This PDF file contains the front matter associated with SPIE Proceedings Volume 7576, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Nanomedicine is beginning to impact the treatment of several diseases and current research efforts include development of integrated nano-constructs (theranostics) which serve as probes for imaging and therapy in addition to delivering macromolecules intracellularly. In cancer, there is a vital unmet need for effective alternative treatments with high specificity and low systemic toxicity. This can be achieved by targeting key molecular markers associated with cancer cells with reduced effective drug doses. Here, we show an innovative proof-of-principle approach for efficient killing of proliferating ovarian cancer cells by inactivating a protein associated with cell proliferation namely, the nuclear Ki-67 protein (pKi-67), using nanotechnology-based photodynamic therapy (PDT). Antibodies against pKi-67 are widely used as prognostic tools for tumor diagnosis. In this work, anti pKi-67 antibodies were first conjugated to fluorescein isothiocyanate (FITC) and then encapsulated inside liposomes. After incubation of OVCAR-5 ovarian cancer cells with these liposomes, confocal microscopy confirmed the localization of the antibodies to the nucleoli of the cells. Irradiation with a 488 nm laser led to a significant loss of cell viability. The specificity of this approach for pKi-67 positive cells was demonstrated in confluent human lung fibroblasts (MRC-5) where only a small population of cells stain positive for pKi-67 and only minimal cell death was observed. Taken together, our findings suggest that pKi-67 targeted with nano-platform is an attractive therapeutic target in cancer therapy.

This paper deals with two potential modes for the treatment of skin cancer-one a novel approach using a squaraine dye and the other using a natural product- the flavonoid fraction of Saraca asoka. Squaraine dye is a photosensitizing agent, which is preferentially taken up and retained by the tumor cells and when irradiated with high power visible light results in the selective destruction of the tumor cells by photodynamic therapy. The uniqueness of this mode of treatment lies in the selective destruction of tumor cells without affecting the neighbouring normal cells, which is much advantageous over radiation therapy now frequently used. The chemopreventive and therapeutic effects of the plant component are explored as well. The experimental models were Swiss albino mice in which skin tumor was induced by DMBA. Marked reduction in tumor volume and burden in the treated groups were observed. The reversal of biochemical enzyme markers like rhodanese, myeloperoxidase, β-D glucuronidase, lactate dehydrogenase, hexokinase and sialic acid to near normal levels were observed in the PDT and flavonoid fraction treated groups. The live photographs of the experimental animals and histopathological data further support the obtained results. The study assumes importance as it combines a traditional treatment mode and a novel aspect in cancer therapy using the same experimental models. Also this is the first report on PDT using a squaraine dye for skin cancer therapy in vivo.

The microdistribution of therapeutic monoclonal antibodies within a tumor is important for determining clinical response. Nonuniform microdistribution predicts therapy failure. Herein, we developed a semiquantitative method for measuring microdistribution of an antibody within a tumor using in situ fluorescence microscopy and sought to modulate the microdistribution by altering the route and timing of antibody dosing. The microdistribution of a fluorescently-labeled antibody, trastuzumab (50-μg and 150-μg intraperitoneal injection (i.p.), and 100-μg intravenous injection (i.v.)) was evaluated in a peritoneal dissemination mouse model of ovarian cancer. In addition, we evaluated the microdistribution of concurrently-injected (30-μg i.p. and 100-μg i.v.) or serial (two doses of 30-μg i.p.) trastuzumab using in situ multicolor fluorescence microscopy. After the administration of 50-μg i.p. and 100-μg i.v. trastuzumab fluorescence imaging showed no significant difference in the central to peripheral signal ratio (C/P ratio) and demonstrated a peripheral-dominant accumulation, whereas administration of 150-μg i.p. trastuzumab showed relatively uniform, central dominant accumulation. With concurrent-i.p.-i.v. injections trastuzumab showed slightly higher C/P ratio than concurrently-injected i.p. trastuzumab. Moreover, in the serial injection study, the second injection of trastuzumab distributed more centrally than the first injection, while no difference was observed in the control group. Our results suggest that injection routes do not affect the microdistribution pattern of antibody in small peritoneal disseminations. However, increasing the dose results in a more uniform antibody distribution within peritoneal nodules. Furthermore, the serial i.p. injection of antibody can modify the microdistribution within tumor nodules. This work has implications for the optimal delivery of antibody based cancer therapies.

Macrophage elastase, also called MMP12, belongs to a family of proteolytic enzymes whose best known physiological function is the remodeling of the extracellular matrix. Under certain pathological conditions, including inflammation, chronic overexpression of MMP12 has been observed and its elevated proteolytic activity has been suggested to be the cause of pulmonary emphysema. However, it was until recently impossible to monitor the activity of MMP12 under disease conditions, mainly due to a lack of detection methods. Recent development of new reporters for monitoring MMP12 activity in living cells, such as LaRee1, provided novel insights into the pathobiology of MMP12 in pulmonary inflammation.1 In the future, these reporters might contribute to improved diagnosis and in finding better treatments for chronic inflammatory lung diseases and emphysema. Our approach for visualizing MMP12 activity is based on peptidic, membrane-targeted FRET (Foerster Resonance Energy Transfer) reporters. Here we describe a set of new reporters containing different fluorophore pairs as well as modifications in the membrane-targeting lipid moiety. We studied the influence of these modifications on reporter performance and the reporter mobility on live cell membranes by FRAP (fluorescence recovery after photobleaching). Finally, we generated several new fluorescently labeled MMP inhibitors based on the peptidic reporter structures as prototypes for future tools to inhibit and monitor MMP activity at the same time.

