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Sample records for vivo fluorescence spectroscopy

  1. In Vivo Fluorescence Correlation and Cross-Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Mütze, Jörg; Ohrt, Thomas; Petrášek, Zdeněk; Schwille, Petra

    In this manuscript, we describe the application of Fluorescence Correlation Spectroscopy (FCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), and scanning FCS (sFCS) to two in vivo systems. In the first part, we describe the application of two-photon standard and scanning FCS in Caenorhabditis elegans embryos. The differentiation of a single fertilized egg into a complex organism in C. elegans is regulated by a number of protein-dependent processes. The oocyte divides asymmetrically into two daughter cells of different developmental fate. Two of the involved proteins, PAR-2 and NMY-2, are studied. The second investigated system is the mechanism of RNA interference in human cells. An EGFP based cell line that allows to study the dynamics and localization of the RNA-induced silencing complex (RISC) with FCS in vivo is created, which has so far been inaccessible with other experimental methods. Furthermore, Fluorescence Cross-Correlation Spectroscopy is employed to highlight the asymmetric incorporation of labeled siRNAs into RISC.

  2. In-vivo pharmacokinetic study of two fluorescein derivatives by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Soulie-Begu, Sylvie; Devoisselle, Jean-Marie; Mordon, Serge R.

    1995-12-01

    We have already demonstrated the ability of fluorescence spectroscopy and imaging to measure the pH of superficial tissues using pH sensitive fluorescent probes. The purpose of this study was to investigate the in vivo behavior of such fluorescent probes. We report the monitoring of tissue fluorescence after injection of two fluorescein derivatives (carboxyfluorescein and biscarboxyethyl-carboxyfluorescein). The in vivo study was performed on anaesthetized adult Wistar rats. After laparotomy, CF or BCECF solution was injected into the penial vein. Fluorescence spectra were recorded during one hour using an optical multichannel analyzer coupled to a CCD camera. Fiber optic was placed alternatively on the liver area or on the skin. Blood samples were collected and fluorescence was measured in vitro. A clear linear relationship between dose and fluorescence intensity was found in liver for these fluorescent markers. Concerning spectral characteristics, it was found that CF and BCECF spectra show a shift compared to in vivo maximum emission peak and BCECF emission peak was different when recorded in the liver and in the skin. Differences of kinetic profiles are also observed between CF and BCECF. The BCECF derivative displays a fluorescence peak in the liver two minutes after injection, while CF fluorescence peak is observed seven minutes after injection. Clearance of skin fluorescence is slower than the plasmatic one indicating that dye elimination in superficial blood vessels does not follow the same pharmacokinetic behavior. Based on these preliminary findings, fluorescence spectroscopy appears as a tool in pharmacokinetic study in situ and in vivo.

  3. In vivo characterization of myocardial infarction using fluorescence and diffuse reflectance spectroscopy

    NASA Astrophysics Data System (ADS)

    Ti, Yalin; Chen, Poching; Lin, Wei-Chiang

    2010-05-01

    We explore the feasibility of using combined fluorescence and diffuse reflectance spectroscopy to characterize a myocardial infarct at different developing stages. An animal study is conducted using rats with surgically induced myocaridal infarction (MI). In vivo fluorescence spectra at 337-nm excitation and diffuse reflectance between 400 and 900 nm are measured from the heart. Spectral acquisition is performed: 1. for normal heart tissue; 2. for the area immediately surrounding the infarct; and 3. for the infarcted tissue itself, one, two, three, and four weeks into MI development. Histological and statistical analyses are used to identify unique pathohistological features and spectral alterations associated with the investigated regions. The main alterations (p<0.05) in diffuse reflectance spectra are identified primarily between 450 and 600 nm. The dominant fluorescence alterations are increases in peak fluorescence intensity at 400 and 460 nm. The extent of these spectral alterations is related to the duration of the infarction. The findings of this study support the concept that optical spectroscopy could be useful as a tool to noninvasively determine the in vivo pathophysiological features of a myocardial infarct and its surrounding tissue, thereby providing real-time feedback to surgeons during various surgical interventions for MI.

  4. Dynamic imaging by fluorescence correlation spectroscopy identifies diverse populations of polyglutamine oligomers formed in vivo.

    PubMed

    Beam, Monica; Silva, M Catarina; Morimoto, Richard I

    2012-07-27

    Protein misfolding and aggregation are exacerbated by aging and diseases of protein conformation including neurodegeneration, metabolic diseases, and cancer. In the cellular environment, aggregates can exist as discrete entities, or heterogeneous complexes of diverse solubility and conformational state. In this study, we have examined the in vivo dynamics of aggregation using imaging methods including fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS), to monitor the diverse biophysical states of expanded polyglutamine (polyQ) proteins expressed in Caenorhabditis elegans. We show that monomers, oligomers and aggregates co-exist at different concentrations in young and aged animals expressing different polyQ-lengths. During aging, when aggregation and toxicity are exacerbated, FCS-based burst analysis and purified single molecule FCS detected a populational shift toward an increase in the frequency of brighter and larger oligomeric species. Regardless of age or polyQ-length, oligomers were maintained in a heterogeneous distribution that spans multiple orders of magnitude in brightness. We employed genetic suppressors that prevent polyQ aggregation and observed a reduction in visible immobile species with the persistence of heterogeneous oligomers, yet our analysis did not detect the appearance of any discrete oligomeric states associated with toxicity. These studies reveal that the reversible transition from monomers to immobile aggregates is not represented by discrete oligomeric states, but rather suggests that the process of aggregation involves a more complex pattern of molecular interactions of diverse intermediate species that can appear in vivo and contribute to aggregate formation and toxicity. PMID:22669943

  5. Noninvasive fluorescence excitation spectroscopy for the diagnosis of oral neoplasia in vivo

    NASA Astrophysics Data System (ADS)

    Ebenezar, Jeyasingh; Ganesan, Singaravelu; Aruna, Prakasarao; Muralinaidu, Radhakrishnan; Renganathan, Kannan; Saraswathy, Thillai Rajasekaran

    2012-09-01

    Fluorescence excitation spectroscopy (FES) is an emerging approach to cancer detection. The goal of this pilot study is to evaluate the diagnostic potential of FES technique for the detection and characterization of normal and cancerous oral lesions in vivo. Fluorescence excitation (FE) spectra from oral mucosa were recorded in the spectral range of 340 to 600 nm at 635 nm emission using a fiberoptic probe spectrofluorometer to obtain spectra from the buccal mucosa of 30 sites of 15 healthy volunteers and 15 sites of 10 cancerous patients. Significant FE spectral differences were observed between normal and well differentiated squamous cell carcinoma (WDSCC) oral lesions. The FE spectra of healthy volunteers consists of a broad emission band around 440 to 470 nm, whereas in WDSCC lesions, a new primary peak was seen at 410 nm with secondary peaks observed at 505, 540, and 580 nm due to the accumulation of porphyrins in oral lesions. The FE spectral bands of the WDSCC lesions resemble the typical absorption spectra of a porphyrin. Three potential ratios (I410/I505, I410/I540, and I410/I580) were calculated from the FE spectra and used as input variables for a stepwise linear discriminant analysis (SLDA) for normal and WDSCC groups. Leave-one-out (LOO) method of cross-validation was performed to check the reliability on spectral data for tissue characterization. The diagnostic sensitivity and specificity were determined for normal and WDSCC lesions from the scatter plot of the discriminant function scores. It was observed that diagnostic algorithm based on discriminant function scores obtained by SLDA-LOO method was able to distinguish WDSCC from normal lesions with a sensitivity of 100% and specificity of 100%. Results of the pilot study demonstrate that the FE spectral changes due to porphyrin have a good diagnostic potential; therefore, porphyrin can be used as a native tumor marker.

  6. Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin.

    PubMed Central

    Masters, B R; So, P T; Gratton, E

    1997-01-01

    Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 PMID:9168018

  7. Fluorescence excitation-emission matrix spectroscopy of vitiligo skin in vivo (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Zhao, Jianhua; Richer, Vincent; Al Jasser, Mohammed; Zandi, Soodabeh; Kollias, Nikiforos; Kalia, Sunil; Zeng, Haishan; Lui, Harvey

    2016-02-01

    Fluorescence signals depend on the intensity of the exciting light, the absorption properties of the constituent molecules, and the efficiency with which the absorbed photons are converted to fluorescence emission. The optical features and appearance of vitiligo have been explained primarily on the basis of reduced epidermal pigmentation, which results in abnormal white patches on the skin. The objective of this study is to explore the fluorescence properties of vitiligo and its adjacent normal skin using fluorescence excitation-emission matrix (EEM) spectroscopy. Thirty five (35) volunteers with vitiligo were acquired using a double-grating spectrofluorometer with excitation and emission wavelengths of 260-450 nm and 300-700 nm respectively. As expected, the most pronounced difference between the spectra obtained from vitiligo lesions compared to normally pigmented skin was that the overall fluorescence was much higher in vitiligo; these differences increased at shorter wavelengths, thus matching the characteristic spectral absorption of epidermal melanin. When comparing the fluorescence spectra from vitiligo to normal skin we detected three distinct spectral bands centered at 280nm, 310nm, and 335nm. The 280nm band may possibly be related to inflammation, whereas the 335 nm band may arise from collagen or keratin cross links. The source of the 310 nm band is uncertain; it is interesting to note its proximity to the 311 nm UV lamps used for vitiligo phototherapy. These differences are accounted for not only by changes in epidermal pigment content, but also by other optically active cutaneous biomolecules.

  8. Two-photon excited fluorescence lifetime imaging and spectroscopy of melanins in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Krasieva, Tatiana B.; Stringari, Chiara; Liu, Feng; Sun, Chung-Ho; Kong, Yu; Balu, Mihaela; Meyskens, Frank L.; Gratton, Enrico; Tromberg, Bruce J.

    2013-03-01

    Changes in the amounts of cellular eumelanin and pheomelanin have been associated with carcinogenesis. The goal of this work is to develop methods based on two-photon-excited-fluorescence (TPEF) for measuring relative concentrations of these compounds. We acquire TPEF emission spectra (λex=1000 nm) of melanin in vitro from melanoma cells, hair specimens, and in vivo from healthy volunteers. We find that the pheomelanin emission peaks at approximately 615 to 625 nm and eumelanin exhibits a broad maximum at 640 to 680 nm. Based on these data we define an optical melanin index (OMI) as the ratio of fluorescence intensities at 645 and 615 nm. The measured OMI for the MNT-1 melanoma cell line is 1.6±0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI=0.5±0.05 and 0.17±0.03, respectively). The measured values are in good agreement with chemistry-based melanin extraction methods. In order to better separate melanin fluorescence from other intrinsic fluorophores, we perform fluorescence lifetime imaging microscopy of in vitro specimens. The relative concentrations of keratin, eumelanin, and pheomelanin components are resolved using a phasor approach for analyzing lifetime data. Our results suggest that a noninvasive TPEF index based on spectra and lifetime could potentially be used for rapid melanin ratio characterization both in vitro and in vivo.

  9. Two-photon excited fluorescence lifetime imaging and spectroscopy of melanins in vitro and in vivo.

    PubMed

    Krasieva, Tatiana B; Stringari, Chiara; Liu, Feng; Sun, Chung-Ho; Kong, Yu; Balu, Mihaela; Meyskens, Frank L; Gratton, Enrico; Tromberg, Bruce J

    2013-03-01

    Changes in the amounts of cellular eumelanin and pheomelanin have been associated with carcinogenesis. The goal of this work is to develop methods based on two-photon-excited-fluorescence (TPEF) for measuring relative concentrations of these compounds. We acquire TPEF emission spectra (λ(ex)=1000  nm) of melanin in vitro from melanoma cells, hair specimens, and in vivo from healthy volunteers. We find that the pheomelanin emission peaks at approximately 615 to 625 nm and eumelanin exhibits a broad maximum at 640 to 680 nm. Based on these data we define an optical melanin index (OMI) as the ratio of fluorescence intensities at 645 and 615 nm. The measured OMI for the MNT-1 melanoma cell line is 1.6 ± 0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI=0.5 ± 0.05 and 0.17 ± 0.03, respectively). The measured values are in good agreement with chemistry-based melanin extraction methods. In order to better separate melanin fluorescence from other intrinsic fluorophores, we perform fluorescence lifetime imaging microscopy of in vitro specimens. The relative concentrations of keratin, eumelanin, and pheomelanin components are resolved using a phasor approach for analyzing lifetime data. Our results suggest that a noninvasive TPEF index based on spectra and lifetime could potentially be used for rapid melanin ratio characterization both in vitro and in vivo.

  10. In vivo detection of epileptic brain tissue using static fluorescence and diffuse reflectance spectroscopy.

    PubMed

    Yadav, Nitin; Bhatia, Sanjiv; Ragheb, John; Mehta, Rupal; Jayakar, Prasanna; Yong, William; Lin, Wei-Chiang

    2013-02-01

    Diffuse reflectance and fluorescence spectroscopy are used to detect histopathological abnormalities of an epileptic brain in a human subject study. Static diffuse reflectance and fluorescence spectra are acquired from normal and epileptic brain areas, defined by electrocorticography (ECoG), from pediatric patients undergoing epilepsy surgery. Biopsy specimens are taken from the investigated sites within an abnormal brain. Spectral analysis reveals significant differences in diffuse reflectance spectra and the ratio of fluorescence and diffuse reflectance spectra from normal and epileptic brain areas defined by ECoG and histology. Using these spectral differences, tissue classification models with accuracy above 80% are developed based on linear discriminant analysis. The differences between the diffuse reflectance spectra from the normal and epileptic brain areas observed in this study are attributed to alterations in the static hemodynamic characteristics of an epileptic brain, suggesting a unique association between the histopathological and the hemodynamic abnormalities in an epileptic brain.

  11. In vivo detection of epileptic brain tissue using static fluorescence and diffuse reflectance spectroscopy

    NASA Astrophysics Data System (ADS)

    Yadav, Nitin; Bhatia, Sanjiv; Ragheb, John; Mehta, Rupal; Jayakar, Prasanna; Yong, William; Lin, Wei-Chiang

    2013-02-01

    Diffuse reflectance and fluorescence spectroscopy are used to detect histopathological abnormalities of an epileptic brain in a human subject study. Static diffuse reflectance and fluorescence spectra are acquired from normal and epileptic brain areas, defined by electrocorticography (ECoG), from pediatric patients undergoing epilepsy surgery. Biopsy specimens are taken from the investigated sites within an abnormal brain. Spectral analysis reveals significant differences in diffuse reflectance spectra and the ratio of fluorescence and diffuse reflectance spectra from normal and epileptic brain areas defined by ECoG and histology. Using these spectral differences, tissue classification models with accuracy above 80% are developed based on linear discriminant analysis. The differences between the diffuse reflectance spectra from the normal and epileptic brain areas observed in this study are attributed to alterations in the static hemodynamic characteristics of an epileptic brain, suggesting a unique association between the histopathological and the hemodynamic abnormalities in an epileptic brain.

  12. Two-photon excited fluorescence spectroscopy and imaging of melanin in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Krasieva, Tatiana B.; Liu, Feng; Sun, Chung-Ho; Kong, Yu; Balu, Mihaela; Meyskens, Frank L.; Tromberg, Bruce J.

    2012-03-01

    The ability to detect early melanoma non-invasively would improve clinical outcome and reduce mortality. Recent advances in two-photon excited fluorescence (TPEF) in vivo microscopy offer a powerful tool in early malignant melanoma diagnostics. The goal of this work was to develop a TPEF optical index for measuring relative concentrations of eumelanin and pheomelanin since ex vivo studies show that changes in this ratio have been associated with malignant transformation. We acquired TPEF emission spectra (λex=1000 nm) of melanin from several specimens, including human hair, malignant melanoma cell lines, and normal melanocytes and keratinocytes in different skin layers (epidermis, papillary dermis) in five healthy volunteers in vivo. We found that the pheomelanin emission peaks at around 620 nm and is blue-shifted from the eumelanin with broad maximum at 640-680nm. We defined "optical melanin index" (OMI) as a ratio of fluorescence signal intensities measured at 645 nm and 615nm. The measured OMI for a melanoma cell line MNT-1 was 1.6+/-0.2. The MNT-46 and MNT-62 lines (Mc1R gene knockdown) showed an anticipated change in melanins production ratio and had OMI of 0.55+/-0.05 and 0.17+/-0.02, respectively, which strongly correlated with HPLC data obtained for these lines. Average OMI measured for basal cells layers (melanocytes and keratinocytes) in normal human skin type I, II-III (not tanned and tanned) in vivo was 0.5, 1.05 and 1.16 respectively. We could not dependably detect the presence of pheomelanin in highly pigmented skin type V-VI. These data suggest that a non-invasive TPEF index could potentially be used for rapid melanin ratio characterization both in vitro and in vivo, including pigmented lesions.

  13. Fluorescence spectroscopy of gastrointestinal tumors: in vitro studies and in vivo clinical applications

    NASA Astrophysics Data System (ADS)

    Angelova, L.; Borisova, E.; Zhelyazkova, Al.; Keremedchiev, M.; Vladimirov, B.; Avramov, L.

