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In vivo selection of phage for the optical imaging of PC-3 human prostate carcinoma in mice.

There is an increasing medical need to detect and spatially localize early and aggressive forms of prostate cancer. Affinity ligands derived from bacteriophage (phage) library screens can be developed to molecularly target prostate cancer with fluorochromes for optical imaging. Toward this goal, we used in vivo phage display and a newly described micropanning assay to select for phage that extravasate and bind human PC-3 prostate carcinoma xenografts in severe combined immune deficiency mice. One resulting phage clone (G1) displaying the peptide sequence IAGLATPGWSHWLAL was fluorescently labeled with the near-infrared fluorophore AlexaFluor 680 and was evaluated both in vitro and in vivo for its ability to bind and target PC-3 prostate carcinomas. The fluorescently labeled phage clone (G1) had a tumor-to-muscle ratio of approximately 30 in experiments. In addition, prostate tumors (PC-3) were readily detectable by optical-imaging methods. These results show proof of principle that disease-specific library-derived fluorescent probes can be rapidly developed for use in the early detection of cancers by optical means.

Animals↗

Three dimensional optical imaging of blood volume and oxygenation in the neonatal brain.

Optical methods provide a means of monitoring cerebral oxygenation in newborn infants at risk of brain injury. A 32-channel optical imaging system has been developed with the aim of reconstructing three-dimensional images of regional blood volume and oxygenation. Full image data sets were acquired from 14 out of 24 infants studied; successful images have been reconstructed in 8 of these infants. Regional variations in cerebral blood volume and tissue oxygen saturation are present in healthy preterm infants. In an infant with a large unilateral intraventricular haemorrhage, a corresponding region of low oxygen saturation was detected. These results suggest that optical tomography may provide an appropriate technique for investigating regional cerebral haemodynamics and oxygenation at the cotside.

Blood Volume↗

Optical imaging of epileptiform events in visual cortex in response to patterned photic stimulation.

In a subset of patients with epilepsy, patterned visual stimuli can trigger clinical seizures. The etiology of this phenomenon, and the complex interaction between functional architecture and epilepsy, were investigated in ferret visual cortex. Optical imaging of intrinsic signals was used to visualize maps of orientation, ocular dominance and spatial frequency. Acute interictal spike foci were then induced within V1 using focal iontophoresis of bicuculline methiodide and optically mapped during presentation of patterned visual stimuli. We found that specific orientations and spatial frequencies could preferentially trigger epileptiform events, depending on the location of the epicenter of the epileptic focus within the columnar architecture of visual cortex. These data support a cortical etiology of the clinical phenomenon of pattern-sensitive epilepsy. We were not able to demonstrate a spatial correlation between the functional architecture maps and the topography of the epileptic focus. These findings implicate short-range rather than long-range horizontal excitatory connections in the lateral spread of interictal spikes, which may be specific to the epilepsy model of acute focal disinhibition. Orientation and spatial frequency maps were severely disturbed in the region of the focus but were unaltered in the surrounding cortex. Thus, optical imaging of intrinsic signals can be used to simultaneously map epilepsy and normal functional anatomy with high spatial resolution.

Action Potentials↗

Evaluation of the human airway with multi-detector x-ray-computed tomography and optical imaging.

Defining the healthy human airway is important in enhancing our understanding of pulmonary disease states such as inflammation and cancer. The structure of the human airway, both static and dynamic, can be assessed using multi-detector CT (MDCT) scanning. This modality also allows for the evaluation of structures outside of the airway. The airway wall can be directly visualized using CCD chip high-resolution color optical imaging through endoscopy allowing bronchial wall evaluation by traditional biopsy methods, as well as by newer optically based strategies. We suggest that these two imaging modalities, MDCT and optical imaging, provide complementary information about the normal airway, and the airway in various diseases. Methods for evaluating the human airway using MDCT images are presented facilitating automatic airway segmentation, branchpoint finding and airway dimension analysis. The airway wall color is objectively evaluated as an important surrogate for airway wall inflammation and cancer formation, and the integration of the color endoscopic information into the MDCT scan data set is currently ongoing. The amalgamation of these two digital imaging modalities appears increasingly useful for enabling biopsy techniques, and for relating structure and function of the airway. In addition, these developments may be progressively more useful in understanding the normal airway structure and function, for defining airway diseases patterns and for guiding biopsy and therapeutic procedures.

