Conventional widefield light microscopy and confocal scanning microscopy have been indispensable for pathology and drug discovery research. Clinical specimens from diseased tissues are examined, new drug candidates are tested on drug targets, and the morphological and molecular biological changes of cells and tissues are observed. High throughput screening of drug candidates requires highly efficient screening instruments. A standard biomedical slide is 1 by 3 inches (25.4 by 76.2 mm) in size. A typical tissue specimen is 10 mm in diameter. To form a high resolution image of the entire specimen, a conventional widefield light microscope must acquire a large number of small images of the specimen, and then tile them together, which is tedious, inefficient and error-prone. A patented new wide field-of-view confocal scanning laser imaging system has been developed for tissue imaging, which is capable of imaging an entire microscope slide without tiling. It is capable of operating in brightfield, reflection and epi-fluorescence imaging modes. Three (red, green and blue (RGB)) lasers are used to produce brightfield and reflection images, and to excite various fluorophores. This new confocal system makes examination of large biomedical specimens more efficient, and makes fluorescence examination of large specimens possible for the first time without tiling. Description of the new confocal technology and applications of the imaging system in pathology and drug discovery research, for example, imaging large tissue specimens, tissue microarrays, and zebrafish sections, are reported in this paper.

The scope of this presentation is a new methodology to correct conventional NIR data for scattering effects. The technique aims at measuring the absorption coefficient of the samples rather than the total attenuation, measured by conventional NIR spectroscopy. The main advantage of this is that the absorption coefficient is independent of the path length of the light inside the sample, and therefore independent of the scattering effects. The measurements in this work were made using a novel system for time-resolved measurements, based on short light continuum pulses generated in an index-guided crystal fibre and a spectrometer-equipped streak camera. The system enables spectral recordings in the wavelength range 500 - 1200 nm with a spectral resolution of 5 nm and a temporal resolution of 30 ps. The evaluation scheme is based on modeling of light transport by diffusion theory, that provides an independent measure of the scattering properties of the samples, that later is used to correct conventional NIR data. This yields a clear advantage over other pre-processing techniques, where scattering effects are estimated and corrected for by using the shape of the measured spectrum only. PLS calibration models shows that, by using the proposed evaluation scheme, the predictive ability is improved by 50 % as compared to models based on conventional NIR data. The method also makes it possible to predict the concentration of active substance in samples with physical properties different from those of the samples included in the calibration model.

Quantum dots (QDs) are optical semiconductor nanocrystals that exhibit stable, bright fluorescence over narrow, size tunable emission bands. The size tunable optical properties of QDs allow multiplexing with multiple emission wavelengths from a single excitation source. QDs may be linked to antibodies, peptides, and nucleic acids for use as fluorescence probes in vitro and in vivo. The electron dense construction of QDs makes it possible to detect QDs with radiographic techniques such as computed tomography. The intravenous injection of QDs may be exploited to optically label brain tumors, potentially leading to improved techniques for surgical biopsy and resection. Macrophage-mediated delivery of QDs to areas of neoplasm and inflammation may represent a novel technique that might be exploited in a variety of surgical situations in which optical feedback control could be useful in aiding the completeness of resection of a lesion or in the accurate localization of pathology for biopsy.

Near-infrared light propagation through living tissue provides promising opportunities for the development of non-invasive imaging techniques for human care. We have developed a Fluorescence-Assisted Resection and Exploration (FLARE) imaging system for surgery. The FLARE system uses invisible near-infrared light to help the surgeon visualize critical structures intraoperatively and in real-time. We present here the continued optimization of our imaging system from a research prototype to an efficient and ergonomic tool to be used during human surgery. New, hands-free operation enables the surgeon to zoom, focus, recall and save images through a footswitch. A LabVIEW curve-fitting algorithm, in combination with stepper motor control, provides auto-focus capability. Cardiac and/or respiratory gating minimizes motion artifacts of moving objects in the surgical field, and permits in-focus imaging during long fluorescence integration times. Automated subtraction of the near-infrared fluorescence signal from background reflections minimizes the effect of ambient illumination and improves the contrast to noise ratio with only moderate effects on intensity precision. Taken together, this study improves several optical components of the FLARE system, and helps ready it for human clinical testing.