    2013-11-01

    The limitations of standard endoscopy for detection and evaluation of cancerous changes in the gastrointestinal tract (GIT) are significant challenges and initiate development of new diagnostic modalities. Therefore many spectral and optical techniques are applied recently into the clinical practice for obtaining qualitatively and quantitatively new data from gastrointestinal neoplasia with different levels of clinical applicability and diagnostic success. Fluorescence imaging has been one of the most promising technologies in this area. The technique is very topical with its practical application in intra-operative, image-guided resection of tumors, because it permits minimal surgery intervention and friendly therapeutic conditions. The investigations presented here are based on in vitro measurements of excitation-emission matrices (EEM) for GIT neoplasia and in vivo measurements in the frames of initial clinical trial for tumor fluorescence spectra detection, applied for introduction of spectroscopic diagnostic system for optical biopsy of GIT tumors in the daily clinical practice of the University Hospital "Queen Jiovanna - ISUL"- Sofia. Autofluorescence and exogenous fluorescence signals are detected from normal mucosa, inflammation, dysphasia and carcinoma and main spectral features are evaluated. The systems and methods developed for diagnosis and monitoring could open new dimensions in diagnostic and real-time tumor resection. This will make the entire procedure more personal, patient friendly and effective and will help for further understanding of the tumor nature.

  14. Delta-ALA-mediated fluorescence spectroscopy of gastrointestinal tumors: comparison of in vivo and in vitro results

    NASA Astrophysics Data System (ADS)

    Vladimirov, B.; Borisova, E.; Avramov, L.

    2007-06-01

    The limitations of standard endoscopy for detection of dysplastic changes of mucosa are significant challenge and initiate development of new photodiagnostic techniques, additional to diagnostic possibilities of standard endoscopic equipment. One of the most widely examined optical modalities is the laser- or light-induced fluorescence spectroscopy (LIFS), because of its rapid and highly sensitive response to early biochemical and morphological changes in biological tissues. In the recent study delta-aminolevulinic acid/protoporphyrin IX is used as fluorescent marker for dysplasia and tumor detection in esophagus and stomach. The δ -ALA is administered per os six hours before measurements at dose 20mg/kg weight. High-power light-emitting diode at 405 nm is used as an excitation source. Special opto-mechanical device is built to use the light guide of standard video-endoscopic system. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer. The fluorescence detected from in vivo tumor sites has very complex spectral origins. It consists of autofluorescence, fluorescence from exogenous fluorophores and re-absorption from the chromophores accumulated in the tissue investigated. Mucosa autofluorescence lies at 450-600 nm region. The fluorescence of PpIX is clearly pronounced at the 630-710 nm region. Deep minima in the tumor fluorescence signals are observed in the region 540-575 nm, related to hemoglobin re-absorption. Such high hemoglobin content is an indication of the tumors vascularization and it is clearly pronounced in all dysplastic and tumor sites investigated. After formalin conservation for in vitro samples hemoglobin absorption is strongly reduced that increases mucous fluorescence signal in green-yellow spectral region. Simultaneously the maxima at 635 nm and 720 nm are reduced.

  15. Fluorescence correlation spectroscopy (FCS): Ultrasensitive measurements of molecular dynamics in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Schwille, Petra

    1998-05-01

    FCS, a technique introduced in 1972 (Magde et al. 1972, Appl. Phys. Lett. 29:705), is currently being employed for ultrasensitive studies of molecular diffusion and aggregation, photophysical dynamics and fast chemical reactions. When performed in confocal geometries with one- or two-photon excitation (Denk et al. 1990, Science 248:73) of photostable dyes, its sensitivity enables us to follow the dynamics of sparse biologically relevant agents such as drugs and pathogens in solution as well as in living cells. Minimally interfering with biological functions of the observed system, FCS allows us to address a variety of important questions in molecular biology. We report that green fluorescent protein (GFP), a popular tool for in-vivo studies, is not only well suited for intracellular FCS, but moreover turned out to be a perfect candidate for the study of intramolecular dynamics. It exhibits a rapid flickering in the 100 μ s time range that we have identified to be due to reversible protonation of the chromophore. Supported by NIH-P41.RR04224, NSF-D1R 8800278, Alexander-von-Humboldt fellowship to PS.

  16. Ex vivo optical coherence tomography and laser induced fluorescence spectroscopy imaging of murine gastrointestinal tract

    NASA Astrophysics Data System (ADS)

    Hariri, Lida; Tumlinson, Alexandre R.; Wade, Norman; Besselsen, David; Utzinger, Urs; Gerner, Eugene; Barton, Jennifer

    2005-04-01

    Optical Coherence Tomography (OCT) and Laser Induced Fluorescence Spectroscopy (LIF) have separately been found to have clinical potential in identifying human gastrointestinal (GI) pathologies, yet their diagnostic capability in mouse models of human disease is unknown. We combine the two modalities to survey the GI tract of a variety of mouse strains and sample dysplasias and inflammatory bowel disease (IBD) of the small and large intestine. Segments of duodenum and lower colon 2.5 cm in length and the entire esophagus from 10 mice each of two colon cancer models (ApcMin and AOM treated A/J) and two IBD models (Il-2 and Il-10) and 5 mice each of their respective controls were excised. OCT images and LIF spectra were obtained simultaneously from each tissue sample within 1 hour of extraction. Histology was used to classify tissue regions as normal, Peyer"s patch, dysplasia, adenoma, or IBD. Features in corresponding regions of OCT images were analyzed. Spectra from each of these categories were averaged and compared via the student's t-test. Features in OCT images correlated to histology in both normal and diseased tissue samples. In the diseased samples, OCT was able to identify early stages of mild colitis and dysplasia. In the sample of IBD, the LIF spectra displayed unique peaks at 635nm and 670nm, which were attributed to increased porphyrin production in the proliferating bacteria of the disease. These peaks have the potential to act as a diagnostic for IBD. OCT and LIF appear to be useful and complementary modalities for imaging mouse models.

  17. In vivo quantification of photosensitizer concentration using fluorescence differential path-length spectroscopy: influence of photosensitizer formulation and tissue location

    NASA Astrophysics Data System (ADS)

    de Visscher, Sebastiaan A. H. J.; Witjes, Max J. H.; Kaščáková, Slávka; Sterenborg, Henricus J. C. M.; Robinson, Dominic J.; Roodenburg, Jan L. N.; Amelink, Arjen

    2012-06-01

    In vivo measurement of photosensitizer concentrations may optimize clinical photodynamic therapy (PDT). Fluorescence differential path-length spectroscopy (FDPS) is a non-invasive optical technique that has been shown to accurately quantify the concentration of Foscan® in rat liver. As a next step towards clinical translation, the effect of two liposomal formulations of mTHPC, Fospeg® and Foslip®, on FDPS response was investigated. Furthermore, FDPS was evaluated in target organs for head-and-neck PDT. Fifty-four healthy rats were intravenously injected with one of the three formulations of mTHPC at 0.15 mg kg-1. FDPS was performed on liver, tongue, and lip. The mTHPC concentrations estimated using FDPS were correlated with the results of the subsequent harvested and chemically extracted organs. An excellent goodness of fit (R2) between FDPS and extraction was found for all formulations in the liver (R2=0.79). A much lower R2 between FDPS and extraction was found in lip (R2=0.46) and tongue (R2=0.10). The lower performance in lip and in particular tongue was mainly attributed to the more layered anatomical structure, which influences scattering properties and photosensitizer distribution.

  18. Nanosecond fluorescence spectroscopy

    SciTech Connect

    Leskovar, B.

    1985-03-01

    This article is a summary of a short course lecture given in conjunction with the 1984 Nuclear Science Symposium. Measuring systems for nanosecond fluorescence spectroscopy using single-photon counting techniques are presented. These involve systems based on relaxation-type spark gap light pulser and synchronously pumped mode-locked dye lasers. Furthermore, typical characteristics and optimization of operating conditions of the critical components responsible for the system time resolution are discussed. A short comparison of the most important deconvolution methods for numerical analysis of experimental data is given particularly with respect to the signal-to-noise ratio of the fluorescence signal. 22 refs., 8 figs.

  19. Fluorescence lifetime spectroscopy of tissue autofluorescence in normal and diseased colon measured ex vivo using a fiber-optic probe

    PubMed Central

    Coda, Sergio; Thompson, Alex J.; Kennedy, Gordon T.; Roche, Kim L.; Ayaru, Lakshmana; Bansi, Devinder S.; Stamp, Gordon W.; Thillainayagam, Andrew V.; French, Paul M. W.; Dunsby, Chris

    2014-01-01

    We present an ex vivo study of temporally and spectrally resolved autofluorescence in a total of 47 endoscopic excision biopsy/resection specimens from colon, using pulsed excitation laser sources operating at wavelengths of 375 nm and 435 nm. A paired analysis of normal and neoplastic (adenomatous polyp) tissue specimens obtained from the same patient yielded a significant difference in the mean spectrally averaged autofluorescence lifetime −570 ± 740 ps (p = 0.021, n = 12). We also investigated the fluorescence signature of non-neoplastic polyps (n = 6) and inflammatory bowel disease (n = 4) compared to normal tissue in a small number of specimens. PMID:24575345

  20. Smartphone fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Yu, Hojoeng; Tan, Yafang; Cunningham, Brian T.

    2014-03-01

    We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of a specific nucleic acid sequences in a liquid test sample. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route towards portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.

  1. Smartphone fluorescence spectroscopy.

    PubMed

    Yu, Hojeong; Tan, Yafang; Cunningham, Brian T

    2014-09-01

    We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of specific nucleic acid sequences in a liquid test sample and compared performance against a conventional laboratory fluorimeter. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route toward portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.

  2. Fluorescence spectroscopy for neoplasms control

    NASA Astrophysics Data System (ADS)

    Bratchenko, I. A.; Kristoforova, Yu. A.; Myakinin, O. O.; Artemyev, D. N.; Kozlov, S. V.; Moryatov, A. A.; Zakharov, V. P.

    2016-04-01

    Investigation of malignant skin tumors diagnosis was performed involving two setups for native tissues fluorescence control in visible and near infrared regions. Combined fluorescence analysis for skin malignant melanomas and basal cell carcinomas was performed. Autofluorescence spectra of normal skin and oncological pathologies stimulated by 457 nm and 785 nm lasers were registered for 74 skin tissue samples. Spectra of 10 melanomas and 27 basal cell carcinomas were registered ex vivo. Skin tumors analysis was made on the basis of autofluorescence spectra intensity and curvature for analysis of porphyrins, lipo-pigments, flavins and melanin. Separation of melanomas and basal cell carcinomas was performed on the basis of discriminant analysis. Overall accuracy of basal cell carcinomas and malignant melanomas separation in current study reached 86.5% with 70% sensitivity and 92.6% specificity.

  3. Fluorescence Spectroscopy in a Shoebox

    NASA Astrophysics Data System (ADS)

    Farooq Wahab, M.

    2007-08-01

    This article describes construction of a simple, inexpensive fluorometer. It utilizes a flashlight or sunlight source, highlighter marker ink, bowl of water with mirror as dispersing element, and colored cellophane sheets as filters. The human eye is used as a detector. This apparatus is used to demonstrate important concepts related to fluorescence spectroscopy. Using ink from a highlighter marker, one can demonstrate the difference between light scattering and fluorescence emission, the need for an intense light source, phenomenon of the Stokes shift, the choice of filters, the preferred geometry of excitation source and emission detector, and the low detection limits that can be achieved by fluorescence measurements. By reflecting the fluorescence emission from a compact disk, it can be seen that the light emitted by molecules is not monochromatic. Furthermore, a spectrofluorometer is constructed using gratings made from a DVD or a CD. The shoebox fluorometer and spectrofluorometer can serve as useful teaching aids in places where commercial instruments are not available, and it avoids the black box problem of modern instruments.

  4. Combined fiber probe for fluorescence lifetime and Raman spectroscopy

    PubMed Central

    Dochow, Sebastian; Ma, Dinglong; Latka, Ines; Bocklitz, Thomas; Hartl, Brad; Bec, Julien; Fatakdawala, Hussain; Marple, Eric; Urmey, Kirk; Wachsmann-Hogiu, Sebastian; Schmitt, Michael; Marcu, Laura; Popp, Jürgen

    2016-01-01

    In this contribution we present a dual modality fiber optic probe combining fluorescence lifetime imaging (FLIm) and Raman spectroscopy for in vivo endoscopic applications. The presented multi-spectroscopy probe enables efficient excitation and collection of fluorescence lifetime signals for FLIm in the UV/visible wavelength region, as well as of Raman spectra in the near-IR for simultaneous Raman/FLIm imaging. The probe was characterized in terms of its lateral resolution and distance dependency of the Raman and FLIm signals. In addition, the feasibility of the probe for in vivo FLIm and Raman spectral characterization of tissue was demonstrated. PMID:26093843

  5. Ultrafast Nonlinear Spectroscopy of Red Fluorescent Proteins

    NASA Astrophysics Data System (ADS)

    Konold, Patrick Eugene

    Red-emitting homologues (RFPs) of the native Green Fluorescent Protein (GFP) with emission wavelengths beyond 650 nm are desirable probes for in vivo imaging experiments. They offer the potential for deeper tissue penetration and lower background scatter given a cleaner spectral window. However, bioimaging applications are hindered by poor photophysics ( e.g. low fluorescence quantum yield, high photobleaching), which limits experimental resolution and represents a significant obstacle towards utilization for low copy-number, long-duration imaging applications. In this thesis, a variety of femtosecond nonlinear electronic spectroscopies were employed jointly with site-directed mutagenesis to investigate the photophysical properties of RFPs. In one study, the molecular mechanism of red emission was pursued in two notable RFPs, mPlum and TagRFP675. Solvation dynamics observed with time-resolved transient grating spectroscopy were interpreted with the aid of molecular dynamics simulations to indicate that their red-emission is correlated with the ability of specific chromophore-sidechain hydrogen-bonding interactions to interconvert between direct and water-mediated states. In a second set of studies, two-dimensional double quantum coherence spectroscopy was used to probe the electronic transitions of mPlum. It was discovered that it displayed a response distinctly different from an organic dye in bulk solvent. Modeling indicate of these spectra indicate the spectral features may be attributed to the existence of multiple high-lying (n>1) excited states. The results provide new insight into the electronic structure of these widely used fluorescent probes.

  6. Intraluminal fluorescence spectroscopy catheter with ultrasound guidance

    NASA Astrophysics Data System (ADS)

    Stephens, Douglas N.; Park, Jesung; Sun, Yang; Papaioannou, Thanassis; Marcu, Laura

    2009-05-01

    We demonstrate the feasibility of a time-resolved fluorescence spectroscopy (TRFS) technique for intraluminal investigation of arterial vessel composition under intravascular ultrasound (IVUS) guidance. A prototype 1.8-mm (5.4 Fr) catheter combining a side-viewing optical fiber (SVOF) and an IVUS catheter was constructed and tested with in vitro vessel phantoms. The prototype catheter can locate a fluorophore in the phantom vessel wall, steer the SVOF in place, perform blood flushing under flow conditions, and acquire high-quality TRFS data using 337-nm wavelength excitation. The catheter steering capability used for the coregistration of the IVUS image plane and the SVOF beam produce a guiding precision to an arterial phantom wall site location of 0.53+/-0.16 mm. This new intravascular multimodal catheter enables the potential for in vivo arterial plaque composition identification using TRFS.

  7. Glucose Recognition in Vitro Using Fluorescent Spectroscopy

    SciTech Connect

    Noronha, G; Heiss, A M; Reilly, J R; Vachon, Jr, D J; Cary, D R; Zaitseva, N P; Reibold, R A; Lane, S M; Peyser, T A; Satcher, J H

    2001-04-25

    Diabetes is a disease that affects over 16 million people in the USA at a cost of 100 billion dollars annually. The ability to regulate insulin delivery in people with Type 1 diabetes is imperative as is the need to manage glucose levels in all people with this disease. Our current method for monitoring glucose is a (FDA approved) minimally invasive enzymatic sensor that can measure glucose levels in vivo for three days. We are focused on developing a noninvasive implantable glucose sensor that will be interrogated by an external device. The material must be robust, easy to process, biocompatible and resistant to biofouling. In this Presentation we will discuss the development of a new polymeric matrix that can recognize physiological levels of glucose in vitro using fluorescent spectroscopy.

  8. NIR fluorescent ytterbium compound for in vivo fluorescence molecular imaging.

    PubMed

    Aita, Kazuki; Temma, Takashi; Kuge, Yuji; Seki, Koh-ichi; Saji, Hideo

    2010-01-01

    We have developed a new NIR fluorescent probe based on an ytterbium(III) (E)-1-(pyridin-2-yl-diazenyl)naphthalen-2-ol (PAN) complex. This probe emits near-infrared luminescence derived from the Yb ion through excitation of the PAN moiety with visible light (lambda(ex)= 530 nm, lambda(em)= 975 nm). The results support the possible utility of the probe for in vivo fluorescence molecular imaging.

  9. New tools for in vivo fluorescence tagging.

    PubMed

    Chapman, Sean; Oparka, Karl J; Roberts, Alison G

    2005-12-01

    Engineering of fluorescent proteins continues to produce new tools for in vivo studies. The current selection contains brighter, monomeric, spectral variants that will facilitate multiplex imaging and FRET, and a collection of optical highlighter proteins that might replace photoactivatable-GFP. These new highlighter proteins, which include proteins that have photoswitchable fluorescence characteristics and a protein whose fluorescence can be repeatedly turned on and off, should simplify refined analyses of protein dynamics and kinetics. Fluorescent protein-based systems have also been developed to allow facile detection of protein-protein interactions in planta. In addition, new tags in the form of peptides that bind fluorescent ligands and quantum dots offer the prospect of overcoming some of the limitations of fluorescent proteins such as excessive size and insufficient brightness.