Many clinical evidences demonstrate that the sites of distant metastasis are not random and certain malignant tumors show a tendency to develop metastases in specific organs (e.g., brain, liver, and lungs). However, an appropriate animal model to characterize the metastatic nature of transplantable human cancer cell lines has not been reported well. Recent advances in bio-luminescent imaging (BLI) technologies have facilitated the quantitative analysis of various cellular processes in vivo. To visualize the fate of tumor progression in the living mice, we are constructing a luciferaseexpressing human cancer cell library (including melanoma, colon, breast, and prostate cancer). Herein we demonstrate that the BLI technology in couple with a fine ultrasonic guidance realizes cancer cell-type dependent metastasis to the specific organs. For example, some melanoma cell lines showed frequent metastasis to brain, lungs, and lymph nodes in the mouse model. Notably, reflecting the clinical features of melanoma, breast, and prostate cancer, some of the cell lines showed preferential metastasis to the brain. Moreover, these cellular resources for BLI allow a high throughput screening for potential anti-cancer drugs. Thus, this BLI-mediated additional strategy with the luciferase-expressing cancer cell resources should promote many translational studies for human cancer therapy.

Near-Infrared (NIR) fluorescence has been valuable in analytical and bioanalytical chemistry. NIR probes and labels have been used for several applications, including hydrophobicity of protein binding sites, DNA sequencing, immunoassays, CE separations, etc. The NIR region (700-1100 nm) has advantages for the spectroscopist due to the inherently lower background interference from the biological matrix and the high molar absorptivities of NIR chromophores. During the studies we report here several NIR dyes were prepared to determine the role of the hydrophobicity of NIR dyes and their charge in binding to amino acids and proteins, e.g., serum albumins. We synthesized NIR dye homologs containing the same chromophore but substituents of varying hydrophobicity. Hydrophobic moieties were represented by alkyl and aryl groups. These NIR dyes of varying hydrophobicity exhibited varying degrees of H-aggregation in aqueous solution indicating that the degree of H-aggregation could be used as an indicator to predict binding characteristics to serum albumins. In order to understand what factors may be important in the binding process, spectral behavior of these varying hydrophobicity dyes were examined in the presence of amino acids. Typical dye structures that exhibit large binding constants to biomolecules were compared in order to optimize applications utilizing non-covalent interactions.

Fluorophore-labeled contrast imaging agents are moving toward clinical use as aids in nodal staging and intraoperative resection of tumors. Near-infrared fluorophores with defined toxicity properties will be needed before these agents can be translated to the clinic. The near-infrared dye IRDye 800CW is frequently used in its N-hydroxysuccinamide (NHS) ester form for labeling these agents. Following conjugation or breakdown of a labeled ligand, excess NHS ester is converted to the carboxylate form. We report here the results of a preliminary toxicity study on IRDye 800CW carboxylate in preparation for its use as a labeling moiety for targeted contrast agents. Male and female Sprague Dawley rats were given a single intravenous or intradermal administration of IRDye 800CW carboxylate; indocyanine green was used as a comparative control. Following administration of varying doses of either the dyes or saline, animals were observed for up to fourteen days during which time, hematological, clinical chemistry, enzymological, and histological testing was performed on animal subgroups. Under the conditions tested, a single administration of IRDye 800CW carboxylate intravenously at dose levels of 1, 5 and 20 mg/kg or 20 mg/kg intradermally produced no pathological evidence of toxicity. A dose of 20 mg/kg was identified as the NOAEL (no observed adverse effect level) following IV or ID routes of administration of IRDye 800CW.

Near infrared spectroscopy possess great potential for in vivo quantitative monitoring of drugs in animal subject. The accuracy of the measurements by near infrared technique should be evaluated by an established method. In this study, a near infrared fluorescence dye, cypate and its conjugation cypate-PEG were used as model drug for in vivo dynamic study. The pharmacokinetics of the model drug in mice subjects were investigated by near infrared spectroscopy and high performance liquid chromatography, respectively. The results from the two techniques were compared. The pharmacokinetic parameters calculated based on the acquired data by DAS software showed that there were no statistical differences between the two methods. The dynamic distribution of the model drugs in mouse model imaged by NIR image system indicated that cypate firstly accumulated in liver and was cleared from the enteron system, while cypate - PEG clearance from the urine system. Results indicated that NIR monitoring technique provide a promising quantitative way for in vivo monitoring the dynamics of drugs in animal subjects.

Iridium complex, a promising organic light-emitting diode material for next generation television and computer displays, emits phosphorescence. Phosphorescence is quenched by oxygen. We used this oxygen-quenching feature for imaging tumor hypoxia. Red light-emitting iridium complex Ir(btp)₂(acac) (BTP) presented hypoxia-dependent light emission in culture cell lines, whose intensity was in parallel with hypoxia-inducible factor (HIF)-1 expression. BTP was further applied to imaging five nude mouse-transplanted tumors. All tumors presented a bright BTP-emitting image as early as 5 min after the injection. The BTP-dependent tumor image peaked at 1 to 2 h after the injection, and was then removed from tumors within 24 h. The minimal BTP image recognition size was at least 2 mm in diameter. By morphological examination and phosphorescence lifetime measurement, BTP is presumed to localize to the tumor cells, not to stay in the tumor microvessels by binding to albumin. The primary problem on suse of luminescent probe for tumor imaging is its weak penetrance to deep tissues from the skin surface. Since BTP is easily modifiable, we made BTP analogues with a longer excitation/emission wavelength to improve the tissue penetrance. One of them, BTPHSA, displayed 560/720 wavelength, and depicted its clear imaging from tumors transplanted over 6-7 mm deep from the skin surface. We suggest that BTP analogues have a vast potential for imaging hypoxic lesions such as tumor tissues.

