This PDF file contains the front matter associated with SPIE-OSA Proceedings Volume 6626, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing.

Photodynamic molecular beacon (PMB) is a novel photodynamic therapy (PDT) concept featuring the precise control of the ability of photosensitizer (PS) to produce singlet oxygen in response to specific cancer-associated biomarkers. It comprises a disease-specific linker, a PS and a singlet oxygen quencher so that the PS's photodynamic toxicity is silenced until the linker interacts with a tumor-associated biomarker. The development of PMB depends on two key factors. The first is the design of a suitable PS-quencher pair to achieve an effective singlet oxygen quenching, minimizing phototoxicity of native PMB in non-targeted (normal) cells. The second is the design of a suitable linker for the choice of target biomarker to achieve a specific photodynamic activation, resulting in selective PDT efficacy in targeted (tumor) cells. These two factors make PMB designs versatile and customizable. In this report, we will focus on the new directions on PMB linker design utilizing two "on-and-off" activation mechanisms. The first one uses a "cleavable" linker that is triggered by fibroblast activation protein or phospholipase. The second one uses an "openable" linker that can hybridize with a tumor-specific mRNA.

Exogenous fluorescent agents such as green fluorescent protein (GFP) have been widely used as biological indicators in bioimaging techniques. Although GFP and its mutants have been used in many applications, their optical properties have not been completely investigated, especially when they are under various environmental conditions. In this research, we developed a spectrum-analyzing system to investigate the fluorescent properties of GFP in the environments of different temperatures. We found that the fluorescent spectrum of GFP consisted of two components that might come from the transitions between different electronic energy states where the quantum efficiencies of the two components varied with different temperature. This effect was expected to come from the thermal effect on the electron populations in the molecular energy states of GFP. Furthermore, GFP was used as fluorescent marker to monitor the infection process of cells by viruses with a dynamic spectral imaging system. The recombinant baculoviruses containing the red and green fluorescent protein gene that can simultaneously produce dual fluorescence were used as vectors in insect cells. The system was used to monitor the spatial distribution of fluorescent spectra of cells infected by virus during the process of infection.

We report the performance of a simple method for making quantitative bioluminescence measurements of a point-like source embedded in small animals. In this method, video reflectometry is first used to obtain an estimate of the in situ optical properties of the tissue containing the bioluminescent source. A 2-dimensional image of the bioluminescence signal emitted from the surface of the animal is then acquired with a CCD. Using the measured optical properties, and a simple diffusion theory model, an inversion algorithm is applied to retrieve the source depth and power from a region of interest of the bioluminescence images. Two major factors determine the accuracy of the reconstruction: tissue heterogeneity and curvature of the imaged surface. The use of measured optical properties to characterize in situ tissue surmounts, to a degree, the heterogeneity problem: post mortem data from rats show that the relative power can be retrieved within a factor of 2 and frequently within 20 %, and the depth within 1.0 mm for implanted depths of 4-10 mm, when the curvature effects were eliminated. For depths shallower than 4 mm, the errors in the retrieved depth are consistently larger.

Fluorescence Molecular Tomography (FMT) has emerged as a powerful tool for monitoring biological functions in vivo in small animals. It provides the means to determine volumetric images of fluorescent protein concentration by applying the principles of diffuse optical tomography. Using different probes tagged to different proteins or cells, different biological functions and pathways can be simultaneously imaged in the same subject. In this work we present a spectral unmixing algorithm capable of separating signal from different probes when combined with the tomographic imaging modality. We show results of two-color imaging when the algorithm is applied to separate fluorescence activity originating from phantoms containing two different fluorophores, namely CFSE and SNARF, with well separated emission spectra, as well as Dsred- and GFP-fused cells in F5-b10 transgenic mice in vivo. The same algorithm can furthermore be applied to tissue-specific spectroscopy data. Spectral analysis of a variety of organs from control, DsRed and GFP F5/B10 transgenic mice showed that fluorophore detection by optical systems is highly tissue-dependent. Spectral data collected from different organs can provide useful insight into experimental parameter optimisation (choice of filters, fluorophores, excitation wavelengths) and spectral unmixing can be applied to measure the tissue-dependency, thereby taking into account localized fluorophore efficiency. Summed up, tissue spectral unmixing can be used as criteria in choosing the most appropriate tissue targets as well as fluorescent markers for specific applications.