  10. Fluorescence spectroscopy applied to orange trees

    NASA Astrophysics Data System (ADS)

    Marcassa, L. G.; Gasparoto, M. C. G.; Belasque, J., Jr.; Lins, E. C.; Dias Nunes, F.; Bagnato, V. S.

    2006-05-01

    In this work, we have applied laser-induced fluorescence spectroscopy to investigate biological processes in orange trees (Citrus aurantium L.). We have chosen to investigate water stress and Citrus Canker, which is a disease caused by the Xanthomonas axonopodis pv. citri bacteria. The fluorescence spectroscopy was investigated by using as an excitation source a 442-nm 15-mW HeCd gas multimode discharge laser and a 532-nm 10-mW Nd3+:YAG laser. The stress manifestation was detected by the variation of fluorescence ratios of the leaves at different wavelengths. The fluorescence ratios present a significant variation, showing the possibility to observe water stress by fluorescence spectrum. The Citrus Canker’s contaminated leaves were discriminated from the healthy leaves using a more complex analysis of the fluorescence spectra. However, we were unable to discriminate it from another disease, and new fluorescence experiments are planned for the future.

  11. Accurate fluorescence quantum yield determination by fluorescence correlation spectroscopy.

    PubMed

    Kempe, Daryan; Schöne, Antonie; Fitter, Jörg; Gabba, Matteo

    2015-04-01

    Here, we present a comparative method for the accurate determination of fluorescence quantum yields (QYs) by fluorescence correlation spectroscopy. By exploiting the high sensitivity of single-molecule spectroscopy, we obtain the QYs of samples in the microliter range and at (sub)nanomolar concentrations. Additionally, in combination with fluorescence lifetime measurements, our method allows the quantification of both static and collisional quenching constants. Thus, besides being simple and fast, our method opens up the possibility to photophysically characterize labeled biomolecules under application-relevant conditions and with low sample consumption, which is often important in single-molecule studies.

  12. Laser-induced fluorescence spectroscopy in tissue local necrosis detection

    NASA Astrophysics Data System (ADS)

    Cip, Ondrej; Buchta, Zdenek; Lesundak, Adam; Randula, Antonin; Mikel, Bretislav; Lazar, Josef; Veverkova, Lenka

    2014-03-01

    The recent effort leads to reliable imaging techniques which can help to a surgeon during operations. The fluorescence spectroscopy was selected as very useful online in vivo imaging method to organics and biological materials analysis. The presented work scopes to a laser induced fluorescence spectroscopy technique to detect tissue local necrosis in small intestine surgery. In first experiments, we tested tissue auto-fluorescence technique but a signal-to-noise ratio didn't express significant results. Then we applied a contrast dye - IndoCyanine Green (ICG) which absorbs and emits wavelengths in the near IR. We arranged the pilot experimental setup based on highly coherent extended cavity diode laser (ECDL) used for stimulating of some critical areas of the small intestine tissue with injected ICG dye. We demonstrated the distribution of the ICG exciter with the first file of shots of small intestine tissue of a rabbit that was captured by high sensitivity fluorescent cam.

  13. In vivo fluorescence spectroscopy and imaging of ALA-induced endogenous porphyrins in skin after Er:YAG ablation of human stratum corneum

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Schneckenburger, Herbert; Boehncke, Wolf-Henning; Hibst, Raimund

    1994-09-01

    Limited regions of human stratum corneum were removed by laser ablation using an Er:YAG laser. Immediately after this procedure, an ointment containing 5-aminolevulinic acid (ALA) was applied topically to the laser-treated and surrounding skin. The time-dependent ALA- induced biosynthesis of protoporphyrin IX was measured by fluorescence detection. Fluorescence in the red spectral region was found to occur in the ablated skin regions only. Time-resolved measurements showed the formation of long-lived fluorophores (16 ns) indicating the presence of ALA-induced monomeric porphyrin. Naturally occurring fluorophores (NAD(P)H, flavins, collagen, elastin) possess shorter fluorescence decay times. Therefore, time-gated measurements in the nanosecond region enable the specific detection of ALA-stimulated porphyrin fluorescence by choosing an appropriate time-window. In addition, detection of backscattered excitation light can be avoided. High-contrast video images of ALA-incubated fluorescent areas were obtained using this novel imaging technique.

  14. Recent advances on in vivo imaging with fluorescent proteins.

    PubMed

    Hoffman, Robert M

    2008-01-01

    In vivo imaging with green fluorescent protein (GFP) and other fluorescent proteins is revolutionizing cancer biology and other fields of in vivo biology (Hoffman, 2005; Hoffman and Yang, 2006a,b,c). Our laboratory pioneered the use of GFP for in vivo imaging in 1997 (Chishima et al., 1997). This chapter highlights recent developments from our laboratory on both macro and micro in vivo imaging by using fluorescent proteins.

  15. Ultraviolet, Visible, and Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Penner, Michael H.

    Spectroscopy in the ultraviolet-visible (UV-Vis) range is one of the most commonly encountered laboratory techniques in food analysis. Diverse examples, such as the quantification of macrocomponents (total carbohydrate by the phenol-sulfuric acid method), quantification of microcomponents, (thiamin by the thiochrome fluorometric procedure), estimates of rancidity (lipid oxidation status by the thiobarbituric acid test), and surveillance testing (enzyme-linked immunoassays), are presented in this text. In each of these cases, the analytical signal for which the assay is based is either the emission or absorption of radiation in the UV-Vis range. This signal may be inherent in the analyte, such as the absorbance of radiation in the visible range by pigments, or a result of a chemical reaction involving the analyte, such as the colorimetric copper-based Lowry method for the analysis of soluble protein.

  16. Differentiating tissue by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Woessner, Stefan; Huen, Julien; Malthan, Dirk

    2004-03-01

    A common problem in several surgical applications is the lack of navigational information. Most often, the only source of information about the location of crucial structures, in relation to the surgical instrument, is the visible and tactile sensory input of the surgeon. In some cases, this leads to time-consuming procedures and a high risk for the patient. Therefore, we developed a spectroscopic sensor system for automatic differentiation between several tissue types. For example in milling processes, a sensor that is able to detect bone in contrast to nerve or vein tissue can be used to control the milling process. We showed exemplarily for the cochlea implant, a typical ENT-surgery, that with the help of our sensor system, the milling of bone can be accelerated without increasing the risk for the patient. It is also possible to use this type of sensor system in the area of medical robotics in soft-tissue applications. With real-time information, a continuous registration can take place, in contrast to a registration that is done using static preoperatively acquired images. We showed that our sensor system can be used to dynamically update the location of the patient in relation to CT or MR-images. In conclusion, we have been able to show that well-known spectroscopy sensors can be used to open new possibilities in medical treatment with and without the use of robotics.

  17. Recent progress in in vivo ESR spectroscopy.

    PubMed

    Takeshita, Keizo; Ozawa, Toshihiko

    2004-09-01

    The generation of free radicals and redox status is related to various diseases and injuries that are related to radiation, aging, ischemia-reperfusion, and other oxidative factors. In vivo electron spin resonance (ESR) spectroscopy is noninvasive and detects durable free radicals in live animals. ESR spectrometers for in vivo measurements operate at a lower frequency (approximately 3.5 GHz, approximately 1 GHz, 700 MHz, and approximately 300 MHz) than usual (9-10 GHz). Several types of resonators have been designed to minimize the dielectric loss of electromagnetic waves caused by water in animal bodies. In vivo ESR spectroscopy and its imaging have been used to analyze radical generation, redox status, partial pressure of oxygen and other conditions in various disease and injury models related to oxidative stress with probes, such as nitroxyl radicals. Through these applications, the clarification of the mechanisms related to oxidative diseases (injuries) and the accumulation of basic data for radiological cancer therapy are now ongoing. In vivo ESR measurement is performed in about 10 laboratories worldwide, including ours. To introduce in vivo ESR spectroscopy to life scientists, this article reviews the recent progress of in vivo ESR spectroscopy in instrumentation and its application to the life sciences.

  18. Multiphoton excitation microscopy of in vivo human skin. Functional and morphological optical biopsy based on three-dimensional imaging, lifetime measurements and fluorescence spectroscopy.

    PubMed

    Masters, B R; So, P T; Gratton, E

    1998-02-01

    Two-photon excitation microscopy has the potential as an effective, noninvasive, diagnostic tool for in vivo examination of human deep tissue structure at the subcellular level. By using infrared photons as the excitation source in two-photon microscopy, a significant improvement in penetration depth can be achieved because of the much lower tissue scattering and absorption coefficients in the infrared wavelengths. Two-photon absorption occurs primarily at the focal point and provides the physical basis for optical sectioning. Multiphoton excitation microscopy at 730 nm was used to image in vivo human skin autofluorescence from the surface to a depth of about 200 microns. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence using 730 nm excitation. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells and provides a functional and morphological optical biopsy.

  19. Fluorescence spectroscopy for wastewater monitoring: A review.

    PubMed

    Carstea, Elfrida M; Bridgeman, John; Baker, Andy; Reynolds, Darren M

    2016-05-15

    Wastewater quality is usually assessed using physical, chemical and microbiological tests, which are not suitable for online monitoring, provide unreliable results, or use hazardous chemicals. Hence, there is an urgent need to find a rapid and effective method for the evaluation of water quality in natural and engineered systems and for providing an early warning of pollution events. Fluorescence spectroscopy has been shown to be a valuable technique to characterize and monitor wastewater in surface waters for tracking sources of pollution, and in treatment works for process control and optimization. This paper reviews the current progress in applying fluorescence to assess wastewater quality. Studies have shown that, in general, wastewater presents higher fluorescence intensity compared to natural waters for the components associated with peak T (living and dead cellular material and their exudates) and peak C (microbially reprocessed organic matter). Furthermore, peak T fluorescence is significantly reduced after the biological treatment process and peak C is almost completely removed after the chlorination and reverse osmosis stages. Thus, simple fluorometers with appropriate wavelength selectivity, particularly for peaks T and C could be used for online monitoring in wastewater treatment works. This review also shows that care should be taken in any attempt to identify wastewater pollution sources due to potential overlapping fluorophores. Correlations between fluorescence intensity and water quality parameters such as biochemical oxygen demand (BOD) and total organic carbon (TOC) have been developed and dilution of samples, typically up to ×10, has been shown to be useful to limit inner filter effect. It has been concluded that the following research gaps need to be filled: lack of studies on the on-line application of fluorescence spectroscopy in wastewater treatment works and lack of data processing tools suitable for rapid correction and extraction of

  20. Plasmon-controlled fluorescence: a new paradigm in fluorescence spectroscopy.

    PubMed

    Lakowicz, Joseph R; Ray, Krishanu; Chowdhury, Mustafa; Szmacinski, Henryk; Fu, Yi; Zhang, Jian; Nowaczyk, Kazimierz

    2008-10-01

    Fluorescence spectroscopy is widely used in biological research. Until recently, essentially all fluorescence experiments were performed using optical energy which has radiated to the far-field. By far-field we mean at least several wavelengths from the fluorophore, but propagating far-field radiation is usually detected at larger macroscopic distances from the sample. In recent years there has been a growing interest in the interactions of fluorophores with metallic surfaces or particles. Near-field interactions are those occurring within a wavelength distance of an excited fluorophore. The spectral properties of fluorophores can be dramatically altered by near-field interactions with the electron clouds present in metals. These interactions modify the emission in ways not seen in classical fluorescence experiments. In this review we provide an intuitive description of the complex physics of plasmons and near-field interactions. Additionally, we summarize the recent work on metal-fluorophore interactions and suggest how these effects will result in new classes of experimental procedures, novel probes, bioassays and devices.

  1. Plasmon-controlled fluorescence: a new paradigm in fluorescence spectroscopy

    PubMed Central

    Lakowicz, Joseph R.; Ray, Krishanu; Chowdhury, Mustafa; Szmacinski, Henryk; Fu, Yi; Zhang, Jian; Nowaczyk, Kazimierz

    2009-01-01

    Fluorescence spectroscopy is widely used in biological research. Until recently, essentially all fluorescence experiments were performed using optical energy which has radiated to the far-field. By far-field we mean at least several wavelengths from the fluorophore, but propagating far-field radiation is usually detected at larger macroscopic distances from the sample. In recent years there has been a growing interest in the interactions of fluorophores with metallic surfaces or particles. Near-field interactions are those occurring within a wavelength distance of an excited fluorophore. The spectral properties of fluorophores can be dramatically altered by near-field interactions with the electron clouds present in metals. These interactions modify the emission in ways not seen in classical fluorescence experiments. In this review we provide an intuitive description of the complex physics of plasmons and near-field interactions. Additionally, we summarize the recent work on metal–fluorophore interactions and suggest how these effects will result in new classes of experimental procedures, novel probes, bioassays and devices. PMID:18810279

  2. Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Krmpot, Aleksandar J.; Nikolić, Stanko N.; Vitali, Marco; Papadopoulos, Dimitrios K.; Oasa, Sho; Thyberg, Per; Tisa, Simone; Kinjo, Masataka; Nilsson, Lennart; Gehring, Walter J.; Terenius, Lars; Rigler, Rudolf; Vukojevic, Vladana

    2015-07-01

    Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.

  3. Ultrasensitive hybridization analysis using fluorescence correlation spectroscopy.

    PubMed

    Kinjo, M; Rigler, R

    1995-05-25

    The hybridization of fluorescently tagged 18mer deoxyribonucleotides with complementary DNA templates was analysed by fluorescence correlation spectroscopy (FCS) in a droplet under an epi-illuminated fluorescence microscope at the level of single molecules. The interaction can be monitored by the change in the translational diffusion time of the smaller (18mer) primer when binding to the bigger (7.5 kb) DNA containing the complementary sequence. The hybridization process in the presence of template M13mp18 ssDNA was monitored in a small volume (2 x 10(-16)I) at various temperatures. The Arrhenius plot of the association rate constant shows that the activation energy was 38.8 kcal/mol, but the hybridization process may involve several components. The titration experiment suggested that approximately 2 primers can be associated with one template DNA at 40 degrees C. Results of a simple homology search for the sequences complementary to the primer indicate the existence of additional sites of lower specificity.

  4. Saturation effects in fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Davis, Lloyd M.; Shen, Guoqing; Ball, David A.

    2005-03-01

    Fluorescence correlation spectroscopy (FCS) could provide a more useful tool for intracellular studies and biological sample characterization if measurement times could be reduced. While an increase in laser power can enable an autocorrelation function (ACF) with adequate signal-to-noise to be acquired within a shorter measurement time, excitation saturation then leads to distortion of the ACF and systematic errors in the measurement results. An empirical method for achieving reduced systematic errors by employing a fitting function with an additional adjustable parameter has been previously introduced for two-photon FCS. Here we provide a unified physical explanation of excitation saturation effects for the three cases of continuous-wave, pulsed one-photon excitation, and two-photon excitation FCS. When the time between laser pulses is longer than the fluorescence lifetime, the signal rate at which excitation saturation occurs is lower for pulsed excitation than for cw excitation, and due to the disparate timescales of the photophysical processes following excitation, it is lower still for two-photon excitation. We use a single-molecule description of FCS to obtain improved analytical ACF fitting functions for the three cases. The fitting functions more accurately account for saturation effects than those previously employed without the need for an additional empirical parameter. Use of these fitting functions removes systematic errors and enables measurements to be acquired more quickly by use of higher laser powers. Increase of background, triplet photophysics, and the cases of scanning FCS and fluorescence cross-correlation spectroscopy are also discussed. Experimental results acquired with a custom built apparatus are presented.

  5. APD detectors for biological fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Mazères, S.; Borrel, V.; Magenc, C.; Courrech, J. L.; Bazer-Bachi, R.

    2006-11-01

    Fluorescence spectroscopy is a very convenient and widely used method for studying the molecular background of biological processes [L. Salomé, J.L. Cazeil, A. Lopez, J.F. Tocanne, Eur. Biophys. J. 27 (1998) 391-402]. Chromophores are included in the structure under study and a flash of laser light induces fluorescence (Fluorescence Recovery After Photo-bleaching), the decay of which yields information on the polarity, the speed of rotation, and the speed of diffusion as well as on the temporal and spatial evolution of interactions between molecular species. The method can even be used to study living cells [J.F. Tocanne, L. Cézanne, A. Lopez, Prog. Lipid Res. 33 (1994) 203-237, L. Cezanne, A. Lopez, F. Loste, G. Parnaud, O. Saurel, P. Demange, J.F. Tocanne, Biochemistry 38 (1999) 2779-2786]. This is classically performed with a PM-based system. For biological reasons a decrease of the excitation of the cells is highly desirable. Because the fluorescence response then becomes fainter a significant improvement in detector capability would be welcome. We present here results obtained with an Avalanche Photo Diode (APD)-based system. The small sensitive area of detection allows a very significant improvement in signal/noise ratio, improvement in gain, and the opening-up of a new parameter space. With these new detectors we can begin the study of information transmission between cells through morphine receptors. This work involves both electronics engineers and biophysicists, so results and techniques in both fields will be presented here.

  6. Excitation–emission matrices and synchronous fluorescence spectroscopy for the diagnosis of gastrointestinal cancers

    NASA Astrophysics Data System (ADS)

    Genova, Ts; Borisova, E.; Penkov, N.; Vladimirov, B.; Zhelyazkova, A.; Avramov, L.