Chlorin-bacteriochlorin dyads as a new class of near-infrared fluorophores were synthesized and spectroscopically characterized. Each dyad is comprised of a chlorin macrocycle (free base or zinc chelate) as an energy donor (and absorber) and a free base bacteriochlorin as an energy acceptor (and emitter). Excitation of the chlorin (λ= 650 nm, zinc chelate; 675 nm, free base) results in fast (5 ps) and nearly quantitative (>99%) energy transfer to the adjacent bacteriochlorin moiety, and consequently bacteriochlorin fluorescence (λ= 760 nm). Thus, each chlorinbacteriochlorin dyad behaves as a single chromophore, with a large effective Stokes shift (85 or 110 nm), a significant fluorescence quantum yield (Φ_f = 0.19), long excited-state lifetime (τ = 5.4 ns), narrow excitation and emission bands (<20 nm), and high chemical stability. Imaging experiments performed using phantoms show that the chlorin-bacteriochlorin dyads exhibit a range of superior properties compare with commercially available imaging dyes. While the latter are six-fold brighter (comparing ε•Φ_f values), the chlorin-bacteriochlorin dyads exhibit narrower excitation and emission bands and larger Stokes shift, therefore allowing more efficient and selective excitation and detection of fluorescence. The high selectivity is further demonstrated with in vivo imaging studies using mice. This selectivity together with the tunability of absorption and emission wavelengths using substituent effects under synthetic control make the chlorin-bacteriochlorin dyads ideal candidates for multicolor imaging applications. In addition, the long fluorescence lifetimes make those probes suitable for lifetime-imaging applications.

Many physiological processes function efficiently within a well-controlled pH range. Higher acidity level has been implicated with a number of systemic pathologies. The potential of pH sensitive fluorescent probes for reporting on biological environments has been widely utilized in a variety of cell studies and has been recently recognized as a powerful technique for in vivo imaging of diseases associated with elevated acidity level. We present several strategies for the development of pH sensitive probes suitable for in vivo imaging. The strategies include incorporation of pH sensitive functionalities in known fluorophores, synthesis of novel pH sensitive skeletons, and design of pH sensitive nanoparticles using acid-degradable polymers.

Intracellular pH of a single cell can be imaged using FLIM of enhanced green fluorescent protein (EGFP). The correlation between the intracellular pH and the fluorescence lifetime of EGFP in HeLa cells is explained by considering the pH-dependent acid-base equilibrium of the p-hydroxybenzylidene-imidazolidinone structure of the chromophore of EGFP. The equilibrium between different forms of chromophore depends on pH of the medium. The equilibrium constant between the neutral and anionic EGFP chromophores in HeLa cells is obtained by analyzing the fluorescence lifetimes observed with different values of intracellular pH. The intracellular pH dependence has been also observed in HeLa cells where enhanced yellow fluorescent protein (EYFP) is expressed. The pH dependence of the fluorescence lifetime of EYFP may result from the pH dependence of the molecular structure of the protein bound ionic form of EYFP or the conformational change of the EYFP chromophore induced by lowering pH. The fluorescence lifetimes both of EGFP and of EYFP are not uniform in the cell. At each pH, for example, the fluorescence lifetime of EGFP located near the outer cell membrane is shorter than those located inside cell, whereas the lifetime of EYFP located near the outer cell membrane is longer than those located inside the cell. These differences are ascribed to the different distribution of the electric field surrounding the fluorescent chromophore in the cells, implying that the chromophores of EGFP and EYFP show the opposite electric field effects of the fluorescence lifetime to each other. The fact that the fluorescence lifetime of BCECF in solution is different from the one observed at the same pH in intact cells of Halobacterium salinarum has been also ascribed to the local field produced by membranes in vivo.

Modern molecular modeling tools are intensively used to gain knowledge of events occurring upon photoexcitation of organic chromophores in the gas-phase, in solution and in protein matrices. We applied quantum mechanical approach to estimate equilibrium geometry configurations as well as positions and intensities of spectral bands for a number of red fluorescent proteins, including the DsRed from Discosoma coral, and its mutants of the so-called mFruits series. As demonstrated in our previous simulations for GFP and blue fluorescent proteins, this strategy was proven to be productive for modeling. The model system is designed as a molecular cluster constructed on the basis of available crystal structures of the related protein. The equilibrium geometry of the cluster is optimized using density functional theory approximations. The vertical excitation energies corresponding to the S₀-S₁ transitions are computed by using the semiempirical ZINDO technique. Mechanisms of photoexcitation, identification of the functional states of the chromophores, elucidation the role of point mutations in the photoreceptor proteins are considered on the basis of simulations.

