sophisticated event processing is handled by software. The system is capable of handling rates up to 40 MHz from the external plate and 200 kHz from the probe.

Extendable, miniaturized multi-modal optical imaging system: cortical hemodynamic observation in freely moving animals

Observation of brain activities in freely moving animals has become an important approach for neuroscientists to understand the correlation between brain function and behavior. We describe an extendable fiber-optic-based multi-modal imaging system that can concurrently carry out laser speckle contrast imaging (LSCI) of blood flow and optical intrinsic signal (OIS) imaging in freely moving animals, and it could be extended to fluorescence imaging. Our imaging system consists of a multi-source illuminator, a fiber multi-channel optical imaging unit, and a head-mounted microscope. The imaging fiber bundle delivers optical images from the head-mounted microscope to the multi-channel optical imaging unit. Illuminating multi-mode fiber bundles transmit light to the head-mounted microscope which has a mass of less than 1.5 g and includes a gradient index lens, giving the animal maximum movement capability. The internal optical components are adjustable, allowing for a change in magnification and field of view. We test the system by observing hemodynamic changes during cortical spreading depression (CSD) in freely moving and anesthetized animals by simultaneous LSCI and dual-wavelength OIS imaging. Hemodynamic parameters were calculated. Significant differences in CSD propagation durations between the two states were observed. Furthermore, it is capable of performing fluorescence imaging to explore animal behavior and the underlying brain functional activity further.

Multimodal imaging with hybrid semiconductor detectors Timepix for an experimental MRI-SPECT system

An increasing number of clinical applications are being based on multimodal imaging systems (MIS), including anatomical (CT, MRI) and functional (PET, SPECT) techniques to provide complex information in a single image. CT with one of the scintigraphic methods (PET or SPECT) is nowadays a combination of choice for clinical practice and it is mostly used in cardiography and tumour diagnostics. Combination with MRI is also being implemented as no radiation dose is imparted to the patient and it is possible to gain higher structural resolution of soft tissues (brain imaging). A major disadvantage of such systems is inability to operate scintillators with photomultipliers (used for detection of γ rays) in presence of high magnetic fields. In this work we present the application of the semiconductor pixel detector for SPECT method in combination with MR imaging. We propose a novel approach based on MRI compatible setup with CdTe pixel sensor Timepix and non-conductive collimator. Measurements were performed on high proton-density (PD) phantom (1H) with an embedded radioisotopic source inside the shielded RF coil by MRI animal scanner (4.7 T). Our results pave the way for a combined MRI-SPECT system. The project was performed in the framework of the Medipix Collaboration.

Chapter 9 - A High-Resolution Multimode Digital Microscope System

This chapter describes the development of a high-resolution, multimode digital imaging system based on a wide-field epifluorescent and transmitted light microscope, and a cooled charge-coupled device (CCD) camera. The three main parts of this imaging system are Nikon FXA microscope, Hamamatsu C4880 cooled CCD camera, and MetaMorph digital imaging system. This chapter presents various design criteria for the instrument and describes the major features of the microscope components-the cooled CCD camera and the MetaMorph digital imaging system. The Nikon FXA upright microscope can produce high resolution images for both epifluorescent and transmitted light illumination without switching the objective or moving the specimen. The functional aspects of the microscope set-up can be considered in terms of the imaging optics, the epi-illumination optics, the transillumination optics, the focus control, and the vibration isolation table. This instrument is somewhat specialized for microtubule and mitosis studies, and it is also applicable to a variety of problems in cellular imaging, including tracking proteins fused to the green fluorescent protein in live cells. The instrument is also valuable for correlating the assembly dynamics of individual cytoplasmic microtubules (labeled by conjugating X-rhodamine to tubulin) with the dynamics of membranes of the endoplasmic reticulum (labeled with DiOC6) and the dynamics of the cell cortex (by differential interference contrast) in migrating vertebrate epithelial cells. This imaging system also plays an important role in the analysis of mitotic mutants in the powerful yeast genetic system Saccharomyces cerevisiae.