Low density lipoproteins (LDL) have long been recognized as a potential delivery system for exogenous agents. Imaging agents or drugs can be attached to LDL through surface loading, protein loading or core loading methods. The LDL delivery system has received considerable attention particularly among cancer biologists as it was observed that numerous cancers over-express the low density lipoprotein receptor (LDLR). In this paper we investigate the utility of LDL to transport optical imaging contrast agents for caner detection. The method of loading fluorophores into the core of LDL is attractive as it behaves like an activatable contrast agent. Surface and protein labeled methods also prove to be effective strategies for tracing LDL nanoparticle activity. The strengths and limitations of the LDL carrier system are discussed and novel approaches for imaging cancer with LDL nanoparticles are highlighted.

Gold nanoparticles exhibit intense and narrow optical extinction bands due to the phenomenon of plasmon resonance making them useful as contrast agents for light-based imaging techniques. Localized heating results from the absorbed light energy, which shows potential for these particles in photothermal therapy as well. The bioconjugation of gold nanoparticles to appropriate antibodies targeted to tumors in vivo, could make highly selective detection and therapy of tumors possible. We have synthesised gold nanorods based on seed mediated protocols using two methods. The first method is based on using a mono-surfactant silver assisted method which produces gold nanorods having plasmon peaks between 670-850 nm within the "optical imaging and therapeutic window". These nanorods have aspect ratios between 2.3 - 3.7. A second method is a silver assisted bi-surfactant method which produce nanorods with peaks in the range of 850-1100 nm having aspect ratios between 5 - 11. Typical concentrations of these particles in aqueous dispersions are in the range of 1x10¹⁰ - 1x10¹¹ particles per mL. We have bioconjugated these gold nanorods with anti-HER2/neu mouse monoclonal antibodies (MAb). Since the as-prepared CTAB-stabilized nanorods were found to be toxic to SKBR3 cells, we decided to coat the gold nanorods with polyethylene glycol (PEG). Characterization and size estimation of the nanoparticles were performed using electron microscopies, optical spectroscopy and confocal microscopy. We present these results and implications for use of these nanoparticles for in vivo biomedical applications.

Non-invasive near infrared fluorescence imaging of mice models is a very attractive tool for fastening the development of new therapeutics. Two classes of labels exist for the near infrared domain: organic dyes and quantum dots (QDs). QDs are inorganic luminescent semi-conductor nano-crystals which display very attractive optical features. They are now commercially available for in vivo mouse tests, and new compositions with less toxic elements are currently being developed. The concept of activatable probes, which fluorescence is activated specifically upon the biological process to be visualized, has also been demonstrated to improve the fluorescence image contrast. The construction of activatable probes based on quantum dot labels has therefore been undertaken. Commercial PEGylated quantum dots bearing around 80-100 amino pending groups are used. Long PEG chains are demonstrated to be essential in order to increase the blood circulation time of the particles and avoid their massive storage into the liver. The amino groups coating the QD surface can be used for their further functionalization by either a tumor-targeting ligand, a cleavable spacer bearing a fluorescence inhibitor I, or both. Functionalization of 80% of the amino groups by the inhibitors I leads to more than 99% fluorescence quenching. Cleavable spacers X-L-S-S-L'-I in which S-S is a disulfide bond cleavable by cell internalization, and X a chemical group for QD grafting have been synthesized. The functionalization of the QD by 12 cleavable spacers leads to more than 85% fluorescence inhibition, which can be recovered upon cleavage of the disulfide bonds.