This paper presents preliminary results on the use of transferrin protein nanospheres (TfpNS) for targeting cancer cells in vitro. Protein nanospheres represent an easily prepared and modifiable nanoplatform for receptor-specific targeting, molecular imaging and gene delivery. Rhodamine B isothiocyanate conjugated TfpNS (RBITC-TfpNS) show significantly enhanced uptake in vitro in SK-MEL-28 human malignant melanoma cells known to overexpress transferrin receptors compared to controls. RBITCTfpNS labeling of the cancer cells is due to transferrin receptor-mediated uptake, as demonstrated by competitive inhibition with native transferrin. Initial fluorescence microscopy studies indicate GFP plasmid can be transfected into melanoma cells via GFP plasmid encapsulated by TfpNS.

Multifunctional silica nanoparticles provide a framework for the attachment of imaging and targeting agents for applications in spectroscopy, microscopy, and biology while simultaneously serving as supports for molecular machines for the controlled release of cargo. The deliberate placing of molecules or other nanoparticles within specific regions of the mesostructure or the surface of the nanoparticles allows for multiple modes of characterization and application. This review focuses on research related to fluorescence and spectroscopic imaging techniques, targeting strategies to increase particle uptake efficiency in cells, and on demand drug delivery regulated by molecular machines.

A dual contrast agent that combines perfluorocarbon droplets and metal nanoparticles has been developed for combined ultrasound and photoacoustic imaging. Metal nanoparticles were incorporated in dodecafluoropentane (DDFP) droplets encapsulated in a bovine serum albumin (BSA) shell. To embed aqueous colloidal suspensions of metal nanoparticles in DDFP, a phase transfer of the plasmonic nanoparticles was completed in two different strategies, a single and a double ligand exchange of capping materials. Emulsion techniques were used to encapsulate phase transferred metal nanoparticles within the DDFP droplets. Spectrophotometry and cryogenic transmission electron microscopy were used to characterize and to confirm successful fabrication of the dual contrast agent.

The size transition from bulk conducting metals to insulating nanoparticles and eventually to single atoms passes through the relatively unexplored few-atom nanocluster region. With dimensions close to the Fermi wavelength, these nanoclusters demonstrate molecule-like properties distinct from bulk metals or atoms, such as discrete and size-tunable electronic transitions which lead to photoluminescence. Current research aims to elucidate the fundamental photophysical properties of metal nanoclusters made by different means and based on different encapsulation agents. Here, we report the study of the photophysical properties, including quantum yields, lifetimes, extinction coefficients, blinking dynamics and sizes, of silver and gold nanoclusters synthesized using oligonucleotides, a protein (bovine serum albumin) and a Good's buffer molecule (MES, 2-(N-morpholino) ethanesulfonic acid) as encapsulation agents. We also investigate the change of photoluminescence as a function of temperature. Furthermore, we show that the fluorescent metal clusters can be used as a donor in forming a resonance energy transfer pair with a commercial organic quencher. These new fluorophores have great potential as versatile tools for a broad range of applications in biological and chemical detection.

Er doped and Yb-Er-Tm codoped ZrO2 nanocrystals of average 80 nm in size were prepared by a sol-gel process with the presence of nonionic (PLURONIC F-127) surfactant, and the up-conversion emission was characterized under IR (980 nm) excitation. The effect of the codoped conditions on the crystalline structure and photoluminescence properties were studied. A strong green emission was produced with 5 mol %, 0.2 mol %, 0.01 mol % of Yb3+-Er3+-Tm3+ codoped ZrO2 respectively. It was prepared Er doped ZrO2 -SiO2 core-shell and SiO2 coated Er doped ZrO2 in 2-propanol and water, respectively. The presence of the silica shell of average of 15 nm in thickness has been confirmed by transmition electron microscopy. Photolumineiscence studies show that the silica shell does not affect the emission when the nanoparticles are excited with 980 nm. The up-converting Yb3+-Er3+-Tm3+ codoped ZrO2 nanocrystal has showed to be a powerful tool to future detection techniques. The viability of the nanoparticles of codoped ZrO2 for biological imaging was confirmed by multiphotonic microscope imaging of cervix tissue with inserted codoped ZrO2 nanoparticles. The cervix tissue has a moderate dysplasia. The nanoparticles were introduced at 80 % of the tissue depth (5 μm) without being functionalized.

Distance dependant coupling of plasmon resonances between closely spaced metal nanoparticles offers an attractive alternative for the imaging of molecular interactions. Here we analyzed interactions between molecular specific gold nanoparticles and live cells using a combination of dark-field reflectance and hyperspectral imaging. The results of optical imaging were correlated with transmittance electron microscopy of cell slices and theoretical simulations of optical properties of gold aggregates. We showed that nanoparticles targeted to epidermal growth factor receptor (EGFR) form closely spaced assemblies in the presence of the target molecule. Our experiments with living cells showed that receptor mediated assembly and plasmon coupling of gold bioconjugates result in a spectral shift of more than 100 nm in plasmon resonance frequency of the nanoparticles giving a very bright red signal. We demonstrated that plasmon coupling can be used for imaging of EGFR activation and trafficking as formation of EGFR dimers and further intracellular uptake in early and late endosomes is associated with progressive color changes from green to red, respectively, with each stage of EGFR cycle being associated with a distinct color of EGFR bound nanoparticles. This approach can allow imaging of molecular interactions ranging from protein pairs to multi-protein complexes with sensitivity and SNR that cannot be currently achieved with any other method.