A reverse complementary multimodal imaging system to visualize microRNA9-involved neurogenesis using peptide targeting transferrin receptor-conjugated magnetic fluorescence nanoparticles

Multimodal imaging systems may eliminate the disadvantages of individual imaging modality by providing complementary information about cellular and molecular activites. In this sutdy, we developed a reverse complementary multimodal imaging system to image microRNAs (miRNA, miR) during neurognesis using transferrin receptor (TfR) and a magnetic fluorescence (MF) nanoparticle-conjugated peptide targeting TfR (MF targeting TfR). Both in vitro and in vivo imaging demonstrated that, in the absence of miR9 during pre-differentiation of P19 cells, the MF targeting TfR nanoparticles greatly targeted TfR and were successfully internalized into P19 cells, resulting in high fluorescence and low MR signals. When the miR9 was highly expressed during neurogenesis of P19 cells, the MF targeting TfR nanoparticles were hardly targeted due to the miR9 function, which represses the expression and functional activity of TfR from the miRNA TfR reproter gene, resulting in low fluorescence and high MR signals. The reverse complementary multimodal miRNA imaging system may serve as a new imaging probe to montior miRNA-involved cellular developments and diseases.

WE-C-217BCD-01: Development of an Integrated Multimodal Imaging System (IMIS) for Surgical Guidance.

To develop an intraoperative multimodal imaging system (IMIS) for surgical guidance, combining gamma ray scintigraphy with a video camera system for simultaneous real-time visible and near infrared fluorescence (NIRf) imaging. The gamma camera component has been tested in a pilot human study for intraoperative SLN biopsy in melanoma patients. To characterize the optical components, the spatial resolution of the visible light video camera, the depth-dependent visualization by the NIRf system of targets containing indocyanine green (ICG) and the excitation radiation rejection of the NIRf system were evaluated in bench-top tests. Spatial resolution was quantified using a USAF test pattern and NIRf performance was evaluated using phantoms containing fluorescent targets in mixtures of fat-emulsion (Intralipid) and india ink to simulate optical transport in human tissue. The visible/NIRf system was also used to image a live anaesthetized mouse model of breast cancer. Intraoperatively the SiPM gamma camera detected 100% of positive lymph nodes. The visible light camera system contrast transfer function is >10% up to 3.2 1p/mm. The NIRf image SNR when imaging a 9.9 mm diameter lesion containing a 20 μM ICG concentration is > 2 for lesion depths up to 8 mm. The current combination of excitation and emission filters results in nearly complete rejection of backscattered light, resulting in a leakage count rate less than 1% of the system dark count rate. The optical component of the IMIS system demonstrates high spatial resolution, excellent rejection of background excitation radiation, and good target SNR for realistic tracer concentration and target depth. It is capable of accurately coregistered video rate visible/NIRf imaging.

Accuracy of In Vivo Multimodal Optical Imaging for Detection of Oral Neoplasia

If detected early, oral cancer is eminently curable. However, survival rates for oral cancer patients remain low, largely due to late-stage diagnosis and subsequent difficulty of treatment. To improve clinicians' ability to detect early disease and to treat advanced cancers, we developed a multimodal optical imaging system (MMIS) to evaluate tissue in situ, at macroscopic and microscopic scales. The MMIS was used to measure 100 anatomic sites in 30 patients, correctly classifying 98% of pathologically confirmed normal tissue sites, and 95% of sites graded as moderate dysplasia, severe dysplasia, or cancer. When used alone, MMIS classification accuracy was 35% for sites determined by pathology as mild dysplasia. However, MMIS measurements correlated with expression of candidate molecular markers in 87% of sites with mild dysplasia. These findings support the ability of noninvasive multimodal optical imaging to accurately identify neoplastic tissue and premalignant lesions. This in turn may have considerable impact on detection and treatment of patients with oral cancer and other epithelial malignancies.

Microneedle-based analysis of the micromechanics of the metaphase spindle assembled in Xenopus laevis egg extracts

To explain how micrometer-sized cellular structures generate and respond to forces, we need to characterize their micromechanical properties. Here we provide a protocol to build and use a dual force-calibrated microneedle-based setup to quantitatively analyze the micromechanics of a metaphase spindle assembled in Xenopus laevis egg extracts. This cell-free extract system allows for controlled biochemical perturbations of spindle components. We describe how the microneedles are prepared and how they can be used to apply and measure forces. A multimode imaging system allows the tracking of microtubules, chromosomes and needle tips. This setup can be used to analyze the viscoelastic properties of the spindle on timescales ranging from minutes to sub-seconds. A typical experiment, along with data analysis, is also detailed. We anticipate that our protocol can be readily extended to analyze the micromechanics of other cellular structures assembled in cell-free extracts. The entire procedure can take 3-4 d.