Several mechanisms of action can be employed for a molecular imaging contrast agent for use with endoscopy. Targeting of cell surface molecules that are up regulated at an early disease stage, with a fluorescent labelled vector is one attractive approach. However, it suffers from the inherent limitation that the concentration of agent available is fundamentally limited by the concentration of receptor molecules available. Simple models indicate that for successful imaging with a targeting approach, the imaging system should be able to adequately image concentrations in the nanomolar region. Such low reporter molecule concentrations have implications for the choice of contrast agent. Target tissue size and location, the tissue native fluorescence contribution, the brightness of the reporter molecule, and photobleaching thresholds are all factors which contribute to the choice of reporter. For endoscopic imaging of millimetre sized target tissue volumes close to the surface Cy5^TM (650-700nm) wavelengths are preferable to Cy3^TM (550-600nm) and Cy7^TM (750-800nm). We have constructed a system optimised for sensitivity by tailoring light delivery, collection, filtering and detection, in order to address the fundamental technical performance limits for endoscopic applications. It is demonstrated through imaging system calibration, phantom based measurement and animal imaging data that low nanomolar concentrations of Cy5 based fluorescent contrast agent in millimetre sized superficial lesions are adequately imaged with a clinically relevant endoscope system in real time. It is concluded that targeting is a technically viable approach for endoscopic applications.

Autofluorescence has been a significant disadvantage when dealing with tomographic imaging of biological samples or tissue phantoms. Consequently, the accurate removal of autofluorescence signal has been a major concern in fluorescence tomography. Here we present a study on three-dimensional mapping and removal of autofluorescence from fluorescence molecular tomography (FMT) data, both for phantoms and small animal in vivo. The technique is based on the recording of tomographic data in multiple spectral regions with different excitation light and on the application of a linear unmixing algorithm for targeting multiple fluorescent probes. Two types of measurements are taken, one with the excitation being in the region of the maximum absorption of the targeted fluorophore and one in a region away from the maximum. The relative strengths of the different spectra are employed to calculate the signal to be removed from the tomographic reconstruction. Autofluorescence spectra are recorded using identical reflection geometry as during the FMT acquisitions allowing for the correct mapping of the autofluorescence signal. Results from phantoms exhibiting different background autofluorescence strengths are presented and discussed. In this work we have also studied in vivo fluorescent activity in mice, involving both subcutaneously implanted fluorescent phantoms and b10 transgenic mice.

We developed a time-resolved scanning system for fluorescence molecular imaging in diffusive media, such as biological tissues. In the present work the system is described and characterized in terms of linearity against optical parameters of the sample and against homogeneously diffuse fluorescent dye. Finally, preliminary measurements performed on phantom are presented, pointing out the ability of our system to produce projective images of the fluorophore distribution into the sample with a 200 fmol sensitivity and to decouple fluorescent amplitude from depth by means of fluorescent transmittance imaging.

Can time-resolved, high-resolution data as acquired by an intensified gated CCD camera (ICCD) aid in the tomographic reconstruction of fluorescence concentration? Usually it is argued that fluorescence is a linear process and thus does not require non-linear, time-dependent reconstructions algorithms, unless absorption and scattering coefficients need to be determined as well. Furthermore, the acquisition of a number of time frames is usually prohibitive for fluorescence measurements, at least in small animals, due to the increased total measurement time. On the other hand, it is obvious that diffusion is less pronounced in images at early gates, due to selective imaging of photons of lower scatter order. This will be the case also for photons emitted by fluorescent sources. Early-gated imaging might increase the contrast in acquired images and could possibly improve fluorescence localization. Herein, we present early gated fluorescence images obtained from phantoms and compare them to continuously acquired data. Increased contrast between background and signal maximum can be observed in time-gated images as compared to continuous data. To make use of the properties exhibited by early gated frames, it is necessary to use a modified reconstruction algorithm. We propose a variant of the well-known Born approximation to the diffusion equation that allows to take into account single time frames. The system matrix for the time-dependent Born approach is more complex to calculate, however the complexity of the actual inverse problem (and the acquisition times) of single-frame reconstructions remains the same as compared to continuous mode.