Nanoparticles such as gold and silver with plasmonic resonances in the near-infrared (NIR) optical region, where soft tissue is the most transparent, are of great interest in biomedical applications. A major roadblock in translation of inorganic nanoparticles to clinical practice for systemic targeting of disease is their nonbiodegradable nature. In addition, gold nanoparticles that absorb in the NIR are typically greater than 50 nm, which is above the threshold size of 5.5 nm required for effective excretion from the body. Here we describe a new class of biodegradable gold nanoparticles with plasmon resonances in the NIR region. The synthesis is based on controlled assembly of very small (less than 5 nm) primary gold particles into nanoclusters with sub-100 nm overall diameter and an intense NIR absorbance. The assembly is mediated by biodegradable polymers, polyethylene glycol (PEG) and polylactic acid (PLA) copolymer, and small capping ligands on the constituent nanoparticles. Nanoclusters deaggregate into sub-5nm primary gold particles upon biodegradation of the polymer binder in live cells over one week, as shown by dark-field reflectance and hyperspectral imaging.

Gold nanoparticles have been shown to possess x-ray attenuation higher than that of clinically used iodine based contrast agents. Additionally, gold nanoparticles have also been used in enhancing the Raman signal through a phenomenon called Surface Enhanced Raman Scattering (SERS). The development of Gold nanoparticle based probes (nanotags) for combined SERS and x-ray computed tomography (CT) are reported. These nanotags comprise quasi-spherical gold nanoparticles encoded using a Raman active molecule and encapsulated by a monolayer of polyethylene glycol with carboxylate functional groups for bioconjugation. The nanotags, made from ~65 nm gold nanoparticles, display large SERS cross-section and x-ray absorption which are used for dual modality imaging of tumor cells in of an orthotropic prostate cancer tumor animal model.

Core-shell nanoparticles with an upconverting phosphorescent, lanthanide core and plasmonic gold shell are employed for Förster Resonance Energy Transfer (FRET). FRET is demonstrated from a highly efficient upconverting fluoride nanoparticle doped with Ytterbium (Yb) and Erbium (Er) ions to Streptavidin conjugated with Tetramethyl rhodamine fluorophore.

Gold nanorods (GNRs) combined with two-photon microscopy were explored for potential application in imaging of oral carcinogenesis. GNRs have been shown to be effective contrast agents for two photon luminescence in that excitation laser powers required for imaging are low compared to traditional fluorophores. Imaging of cells, ex vivo tissues, and in vivo oral mucosa labeled with GNRs was performed to evaluate potential advantages of these agents in molecular imaging of epithelial carcinogenesis. Powers required to elicit a two-photon luminescence signal from GNRs were determined for cells as well as normal and malignant transformed lesions, 24 hours following injection of GNRs in a hamster model for oral cancer. The strength of the detected emission as the function of the average incident laser power was measured in tissues with and without GNRs to compare the sensitivity of GNRs against tissue autofluorescence. Finally, in vivo imaging was performed immediately following GNR injection to establish the ability to image microvasculature at low incident powers. The pilot study demonstrated uptake of GNRs by cells and in tissues yielding bright fluorescence signals using significantly lower incident powers than those needed to excite tissue autofluorescence. The in vivo imaging aspect of the study demonstrated the localization of GNRs within the microvasculature of the oral cancer model. These preliminary studies demonstrated the ability of GNRs to function as photostable, high contrast imaging agents and suggest that GNRs and multi-photon imaging have great potential for applications in the field of molecular imaging and early detection of cancer.

In this communication, we show that off-axis digital holography combined to phase-shifting acquisition of holograms is an effective microscopic tool to fully localize, in three dimensions, transmembrane receptors of living cells tagged with Gold nanocolloids. Gold nanoparticles, known for their interesting optical properties as well as for their noncytotoxicity are used here as biomarkers to target the cellular receptors.

Nanoparticles are being researched as a noninvasive method for selectively killing cancer cells. With particular antibody coatings on nanoparticles, they attach to the abnormal cells of interest (cancer or otherwise). Once attached, nanoparticles can be heated with UV/visible/IR or RF pulses, heating the surrounding area of the cell to the point of death. Researchers often use single-pulse or multipulse lasers when conducting nanoparticle ablation research. In the present paper, we are conducting an analysis to determine if the multipulse mode has any advantage in heating of spherical metal nanoparticles (such as accumulative heating effect). The laser heating of nanoparticles is very sensitive to the time structure of the incident pulsed laser radiation, the time interval between the pulses, and the number of pulses used in the experiments. We perform time-dependent simulations and detailed analyses of the different nonstationary pulsed laser-nanoparticle interaction modes, and show the advantages and disadvantages of multipulse (set of short pulses) and single-pulse laser heating of nanoparticles. A comparative analysis for both radiation modes (single-pulse and multipulse) are discussed for laser heating of metal nanotargets on nanosecond, picosecond and femtosecond time scales to make recommendations for efficient laser heating of nanomaterials in the experiments.