Bronchoscopy↗

Model-based parameter recovery from uncalibrated optical images.

We propose a novel method for quantitative interpretation of uncalibrated optical images which is derived explicitly from an analysis of the image formation model. Parameters characterising the tissue are recovered from images acquired using filters optimised to minimise the error. Preliminary results are shown for the skin, where the technique was successfully applied to aid the diagnosis and interpretation of non-melanocytic skin cancers and acne; and for the more challenging ocular fundus, for mapping of the pigment xanthophyll.

Algorithms↗

Quatrefoil reentry in myocardium: an optical imaging study of the induction mechanism.

INTRODUCTION: The "critical point hypothesis" for induction of ventricular fibrillation has previously been extended to infer the coexistence of four critical points, and hence four simultaneous spiral reentries or a quatrefoil reentry, resulting from only one premature stimulus delivered to the same location as the pacing stimulus. An optical imaging technique was used to explore its existence and to study the induction mechanism of this peculiar reentry pattern. METHODS AND RESULTS: In 16 isolated, Langendorff-perfused rabbit hearts, high-speed optical imaging at 133 or 267 frames/sec was performed to observe the induced response with a unipolar point electrode. A novel quatrefoil-shaped reentry pattern consisting of two pairs of opposing rotors was created by delivering long stimuli during the vulnerable phase. Successful induction occurred in a narrow range of coupling intervals. A dogbone pattern of virtual electrodes was established during the premature stimulus. Propagating wavefronts launched from the virtual anodes immediately after the termination of S2. The alternating blocking and conducting effects of the virtual electrodes, as well as the boundary between virtual cathode and virtual anode, provided the necessary pathways for quatrefoil reentry. Propagation directions of the reentrant spiral wavefronts reversed with a reversal in S2 polarity. Quatrefoil reentries were not sustained and lasted 1 to 4 complete cycles. CONCLUSION: The initiation of quatrefoil reentry followed anodal- or cathodal-break stimulation as a result of local symmetrical enhancement of the dispersion of tissue excitability. The "critical point hypothesis" provides the minimum topology required for this type of reentry; the "graded response hypothesis" can be viewed as providing a more detailed explanation of how this topology is actually realized. Triggering mechanisms due to the "break" mode of stimulation also posits a new mechanism for defibrillation.

Animals↗

Simultaneous optical imaging of intracellular Cl- in neurons in different layers of rat neocortical slices: advantages and limitations.

Simultaneous recording of changes in intracellular Cl- concentration ([Cl-]i) in individual neurons situated in different layers (e.g. II/III-VI) of neocortical slices was found to be feasible by means of optical fluorescence measurements using 6-methoxy-N-ethylquinolinium iodide (MEQ). Gamma-aminobutyric acid (GABA) caused a measurable increase in [Cl-]i in adult neocortical neurons, but a decrease in immature neurons. Developmental changes in the function of the Cl- pump and cation-Cl- co-transporters were evaluated using inhibitors such as furosemide (FURO), ethacrynic acid (ETA), and bumetanide (BMT). However, it was found that these inhibitors absorb and/or emit light of the wavelength that is used for the optical imaging of MEQ. In addition, quenching of MEQ fluorescence by Cl- and leakage of loaded MEQ was significantly enhanced at a higher temperature, which will limit experimentation at > 30 degrees C. Estimation of [Cl-]i in individual neurons in slices was made possible by calibrating intracellular MEQ fluorescence signals at known Cl- concentrations ([Cl-]) in the presence of tributyltin, a Cl(-)-OH- antiporter, nigericin, a K+-H+ antiporter, and KSCN. This enables comparison of [Cl-]i between neurons in different slices. Thus, optical imaging of [Cl-]i in brain slices can provide valuable spatial information about [Cl-]i dynamics and homeostasis, although it should be emphasized that the technique does have some limitations.

Animals↗

Isofrequency band-like zones of activation revealed by optical imaging of intrinsic signals in the cat primary auditory cortex.