Isotope studies provide valuable data about an organ's function in vivo. Thanks to positron emission tomography (PET) using the radiolabeled natural metabolites, such as [18F]-2-fluoro-deoxy-d-glucose (FDG), biological and physiological meaning of nuclear medicine scans has been considerably increased. Therefore it is of interest to elucidate the possibilities of the technique in a study of some natural metabolites like glycine influencing the blood microcirculation. Glycine, as a medicine, was recently shown to have a positive therapeutic effect in the treatment of patients with ischemic stroke and some other neurological disorders based on vascular disturbances. By previous direct biomicroscopic investigations of pial microvessels in laboratory rats an expressed vasodilatory effect of topically applied glycine was proved. The arterioles diameters depending on initial size have been increased by 200-250% for arterioles of 20-40 μm and by 150-200% for arterioles of 50-80 μm. The PET images were acquired before and after sublingual application of glycine (200 mg). The quantitative analysis of FDG volume concentration (Bq/ml) in the rat brain demonstrated that, in studies after glycine administration, maximal, minimal and mean FDG volume concentration in the brain increased by 200-250% in comparison with the baseline data. Thus, our results revealing evident correlation between FDG-PET images and direct biomicroscopic observations confirm the great potential of molecular imaging techniques to explore in vivo process in the brain.

We present a multiresolution transform-based method for the extraction of moving filament trajectories from single molecule motility data. Noise-corrupted fluorescence image series are denoised using the multiscale median transform and trajectories are detected in the denoised data set. The presented method reduces noise more efficiently than 2D-anisotropic diffusion and several wavelet based techniques. Fibre trajectories are extracted by segmentation of the denoised image stacks and non-crossing trajectories are unambiguously identified combining the information of 2D (XY) and 3D (XYT) segmentation. The algorithm is applied and evaluated using experimental data sets - image sequences of fluorescently labeled F-actin molecules and their 2D-trajectories on a myosin coated surface. This so-called 'motility assay' is used to analyse kinetics, biochemical regulation and pharmacological modulation of these biologically relevant molecules. The presented method improves signal-to-background discrimination, facilitates filament identification and finally, may contribute to significantly improve the performance of this assay.

Capabilities of tumor detection by different optical methods can be significantly improved by labeling of tumors with fluorescent markers. Creation of tumor cell lines transfected with fluorescent proteins provides the possibility not only to detect tumor, but also to conduct the intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Kor and human embryonic kidney HEK-293 Phoenix were transfected with DsRed-Express and Turbo-RFP genes. Emission of RFP in the long-wave optical range permits detection of the deeply located tumors, which is essential for whole-body imaging. Only special tools for turbid media imaging, such as fluorescent diffusion tomography (FDT), enable noninvasive investigation of the internal structure of biological tissue. FDT setup for monitoring of tumor growth in small animals has been created. An animal is scanned in the transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser with second harmonic generation at the 532 nm wavelength. An optimizing algorithm for scanning of an experimantal animal is suggested. In vivo experiments were conducted immediately after the subcutaneously injection of fluorescing cells into small animals. It was shown that FDT method allows to detect the presence of fluorescent cells in small animals and can be used for monitoring of tumor growth and anticancer drug responce.

We introduce a novel approach for calibrating an axis of rotation in a 3D optical metrology system. The system uses a stereo camera pair, along with rotation and translation stages for obtaining a 3D model of the surface of small animals. The metrology system will be part of a fully non-contact diffuse optical tomography (DOT) scanner for small animal imaging. The rotation axis calibration technique is based on measuring, with the stereo pair, the 3D position of a small ball as it is moved by the rotation stage (turntable). Our system has the advantage of using the tomograph's laser beam to measure the outer shape of the subject, thereby reducing overall system complexity, and allowing simultaneous surface and DOT measurements. Additionnaly, the exact position where laser light penetrates the animal is measured, while traditionally, this information is indirectly inferred with less accuracy. This information plays an important role in a tomographic reconstruction algorithm. Our new approach for the calibration of the rotation axis is compared to another technique we previously developed, where a checkerboard pattern is tracked instead of a ball. We present measurements of a reference shape and a small animal taken by our system.