    2016-06-01

    We report the development of an improved fluorescence technique for cancer diagnostics in the gastrointestinal tract. We investigate the fluorescence of ex vivo colorectal (cancerous and healthy) tissue samples using excitation–emission matrix (EEM) and synchronous fluorescence spectroscopy (SFS) steady-state approaches. The obtained results are processed for revealing characteristic fluorescence spectral features with a valuable diagnostic meaning. The main tissue fluorophores, contributing to the observed fluorescence, are tyrosine, tryptophan, NADH, FAD, collagen and elastin. Based on the results of the Mann–Whitney test as useful parameters for differentiation of gastrointestinal cancer from normal mucosa, we suggest using excitation wavelengths in the range 300 – 360 nm for fluorescence spectroscopy and wavelengths intervals of 60 nm and 90 nm for SFS.

  7. Excitation-emission matrices and synchronous fluorescence spectroscopy for the diagnosis of gastrointestinal cancers

    NASA Astrophysics Data System (ADS)

    Genova, Ts; Borisova, E.; Penkov, N.; Vladimirov, B.; Zhelyazkova, A.; Avramov, L.

    2016-06-01

    We report the development of an improved fluorescence technique for cancer diagnostics in the gastrointestinal tract. We investigate the fluorescence of ex vivo colorectal (cancerous and healthy) tissue samples using excitation-emission matrix (EEM) and synchronous fluorescence spectroscopy (SFS) steady-state approaches. The obtained results are processed for revealing characteristic fluorescence spectral features with a valuable diagnostic meaning. The main tissue fluorophores, contributing to the observed fluorescence, are tyrosine, tryptophan, NADH, FAD, collagen and elastin. Based on the results of the Mann-Whitney test as useful parameters for differentiation of gastrointestinal cancer from normal mucosa, we suggest using excitation wavelengths in the range 300 - 360 nm for fluorescence spectroscopy and wavelengths intervals of 60 nm and 90 nm for SFS.

  8. Fluorescence spectroscopy to assess apoptosis in myocardium

    NASA Astrophysics Data System (ADS)

    Ranji, Mahsa; Matsubara, Muneaki; Grosso, Michael A.; Jaggard, Dwight L.; Chance, Britton; Gorman, Robert C.; Gorman, Joseph H., III

    2007-02-01

    Apoptosis induced mitochondrial destruction and dysfunction has been shown to play an important role in the pathogenesis of both acute cardiac ischemia-reperfusion injury and chronic myocardial infarction-induced ventricular remodeling. Unfortunately this understanding has not translated into effective therapeutic strategies for either condition-mostly due to an inability to assess mitochondrial dysfunction/apoptosis effectively in humans. All current measures of apoptosis are pseudo-quantitative and require invasive tissue biopsy. Our group has developed an optical, non-tissue destructive catheter based device that allows the quantitative regional assessment of this pathological process in vivo. This instrument has been designed to acquire fluorescence signals of intrinsic mitochondrial fluorophores, Nicotinamide Adenine Dinucleotide (NAD) and Flavoprotein (FP). The normalized ratio of these fluorophores (FP/FP+NADH) called the redox ratio, is an indicator of the in vivo mitochondrial dysfunction. 1-3 We have demonstrated in a rabbit reperfusion model of apoptotic myocyte injury that this redox ratio is drastically increased which is consistent with profound apoptosis-induced "unhinging" of the mitochondrial respiratory function.

  9. Optical biopsy fiber-based fluorescence spectroscopy instrumentation

    NASA Astrophysics Data System (ADS)

    Katz, Alvin; Ganesan, Singaravelu; Yang, Yuanlong; Tang, Gui C.; Budansky, Yury; Celmer, Edward J.; Savage, Howard E.; Schantz, Stimson P.; Alfano, Robert R.

    1996-04-01

    Native fluorescence spectroscopy of biomolecules has emerged as a new modality to the medical community in characterizing the various physiological conditions of tissues. In the past several years, many groups have been working to introduce the spectroscopic methods to diagnose cancer. Researchers have successfully used native fluorescence to distinguish cancerous from normal tissue samples in rat and human tissue. We have developed three generations of instruments, called the CD-scan, CD-ratiometer and CD-map, to allow the medical community to use optics for diagnosing tissue. Using ultraviolet excitation and emission spectral measurements on both normal and cancerous tissue of the breast, gynecology, colon, and aerodigestive tract can be separated. For example, from emission intensities at 340 nm to 440 nm (300 nm excitation), a statistically consistent difference between malignant tissue and normal or benign tissue is observed. In order to utilize optical biopsy techniques in a clinical setting, the CD-scan instrument was developed, which allows for rapid and reliable in-vitro and in-vivo florescence measurements of the aerodigestive tract with high accuracy. The instrumentation employs high sensitivity detection techniques which allows for lamp excitation, small diameter optical fiber probes; the higher spatial resolution afforded by the small diameter probes can increase the ability to detect smaller tumors. The fiber optic probes allow for usage in the aerodigestive tract, cervix and colon. Needle based fiber probes have been developed for in-vivo detection of breast cancer.

  10. Detecting Nanodomains in Living Cell Membrane by Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    He, Hai-Tao; Marguet, Didier

    2011-05-01

    Cell membranes actively participate in numerous cellular functions. Inasmuch as bioactivities of cell membranes are known to depend crucially on their lateral organization, much effort has been focused on deciphering this organization on different length scales. Within this context, the concept of lipid rafts has been intensively discussed over recent years. In line with its ability to measure diffusion parameters with great precision, fluorescence correlation spectroscopy (FCS) measurements have been made in association with innovative experimental strategies to monitor modes of molecular lateral diffusion within the plasma membrane of living cells. These investigations have allowed significant progress in the characterization of the cell membrane lateral organization at the suboptical level and have provided compelling evidence for the in vivo existence of raft nanodomains. We review these FCS-based studies and the characteristic structural features of raft nanodomains. We also discuss the findings in regards to the current view of lipid rafts as a general membrane-organizing principle.

  11. Validation of a time-resolved fluorescence spectroscopy apparatus in a rabbit atherosclerosis model

    NASA Astrophysics Data System (ADS)

    Fang, Qiyin; Jo, Javier A.; Papaioannou, Thanassis; Dorafshar, Amir; Reil, Todd; Qiao, Jian-Hua; Fishbein, Michael C.; Freischlag, Julie A.; Marcu, Laura

    2004-07-01

    Time-resolved laser-induced fluorescence spectroscopy (tr-LIFS) has been studied as a potential tool for in vivo diagnosis of atherosclerotic lesions. This study is to evaluate the potential of a compact fiber-optics based tr-LIFS instrument developed in our laboratory for in vivo analysis of atherosclerotic plaque composition. Time-resolved fluorescence spectroscopy studies were performed in vivo on fifteen New Zealand White rabbits (atherosclerotic: N=8, control: N=7). Time-resolved fluorescence spectra were acquired (range: 360-600 nm, increment: 5 nm, total acquisition time: 65 s) from normal aorta wall and lesions in the abdominal aorta. Data were analyzed in terms of fluorescence emission spectra and wavelength specific lifetimes. Following trichrome staining, tissue specimens were analyzed histopathologically in terms of intima/media thickness and biochemical composition (collagen, elastin, foam cells, and etc). Based on intimal thickness, the lesions were divided into thin and thick lesions. Each group was further separated into two categories: collagen rich lesions and foam cell rich lesions based on their biochemical composition. The obtained spectral and time domain fluorescence signatures were subsequently correlated to the histopathological findings. The results have shown that time-domain fluorescence spectral features can be used in vivo to separate atherosclerotic lesions from normal aorta wall as well discrimination within certain types of lesions.

  12. In vivo imaging with near-infrared fluorescence lifetime contrast

    NASA Astrophysics Data System (ADS)

    Akers, Walter J.; Berezin, Mikhail Y.; Lee, Hyeran; Achilefu, Samuel

    2009-02-01

    Fluorescence imaging is a mainstay of biomedical research, allowing detection of molecular events in both fixed and living cells, tissues and whole animals. Such high resolution fluorescence imaging is hampered by unwanted signal from intrinsic background fluorescence and scattered light. The signal to background ratio can be improved by using extrinsic contrast agents and greatly enhanced by multispectral imaging methods. Unfortunately, these methods are insufficient for deep tissue imaging where high contrast and speedy acquisition are necessary. Fluorescence lifetime (FLT) is an inherent characteristic of each fluorescent species that can be independent of intensity and spectral properties. Accordingly, FLT-based detection provides an additional contrast mechanism to optical measurements. This contrast is particularly important in the near-infrared (NIR) due to relative transparency of tissue as well as the broad absorption and emission spectra of dyes that are active in this region. Here we report comparative analysis of signal distribution of several NIR fluorescent polymethine dyes in living mice and their correlations with lifetimes obtained in vitro using solution models. The FLT data obtained from dyes dissolved in serum albumin solution correlated well with FLTs measured in vivo. Thus the albumin solution model could be used as a good predictive model for in vivo FLT behavior of newly developed fluorescent reporters. Subsequent experiments in vivo, including monitoring slow release kinetics and detecting proteinuria, demonstrate the complementary nature of FLT for fluorescence intensity imaging.

  13. Comparison of in vivo and ex vivo imaging of the microvasculature with 2-photon fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Steinman, Joe; Koletar, Margaret; Stefanovic, Bojana; Sled, John G.

    2016-03-01

    This study evaluates 2-Photon fluorescence microscopy of in vivo and ex vivo cleared samples for visualizing cortical vasculature. Four mice brains were imaged with in vivo 2PFM. Mice were then perfused with a FITC gel and cleared in fructose. The same regions imaged in vivo were imaged ex vivo. Vessels were segmented automatically in both images using an in-house developed algorithm that accounts for the anisotropic and spatially varying PSF ex vivo. Through non-linear warping, the ex vivo image and tracing were aligned to the in vivo image. The corresponding vessels were identified through a local search algorithm. This enabled comparison of identical vessels in vivo/ex vivo. A similar process was conducted on the in vivo tracing to determine the percentage of vessels perfused. Of all the vessels identified over the four brains in vivo, 98% were present ex vivo. There was a trend towards reduced vessel diameter ex vivo by 12.7%, and the shrinkage varied between specimens (0% to 26%). Large diameter surface vessels, through a process termed 'shadowing', attenuated in vivo signal from deeper cortical vessels by 40% at 300 μm below the cortical surface, which does not occur ex vivo. In summary, though there is a mean diameter shrinkage ex vivo, ex vivo imaging has a reduced shadowing artifact. Additionally, since imaging depths are only limited by the working distance of the microscope objective, ex vivo imaging is more suitable for imaging large portions of the brain.

  14. In Vivo Metal Ion Imaging Using Fluorescent Sensors.

    PubMed

    Van de Bittner, Genevieve C; Hirayama, Tasuku

    2016-01-01

    In vivo imaging in living animals provides the ability to monitor alterations of signaling molecules, ions, and other biological components during various life stages and in disease. The data gained from in vivo imaging can be used for biological discovery or to determine elements of disease progression and can inform the development and translation of therapeutics. Herein, we present theories behind small-molecule, fluorescent, metal ion sensors as well as the methods for their successful application to in vivo metal ion imaging, including ex vivo validation. PMID:27283424

  15. Laser Induced Fluorescence Spectroscopy of Soft Tissues of the Oral Cavity

    NASA Astrophysics Data System (ADS)

    Patil, Ajeetkumar; Unnikrishnan, V. K.; Bernard, Rodney; Pai, Keerthilatha M.; Ongole, Ravikiran; Kartha, V. B.; Chidangil, Santhosh

    2011-07-01

    The present study deals with the in vivo measurement of auto-fluorescence from different anatomical sites of oral cavities of healthy volunteers, using a homebuilt Laser Induced Fluorescence (LIF) Spectroscopy setup. Excitation wave length of 325 nm from a He-Cd laser was used as the source. From the 7 anatomical sites (say buccal mucosa, tongue, palate etc) of each oral cavity of 113 subjects, 1266 fluorescence spectra were recorded. The spectra were analysed using Principal Component Analysis (PCA) to see the correlation between different sites.

  16. Combined fiber probe for fluorescence lifetime and Raman spectroscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Dochow, Sebastian; Ma, Dinglong; Latka, Ines; Bocklitz, Thomas; Hartl, Brad; Bec, Julien; Fatakdawala, Hussain; Wachsmann-Hogiu, Sebastian; Marple, Eric; Urmey, Kirk; Schmitt, Michael; Marcu, Laura; Popp, Jürgen

    2016-03-01

    Raman spectroscopy has been proven to have tremendous potential as biomedical analytical tool for spectroscopic disease diagnostics. The use of fiberoptic coupled Raman spectroscopy systems can enable in-vivo characterization of suspicious lesions. However, Raman spectroscopy has the drawback of rather long acquisition times of several hundreds of milliseconds which makes scanning of larger regions quite challenging. By combining Raman spectroscopy with a fast imaging technique this problem can be alleviate in part. Fluorescence lifetime imaging (FLIm) offers a great potential for such a combination. FLIm can allow for fast tissue area pre-segmentation and location of the points for Raman spectra acquisition. Here, we introduce an optical fiber probe combining FLIm and Raman spectroscopy with an outer diameter of 2 mm. Fluorescence is generated via excitation with a fiber laser at 355 nm. The fluorescence emission is spectrally resolved using a custom-made wavelength-selection module (WSM). The Raman excitation power at 785 nm was set to 50 mW for the in-vivo measurements to prevent sample drying. The lateral probe resolution was determined to be <250 μm for both modalities. This value was taken as step size for several raster scans of different tissue types which were conducted to show the overlap of both modalities under realistic conditions. Finally the probe was used for in vivo raster scans of a rat's brain and subsequently to acquire FLIm guided Raman spectra of several tissues in and around the craniotomy.

  17. In vivo Raman spectroscopy of cervix cancers

    NASA Astrophysics Data System (ADS)

    Rubina, S.; Sathe, Priyanka; Dora, Tapas Kumar; Chopra, Supriya; Maheshwari, Amita; Krishna, C. Murali

    2014-03-01

    Cervix-cancer is the third most common female cancer worldwide. It is the leading cancer among Indian females with more than million new diagnosed cases and 50% mortality, annually. The high mortality rates can be attributed to late diagnosis. Efficacy of Raman spectroscopy in classification of normal and pathological conditions in cervix cancers on diverse populations has already been demonstrated. Our earlier ex vivo studies have shown the feasibility of classifying normal and cancer cervix tissues as well as responders/non-responders to Concurrent chemoradiotherapy (CCRT). The present study was carried out to explore feasibility of in vivo Raman spectroscopic methods in classifying normal and cancerous conditions in Indian population. A total of 182 normal and 132 tumor in vivo Raman spectra, from 63 subjects, were recorded using a fiberoptic probe coupled HE-785 spectrometer, under clinical supervision. Spectra were acquired for 5 s and averaged over 3 times at 80 mW laser power. Spectra of normal conditions suggest strong collagenous features and abundance of non-collagenous proteins and DNA in case of tumors. Preprocessed spectra were subjected to Principal Component-Linear Discrimination Analysis (PCLDA) followed by leave-one-out-cross-validation. Classification efficiency of ~96.7% and 100% for normal and cancerous conditions respectively, were observed. Findings of the study corroborates earlier studies and suggest applicability of Raman spectroscopic methods in combination with appropriate multivariate tool for objective, noninvasive and rapid diagnosis of cervical cancers in Indian population. In view of encouraging results, extensive validation studies will be undertaken to confirm the findings.

  18. Structured illumination fluorescence correlation spectroscopy for velocimetry in Zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Pozzi, Paolo; Rossetti, Leone; Sironi, Laura; Freddi, Stefano; D'Alfonso, Laura; Caccia, Michele; Bouzin, Margaux; Collini, Maddalena; Chirico, Giuseppe

    2013-02-01

    The vascular system of Zebrafish embryos is studied by means of Fluorescence Correlation and Image Correlation Spectroscopy. The long term project addresses biologically relevant issues concerning vasculogenesis and cardiogenesis and in particular mechanical interaction between blood flow and endothelial cells. To this purpose we use Zebrafish as a model system since the transparency of its embryos facilitates morphological observation of internal organs in-vivo. The correlation analysis provides quantitative characterization of fluxes in blood vessels in vivo. We have pursued and compared two complementary routes. In a first one we developed a two-spots two-photon setup in which the spots are spaced at adjustable micron-size distances (1-40 μm) along a vessel and the endogenous (autofluorescence) or exogenous (dsRed transgenic erythrocytes) signal is captured with an EM-CCD and cross-correlated. In this way we are able to follow the morphology of the Zebrafish embryo, simultaneously measure the heart pulsation, the velocity of red cells and of small plasma proteins. These data are compared to those obtained by image correlations on Zebrafish vessels. The two methods allows to characterize the motion of plasma fluids and erythrocytes in healthy Zebrafish embryos to be compared in the future to pathogenic ones.

  19. Quantitative Determination of DNA-Ligand Binding Using Fluorescence Spectroscopy

    ERIC Educational Resources Information Center

    Healy, Eamonn F.

    2007-01-01

    The effective use of fluorescence spectroscopy for determining the binding of the intercalcating agent crhidium bromide to DNA is being described. The analysis used simple measurement techniques and hence can be easily adopted by the students for a better understanding.

  20. Native fluorescence spectroscopy of thymus and fat tissues

    NASA Astrophysics Data System (ADS)

    Tang, Gui C.; Oz, Mehmet C.; Reid, V.; Steinglass, K.; Ginsberg, Mark D.; Jacobowitz, Larry; Alfano, Robert R.