Fluorescence lifetime is a relatively new contrast mechanism for optical imaging in living subjects that relies on intrinsic properties of fluorophores rather than concentration dependent intensity. Drawing upon the success of fluorescence lifetime imaging microscopy (FLIM) for investigation of protein-protein interactions and intracellular physiology, in vivo fluorescence lifetime imaging (FLI) promises to dramatically increase the utility of fluorescencebased imaging in preclinical and clinical applications. Intrinsic fluorescence lifetime measurements in living tissues can distinguish pathologies such as cancer from healthy tissue. Unfortunately, intrinsic FLT contrast is limited to superficial measurements. Conventional intensity-based agents have been reported for measuring these phenomena in vitro, but translation into living animals is difficult due to optical properties of tissues. For this reason, contrast agents that can be detected in the near infrared (NIR) wavelengths are being developed by our lab and others to enhance the capabilities of this modality. FLT is less affected by concentration and thus is better for detecting small changes in physiology, as long as sufficient fluorescence signal can be measured. FLT can also improve localization of signals for improved deep tissue imaging. Examples of the utility of exogenous contrast agents will be discussed, including applications in monitoring physiologic functions, controlled drug release and cancer biology. Instrumentation for FLI will also be discussed, including planar and diffuse optical imaging in time and frequency domains. Future applications will also be discussed that are being developed in this exciting field that complement other optical modalities.

Applications of fluorescence spectroscopy that enable the real-time or rapid detection of fecal contamination on beef carcasses and the presence of central nervous system tissue in meat products are discussed. The former is achieved by employing spectroscopic signatures of chlorophyll metabolites; the latter, by exploiting the characteristic structure and intensity of lipofuscin in central nervous system tissue. The success of these techniques has led us to investigate the possibility of diagnosing scrapie in sheep by obtaining fluorescence spectra of the retina. Crucial to this diagnosis is the ability to obtain baseline correlations of lipofuscin fluorescence with age. A murine model was employed as a proof of principle of this correlation.

The drug-binding site subdomain IIA of human serum albumin (HSA) was characterized by absorption and fluorescence spectroscopy using 7-hydroxyquinoline (7-HQ) as a local reporter. The spectra of 7-HQ in solution indicate that a ztitterionic tautomer is stabilized by water in the ground state and produces a unique absorption peak at 400 nm and a fluorescence peak at 510 nm. By examining the spectral change in binary mixtures of water and 1,4-dioxane, three water molecules were estimated to stabilize this tautomer through direct interactions with the polar regions of the molecule. When 7-HQ is mixed with HSA, a reduction in the absorbance of the zwitterionic tautomer was observed which indicates a less polar environment around the molecule. The 7-HQ molecule is found to specifically bind in subdomain IIA of HSA and causes a reduction in the fluorescence intensity of the Trp-214 residue which is located in the same binding site. The reduction in the fluorescence of Trp-214 is due to energy transfer from the Trp-214 residue to the 7- HQ probe. The distance between Trp-214 and the probe was calculated using Förster theory for energy transfer to be 1.95 nm. This distance and the calculated quenching rate constant using a Stern-Valmer plot (k_q = 3.04 x 10¹² M^-1s^-1) both point to a static quenching mechanism. The binding constant and the number of binding sites of the complex were also estimated and the calculations show that the 7-HQ probe binds only in subdomain IIA. The change in the fluorescence intensity of HSA in the presence of the probe indicates that the 7-HQ molecule selectively interacts with the Trp-214 residue which results in partial unmasking of the fluorescence due to the Tyr-263 residue (located in the same site). A much stronger fluorescence from Tyr-263 is observed when HSA is chemically unfolded by 6.0 M GdnHCl. 7- HQ is found to still bind in subdomain IIA in the unfolded state of HSA and causes a reduction in the fluorescence intensities of both Trp-214 and Tyr-263. The present results propose 7-HQ as a useful photophysical probe in studying binding sites in proteins and exploring their hydrophobic environment.

In the present study the photophysical properties of DY-635B, a cyanine dye, bound to streptavidin were characterized in detail. Special emphasis was given to i) the alterations in the intrinsic photophysical characteristics of the dye due to (un)specific interactions with streptavidin and ii) the evaluation of interaction between the fluorescence probe and streptavidin in the presence of unlabeled biotin. Fluorescence correlation spectroscopy (FCS) and time-resolved anisotropy experiments were carried out in the presence of excess biotin to monitor also a possible cooperative effect on the fluorescence behavior of DY-635B. Based on the evaluation of FCS and time-resolved anisotropy data it is shown that due to binding to streptavidin the rotational freedom of DY-635B is restricted. This restriction is further increased by additional biotin indicating that the biotin binding is altering the tertiary structure of streptavidin. The intrinsic photophysical deactivation processes of DY-635B are changed as well. From FCS measurements it is concluded that due to the specific interaction of DY-635B and streptavidin, the deactivation via a "dark state" becomes less effective, shown as an increase of the corresponding decay time τR.

A Smart pH Cuvette is developed by coating the inner surface with pH sensitive thin film. The coating is a hydroscopic sol-gel material doped with colorimetric pH indicator dye sensitive to the pH of analyte solutions in biological range. Ocean optics miniaturized spectrometers are used for signal detection and analysis, along with multimode optical fibers. This new pH sensing arrangement yields an inexpensive solution for monitoring the pH of samples for biological applications. The Smart pH Cuvettes provide a resolution of 0.01 pH units, an accuracy of 1% of the reading, and 90% response in less than 10 seconds.