Neurons of similar frequency preference are arranged in isofrequency bands (IFBs) across the primary auditory cortex (AI) of many mammals. Across the AI of the cat, one of the most frequently studied species for auditory anatomy and function, we demonstrate IFB-like responses using optical imaging of intrinsic signals (OIS). Optically defined activations were extensively elongated along the dorsoventral axis of AI (the ratio of the major and minor axes was approximately 2:1), and systematically shifted as a function of stimulus frequency. The elongation of this IFB-like zone was more conspicuous at higher frequencies. In the ventral sector of the imaged field, the IFB-like zones of activation evoked at different pure tone frequencies tended to overlap extensively. Electrophysiological recording from loci within the optically defined zones of activation revealed matched responses to the frequencies used for optical imaging at 65% of these loci. The dorsoventral orientation of these zones of activation was also closely matched with the orientation of tangentially spreading intrinsic axon terminals, as revealed anatomically. The visualization of IFB-like architecture and tonotopic organization by OIS provides a basic framework for investigating the relationships of different spectral channels and between multiple acoustic parameters at a neuronal population level.

Acoustic Stimulation↗

A new approach to optical imaging applied to rat barrel cortex.

Several groups have described using intrinsic optical imaging to form images of activity patterns in the cortex. Because the signal is small, the general approach has been to use expensive camera equipment with a high dynamic range to make these measurements. However, by using signal averaging to compensate for lower dynamic range, images can be obtained using equipment already available in many laboratories. This modified technique has been used for imaging activity in 'barrel' cortex of the rat. A map of the representation of a single whisker as determined by the imaging technique corresponded well with a similar map made using standard electrophysiology. A map of several whiskers was made by overlaying images of single-whisker representations. The details of the images differ from those previously described. Possible mechanisms for the signal are discussed.

Animals↗

Optically imageable metastatic model of human breast cancer.

We report an optically imageable orthotopic metastatic nude mouse model of the human breast cancer MDA-MB-435 expressing green fluorescent protein (GFP). We demonstrate fluorescent imaging of primary and metastatic growth in live tissue and in intact animals. Fragments of tumor tissue expressing GFP were sutured into the pocket in the right second mammary gland. Tumor tissue was strongly fluorescent, enabling whole-body imaging of tumor growth by week 5. Neovascularization of the primary tumor was also visualized by whole-body imaging by contrast of the vessels to the fluorescent tumor. At autopsy, the MDA-MB-435-GFP was found to have metastasized to various organs, including the lung in 55% of the animals, the lymph nodes in 15% of the animals including axillary nodes, and the liver in 10% of the animals. These metastases could be visualized in fresh tissue by fluorescent imaging. Detailed fluorescence analysis visualized extensive metastasis in the thoracic cavity and the lymphatic system. Large metastatic nodules in the lung involved most of the pulmonary parenchyma in all lobes. Lymph node metastasis was found mainly in the axillary area. In the liver, fluorescent macroscopic metastatic nodules were found under the capsule. The metastatic pattern in the model thus reflected clinical metastatic breast cancer and provides a powerful model for drug discovery for this disease.

Adult↗

Noninvasive optical imaging by speckle ensemble.

We propose a new method imaging through scattering media. An object hidden between two biological tissues (chicken breast) is reconstructed from any speckled images obtained from the output of a multichannel optical imaging system. The effect of multiple imaging is achieved with a microlens array. Each lens is the array projects a different speckled image onto a digital camera. The set of speckled images from the entire array is first shifted to a common center and then accumulated into a single average picture.

Animals↗

Noninvasive optical imaging of ovarian metastases using Cy5-labeled RAFT-c(-RGDfK-)4.