We have developed a software platform for multimodal integration and visualization of diffuse optical tomography (DOT) and magnetic resonance imaging (MRI) of breast cancer. The image visualization platform allows multimodality 3D image visualization and manipulation of datasets, such as a variety of 3D rendering technique, and the ability to simultaneously control multiple fields of view. This platform enables quantitative and qualitative analysis of structural and functional diagnostic data, using both conventional & molecular imaging. The functional parameters, together with morphological parameters from MR can be suitably combined and correlated to the absolute diagnosis from histopathology. Fusion of the multimodal datasets will eventually lead to a significant improvement in the sensitivity and specificity of breast cancer detection. Fusion may also allow a priori structural information derived from MRI to be incorporated into the reconstruction of diffuse optical tomography images. We will present the early results of image visualization and registration on multimodal breast cancer data, DOT and MRI.

The fluorescence correlation spectroscopy (FCS) has become a powerful tool that entails the information about molecules at very minute concentrations in the biological system. With the advances in the laser technology and confocal microscopy, the applications of FCS have been extended to the studies of protein dynamics in living cells as well as drug-screening. Two assumptions are made in FCS: the biological system under study is in its equilibrium state and the molecules diffuse within the system freely according to Brownian motions. Fluorescence intensity fluctuations occur when the fluorescent molecule moving in and out of the confocal microscopy defined detection volume in which bursts of photons are emitted. Based on the assumptions above, the statistical-physics-based autocorrelation function of the fluorescence intensity fluctuations was formulated by Aragon and Pecora, which provides the information about the dynamics of the fluorescent macromolecules in the system. However, in this manuscript, we demonstrate that the temporal autocorrelation function of Aragon and Pecora was inadequately derived due to the fact that the process of the positions of a molecule in the system over time is not a stationary Gaussian process. Efforts are made here to derive a new version of the autocorrelation function of the temporal fluorescence intensity fluctuation. The fit of the new autocorrelation function will be compared with that of Aragon and Pecora.

We report on the optical characterization and measurement of oxygen singlet quantum yield of Chlorophyll and Chlorin e₆ in water-ethanol mixtures by direct observation of thermal relaxation using time resolved thermal lens method. The analysis of the time-resolved curve allows the determination of the quantum yield of singlet oxygen formation. The quantum yield is deduced from the relative magnitudes of the fast and slow components.

The development of highly sensitive fluorescence molecular probes in combination with innovative optical techniques plays a pivotal role in the advancement of non-invasive optical imaging of human pathologic conditions. Fluorochromes attached to various ligands have been used for detection of nucleic acid hybridization, in drug discovery, detection of molecular interactions, and for deciphering biological pathways. To advance this technique to an application that could be relevant to clinical study requires the development of near-infrared (NIR) fluorescent dyes since imaging in this range of wavelength (700-900 nm) allows light to penetrate several centimeters into the tissue and reducing the interference from biological background. In addition, the wavelength of NIR optical probes lies in the oscillation wavelength of a semiconductor laser. Therefore, it is suitable for imaging using laser beam as a source of light. We describe herein the design and synthesis of specific peptide-based fluorescence beacons for detection of cancer. Besides the NIR feature, the dyes possess some other important properties for biological application such as stability in an array of chemical milieu, water solubility, good quantum yield, and a handle for bioconjugation. In order to increase the selectivity during bioconjugation, the cyclic polymethine cyanine dyes were developed using different approaches. The stability of these dyes was demonstrated by labeling a peptide epitope via solid phase peptide chemistry. The in vivo optical imaging showed potential and broad application of these dyes in developing molecular-based beacons for cancer detection.