    1993-08-01

    Fluorescence spectroscopy of the human thymus gland and surrounding mediastinal fat were measured to evaluate this approach in distinguishing between thymus and fat tissues during therapeutic surgery for myasthenia gravis disease.

  1. One-pot green hydrothermal synthesis of fluorescent nitrogen-doped carbon nanodots for in vivo bioimaging.

    PubMed

    Kuo, Tsung-Rong; Sung, Shuo-Yuan; Hsu, Chun-Wei; Chang, Chih-Jui; Chiu, Tai-Chia; Hu, Cho-Chun

    2016-01-01

    One-pot green synthesis of fluorescent nitrogen-doped carbon nanodots (CNDs) was developed by hydrothermal treatments of biocompatible polyvinylpyrrolidone (PVP) and glycine. The fluorescent nitrogen-doped CNDs exhibited excellent water solubility, low cytotoxicity, and good salt stability for biological imaging. UV-vis spectroscopy, fluorescence spectroscopy, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) spectroscopy, and Raman spectroscopy were applied to confirm the optical and structural characteristics of the CNDs. Fluorescence of the CNDs was tunable from 417 to 450 nm adjusted by different excitation energy. Fluorescent quantum yield of the CNDs (21.43%) was significantly increased ~47.59% in comparison to that of the CNDs (14.52%) without nitrogen doping by glycine. In the in vivo imaging system (IVIS), fluorescence signal of the nitrogen-doped CNDs was obviously observed in the lungs at 12- and 24-h post-injection. Our work has shown the potential applications of the nitrogen-doped CNDs in fluorescence imaging in vivo.

  2. Imaging cellular dynamics in vivo with multicolor fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Hoffman, Robert M.

    2005-04-01

    The new field of in vivo cell biology is being developed with multi-colored fluorescent proteins. With the use of fluorescent proteins, the behavior of individual cells can be visualized in the living animal. An example of the new cell biology is dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP)-expressing transgenic mice. These models show with great clarity the details of the tumor-stroma cell-cell interaction especially tumor-induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts and macrophages. Another example is the color-coding of cells with RFP or GFP such that both cell types and their interaction can be simultaneously visualized in vivo. Stem cells can also be visualized and tracked in vivo with fluorescent proteins. Mice, in which the regulatory elements of the stem-cell marker nestin drive GFP expression, can be used to visualize hair follicle stem cells including their ability to form hair follicles as well as blood vessels. Dual-color cells expressing GFP in the nucleus and RFP in the cytoplasm enable real-time visualization of nuclear-cytoplasm dynamics including cell cycle events and apoptosis. Dual-color cells also enable the in vivo imaging of cell and nuclear deformation as well as trafficking in capillaries in living animals. Multiple-color labeling of cells will enable multiple events to be simultaneously visualized in vivo including cell-cell interaction, gene expression, ion fluxes, protein and organelle trafficking, chromosome dynamics and numerous other processes currently still studied in vitro.

  3. Fluorescence correlation spectroscopy using quantum dots: advances, challenges and opportunities.

    PubMed

    Heuff, Romey F; Swift, Jody L; Cramb, David T

    2007-04-28

    Semiconductor nanocrystals (quantum dots) have been increasingly employed in measuring the dynamic behavior of biomacromolecules using fluorescence correlation spectroscopy. This poses a challenge, because quantum dots display their own dynamic behavior in the form of intermittent photoluminescence, also known as blinking. In this review, the manifestation of blinking in correlation spectroscopy will be explored, preceded by an examination of quantum dot blinking in general.

  4. Multiphoton cascade absorption in single molecule fluorescence saturation spectroscopy.

    PubMed

    Winckler, Pascale; Jaffiol, Rodolphe

    2013-05-01

    Saturation spectroscopy is a relevant method to investigate photophysical parameters of single fluorescent molecules. Nevertheless, the impact of a gradual increase, over a broad range, of the laser excitation on the intramolecular dynamics is not completely understood, particularly concerning their fluorescence emission (the so-called brightness). Thus, we propose a comprehensive theoretical and experimental study to interpret the unexpected evolution of the brightness with the laser power taking into account the cascade absorption of two and three photons. Furthermore, we highlight the key role played by the confocal observation volume in fluorescence saturation spectroscopy of single molecules in solution.

  5. In vivo lipidomics using single-cell Raman spectroscopy

    PubMed Central

    Wu, Huawen; Volponi, Joanne V.; Oliver, Ann E.; Parikh, Atul N.; Simmons, Blake A.; Singh, Seema

    2011-01-01

    We describe a method for direct, quantitative, in vivo lipid profiling of oil-producing microalgae using single-cell laser-trapping Raman spectroscopy. This approach is demonstrated in the quantitative determination of the degree of unsaturation and transition temperatures of constituent lipids within microalgae. These properties are important markers for determining engine compatibility and performance metrics of algal biodiesel. We show that these factors can be directly measured from a single living microalgal cell held in place with an optical trap while simultaneously collecting Raman data. Cellular response to different growth conditions is monitored in real time. Our approach circumvents the need for lipid extraction and analysis that is both slow and invasive. Furthermore, this technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of impermeability, toxicity, and specificity of the fluorescent probes common in currently used protocols. Although the single-cell Raman spectroscopy demonstrated here is focused on the study of the microalgal lipids with biofuel applications, the analytical capability and quantitation algorithms demonstrated are applicable to many different organisms and should prove useful for a diverse range of applications in lipidomics. PMID:21310969

  6. Clinical applications of in vivo fluorescence confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Oh, Chilhwan; Park, Sangyong; Kim, Junhyung; Ha, Seunghan; Park, Gyuman; Lee, Gunwoo; Lee, Onseok; Chun, Byungseon; Gweon, Daegab

    2008-02-01

    Living skin for basic and clinical research can be evaluated by Confocal Laser Scanning Microscope (CLSM) non-invasively. CLSM imaging system can achieve skin image its native state either "in vivo" or "fresh biopsy (ex vivo)" without fixation, sectioning and staining that is necessary for routine histology. This study examines the potential fluorescent CLSM with a various exogenous fluorescent contrast agent, to provide with more resolution images in skin. In addition, in vivo fluorescent CLSM researchers will be extended a range of potential clinical application. The prototype of our CLSM system has been developed by Prof. Gweon's group. The operating parameters are composed of some units, such as illuminated wavelength 488 nm, argon illumination power up to 20mW on the skin, objective lens, 0.9NA oil immersion, axial resolution 1.0μm, field of view 200μm x 100μm (lateral resolution , 0.3μm). In human volunteer, fluorescein sodium was administrated topically and intradermally. Animal studies were done in GFP transgenic mouse, IRC mouse and pig skin. For imaging of animal skin, fluorescein sodium, acridine orange, and curcumine were used for fluorescein contrast agent. We also used the GFP transgenic mouse for fluorescein CLSM imaging. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. Curcumin is a yellow food dye that has similar fluorescent properties to fluorescein sodium. Acridin Orange can be highlight nuclei in viable keratinocyte. In vivo CLSM of transgenic GFP mouse enable on in vivo, high resolution view of GFP expressing skin tissue. GFP signals are brightest in corneocyte, kertinocyte, hair and eccrine gland. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. In

  7. Assessment of skin flap viability using visible diffuse reflectance spectroscopy and auto-fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhu, Caigang; Chen, Shuo; Chui, Christopher Hoe-Kong; Liu, Quan

    2012-12-01

    The accurate assessment of skin flap viability is vitally important in reconstructive surgery. Early identification of vascular compromise increases the change of successful flap salvage. The ability to determine tissue viability intraoperatively is also extremely useful when the reconstructive surgeon must decide how to inset the flap and whether any tissue must be discarded. Visible diffuse reflectance and auto-fluorescence spectroscopy, which yield different sets of biochemical information, have not been used in the characterization of skin flap viability simultaneously to our best knowledge. We performed both diffuse reflectance and fluorescence measurements on a reverse MacFarlane rat dorsal skin flap model to identify the additional value of auto-fluorescence spectroscopy to the assessment of flap viability. Our result suggests that auto-fluorescence spectroscopy appears to be more sensitive to early biochemical changes in a failed flap than diffuse reflectance spectroscopy, which could be a valuable complement to diffuse reflectance spectroscopy for the assessment of flap viability.

  8. Noncontact point spectroscopy guided by two-channel fluorescence imaging in a hamster cheek pouch model

    NASA Astrophysics Data System (ADS)

    Yang, Victor X.; Yeow, Jenny; Lilge, Lothar D.; Kost, James; Mang, Thomas S.; Wilson, Brian C.

    1999-07-01

    A system for in vivo, fluorescence image-guided, non-contact point fluorescence spectroscopy is presented. A 442 nm HeCd laser is used as the fluorescence excitation source. An intensified CCD serves as the detector for both imaging and spectroscopy, on which two regions of 300 X 300 pixels were used for green (500 +/- 18 nm) and red (630 +/- 18 nm) imaging channels, and a strip of 600 X 120 pixels are used for emission spectroscopy (450 - 750 nm). At a working distance of 40 mm, the system has a spatial resolution of 0.16 mm and a spectral resolution of 5 nm. System performance is demonstrated in a carcinogenesis model in hamsters, where tumors were induced by painting DMBA in the cheek pouch. Autofluorescence and Photofrin-induced fluorescence measurements were performed every 2 weeks during the 18 weeks of tumor induction. Punch biopsies on selected animals were taken for histological staging. The results show that autofluorescence fluorescence can distinguish dysplasia from normal mucosal tissue model, utilizing the peak red intensity (or the red-to-green intensity ratio). Photofrin-induced fluorescence was superior to autofluorescence for differentiating high grade dysplasia from invasive cancer.

  9. Absorption and fluorescence spectroscopy on a smartphone

    NASA Astrophysics Data System (ADS)

    Hossain, Md. Arafat; Canning, John; Cook, Kevin; Ast, Sandra; Rutledge, Peter J.; Jamalipour, Abbas

    2015-07-01

    A self-powered smartphone-based field-portable "dual" spectrometer has been developed for both absorption and fluorescence measurements. The smartphone's existing flash LED has sufficient optical irradiance to undertake absorption measurements within a 3D-printed case containing a low cost nano-imprinted polymer diffraction grating. A UV (λex ~ 370 nm) and VIS (λex ~ 450 nm) LED are wired into the circuit of the flash LED to provide an excitation source for fluorescence measurements. Using a customized app on the smartphone, measurements of absorption and fluorescence spectra are demonstrated using pH-sensitive and Zn2+-responsive probes. Detection over a 300 nm span with 0.42 nm/pixel spectral resolution is demonstrated. Despite the low cost and small size of the portable spectrometer, the results compare well with bench top instruments.

  10. In-vitro bacterial identification using fluorescence spectroscopy with an optical fiber system

    NASA Astrophysics Data System (ADS)

    Spector, Brian C.; Werkhaven, Jay A.; Smith, Dana; Reinisch, Lou

    2000-05-01

    Acute otitis media (AOM) remains a source of significant morbidity in children. With the emergence of antibiotic resistant strains of bacteria, tympanocentesis has become an important method of bacterial identification in the setting of treatment failures. Previous studies described a prototype system for the non-invasive fluorescence identification of bacteria in vitro. We demonstrate the addition of an optical fiber to allow for the identification of a specimen distant to the spectrofluorometer. Emission spectra from three bacteria, Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus were successfully obtained in vitro. This represents a necessary step prior to the study of in vivo identification of bacteria in AOM using fluorescence spectroscopy.

  11. Spectroscopy detection of green and red fluorescent proteins in genetically modified plants using a fiber optics system

    NASA Astrophysics Data System (ADS)

    Liew, Oi Wah; Asundi, Anand K.; Chen, Jun-Wei; Chew, Yiwen; Yu, Shangjuan; Yeo, Gare H.

    2001-05-01

    In this paper, fiber optic spectroscopy is developed to detect and quantify recombinant green (EGFP) and red (DsRED) fluorescent proteins in vitro and in vivo. The bacterial expression vectors carrying the coding regions of EGFP and DsRED were introduced into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and serially diluted for quantitative analysis by fiber optic spectroscopy. Fluorescence at the appropriate emission wavelengths could be detected up to 64X dilution for EGFP and 40X dilution for DsRED. To determine the capability of spectroscopy detection in vivo, transgenic potato hairy roots expressing EGFP and DsRED were regenerated. This was achieved by cloning the EGFP and DsRED genes into the plant binary vector, pTMV35S, to create the recombinant vectors pGLOWGreen and pGLOWRed. These latter binary vectors were introduced into Agrobacterium rhizogenes strain A4T. Infection of potato cells with transformed agrobacteria was used to insert the fluorescent protein genes into the potato genome. Genetically modified potato cells were then regenerated into hairy roots. A panel of transformed hairy roots expressing varying levels of fluorescent proteins was selected by fluorescence microscopy. We are now assessing the capability of spectroscopic detection system for in vivo quantification of green and red fluorescence levels in transformed roots.

  12. Pancreatic tissue assessment using fluorescence and reflectance spectroscopy

    NASA Astrophysics Data System (ADS)

    Chandra, Malavika; Heidt, David; Simeone, Diane; McKenna, Barbara; Scheiman, James; Mycek, Mary-Ann

    2007-07-01

    The ability of multi-modal optical spectroscopy to detect signals from pancreatic tissue was demonstrated by studying human pancreatic cancer xenografts in mice and freshly excised human pancreatic tumor tissue. Measured optical spectra and fluorescence decays were correlated with tissue morphological and biochemical properties. The measured spectral features and decay times correlated well with expected pathological differences in normal, pancreatitis and adenocarcinoma tissue states. The observed differences between the fluorescence and reflectance properties of normal, pancreatitis and adenocarcinoma tissue indicate a possible application of multi-modal optical spectroscopy to differentiating between the three tissue classifications.

  13. Quantitative fluorescence tomography using a trimodality system: in vivo validation

    NASA Astrophysics Data System (ADS)

    Lin, Yuting; Barber, William C.; Iwanczyk, Jan S.; Roeck, Werner W.; Nalcioglu, Orhan; Gulsen, Gultekin

    2010-07-01

    A fully integrated trimodality fluorescence, diffuse optical, and x-ray computed tomography (FT/DOT/XCT) system for small animal imaging is reported in this work. The main purpose of this system is to obtain quantitatively accurate fluorescence concentration images using a multimodality approach. XCT offers anatomical information, while DOT provides the necessary background optical property map to improve FT image accuracy. The quantitative accuracy of this trimodality system is demonstrated in vivo. In particular, we show that a 2-mm-diam fluorescence inclusion located 8 mm deep in a nude mouse can only be localized when functional a priori information from DOT is available. However, the error in the recovered fluorophore concentration is nearly 87%. On the other hand, the fluorophore concentration can be accurately recovered within 2% error when both DOT functional and XCT structural a priori information are utilized together to guide and constrain the FT reconstruction algorithm.

  14. Ex Vivo Fluorescence Molecular Tomography of the Spine

    PubMed Central

    Pimpalkhare, Monish; Chen, Jin; Venugopal, Vivek; Intes, Xavier

    2012-01-01

    We investigated the potential of fluorescence molecular tomography to image ex vivo samples collected from a large animal model, in this case, a dog spine. Wide-field time-gated fluorescence tomography was employed to assess the impact of multiview acquisition, data type, and intrinsic optical properties on the localization and quantification accuracy in imaging a fluorescent inclusion in the intervertebral disk. As expected, the TG data sets, when combining early and late gates, provide significantly better performances than the CW data sets in terms of localization and quantification. Moreover, the use of multiview imaging protocols led to more accurate localization. Additionally, the incorporation of the heterogeneous nature of the tissue in the model to compute the Jacobians led to improved imaging performances. This preliminary imaging study provides a proof of concept of the feasibility of quantitatively imaging complex ex vivo samples nondestructively and with short acquisition times. This work is the first step towards employing optical molecular imaging of the spine to detect and characterize disc degeneration based on targeted fluorescent probes. PMID:23197973

  15. Optical spectroscopy for differentiation of liver tissue under distinct stages of fibrosis: an ex vivo study

    NASA Astrophysics Data System (ADS)

    Fabila, D. A.; Hernández, L. F.; de la Rosa, J.; Stolik, S.; Arroyo-Camarena, U. D.; López-Vancell, M. D.; Escobedo, G.

    2013-11-01

    Liver fibrosis is the decisive step towards the development of cirrhosis; its early detection affects crucially the diagnosis of liver disease, its prognosis and therapeutic decision making. Nowadays, several techniques are employed to this task. However, they have the limitation in estimating different stages of the pathology. In this paper we present a preliminary study to evaluate if optical spectroscopy can be employed as an auxiliary tool of diagnosis of biopsies of human liver tissue to differentiate the fibrosis stages. Ex vivo fluorescence and diffuse reflectance spectra were acquired from biopsies using a portable fiber-optic system. Empirical discrimination algorithms based on fluorescence intensity ratio at 500 nm and 680 nm as well as diffuse reflectance intensity at 650 nm were developed. Sensitivity and specificity of around 80% and 85% were respectively achieved. The obtained results show that combined use of fluorescence and diffuse reflectance spectroscopy could represent a novel and useful tool in the early evaluation of liver fibrosis.