Our group has developed a new molecular tool based on the use of a regioselectively addressable, functionalized template (RAFT) scaffold, where four cyclic (Arg-Gly-Asp) (cRGD) peptide motifs were grafted. The aim of this study was to determine whether RAFT-c(-RGDfK-)4 combined with optical imaging could allow noninvasive detection of deep ovarian metastases. Human ovarian adenocarcinoma IGROV1 cells expressing low levels of integrin alphaVbeta3 (the main receptor for the cRGD peptide) were used for in vitro and in vivo assays in combination with Cy5-labeled RAFT-c(-RGDfK-)4, cRGD, or RAFT-c(-RbetaADfK-)4. In vivo fluorescence imaging was performed on subcutaneous (SC) tumors and intraperitoneal IGROV1 metastases in nude mice. The accumulation of RGD-Cy5 conjugates in cultured cells or in tumor tissues was examined using confocal laser scanning microscopy. RAFT-c(-RGDfK-)4 exhibited stronger staining in vitro, enhanced tumor-to-background ratio for sc tumors, and allowed early detection of 1- to 5-mm large intraabdominal nodules using noninvasive optical imaging. Histological study revealed that RAFT-c(-RGDfK-)4 accumulated into tumor neovasculature but also into tumor cells. Our data demonstrate that a Cy5-labeled RAFT-c(-RGDfK-)4 is an efficient optical probe for early and noninvasive tumor detection.

Animals↗

Gadolinium-rhodamine nanoparticles for cell labeling and tracking via magnetic resonance and optical imaging.

A novel dual-labeled nanoparticle for use in labeling and tracking cells in vivo is described. We report the construction and characterization of these gadolinium-rhodamine nanoparticles. These particles are constructed from lipid monomers with diacetylene bonds that are sonicated and photolyzed to form polymerized nanoparticles. Cells are efficiently labeled with these nanoparticles. We have inoculated labeled tumor cells subcutaneouosly into the flanks of C3H mice and have been able to image these labeled tumor cells via MRI and optical imaging. Furthermore, the labeled tumor cells can be visualized via fluorescent microscopy after tissue biopsy. Our results suggest that these nanoparticles could be used to track cells in vivo. This basic platform can be modified with different fluorophores and targeting agents for studying metastisic cell, stem cell, and immune cell trafficking among other applications.

Animals↗

Optical imaging in medicine: II. Modelling and reconstruction.

The desire for a diagnostic optical imaging modality has motivated the development of image reconstruction procedures involving solution of the inverse problem. This approach is based on the assumption that, given a set of measurements of transmitted light between pairs of points on the surface of an object, there exists a unique three-dimensional distribution of internal scatterers and absorbers which would yield that set. Thus imaging becomes a task of solving an inverse problem using an appropriate model of photon transport. In this paper we examine the models that have been developed for this task, and review current approaches to image reconstruction. Specifically, we consider models based on radiative transfer theory and its derivatives, which are either stochastic in nature (random walk, Monte Carlo, and Markov processes) or deterministic (partial differential equation models and their solutions). Image reconstruction algorithms are discussed which are based on either direct backprojection, perturbation methods, nonlinear optimization, or Jacobian calculation. Finally we discuss some of the fundamental problems that must be addressed before optical tomography can be considered to be an understood problem, and before its full potential can be realized.

Diagnostic Imaging↗

Three-dimensional Bayesian optical image reconstruction with domain decomposition.

Most current efforts in near-infrared optical tomography are effectively limited to two-dimensional reconstructions due to the computationally intensive nature of full three-dimensional (3-D) data inversion. Previously, we described a new computationally efficient and statistically powerful inversion method APPRIZE (automatic progressive parameter-reducing inverse zonation and estimation). The APPRIZE method computes minimum-variance estimates of parameter values (here, spatially variant absorption due to a fluorescent contrast agent) and covariance, while simultaneously estimating the number of parameters needed as well as the size, shape, and location of the spatial regions that correspond to those parameters. Estimates of measurement and model error are explicitly incorporated into the procedure and implicitly regularize the inversion in a physically based manner. The optimal estimation of parameters is bounds-constrained, precluding infeasible values. In this paper, the APPRIZE method for optical imaging is extended for application to arbitrarily large 3-D domains through the use of domain decomposition. The effect of subdomain size on the performance of the method is examined by assessing the sensitivity for identifying 112 randomly located single-voxel heterogeneities in 58 3-D domains. Also investigated are the effects of unmodeled heterogeneity in background optical properties. The method is tested on simulated frequency-domain photon migration measurements at 100 MHz in order to recover absorption maps owing to fluorescent contrast agent. This study provides a new approach for computationally tractable 3-D optical tomography.