Anatomically accurate hard priors for transrectal electrical impedance tomography (TREIT) of the prostate

Current prostate biopsy procedures entail sampling tissues at template-based locations that are not patient specific. Ultrasound (US)-coupled transrectal electrical impedance tomography (TREIT), featuring an endorectal US probe retrofitted with electrodes, has been developed for prostate imaging. This multi-modal imaging system aims to identify suspicious tumor regions based on their electrical properties and ultimately provide additional patient-specific locations where to take biopsy samples. Unfortunately, the open-domain geometry associated with TREIT results in a severely ill-posed problem due to the small number of measurements and unbounded imaging domain. Furthermore, reconstructing contrasts within the prostate volume is challenging because the conductivity differences between the prostate and surrounding tissues are much larger than the conductivity differences between benign and malignant tissues within the prostate. To help overcome these problems, anatomically accurate hard priors can be employed to limit estimation of the electrical property distribution to within the prostate volume; however, this requires the availability of structural information. Here, a method that extracts the prostate surface from US images and incorporates this surface into the image reconstruction algorithm has been developed to enable estimation of electrical parameters within the prostate volume. In this paper, the performance of this algorithm is evaluated against a more traditional EIT algorithm that does not use anatomically accurate structural information, in the context of numerical simulations and phantom experiments. The developed anatomically accurate hard-prior algorithm demonstrably identifies contrasts within the prostate volume while an algorithm that does not rely on anatomically accurate structural information is unable to localize these contrasts. While inclusions are identified in the correct locations, they are found to be smaller in size than the actual object due to the rapid decay in sensitivity at increasing distances from the probe surface. Despite this, identifying the size of the inclusion accurately may not be essential for biopsy guidance in a clinical setting; instead, knowledge of the general vicinity of a cancerous lesion may be sufficient for suggesting and guiding clinicians to extract additional biopsy cores.

Abstract 3566: Multimodal optical imaging for detection of oral neoplasia

Abstract Despite advances in treatment methods the 5-year survival rate for patients with oral cancer in the United States is still only 61%, in part due to late stage diagnosis and subsequent difficulty of surgical treatment for advanced disease. To improve the ability of clinicians to detect early stage disease and to treat advanced cancers, we developed a multimodal optical imaging system (MMIS) to evaluate tissue structure in vivo at macroscopic and microscopic scales. The MMIS noninvasively measures changes associated with the development of precancer and cancer, including alterations in tissue autofluorescence properties (due to loss of collagen integrity, epithelial thickening, metabolic changes, and microvascularization) and alterations in tissue morphology (nuclear enlargement and crowding within the epithelium). The macroscopic imaging component of the MMIS acquires autofluorescence images of tissue with an excitation wavelength of 405 nm, a field of view of 45 mm, and spatial resolution of 0.1 mm. Loss of autofluorescence intensity is quantified by calculating the normalized red-to-green ratio in widefield autofluorescence images. Regions of tissue that display reduced blue-green autofluorescence are examined further using the high-resolution imaging component of the MMIS, which is a compact fluorescence microscope coupled to a fiber-optic imaging probe with a 720 μm diameter field-of-view and 4.4 μm spatial resolution. High-resolution imaging is performed at an excitation wavelength of 455 nm following topical application of proflavine, a fluorescent contrast agent which predominantly stains cell nuclei. Alterations in nuclear size and crowding are quantified by calculating the mean nuclear-to-cytoplasm ratio in high-resolution images. The MMIS was used to measure 100 sites in 30 patients undergoing surgery for oral cancer or precancer. Histopathology results from tissue specimens collected from the measured sites were used as the gold standard. Tissue specimens with a histopathologic diagnosis of dysplasia (mild, moderate, or severe) were further evaluated for molecular alterations by immunohistochemical staining for the markers Ki-67, p63 and PHH3. The MMIS correctly classified 98% of pathologically confirmed normal tissue sites and 95% of sites graded as moderate dysplasia, severe dysplasia, or cancer. MMIS measurements also correlated with molecular markers in 87% of sites with mild dysplasia. Following stratification of mild dysplasia sites by biomarker status, multimodal optical imaging classified all 100 measured sites with 93% sensitivity and 96% specificity. These findings support the ability of MMIS to guide the clinician in identifying neoplastic tissue, and potentially those sites with elevated risk of malignant transformation. This would have considerable impact on the detection and treatment of oral cancer and other epithelial malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3566. doi:1538-7445.AM2012-3566