The antiphotooxidative properties of boldine and chloride berberine were studied by time-resolved thermal lensing technique. These compounds belong to isoquinoline alkaloids possessing interesting biological activity (e.g. antibacterial, antimalarial, antitumor). Antiphotooxidative properties of the alkaloids were studied by mechanism of energy transference between powerful oxidizing agents such as singlet oxygen. Singlet oxygen was produced by energy transfer from chlorophyll-sensitized photooxidation of oil by exposure of high light intensities like laser. The lifetimes of singlet oxygen in dimethylsulfoxide, methanol and water were determined to confirm the assignment of the singlet molecular oxygen O₂ (¹Δ_g) in the experiments. In order to understand the effect of the alkaloids on active oxygen species, we carried out in detail an analysis of the thermal lensing signal. It was shown that the alkaloids can act as quenchers of singlet oxygen. To demonstrate the ability of the alkaloids to act efficient singlet oxygen acceptors, we have measured the fluorescence spectra of the studied alkaloids in the presence and in the absence of singlet oxygen. The antiphotooxidative activity of boldine and chloride berberine can be explained by the ability to quench singlet oxygen.

Energy transfer from organic fluorophores to small metal nanoparticles is being used as a molecular beacon tool to monitor the kinetic processes of the hammerhead ribozyme. This marks the first time that nanomaterials have been used to monitor ribozyme kinetics. The quantum efficiency of energy transfer from the fluorophore to the gold nanoparticle follows a distance dependence behavior, which allows the real-time characterization of ribozyme complex structure and cleavage kinetics. The rate of cleavage for our ribozyme at pH=6.5 and 37°C is measured to be on the order of 10^-2 min^-1, which is the correct order of magnitude for similar ribozymes at this pH in the literature.

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystals QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies. Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.

OBJECTIVE: Optical spectroscopic tools exist that allow open surgical and minimally invasive assays of intrinsic tissue optics. Optical detection of cellular and tissue viability may offer a minimally invasive way to assess tumor responsiveness to chemotherapies. We report on an optical spectroscopic change that precedes apoptotic cell death and appears related to NAD(P)H autofluorescence. METHODS: The cell lines SW 480 and U87-MG were grown in culture and treated with cisplatin 100 μg/ml and tamoxifen 10 μM, respectively. Fluorescence spectroscopy at 355 nm excitation and 460 nm emission were collected. MTS assays were used to determine cell viability. Cell lysates were analyzed for NAD(P)H concentrations by mass spectroscopy. RESULTS: Autoflourescence at 355 nm excitation and 460 nm emission declines markedly despite normalization for cell number and total protein concentration after treatment with tamoxifen or cisplatin. The autofluorescence drop precedes the loss of cell viability as measured by MTS assay. For example, the relative viability of the U87-MG cell treated with tamoxifen at hours 0, 8, 12 and 24 of treatment was 100 ± 6, 85 ± 6, 53 ± 9 and 0 ± 3. The relative fluorescence at the same time points were 100 ± 2, 57 ± 6, 47 ± 3, and 0 ± 1. TUNNEL assays confirm that cell death is via apoptosis. The key cellular fluorophore at these wavelengths is NAD(P)H. Mass spectroscopic analysis of cell lysates at these time points reveals a drop in NAD(P)H concentrations that is parallel to the loss of fluorescence signal. CONCLUSIONS: NAD(P)H autofluoresence decline precedes apoptotic cell death. This may allow the design of minimally invasive spectroscopic tools to monitor chemotherapeutic response.

Introduction: Brain tumor margin detection remains a challenging problem in the operative resection of gliomas. A novel nanoparticle, a PEGylated quantum dot, has been shown to be phagocytized by macrophages in vivo. This feature may allow quantum dots to co-localize with brain tumors and serve as an optical aid in the surgical resection of brain tumors. Methods: Sprague-Daly rats were injected intracranially with C6 gliosarcoma cell lines to establish tumors. Two weeks after implantation of brain tumors, PEGylated quantum dots emitting at 705 nm (PEG-705 QD) were injected via the tail vein. Twenty-four hours post PEG-705 QD injection, the animals were sacrificed and their tissues examined. Results: PEGylated quantum dots are avidly phagocytized by macrophages and are taken up by liver, spleen and lymph nodes. Macrophages and microglia co-localize with glioma cells, carrying the optical nanoparticle, the quantum dot. Excitation of the PEG-705 quantum dots gives off a deep red fluorescence detectable with charge coupled device (CCD) cameras, optical spectroscopy units, and in dark field fluorescence microscopy. Conclusions: PEG-705QDs co-localize with brain tumors and may serve as an optical adjunct to aid in the operative resection of gliomas. The particles may be visualized in surgery with CCD cameras or detected by optical spectroscopy.