Bayes Theorem↗

Macromolecular contrast agents for optical imaging of tumors: comparison of indotricarbocyanine-labeled human serum albumin and transferrin.

Macromolecules accumulate in solid tumors and can thus be used as carriers for the delivery of attached contrast agents to tumors. We report the synthesis and use of serum protein-dye conjugates consisting of transferrin (Tf) or human serum albumin (HSA) and an indotricarbocyanine (ITCC) derivative as contrast agents for the optical imaging of tumors. The compounds were characterized with respect to their photophysical properties and tested in vitro for their ability to bind to tumor cells and in vivo for their potential to delineate experimental tumors. In contrast to HAS-ITTC, Tf-ITCC showed receptor-mediated uptake by HT29 human colon cancer cells in vitro. After intravenous injection into HT29 tumor-bearing nude mice both compounds induced increased fluorescence contrast of tumors in vivo. After 24 h the contrast between tumor and normal tissue was significantly higher for Tf-ITCC than for HAS-ITCC. Dye-induced fluorescence was found to be predominantly located in perinecrotic areas of the tumor. Furthermore, Tf-ITCC produced fluorescence of viable tumor cells, whereas HAS-ITCC fluorescence was recorded along connective tissue. We conclude that ITCC-labeled Tf and HSA can serve as macromolecular contrast agents for the optical imaging of tumors, with Tf-ITCC showing higher efficiency.

Animals↗

Recent advances in diffuse optical imaging.

We review the current state-of-the-art of diffuse optical imaging, which is an emerging technique for functional imaging of biological tissue. It involves generating images using measurements of visible or near-infrared light scattered across large (greater than several centimetres) thicknesses of tissue. We discuss recent advances in experimental methods and instrumentation, and examine new theoretical techniques applied to modelling and image reconstruction. We review recent work on in vivo applications including imaging the breast and brain, and examine future challenges.

Diffusion↗

Benign versus malignant breast masses: optical differentiation with US-guided optical imaging reconstruction.

PURPOSE: To investigate prospectively the feasibility of using optical tomography with ultrasonographic (US) localization to differentiate malignant from benign breast masses and to compare optical tomography with color Doppler US. MATERIALS AND METHODS: The study was approved by the local internal review board committee and by the Human Subjects Research Review Board of Army Medical Research and Materiel Command. Signed informed consent was obtained, and the study was HIPAA compliant. Between May 2003 and March 2004, 65 consecutive women (mean age, 51 years; age range, 24-80 years) with 81 breast lesions underwent US-guided biopsy and were scanned with a combined imager. The hand-held probe, which consisted of a centrally located US transducer surrounded by near-infrared sensors, was used to simultaneously acquire coregistered US images and optical data. The lesion location obtained at US was used to guide optical imaging reconstruction. Light absorption was measured at two wavelengths. From these measurements, tumor angiogenesis was assessed on the basis of calculated total hemoglobin concentration. A Student t distribution was used to calculate the statistical significance of mean maximum and mean average hemoglobin concentrations obtained in malignant and benign lesion groups, and P < .001 was considered to indicate a statistically significant difference. RESULTS: Biopsy results revealed eight early stage invasive carcinomas (malignant group) and 73 benign lesions (benign group). The mean maximum and mean average hemoglobin concentrations in the malignant group were 122 micromol/L +/- 26.8 (+/- standard deviation) and 88 micromol/L +/- 24.5, respectively. The mean maximum and mean average hemoglobin concentrations in the benign group were 55 micromol/L +/- 24.8 and 38 micromol/L +/- 17.4, respectively. Both the maximum and average total hemoglobin concentrations were significantly higher in the malignant group compared with the benign group (P < .001). When a maximum hemoglobin concentration of 95 micromol/L was used as the threshold value, the sensitivity, specificity, positive predictive value, and negative predictive value of optical tomography were 100%, 96%, 73%, and 100%, respectively, and the sensitivity, specificity, positive predictive value, and negative predictive value of color Doppler US were 63%, 69%, 19%, and 94%, respectively. CONCLUSION: Findings indicate that optical tomography with US localization is feasible for differentiating benign and early stage malignant breast lesions.

Adult↗