  16. Tracking-FCS: Fluorescence correlation spectroscopy of individual particles

    NASA Astrophysics Data System (ADS)

    Berglund, Andrew J.; Mabuchi, Hideo

    2005-10-01

    We exploit recent advances in single-particle tracking to perform fluorescence correlation spectroscopy on individual fluorescent particles, in contrast to traditional methods that build up statistics over a sequence of many measurements. By rapidly scanning the focus of an excitation laser in a circular pattern, demodulating the measured fluorescence, and feeding these results back to a piezoelectric translation stage, we track the Brownian motion of fluorescent polymer microspheres in aqueous solution in the plane transverse to the laser axis. We discuss the estimation of particle diffusion statistics from closed-loop position measurements, and we present a generalized theory of fluorescence correlation spectroscopy for the case that the motion of a single fluorescent particle is actively tracked by a time-dependent laser intensity. We model the motion of a tracked particle using Ornstein-Uhlenbeck statistics, using a general theory that contains a umber of existing results as specific cases. We find good agreement between our theory and experimental results, and discuss possible future applications of these techniques to passive, single-shot, single-molecule fluorescence measurements with many orders of magnitude in time resolution.

  17. The development of attenuation compensation models of fluorescence spectroscopy signals

    NASA Astrophysics Data System (ADS)

    Dremin, Victor V.; Zherebtsov, Evgeny A.; Rafailov, Ilya E.; Vinokurov, Andrey Y.; Novikova, Irina N.; Zherebtsova, Angelina I.; Litvinova, Karina S.; Dunaev, Andrey V.

    2016-04-01

    This study examines the effect of blood absorption on the endogenous fluorescence signal intensity of biological tissues. Experimental studies were conducted to identify these effects. To register the fluorescence intensity, the fluorescence spectroscopy method was employed. The intensity of the blood flow was measured by laser Doppler flowmetry. We proposed one possible implementation of the Monte Carlo method for the theoretical analysis of the effect of blood on the fluorescence signals. The simulation is constructed as a four-layer skin optical model based on the known optical parameters of the skin with different levels of blood supply. With the help of the simulation, we demonstrate how the level of blood supply can affect the appearance of the fluorescence spectra. In addition, to describe the properties of biological tissue, which may affect the fluorescence spectra, we turned to the method of diffuse reflectance spectroscopy (DRS). Using the spectral data provided by the DRS, the tissue attenuation effect can be extracted and used to correct the fluorescence spectra.

  18. "FluSpec": A Simulated Experiment in Fluorescence Spectroscopy

    ERIC Educational Resources Information Center

    Bigger, Stephen W.; Bigger, Andrew S.; Ghiggino, Kenneth P.

    2014-01-01

    The "FluSpec" educational software package is a fully contained tutorial on the technique of fluorescence spectroscopy as well as a simulator on which experiments can be performed. The procedure for each of the experiments is also contained within the package along with example analyses of results that are obtained using the software.

  19. Fluorescence spectroscopy: a rapid tool for analyzing dairy products.

    PubMed

    Andersen, Charlotte Møller; Mortensen, Grith

    2008-02-13

    This paper gives a critical evaluation of the use of fluorescence spectroscopy for measuring chemical and physical changes in dairy products caused by processing and storage. Fluorescence spectroscopy is able to determine various properties of foods without use of chemicals and time-consuming sample preparation. This is shown by examples where the measurement of a given chemical parameter has been appropriately described and validated, as well as situations showing potential applications, but where further research and validation is required. The interpretation of fluorescence spectroscopic data is complex due to absorbance by other molecular groups, changes caused by variation in the sample matrix, etc. It is illustrated how advanced data analytical techniques are required to obtain optimal interpretation of the data. Even though the review focuses on examples from the dairy industry, the principles are broader and can be applied to other fields of food and agricultural research.

  20. Fluorescence spectroscopy: a rapid, noninvasive method for measurement of skin surface thickness of topical agents.

    PubMed

    Rhodes, L E; Diffey, B L

    1997-01-01

    We report the quantification of skin surface thickness of topical agents by in vivo fluorescence spectroscopy, and demonstrate its potential uses for assessment of application technique and substantivity. A series of studies were performed on forearm skin of eight normal subjects using three creams which have intrinsic fluorescence: a sunscreen (Neutrogena SPF15 waterproof cream), an antiseptic (Hewlett's cream) and a steroid (Trimovate (clobetasone butyrate) cream). Initially, the dose-response relationship was established for each agent by applying a series of five doses (0.5-8 microliters/cm2) and measuring cream fluorescence using appropriate excitation and emission wavelengths. Next, the influence of application technique was examined by comparing light application of cream with firm rubbing. Substantivity of the three creams was assessed on dry skin by taking fluorescence measurements over 8 h. Finally, water resistance of 2 microliters/cm2 of sunscreen and antiseptic cream were compared by measuring fluorescence after each of four water immersions. The fluorescence intensity was strongly correlated with the logarithm of surface density. r = 1.0, 0.92 and 0.98 for sunscreen, antiseptic and steroid creams, respectively, allowing derivation of a simple expression for equivalent thickness. Surface thickness of each cream was lower following firm rubbing compared with light application (P < 0.01). The rate constants for reduction of surface density of the three creams with time on dry skin were not significantly different. However, on washed skin, the rate constant was higher for Hewlett's than Neutrogena cream (0.503 and 0.243 h. respectively, P = 0.02), with a higher rate for each cream on wet compared with dry skin (P < 0.001). Hence, fluorescence spectroscopy is a simple, rapid method for measurement of cream thickness in vivo. The many potential applications in dermatology include quantitative assessment of application technique and substantivity of topical

  1. MRI-coupled spectrally-resolved fluorescence tomography for in vivo imaging

    NASA Astrophysics Data System (ADS)

    Davis, Scott C.; Gibbs-Strauss, Summer L.; Tuttle, Stephen B.; Jiang, Shudong; Springett, Roger; Dehghani, Hamid; Pogue, Brian W.; Paulsen, Keith D.

    2008-02-01

    A unique fluorescence imaging system incorporates multi-channel spectrometer-based optical detection directly into clinical MRI for simultaneous MR and spectrally-resolved fluorescence tomography acquisition in small animal and human breast-sized volumes. A custom designed MRI rodent coil adapted to accommodate optical fibers in a circular geometry for contact mode acquisition provides small animal imaging capabilities, and human breast-sized volumes are imaged using a clinical breast coil modified with an optical fiber patient array. Spectroscopy fibers couple light emitted from the tissue surface to sixteen highly sensitive CCD-based spectrometers operating in parallel. Tissue structural information obtained from standard and contrast enhanced T1-weighted images is used to spatially constrain the diffuse fluorescence tomography reconstruction algorithm, improving fluorescence imaging capabilities qualitatively and quantitatively. Simultaneous acquisition precludes the use of complex co-registration processes. Calibration procedures for the optical acquisition system are reviewed and the imaging limits of the system are investigated in homogeneous and heterogeneous gelatin phantoms containing Indocyanine Green (ICG). Prior knowledge of fluorescence emission spectra is used to de-couple fluorescence emission from residual excitation laser cross-talk. Preliminary in vivo data suggests improved fluorescence imaging in mouse brain tumors using MR-derived spatial priors. U-251 human gliomas were implanted intracranially into nude mice and combined contrast enhanced MRI/fluorescence tomography acquisition was completed at 24 hour intervals over the course of 72 hours after administration of an EGFR targeted NIR fluorophore. Reconstructed images demonstrate an inability to recover reasonable images of fluorescence activity without the use of MRI spatial priors.

  2. In vivo Diagnosis of Cervical Intraepithelial Neoplasia Using 337-nm- Excited Laser-Induced Fluorescence

    NASA Astrophysics Data System (ADS)

    Ramanujam, N.; Mitchell, M. F.; Mahadevan, A.; Warren, S.; Thomsen, S.; Silva, E.; Richards-Kortum, R.

    1994-10-01

    Laser-induced fluorescence at 337-nm excitation was used in vivo to differentiate neoplastic [cervical intraepithelial neoplasia (CIN)], nonneoplastic abnormal (inflammation and human papilloma viral infection), and normal cervical tissues. A colposcope (low-magnification microscope used to view the cervix with reflected light) was used to identify 66 normal and 49 abnormal (5 inflammation, 21 human papilloma virus infection, and 23 CIN) sites on the cervix in 28 patients. These sites were then interrogated spectroscopically. A two-stage algorithm was developed to diagnose CIN. The first stage differentiated histologically abnormal tissues from colposcopically normal tissues with a sensitivity, specificity, and positive predictive value of 92%, 90%, and 88%, respectively. The second stage differentiated preneoplastic and neoplastic tissues from nonneoplastic abnormal tissues with a sensitivity, specificity, and positive predictive value of 87%, 73%, and 74%, respectively. Spectroscopic differences were consistent with a decrease in the absolute contribution of collagen fluorescence, an increase in the absolute contribution of oxyhemoglobin attenuation, and an increase in the relative contribution of reduced nicotinamide dinucleotide phosphate [NAD(P)H] fluorescence as tissue progresses from normal to abnormal in the same patient. These results suggest that in vivo fluorescence spectroscopy of the cervix can be used to diagnose CIN at colposcopy.

  3. Targeted thiazole orange derivative with folate: synthesis, fluorescence and in vivo fluorescence imaging.

    PubMed

    Fei, Xuening; Gu, Yingchun; Wang, Yiqi; Meng, Qingyang; Zhang, Baolian

    2010-10-11

    A Thiazole Orange conjugated with folate derivative was synthesized in two steps. Firstly, folate was coupled with 1-(3-aminopropyl)-4-methylquinolinium bromide to afford folate-methylquinolinium bromide, which then reacted with benzothiazolium to obtain the title folate-conjugated compound. The compound was evaluated by ¹H-NMR MS, TG/DTA and fluorescence spectroscopic methods. The title compound could selectively target folate receptor expressing tumors according to the in vivo fluorescence imaging preliminarily performed on nude mice with breast tumors.

  4. Laser Induced Fluorescence Spectroscopy of Cobalt Monoboride

    NASA Astrophysics Data System (ADS)

    Pang, H. F.; Ng, Y. W.; Cheung, A. S.-C.

    2011-06-01

    Laser induced fluorescence spectrum of cobalt monoboride (CoB) in the visible region between 465 and 560 nm has been observed. CoB molecule was produced by the reaction of laser ablated cobalt atom and diborane (B_2H_6) seeded in argon. Over twenty five vibronic bands have been recorded, and both Co10B and Co11B isotopic species have been observed and analyzed. Preliminary analysis of the rotational lines showed that the observed vibronic bands belong to two categories namely: the Ω' = 2 - Ω'' = 2 and the Ω' = 3 - Ω'' = 3 transitions, which indicated the ground state of CoB is consistent with an assignment of a ^3Δ_i state predicted from ab initio calculations. Unresolved hyperfine structure arising from the Co nucleus (I = 7/2) causes a broadening of spectral lines. This work represents the first experimental investigation of the spectrum of the CoB molecule. Financial support from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. HKU 701008P) is gratefully acknowledged.

  5. Assembly and characterization of a fluorescence lifetime spectroscopy system for skin lesions diagnostic

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Texiera Rosa, Ramon Gabriel; Pratavieira, Sebastião.; D´Almeida, Camila de Paula; Kurachi, Cristina

    2015-06-01

    The fluorescence spectra and fluorescence lifetime analysis in biological tissues has been presented as a technique of a great potential for tissue characterization for diagnostic purposes. The objective of this study is to assemble and characterize a fluorescence lifetime spectroscopy system for diagnostic of clinically similar skin lesions in vivo. The fluorescence lifetime measurements were performed using the Time Correlated Single Photon Counting (Becker & Hickl, Berlin, Germany) technique. Two lasers, one emitting at 378 nm and another at 445 nm, are used for excitation with 20, 50 and 80 MHz repetition rate. A bifurcated optical fiber probe conducts the excitation light to the sample, the collected light is transmitted through bandpass filters and delivered to a hybrid photomultiplier tube detector. The fluorescence spectra were obtained by using a portable spectrometer (Ocean Optics USB-2000-FLG) with the same excitation sources. An instrument response function of about 300 ps was obtained and the spectrum and fluorescence lifetime of a standard fluorescent molecule (Rhodamine 6G) was measured for the calibration of the system ((4.1 +/- 0.3) ns). The assembled system was considered robust, well calibrated and will be used for clinical measurements of skin lesions.

  6. Deep tissue fluorescence imaging and in vivo biological applications

    NASA Astrophysics Data System (ADS)

    Crosignani, Viera; Dvornikov, Alexander; Aguilar, Jose S.; Stringari, Chiara; Edwards, Robert; Mantulin, William W.; Gratton, Enrico

    2012-11-01

    We describe a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. Compared to conventional two-photon microscopy, greater imaging depth is achieved by more efficient harvesting of fluorescence photons propagating in multiple-scattering media. The system maintains the conventional two-photon microscopy scheme for excitation. However, for fluorescence collection the detection system harvests fluorescence photons directly from a wide area of the turbid sample. The detection scheme relies on a wide area detector, minimal optical components and an emission path bathed in a refractive-index-matching fluid that minimizes emission photon losses. This detection scheme proved to be very efficient, allowing us to obtain high resolution images at depths up to 3 mm. This technique was applied to in vivo imaging of the murine small intestine (SI) and colon. The challenge is to image normal and diseased tissue in the whole live animal, while maintaining high resolution imaging at millimeter depth. In Lgr5-GFP mice, we have been successful in imaging Lgr5-eGFP positive stem cells, present in SI and colon crypt bases.

  7. Synthesis of fluorescent dye-tagged nanomachines for single-molecule fluorescence spectroscopy.

    PubMed

    Vives, Guillaume; Guerrero, Jason M; Godoy, Jazmin; Khatua, Saumyakanti; Wang, Yu-Pu; Kiappes, J L; Link, Stephan; Tour, James M

    2010-10-01

    In an effort to elucidate the mechanism of movement of nanovehicles on nonconducting surfaces, the synthesis and optical properties of five fluorescently tagged nanocars are reported. The nanocars were specifically designed for studies by single-molecule fluorescence spectroscopy and bear a tetramethylrhodamine isothiocyanate fluorescent tag for excitation at 532 nm. The molecules were designed such that the arrangement of their molecular axles and p-carborane wheels relative to the chassis would be conducive to the control of directionality in the motion of these nanovehicles.

  8. Fluorescence lifetime imaging microscopy of nanodiamonds in vivo

    NASA Astrophysics Data System (ADS)

    Kuo, Yung; Hsu, Tsung-Yuan; Wu, Yi-Chun; Hsu, Jui-Hung; Chang, Huan-Cheng

    2013-03-01

    The negatively charged nitrogen-vacancy (NV-) center in bulk diamond is a photostable fluorophore with a radiative lifetime of 11.6 ns at room temperature. The lifetime substantially increases to ~20 ns for diamond nanoparticles (size ~ 100 nm) suspended in water due to the change in refractive index of the surrounding medium of the NV- centers. This fluorescence decay time is much longer than that (typically 1 - 4 ns) of endogenous and exogenous fluorophores commonly used in biological imaging, making it possible to detect NV--containing nanodiamonds in vivo at the single particle level by fluorescence lifetime imaging microscopy (FLIM). We demonstrate the feasibility of this approach using Caenorhabditis elegans (C. elegans) as a model organism.

  9. Emerging in vivo analyses of cell function using fluorescence imaging (*).

    PubMed

    Lippincott-Schwartz, Jennifer

    2011-01-01

    Understanding how cells of all types sense external and internal signals and how these signals are processed to yield particular responses is a major goal of biology. Genetically encoded fluorescent proteins (FPs) and fluorescent sensors are playing an important role in achieving this comprehensive knowledge base of cell function. Providing high sensitivity and immense versatility while being minimally perturbing to a biological specimen, the probes can be used in different microscopy techniques to visualize cellular processes on many spatial scales. Three review articles in this volume discuss recent advances in probe design and applications. These developments help expand the range of biochemical processes in living systems suitable for study. They provide researchers with exciting new tools to explore how cellular processes are organized and their activity regulated in vivo.

  10. Fluorescence molecular tomography resolves protease activity in vivo.

    PubMed

    Ntziachristos, Vasilis; Tung, Ching-Hsuan; Bremer, Christoph; Weissleder, Ralph

    2002-07-01

    Systematic efforts are under way to develop novel technologies that would allow molecular sensing in intact organisms in vivo. Using near-infrared fluorescent molecular beacons and inversion techniques that take into account the diffuse nature of photon propagation in tissue, we were able to obtain three-dimensional in vivo images of a protease in orthopic gliomas. We demonstrate that enzyme-activatable fluorochromes can be detected with high positional accuracy in deep tissues, that molecular specificities of different beacons towards enzymes can be resolved and that tomography of beacon activation is linearly related to enzyme concentration. The tomographic imaging method offers a range of new capabilities for studying biological function; for example, identifying molecular-expression patterns by multispectral imaging or continuously monitoring the efficacy of therapeutic drugs.

  11. Fluorescence correlation spectroscopy: Diagnostics for sparse molecules

    PubMed Central

    Maiti, Sudipta; Haupts, Ulrich; Webb, Watt W.

    1997-01-01

    The robust glow of molecular fluorescence renders even sparse molecules detectable and susceptible to analysis for concentration, mobility, chemistry, and photophysics. Correlation spectroscopy, a statistical-physics-based tool, gleans quantitative information from the spontaneously fluctuating fluorescence signals obtained from small molecular ensembles. This analytical power is available for studying molecules present at minuscule concentrations in liquid solutions (less than one nanomolar), or even on the surfaces of living cells at less than one macromolecule per square micrometer. Indeed, routines are becoming common to detect, locate, and examine individual molecules under favorable conditions. PMID:9342306

  12. Femtosecond broadband fluorescence upconversion spectroscopy: Improved setup and photometric correction

    SciTech Connect

    Zhang, X.-X.; Wuerth, C.; Resch-Genger, U.; Zhao, L.; Ernsting, N. P.; Sajadi, M.