A smart Oxygen Cuvette is developed by coating the inner surface of a cuvette with oxygen sensitive thin film material. The coating is glass like sol-gel based sensor that has an embedded ruthenium compound in the glass film. The fluorescence of the ruthenium is quenched depending on the oxygen level. Ocean Optics phase fluorometer, NeoFox is used to measure this rate of fluorescence quenching and computes it for the amount of oxygen present. Multimode optical fibers are used for transportation of light from an LED source to cuvette and from cuvette to phase fluorometer. This new oxygen sensing system yields an inexpensive solution for monitoring the dissolved oxygen in samples for biological and medical applications. In addition to desktop fluorometers, smart oxygen cuvettes can be used with the Ocean Optics handheld Fluorometers, NeoFox Sport. The Smart Oxygen Cuvettes provide a resolution of 4PPB units, an accuracy of less than 5% of the reading, and 90% response in less than 10 seconds.

Nucleic acids experience a variety of perturbations. These may include strand cleavage and ligation, local conformational changes, base flipping, as well as structural and environmental perturbations that are induced upon protein and low MW ligand binding. Since the native nucleobases are practically non-emissive, synthetic fluorescent nucleoside analogs that are sensitive to their local environment have become powerful tools for investigating nucleic acids structure, dynamics, recognition and damage. Our criteria for "ideal" fluorescent nucleoside analogs include: (a) high structural similarity to the native nucleobases to faithfully mimic their size and shape, as well as hybridization and recognition properties; (b) red shifted absorption (> 290 nm) to minimize overlap with the absorption of the natural bases; (c) red shifted emission (preferably in the visible range); (d) reasonable emission quantum efficiency; and, importantly, (e) sensitivity/responsiveness of one or all photophysical parameters (λem and/or ΦF, τ) to changes in the probe's microenvironment. The design and synthesis of new fluorescent isosteric nucleobase analogs and their utilization for the fabrication of "real-time" fluorescence-based discovery and detection assays are outlined.

We have investigated the photophysical properties of Cy3-labeled DNA. Results show that the fluorescence efficiency of the dye changes dramatically upon covalent attachment, and that factors such as DNA sequence, or whether the dye is next to a single- or double-stranded region, also play a crucial role. Such dramatic dependence on the microscopic environment of the probe is due to the existence of an activated photoisomerization process that competes with fluorescence emission to deactivate the excited state. Cy3-DNA interactions, which depend on DNA sequence and the flexibility of the biopolymer, restrict the ability of the dye to photoisomerize and therefore increase the lifetime of the excited state. In this manuscript, we review the results of our most recent studies, and discuss the consequences of the photophysical behavior of Cy3 in biophysical research.

Fluorescence imaging is a valuable tool for the study of living systems. It can be used with good resolution from the micro- to the macroscopic range. However, for macroscopic use in living animals or humans, fluorescent probes must overcome several obstacles such as aqueous solubility, suitable circulating lifetime and clearance. Fluorescent probes should also display high molar extinction coefficient and fluorescence quantum yield. In this article, we report the encapsulation of five hydrophobic or amphiphilic fluorophores (DiO, DiI, DiD, DiR and ICG) with emission wavelength ranging from 500 to 800 nm, in long-circulating Lipid NanoParticles (LNP). Loading of these commercially available indocyanines in LNP is highly efficient (from 77 to 97 %), and fluorescence quantum yields range from 7 to 53%, depending on the dye, in the standard formulation (50 nm diameter nanoparticles). Given the wide range of wavelengths covered and the stability of particle dispersion in aqueous buffer, dye-loaded LNP should be a valuable tool for both in vivo and in vitro fluorescence imaging.

Elucidating the role of calcium fluctuations at the cellular level is essential to gain insight into more complex signaling and metabolic activity within tissues. Recent developments in optical monitoring of calcium transients suggest that cells integrate and transmit information through large networks. Thus, monitoring calcium transients in these populations is important for identifying normal and pathological states of a variety of systems. Though optical techniques can be used to image calcium fluxes using fluorescent probes, depth penetration limits the information that can be acquired from tissues in vivo. Alternatively, the calcium-sensitive dye arsenazo III is useful for optical techniques that rely on absorption of light rather than fluorescence for image contrast. We report on the use of arsenazo III for detection of calcium using photoacoustics, a deeply penetrating imaging technique in which an ultrasound signal is generated following localized absorption of light. The absorbance properties of the dye in the presence of calcium were measured directly using UV-Vis spectrophotometry. For photoacoustic studies, a phantom was constructed to monitor the change in absorbance of 25 μM arsenazo III at 680 nm in the presence of calcium. Subsequent results demonstrated a linear increase in photoacoustic signal as calcium in the range of 1 - 20 μM complexed with the dye, followed by saturation of the signal as increasing amounts of calcium were added. For delivery of the dye to tissue preparations, a liposomal carrier was fabricated and characterized. This work demonstrates the feasibility of using arsenazo III for photoacoustic monitoring of calcium transients in vivo.

In this paper, we present a new facile and environmental friendly method to prepare water-soluble near-infrared (NIR)-emitting PbS quantum dots (QDs) at room temperature under ambient conditions, using dihydrolipoic acid (DHLA) as a stabilizer. The photoluminescence (PL) emissions of the prepared DHLA-capped PbS QDs are tunable between 870 and 1010 nm. A PL quantum yield (QY) of ~10% can be achieved under optimized conditions without any post-preparative treatment. Here, we further use the produced DHLA-capped PbS QDs for NIR fluorescence imaging in a mouse model. The obtained experimental results showed that the NIR fluorescence of the PbS QDs in living tissues generated from the excitation with semiconductor laser (λmax=765.9 nm) could penetrate living tissues and be detected easily by the noninvasive in vivo NIR fluorescence imaging system. In addition, the preliminary studies on the cytotoxicity and in vivo toxicity of the QDs also indicates fully that these water-soluble DHLA-capped PbS QDs are very lowly toxic, and as such they should have greater potential in biological and medical applications especially in noninvasive in vivo fluorescence imaging of mice, compared to other existing highly toxic aqueous NIR-emitting quantum dots (CdTe, HgTe, etc).