Accurate calculation of internal fluence excited in tissue from an optical source can be used for predicting the performance of fluorescent contrast agents for clinical applications. Solutions of excitation fluence for a steady-state Monte Carlo model and a finite element implementation of the 3d diffusion equation have been compared up to depths of 20mm from a point source located on top of a homogeneous cylindrical phantom for a range of reduced scattering-to-absorption ratios. Differences between the fluence calculated by Monte Carlo and diffusion model is found to be dependent on the transport mean free path (mfp), size of the phantom in relation to the penetration depth, distance from the source and mesh resolution. The differences are small at depths ~ mfp and peak at depths ~2mfp. The differences should ideally reduce to zero at large depths but the magnitude of the differences tend to increase due to the finite boundary in the diffusion model. As an example, for a mfp = 0.817mm similar in magnitude to mesh resolution, diffusion fluence at 1mm, 2mm, 10mm and 14mm is 76%, 59%, 66% and 63% respectively of Monte Carlo fluence. For large mfp's characteristic of non- diffusive regimes, diffusion model overestimates fluence at distances less than one mfp. This work demonstrates that mean free path and mesh resolution are the critical parameters that distinguish the performance of Monte Carlo and diffusion models to define error margins that could be utilized for predictive assessment of imageability of fluorescent agents using the diffusion model.

A diffusion approximation to the radiative transfer in a medium with varying refractive index has been proposed as a theoretical model for the ultrasonic tagging of fluorescence or FluoroSound, in a scattering medium. It has been found that the diffuse modulation is a defocusing effect. Defocusing is related to scatter - more the scatter, more the defocusing and there exists a component of the defocusing effect of scatter at the ultrasonic frequency. This is in contrast to the modulation for ballistic photons that originates in the focusing effect of the acoustic lens created by the ultrasonic wave. Simulations with circular phantoms of 1.5 and 2.0cm radius have shown that defocusing is minimum when the acoustic lens is midway between the source and the detector. These results are consistent with physics and demonstrate the capability of the model to function as a predictive tool for FluoroSound instrument design. Both ballistic and diffuse FluoroSound signatures can help in the simultaneous localization of the anomaly and determination of its optical properties. As an adjunct, optimally designed ultrasound beams can be also used to enhance diffuse photon modulation signal through acoustic guidance. Optical properties provide a way to discriminate between normal and diseased tissue. FluoroSound could therefore potentially achieve a fusion of anatomical and functional information non-invasively in a single measurement. The additional information made available by this method will improve the speed and accuracy of optical imaging as a tool in the identification and validation of targets.

Real time investigation of cerebral blood flow (CBF), and oxy/deoxy hemoglobin volume (HbO,HbR) dynamics has been difficult until recently due to limited spatial and temporal resolution of techniques like laser Doppler flowmetry and MRI. This is especially true for studies of disease models in small animals, owing to the fine structure of the cerebral vasculature. The combination of laser speckle flowmetry (LSF) and multi-spectral reflectance imaging (MSRI) yields high resolution spatio-temporal maps of hemodynamic changes in response to events such as sensory stimuli or arterial occlusion. Ischemia was induced by distal occlusion of the medial cerebral artery (dMCAO). Rapid changes in CBF, HbO, and HbR during the acute phase were captured with high temporal and spatial resolution through the intact skull. Hemodynamic changes that were correlated with vasoconstrictive events, peri-infarct spreading depressions (PISD), were observed. These experiments demonstrate the utility of LSF and Multi-spectral reflectance imaging (MSRI) in mouse disease models.

We discuss the application of time domain fluorescence techniques to the recovery of targets embedded in several cm thick biological tissue. Considering the general time domain problem first, a singular value analysis is used to study the optimal use of multiple frequency components extracted from time domain data. Furthermore, a computationally efficient algorithm is presented to tomographically reconstruct fluorophore locations using their decay amplitudes and validated using phantom experiments. The reconstruction algorithm presented here has wide applicability for non-invasive, diagnostic fluorescence imaging in small animals and other biological systems, given that fluorescence lifetime is a sensitive indicator of local tissue environment and elementary interactions at the molecular level.