    2011-06-15

    A setup for fluorescence upconversion spectroscopy (FLUPS) is described which has 80 fs temporal response (fwhm) for emission in the spectral range 425-750 nm. Broadband phase matching is achieved with tilted gate pulses at 1340 nm. Background from harmonics of the gate pulse is removed and sensitivity increased compared to previous designs. Photometric calibration of the upconversion process is performed with a set of fluorescent dyes. For Coumarin 153 in methanol the peak position, bandwidth, and asymmetry depending on delay time are reported.

  13. Histologic differences between orthotopic xenograft pancreas models affect Verteporfin uptake measured by fluorescence microscopy and spectroscopy

    NASA Astrophysics Data System (ADS)

    O'Hara, Julia A.; Samkoe, Kimberley S.; Chen, Alina; Isabelle, Martin; Hoopes, P. J.; Hasan, Tayyaba; Pogue, Brian W.

    2012-02-01

    Photodynamic therapy (PDT) that uses the second generation photosensitizer, verteporfin (VP), is a developing therapy for pancreatic cancer. The optimal timing of light delivery related to VP uptake and distribution in pancreatic tumors will be important information to obtain to improve treatment for this intractable disease. In this work we examined uptake and distribution of VP in two orthotopic pancreatic tumors with different histological structure. ASPC-1 (fast-growing) and Panc-1 (slower growing) tumors were implanted in SCID mice and studied when tumors were approximately 100mm3. In a pilot study, these tumors had been shown to differ in uptake of VP using lightinduced fluorescence spectroscopy (LIFS) in vivo and fluorescence imaging ex vivo and that work is extended here. In vivo fluorescence mean readings of tumor and liver increased rapidly up to 15 minutes after photosensitizer injection for both tumor types, and then continued to increase up to 60 minutes post injection to a higher level in ASPC-1 than in Panc-1. There was variability among animals with the same tumor type, in both liver and tumor uptake and no selectivity of tumor over liver. In this work we further examined VP uptake at multiple time points in relation to microvascular density and perfusion, using DiOC7 (to mark blood vessels) and VP fluorescence in the same tissue slices. Analysis of DiOC7 fluorescence indicates that AsPC-1 and Panc-1 have different vascular densities but AsPC-1 vasculature is more perfusive. Analysis of colocalized DiOC7 and VP fluorescence showed ASPC-1 with higher accumulation of VP 3 hrs after injection and more VP at a distance from blood vessels compared to Panc-1. This work shows the need for techniques to analyze photosensitizer distribution in order to optimize photodynamic therapy as an effective treatment for pancreatic tumors.

  14. Fluorescence suppression using micro-scale spatially offset Raman spectroscopy.

    PubMed

    Conti, Claudia; Botteon, Alessandra; Colombo, Chiara; Realini, Marco; Matousek, Pavel

    2016-09-21

    We present a new concept of fluorescence suppression in Raman microscopy based on micro-spatially offset Raman spectroscopy which is applicable to thin stratified turbid (diffusely scattering) matrices permitting the retrieval of the Raman signals of sublayers below intensely fluorescing turbid over-layers. The method is demonstrated to yield good quality Raman spectra with dramatically suppressed fluorescence backgrounds enabling the retrieval of Raman sublayer signals even in situations where conventional Raman microscopy spectra are fully overwhelmed by intense fluorescence. The concept performance was studied theoretically using Monte Carlo simulations indicating the potential of up to an order or two of magnitude suppression of overlayer fluorescence backgrounds relative to the Raman sublayer signals. The technique applicability was conceptually demonstrated on layered samples involving paints, polymers and stones yielding fluorescence suppression factors between 12 to above 430. The technique has potential applications in a number of analytical areas including cultural heritage, archaeology, polymers, food, pharmaceutical, biological, biomedical, forensics and catalytic sciences and quality control in manufacture.

  15. Fluorescence suppression using micro-scale spatially offset Raman spectroscopy.

    PubMed

    Conti, Claudia; Botteon, Alessandra; Colombo, Chiara; Realini, Marco; Matousek, Pavel

    2016-09-21

    We present a new concept of fluorescence suppression in Raman microscopy based on micro-spatially offset Raman spectroscopy which is applicable to thin stratified turbid (diffusely scattering) matrices permitting the retrieval of the Raman signals of sublayers below intensely fluorescing turbid over-layers. The method is demonstrated to yield good quality Raman spectra with dramatically suppressed fluorescence backgrounds enabling the retrieval of Raman sublayer signals even in situations where conventional Raman microscopy spectra are fully overwhelmed by intense fluorescence. The concept performance was studied theoretically using Monte Carlo simulations indicating the potential of up to an order or two of magnitude suppression of overlayer fluorescence backgrounds relative to the Raman sublayer signals. The technique applicability was conceptually demonstrated on layered samples involving paints, polymers and stones yielding fluorescence suppression factors between 12 to above 430. The technique has potential applications in a number of analytical areas including cultural heritage, archaeology, polymers, food, pharmaceutical, biological, biomedical, forensics and catalytic sciences and quality control in manufacture. PMID:27338230

  16. Fluorescence spectroscopy of excitation transfer in Photosystem 1

    SciTech Connect

    Mukerji, I.

    1990-12-01

    This thesis centers on the study of excitation transfer in a photosynthetic antenna array. The spectroscopic properties of two pigment-protein complexes were investigated. These complexes, isolated from higher plants, display an unusual temperature dependent fluorescence behavior. The author have chosen to study this fluorescence behavior with respect to energy transfer to the reaction center and in an isolated intact antenna preparation. A Photosystem 1 complex, PSI-200, was isolated from spinach. We have characterized this system by both steady state and time-resolved fluorescence spectroscopy. Fluorescence polarization measurements indicate that this emission arises from pigments which absorb in the long wavelength region of the spectrum and comprise a relatively small portion of the antenna population. Comparison of spectral characteristics were made with a PSI complex isolated from the thermophilic cyanobacterium, Synechococcus, sp. To address the role of Chl b in stimulating long wavelength fluorescence and the temperature dependence of the system, we have studied the energy transfer dynamics in an antenna complex, LHC-I isolated from PSI-200. Kinetic measurements indicate that initially absorbed excitation is rapidly redistributed to longer wavelength emitting pigments within 40 ps. The temperature dependence of F685 results from increased back transfer from long wavelength emitters to F685. We suggest that changes in excitation transfer between the various emitting species and a non-radiative fluorescence quenching mechanism account for the temperature dependence of the system. 144 refs., 50 figs., 3 tabs.

  17. Quantitative Tissue Spectroscopy of Near Infrared Fluorescent Nanosensor Implants.

    PubMed

    Iverson, Nicole M; Bisker, Gili; Farias, Edgardo; Ivanov, Vsevolod; Ahn, Jiyoung; Wogan, Gerald N; Strano, Michael S

    2016-05-01

    Implantable, near infrared (nIR) fluorescent nanosensors are advantageous for in vivo monitoring of biological analytes since they can be rendered selective for a particular target molecule while utilizing their unique optical properties and the nIR tissue transparency window for information transfer without an internal power source or telemetry. However, basic questions remain regarding the optimal encapsulation platform, geometrical properties, and concentration ranges required for high signal to noise ratio and effective detection through biological tissue. In this work, we systematically explore these variables quantitatively to optimize the performance of such optical nanosensors for biomedical applications. We investigate both alginate and polyethylene glycol (PEG) as model hydrogel systems, encapsulating d(GT)15 ssDNA-wrapped single-walled carbon nanotubes (SWNT) as model fluorescent nanoparticle sensors, responsive to riboflavin. Hydrogel sensors implanted 0.5 mm into thick tissue samples exhibit 50% reduction of initial fluorescence intensity, allowing an optical detection limit of 5.4 mm and 5.1 mm depth in tissue for alginate and PEG gels, respectively, at a SWNT concentration of 10 mg L(-1), and 785 nm laser excitation of 80 mW and 30 s exposure. These findings are supported with in vivo nIR fluorescent imaging of SWNT hydrogels implanted subcutaneously in mice. For the case of SWNT, we find that the alginate system is preferable in terms of emission intensity, sensor response, rheological properties, and shelf life. PMID:27305824

  18. Multiphoton microscopy, fluorescence lifetime imaging and optical spectroscopy for the diagnosis of neoplasia

    NASA Astrophysics Data System (ADS)

    Skala, Melissa Caroline

    2007-12-01

    the ultraviolet to visible wavelength range indicated that the most diagnostic optical signals originate from sub-surface tissue layers. Optical properties extracted from these spectroscopy measurements showed a significant decrease in the hemoglobin saturation, absorption coefficient, reduced scattering coefficient and fluorescence intensity (at 400 nm excitation) in neoplastic compared to normal tissues. The results from these studies indicate that multiphoton microscopy and optical spectroscopy can non-invasively provide information on tissue structure and function in vivo that is related to tissue pathology.

  19. Principles and applications of fluorescence lifetime correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Beranová, Lenka; Humpolícková, Jana; Hof, Martin

    2009-05-01

    Two fluorescence spectroscopy concepts, fluorescence correlation spectroscopy and time correlated single photon counting (TCSPC) are employed in fluorescence lifetime correlation spectroscopy (FLCS) - a relatively new technique with several experimental benefits. In FLCS experiments, pulsed excitation is used and data are stored in a special time-tagged time-resolved mode. Mathematical treatment of TCSPC decay patterns of distinct fluorophores and their mixture enables to calculate autocorrelation functions of each of the fluorophores and thus their diffusion properties and concentrations can be determined separately. Moreover, crosscorrelation of the two signals can be performed and information on interaction of the species can be obtained. This technique is particularly helpful for distinguishing different states of the same fluorophore in different microenvironments. The first application of that concept represents the simultaneous determination of two-dimensional diffusion in planar lipid layers and three-dimensional vesicle diffusion in bulk above the lipid layers. The lifetime in both investigated systems differed because the lifetime of the dye is considerably quenched in the layer near the light-absorbing surface. This concept was also used in other applications: a) investigation of a conformational change of a labeled protein, b) detection of small amounts of labeled oligonucleotides bound to metal particles or c) elucidation of the compaction mechanism of different sized labeled DNA molecules. Moreover, it was demonstrated that FLCS can help to overcome some FCS experimental drawbacks.

  20. Fluorescence spectroscopy using indocyanine green for lymph node mapping

    NASA Astrophysics Data System (ADS)

    Haj-Hosseini, Neda; Behm, Pascal; Shabo, Ivan; Wârdell, Karin

    2014-02-01

    The principles of cancer treatment has for years been radical resection of the primary tumor. In the oncologic surgeries where the affected cancer site is close to the lymphatic system, it is as important to detect the draining lymph nodes for metastasis (lymph node mapping). As a replacement for conventional radioactive labeling, indocyanine green (ICG) has shown successful results in lymph node mapping; however, most of the ICG fluorescence detection techniques developed are based on camera imaging. In this work, fluorescence spectroscopy using a fiber-optical probe was evaluated on a tissue-like ICG phantom with ICG concentrations of 6-64 μM and on breast tissue from five patients. Fiber-optical based spectroscopy was able to detect ICG fluorescence at low intensities; therefore, it is expected to increase the detection threshold of the conventional imaging systems when used intraoperatively. The probe allows spectral characterization of the fluorescence and navigation in the tissue as opposed to camera imaging which is limited to the view on the surface of the tissue.

  1. Electrostatic Interactions of Fluorescent Molecules with Dielectric Interfaces Studied by Total Internal Reflection Fluorescence Correlation Spectroscopy

    PubMed Central

    Blom, Hans; Hassler, Kai; Chmyrov, Andriy; Widengren, Jerker

    2010-01-01

    Electrostatic interactions between dielectric surfaces and different fluorophores used in ultrasensitive fluorescence microscopy are investigated using objective-based Total Internal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS). The interfacial dynamics of cationic rhodamine 123 and rhodamine 6G, anionic/dianionic fluorescein, zwitterionic rhodamine 110 and neutral ATTO 488 are monitored at various ionic strengths at physiological pH. As analyzed by means of the amplitude and time-evolution of the autocorrelation function, the fluorescent molecules experience electrostatic attraction or repulsion at the glass surface depending on their charges. Influences of the electrostatic interactions are also monitored through the triplet-state population and triplet relaxation time, including the amount of detected fluorescence or the count-rate-per-molecule parameter. These TIR-FCS results provide an increased understanding of how fluorophores are influenced by the microenvironment of a glass surface, and show a promising approach for characterizing electrostatic interactions at interfaces. PMID:20386645

  2. In vivo imaging of tumor angiogenesis using fluorescence confocal videomicroscopy.

    PubMed

    Fitoussi, Victor; Faye, Nathalie; Chamming's, Foucauld; Clement, Olivier; Cuenod, Charles-Andre; Fournier, Laure S

    2013-01-01

    Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained. We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 μm in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools. The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

  3. In vivo imaging of tumor angiogenesis using fluorescence confocal videomicroscopy.

    PubMed

    Fitoussi, Victor; Faye, Nathalie; Chamming's, Foucauld; Clement, Olivier; Cuenod, Charles-Andre; Fournier, Laure S

    2013-01-01

    Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained. We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 μm in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools. The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability. PMID:24056503

  4. Stark Spectroscopy of Rubrene. II. Stark Fluorescence Spectroscopy and Fluorescence Quenching Induced by an External Electric Field.

    PubMed

    Iimori, Toshifumi; Ito, Ryuichi; Ohta, Nobuhiro

    2016-07-21

    We report Stark fluorescence spectroscopy investigation of rubrene dispersed in a poly(methyl methacrylate) film. The features of the fluorescence spectrum are analogous to those in solutions. In the Stark fluorescence spectrum, the decrease of the fluorescence quantum yield in the presence of an external electric field is observed. This result shows that the yield of nonradiative decay processes is increased by the application of an external electric field. It is known that the fluorescence quantum yield for rubrene, which is nearly unity at room temperature, depends on temperature, and a major nonradiative decay process in photoexcited rubrene is ascribed to a thermally activated intersystem crossing (ISC). Equations that express the field-induced fluorescence quenching in terms of the molecular parameters are derived from the ensemble average of electric field effects on the activation energy of the reaction rate constant in random orientation systems. The molecular parameters are then extracted from the observed data. It is inferred that the field-induced increase in the yield of other intramolecular and intermolecular photophysical processes in addition to the ISC should be taken into account. PMID:27341859

  5. Wide-field in vivo background free imaging by selective magnetic modulation of nanodiamond fluorescence

    PubMed Central

    Sarkar, Susanta K.; Bumb, Ambika; Wu, Xufeng; Sochacki, Kem A.; Kellman, Peter; Brechbiel, Martin W.; Neuman, Keir C.

    2014-01-01

    The sensitivity and resolution of fluorescence-based imaging in vivo is often limited by autofluorescence and other background noise. To overcome these limitations, we have developed a wide-field background-free imaging technique based on magnetic modulation of fluorescent nanodiamond emission. Fluorescent nanodiamonds are bright, photo-stable, biocompatible nanoparticles that are promising probes for a wide range of in vitro and in vivo imaging applications. Our readily applied background-free imaging technique improves the signal-to-background ratio for in vivo imaging up to 100-fold. This technique has the potential to significantly improve and extend fluorescent nanodiamond imaging capabilities on diverse fluorescence imaging platforms. PMID:24761300

  6. Synchronous fluorescence spectroscopy for analysis of wine and wine distillates

    NASA Astrophysics Data System (ADS)

    Andreeva, Ya.; Borisova, E.; Genova, Ts.; Zhelyazkova, Al.; Avramov, L.

    2015-01-01

    Wine and brandies are multicomponent systems and conventional fluorescence techniques, relying on recording of single emission or excitation spectra, are often insufficient. In such cases synchronous fluorescence spectra can be used for revealing the potential of the fluorescence techniques. The technique is based on simultaneously scanning of the excitation and emission wavelength with constant difference (Δλ) maintained between them. In this study the measurements were made using FluoroLog3 spectrofluorimeter (HORIBA Jobin Yvon, France) and collected for excitation and emission in the wavelength region 220 - 700 nm using wavelength interval Δλ from 10 to 100 nm in 10 nm steps. This research includes the results obtained for brandy and red wine samples. Fluorescence analysis takes advantage in the presence of natural fluorophores in wines and brandies, such as gallic, vanillic, p-coumaric, syringic, ferulic acid, umbelliferone, scopoletin and etc. Applying of synchronous fluorescence spectroscopy for analysis of these types of alcohols allows us to estimate the quality of wines and also to detect adulteration of brandies like adding of a caramel to wine distillates for imitating the quality of the original product aged in oak casks.

  7. Fluorescence Spectroscopy of Gas-phase Polycyclic Aromatic Hydrocarbons

    NASA Technical Reports Server (NTRS)

    Thomas, J. D.; Witt, A. N.