Photodynamic therapy uses laser, LED or lamp light sources in combination with dyes - exogenous photosensitizers for the enhancement and localization of photodynamic effects within the human body. We are developing a new approach of improvement of the efficiency of antimicrobial phototherapy via combined application of photosensitizers and the photocatalysts to pathogenic microorganisms. The main goal of the paper is to conduct experiments to study the action of nanodyes, based on mixtures of nanoparticles and photosensitizers, in combination with LED irradiation of pathogens.

We have developed a fluorescent ruthenium metalloglycocluster as a powerful molecular probe for evaluating a binding event between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analysis. The fluorescent ruthenium metalloglycoclusters, [Ru(bpy-2Gal)₃] and [Ru(bpy-2Glc)₃], possess clustered galactose and glucose surrounding the ruthenium center. Changes in FE and FP of these metalloglycoclusters were measured by adding each lectin (Peanut agglutinin (PNA), Ricinus communis agglutinin 120 (RCA), Concanavalin A (ConA), or Wheat germ agglutinin (WGA)) or tetanus toxin c-fragment (TCF). Following the addition of PNA, the FE spectrum of [Ru(bpy- 2Gal)₃] showed new emission peak and the FP value of [Ru(bpy-2Gal)₃] increased. Similarly, the FE spectrum of [Ru(bpy-2Glc)₃] showed new emission peak and the FP value increased following the addition of ConA. Since other combinations of the metalloglycoclusters and lectin caused little change, specific bindings of galactose to PNA and glucose to ConA were proved by the FE and FP measurement. From nonlinear least-squares fitting, dissociation constants (K_d) of [Ru(bpy-2Gal)₃] to PNA was 6.1 μM, while the Kd values of [Ru(bpy)2(bpy-2Gal)] to PNA was ca. 10^-4 M. Therefore, the clustered carbohydrates were proved to increase affinity to lectins. Furthermore, the FP measurements proved specific binding of [Ru(bpy-2Gal)₃] to TCF.

Photodynamic Therapy (PDT) using a sensitizing drug is recognized as a promising medical technique for cancer treatment. It is a two step process that requires the administration of a photosensitizer followed by light exposure to treat a disease. Following light exposure the photosensitizer is excited to a higher energy state which generates free radicals and singlet oxygen. The present study was carried out to assess the oxidative damage induced by bis (3, 5-diiodo-2, 4, 6- trihydroxyphenyl) squaraine in skin tumor tissues of mice with/ without light treatment. Skin tumor was induced using 7, 12-Dimethyl Benz(a)anthracene and croton oil. The tumor bearing mice were given an intraperitoneal injection with the squaraine dye. After 24h, the tumor area of a few animals injected with the dye, were exposed to visible light from a 1000 W halogen lamp and others kept away from light. All the mice were sacrificed one week after the PDT treatment and the oxidative profile was analyzed (TBARS, SOD, catalase, GSH, GPx and GR) in tumor/ skin tissues. The dye induces oxidative stress in the tumor site only on illumination and the oxidative status of the tumor tissue was found to be unaltered in the absence of light. The results of the study clearly shows that the tumor destruction mediated by PDT using bis (3, 5-diiodo-2, 4, 6-trihydroxyphenyl) squaraine as a photosensitizer is due to the generation of reactive oxygen species, produced by the light induced changes in the dye.

Indocyanine green (ICG) is an FDA approved near infrared dye used in assessment of hepatic function and ophthalmological vascular imaging. However, given the rapid clearance of ICG from the blood stream, its imaging and phototherapeutic applications remain very limited. As a potential method to increase circulation time of ICG, and extend its clinical applications, we have encapsulated ICG within polymeric based nanoconstructs whose surface can be coated with various materials including polyethylene glycol (PEG). To gain an understanding of the interaction between ICG-containing nanocapsules (ICG-NCs) and vascular cells, we are characterizing the uptake of the nanocapsules coated with various materials by human peripheral blood monocytes and human spleen macrophages using fluorescence microscopy. Results of these studies will be useful in identifying the appropriate coating material that will result in increased circulation time of ICG-NCs within the vasculature.

Surface enhanced Raman scattering (SERS) is now a well-established technique to greatly amplify the normally weak Raman scattering signals. The amplification is achieved by using SERS substrates - specially structured metallic substrates with nano-scale morphological features. One of the most widely used methods for SERS amplification employs nanoparticles of silver or gold either in colloidal suspension or in dry-drop form. In such substrates SERS amplification factors (AF) exceeding 10¹² have been reported. The reproducibility of the colloid-based substrates, however, is a problem. The lack of reproducibility can be caused by a variety of factors that can change the interparticle distances. In this paper we show that thermal annealing of SERS substrates fabricated using commercially available nano-particle inks can be used to create thermally stable substrates with high reproducibility. It appears that thermal annealing destroys the unstable hot-spots with very high AF's but still leaves the sample with high AF sites yielding spatially averaged substrate AF's exceeding 10⁸.