    2006-01-01

    The purpose of this investigation was to produce fluorescence spectra of polycyclic aromatic hydrocarbon (PAH) molecules in the gas-phase for comparison with blue luminescence (BL) emission observed in astrophysical sources Vijh et al. (2004, 2005a,b). The BL occurs roughly from 350 to 450 nm, with a sharp peak near 380 nm. PAHs with three to four rings, e.g. anthracene and pyrene, were found to produce luminescence in the appropriate spectral region, based on existing studies. Relatively few studies of the gas-phase fluorescence of PAHs exist; those that do exist have dealt primarily with the same samples commonly available for purchase such as pyrene and anthracene. In an attempt to understand the chemistry of the nebular environment we also obtained several nitrogen substituted PAHs from our colleagues at NASA Ames. In order to simulate the astrophysical environment we also took spectra by heating the PAHs in a flame. The flame environment counteracts the formation of eximers and permits the spectroscopy of free-flying neutral molecules. Experiments with coal tar demonstrate that fluorescence spectroscopy reveals primarily the presence of the smallest molecules, which are most abundant and which possess the highest fluorescence efficiencies. One gas-phase PAH that seems to fit the BL spectrum most closely is phenanthridine. In view of the results from the spectroscopy of coal tar, a compound containing a mixture of PAHs ranging from small to very large PAH molecules, we can not preclude the presence of larger PAHs in interstellar sources exhibiting BL.

  8. Two-dimensional fluorescence spectroscopy of laser-produced plasmas.

    PubMed

    Harilal, S S; LaHaye, N L; Phillips, M C

    2016-08-01

    We use a two-dimensional laser-induced fluorescence spectroscopy technique to measure the coupled absorption and emission properties of atomic species in plasmas produced via laser ablation of a solid aluminum target at atmospheric pressure. Emission spectra from the Al I 394.4 nm and Al I 396.15 nm transitions are measured while a frequency-doubled, continuous wave (cw) Ti:sapphire laser is tuned across the Al I 396.15 nm transition. The resulting two-dimensional spectra show the energy coupling between the two transitions via increased emission intensity for both transitions during resonant absorption of the cw laser at one transition. Time-delayed, gated detection of the emission spectrum is used to isolate resonantly excited fluorescence emission from thermally excited emission from the plasma. In addition, the tunable cw laser measures the absorption spectrum of the Al transition with ultrahigh resolution after the plasma has cooled, resulting in narrower spectral linewidths than observed in emission spectra. Our results highlight that fluorescence spectroscopy employing cw laser re-excitation after pulsed laser ablation combines benefits of both traditional emission and absorption spectroscopic methods. PMID:27472615

  9. Microenviromental investigation of polymer-bound fluorescent chelator by fluorescence microscopy and optical spectroscopy.

    PubMed

    Wang, Y; Astilean, S; Haran, G; Warshawsky, A

    2001-09-01

    8-Hydroxyquinoline-5-sulfonic acid (HQS) was immobilized onto a strong-base anion-exchange resin AG MP-1 for the purpose of microenvironment investigation, resin characterization, and possibly sensing cadmium. The maximum loading of HQS was found to be 0.9340 mmol/g of AG MP-1. A plateau for Cd complex capacity was already obtained for 0.5500 mmol of HQS/g of AG MP-1. A minicolumn experiment showed an influence of influent Cd concentration on column capacity. IR and Raman spectra proved an electrostatic mode for HQS immobilization and Cd complex formation. UV spectroscopy showed significant differences between solution and solid state for both HQS and Cd complex. A fluorescence microscopy technique was used for fluorescence spectral measurement, microdistribution imaging, and study of photobleaching of HQS and the HQS-Cd complex in the resin phase. The fluorescence of immobilized HQS was found to be red-shifted with regard to the solid-state HQS. The microdistribution of uncomplexed and Cd-complexed AG MP-1-HQS was directly visualized by fluorescence imaging, showing a nonuniform distribution. Cadmium complexation modifies the fluorescence emission of uncomplexed AG MP-1-HQS, exhibiting an increased and red-shifted emission. Significant photobleaching of the fluorescence from the Cd complex was recorded, indicating the occurrence of photochemical reactions within the microenvironment of the resin phase.

  10. Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy.

    PubMed

    Widengren, Jerker; Chmyrov, Andriy; Eggeling, Christian; Löfdahl, Per-Ake; Seidel, Claus A M

    2007-01-25

    Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1-20 ms), and demonstrate its generation via both one- and multistep excitation processes

  11. Quantification of osmotic water transport in vivo using fluorescent albumin.

    PubMed

    Morelle, Johann; Sow, Amadou; Vertommen, Didier; Jamar, François; Rippe, Bengt; Devuyst, Olivier

    2014-10-15

    Osmotic water transport across the peritoneal membrane is applied during peritoneal dialysis to remove the excess water accumulated in patients with end-stage renal disease. The discovery of aquaporin water channels and the generation of transgenic animals have stressed the need for novel and accurate methods to unravel molecular mechanisms of water permeability in vivo. Here, we describe the use of fluorescently labeled albumin as a reliable indicator of osmotic water transport across the peritoneal membrane in a well-established mouse model of peritoneal dialysis. After detailed evaluation of intraperitoneal tracer mass kinetics, the technique was validated against direct volumetry, considered as the gold standard. The pH-insensitive dye Alexa Fluor 555-albumin was applied to quantify osmotic water transport across the mouse peritoneal membrane resulting from modulating dialysate osmolality and genetic silencing of the water channel aquaporin-1 (AQP1). Quantification of osmotic water transport using Alexa Fluor 555-albumin closely correlated with direct volumetry and with estimations based on radioiodinated ((125)I) serum albumin (RISA). The low intraperitoneal pressure probably accounts for the negligible disappearance of the tracer from the peritoneal cavity in this model. Taken together, these data demonstrate the appropriateness of pH-insensitive Alexa Fluor 555-albumin as a practical and reliable intraperitoneal volume tracer to quantify osmotic water transport in vivo.

  12. Long-Term Retention of Fluorescent Quantum Dots In Vivo

    NASA Astrophysics Data System (ADS)

    Ballou, Byron; Ernst, Lauren A.; Andreko, Susan; Eructiez, Marcel P.; Lagerholm, B. Christoffer; Waggoner, Alan S.

    Quantum dots that emit in the near-infrared can be used in vivo to follow circulation, to target the reticuloendothelial system, and to map lymphatic drainage from normal tissues and tumors. We have explored the role of surface charge and passivation by polyethylene glycol in determining circulating lifetimes and sites of deposition. Use of long polyethylene glycol polymers increases circulating lifetime. Changing surface charge can partially direct quantum dots to the liver and spleen, or the lymph nodes. Quantum dots are cleared in the order liver > spleen > bone marrow > lymph nodes. Quantum dots retained by lymph nodes maintained fluorescence for two years, suggesting either that the coating is extremely stable or that some endosomes preserve quantum dot function. We also explored migration from tumors to sentinel lymph nodes using tumor models in mice; surface charge and size make little difference to transport from tumors. Antibody and Fab-conjugates of polymer-coated quantum dots failed to target tumors in vivo, probably because of size.

  13. Frequency-domain fluorescence spectroscopy of human stratum corneum

    NASA Astrophysics Data System (ADS)

    Garrison, Michael D.; Potts, Russell O.; Abraham, William

    1994-08-01

    The intercellular lipid lamellae of mammalian stratum corneum (SC) constitute the major barrier to percutaneous penetration of drugs and other solute molecules. In order to understand the barrier property of skin on a molecular level, we have initiated fluorescence spectroscopic investigation of the membranous structures of the SC and related model systems using the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Incorporated into distearoylphosphatidylcholine and stratum corneum bilayers, DPH fluorescence reflected the change in lipid structure under thermal and chemical perturbations. Using a multiharmonic frequency approach, we measured the fluorescence lifetime and rotational correlation times for DPH in these systems. Our data indicated that a biexponential decay ((tau) 1 approximately equals 9 ns, (tau) 2 approximately equals 1.5 ns) described the intensity decay, while a hindered rotor model ((phi) approximately equals 5 ns, r(infinity ) approximately equals 0.3) described the anisotropy decay. These parameters reported the known thermotropic phase transition in porcine stratum corneum, and the influence of the penetration enhancer oleic acid in human epidermis. Thus, we have shown frequency- domain fluorescence spectroscopy to be a facile and powerful tool for monitoring the permeability of a solid tissue such as the SC.

  14. Brain cancer probed by native fluorescence and stokes shift spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhou, Yan; Liu, Cheng-hui; He, Yong; Pu, Yang; Li, Qingbo; Wang, Wei; Alfano, Robert R.

    2012-12-01

    Optical biopsy spectroscopy was applied to diagnosis human brain cancer in vitro. The spectra of native fluorescence, Stokes shift and excitation spectra were obtained from malignant meningioma, benign, normal meningeal tissues and acoustic neuroma benign tissues. The wide excitation wavelength ranges were used to establish the criterion for distinguishing brain diseases. The alteration of fluorescence spectra between normal and abnormal brain tissues were identified by the characteristic fluorophores under the excitation with UV to visible wavelength range. It was found that the ratios of the peak intensities and peak position in both spectra of fluorescence and Stokes shift may be used to diagnose human brain meninges diseases. The preliminary analysis of fluorescence spectral data from cancer and normal meningeal tissues by basic biochemical component analysis model (BBCA) and Bayes classification model based on statistical methods revealed the changes of components, and classified the difference between cancer and normal human brain meningeal tissues in a predictions accuracy rate is 0.93 in comparison with histopathology and immunohistochemistry reports (gold standard).

  15. An analog filter approach to frequency domain fluorescence spectroscopy

    DOE PAGES

    Trainham, Clifford P.; O'Neill, Mary D.; McKenna, Ian J.

    2015-10-01

    The rate equations found in frequency domain fluorescence spectroscopy are the same as those found in electronics under analog filter theory. Laplace transform methods are a natural way to solve the equations, and the methods can provide solutions for arbitrary excitation functions. The fluorescence terms can be modeled as circuit components and cascaded with drive and detection electronics to produce a global transfer function. Electronics design tools such as Spicea can be used to model fluorescence problems. In applications, such as remote sensing, where detection electronics are operated at high gain and limited bandwidth, a global modeling of the entiremore » system is important, since the filter terms of the drive and detection electronics affect the measured response of the fluorescence signals. Furthermore, the techniques described here can be used to separate signals from fast and slow fluorophores emitting into the same spectral band, and data collection can be greatly accelerated by means of a frequency comb driver waveform and appropriate signal processing of the response.« less

  16. Fluorescence spectroscopy for endogenous porphyrins in human facial skin

    NASA Astrophysics Data System (ADS)

    Seo, I.; Tseng, S. H.; Cula, G. O.; Bargo, P. R.; Kollias, N.

    2009-02-01

    The activity of certain bacteria in skin is known to correlate to the presence of porphyrins. In particular the presence of coproporphyrin produced by P.acnes inside plugged pores has been correlated to acne vulgaris. Another porphyrin encountered in skin is protoporphyrin IX, which is produced by the body in the pathway for production of heme. In the present work, a fluorescence spectroscopy system was developed to measure the characteristic spectrum and quantify the two types of porphyrins commonly present in human facial skin. The system is comprised of a Xe lamp both for fluorescence excitation and broadband light source for diffuse reflectance measurements. A computer-controlled filter wheel enables acquisition of sequential spectra, first excited by blue light at 405 nm then followed by the broadband light source, at the same location. The diffuse reflectance spectrum was used to correct the fluorescence spectrum due to the presence of skin chromophores, such as blood and melanin. The resulting fluorescence spectra were employed for the quantification of porphyrin concentration in a population of healthy subjects. The results show great variability on the concentration of these porphyrins and further studies are being conducted to correlate them with skin conditions such as inflammation and acne vulgaris.

  17. An Analog Filter Approach to Frequency Domain Fluorescence Spectroscopy.

    PubMed

    Trainham, R; O'Neill, M; McKenna, I J

    2015-11-01

    The rate equations found in frequency domain fluorescence spectroscopy are the same as those found in electronics under analog filter theory. Laplace transform methods are a natural way to solve the equations, and the methods can provide solutions for arbitrary excitation functions. The fluorescence terms can be modelled as circuit components and cascaded with drive and detection electronics to produce a global transfer function. Electronics design tools such as SPICE can be used to model fluorescence problems. In applications, such as remote sensing, where detection electronics are operated at high gain and limited bandwidth, a global modelling of the entire system is important, since the filter terms of the drive and detection electronics affect the measured response of the fluorescence signals. The techniques described here can be used to separate signals from fast and slow fluorophores emitting into the same spectral band, and data collection can be greatly accelerated by means of a frequency comb driver waveform and appropriate signal processing of the response. The simplification of the analysis mathematics, and the ability to model the entire detection chain, make it possible to develop more compact instruments for remote sensing applications. PMID:26429345

  18. An analog filter approach to frequency domain fluorescence spectroscopy

    SciTech Connect

    Trainham, Clifford P.; O'Neill, Mary D.; McKenna, Ian J.

    2015-10-01

    The rate equations found in frequency domain fluorescence spectroscopy are the same as those found in electronics under analog filter theory. Laplace transform methods are a natural way to solve the equations, and the methods can provide solutions for arbitrary excitation functions. The fluorescence terms can be modeled as circuit components and cascaded with drive and detection electronics to produce a global transfer function. Electronics design tools such as Spicea can be used to model fluorescence problems. In applications, such as remote sensing, where detection electronics are operated at high gain and limited bandwidth, a global modeling of the entire system is important, since the filter terms of the drive and detection electronics affect the measured response of the fluorescence signals. Furthermore, the techniques described here can be used to separate signals from fast and slow fluorophores emitting into the same spectral band, and data collection can be greatly accelerated by means of a frequency comb driver waveform and appropriate signal processing of the response.

  19. The study of blue LED to induce fluorescence spectroscopy and fluorescence imaging for oral carcinoma detection

    NASA Astrophysics Data System (ADS)

    Zheng, Longjiang; Hu, Yuanting

    2009-07-01

    Fluorescence spectroscopy and fluorescence imaging diagnosis of malignant lesions provides us with a new method to diagnose diseases in precancerous stage. Early diagnosis of disease has significant importance in cancer treatment, because most cancers can be cured well in precancerous, especially when the diffusion of cancer is limited in a restricted region. In this study, Golden hamster models were applied to 5% 9, 10 dimethyl-1, 2-benzanthracene (DMBA) to induce hamster buccal cheek pouch carcinoma three times a week. Rose Bengal, which has been used in clinican for years and avoids visible side-effect to human was chosen as photosensitizer. 405 nm blue LED was used to induce the fluorescence of photosensitizer. After topical application of photosensitizer, characteristic red emission fluorescence peak was observed around 600nm. Similar, normal oral cavity has special luminescence around 480nm. Fluorescence spectroscopy technology is based on analysing emission peaks of photosensitizer in the areas of oral carcinoma, moreover, red-to-green (IR/IG) intensity ratio is also applied as a diagnostic algorithm. A CCD which is connected with a computer is used to take pictures at carcinoma areas through different filters. Fluorescence images from normal hamster buccal cheek pouch are compared with those from carcinogen-induced models of carcinoma, and morphological differences between normal and lesion tissue can be distinguished. The pictures are analyzed by Matlab and shown on the screen of computer. This paper demonstrates that Rose Bengal could be used as photosensitizer to detect oral carcinoma, and blue LED as excitation source could not only have a good effect to diagnose oral carcinoma, but also decrease cost greatly.

  20. In vivo fluorescence imaging of lysosomes: a potential technique to follow dye accumulation in the context of PDT?

    NASA Astrophysics Data System (ADS)

    Devoisselle, Jean-Marie; Mordon, Serge R.; Soulie-Begu, Sylvie

    1995-03-01

    Lysosomes and intracellular acidic compartments seem to play an important role in the context of PDT. Some photosensitizers are localized in the lysosomes of tumor-associated macrophages. Liposomes, which are lysosomotropic drug carriers, are used to deliver photosensitizers in tumors. Liposomes are taken up by the liver cells after intravenous injection. Intracellular pathway and liposomes localization in the different liver cells require sacrifice of the animals, cell separation, and observation by electronic microscopy. Little is known about liposomes kinetic uptake by the acidic intracellular compartments in vivo. We propose in this study a new method to follow liposomes uptake in the liver in vivo using a fluorescent pH-sensitive probe. We have already demonstrated the ability of fluorescence spectroscopy and imaging using a pH-dependent probe to monitor pH in living tissues. As pH of lysosome is very low, the kinetic of liposome uptake in this intracellular acidic compartment is followed by monitoring the pH of the whole liver in vivo and ex vivo. Liposomes-encapsulated carboxyfluorescein are prepared by the sonication procedure. Carboxyfluorescein is used at high concentration (100 mM) in order to quench its fluorescence. Liposomes are injected to Wistar rats into the peinil vein. After laparotomy, fluorescence spectra and images are recorded during two hours. Results show a rapid fluorescence increase followed by a slow phase of fluorescence decrease. pH decreases from physiological value to 6.0. After sacrifice and flush with cold saline solution, pH of liver ex vivo is found to be 5.0 - 5.5. These data show a rapid clearance of released dye and an uptake of liposomes by the liver cells and, as liposomes penetrate in the acidic compartment, dye is released from liposomes and is delivered in lysosomes leading to the decrease of pH.

  1. Terahertz spectroscopy of pigmentary skin nevi in vivo

    NASA Astrophysics Data System (ADS)

    Zaitsev, K. I.; Chernomyrdin, N. V.; Kudrin, K. G.; Reshetov, I. V.; Yurchenko, S. O.

    2015-09-01

    Pigmentary skin nevi are studied in vivo using terahertz pulsed spectroscopy. Dielectric parameters of healthy skin and dysplastic and nondysplastic nevi are reconstructed and analyzed. The fact that complex permittivities of the samples substantially differ in the terahertz spectral range can be used for early noninvasive diagnostics of dysplastic nevi, which are precursors of melanoma (the most dangerous skin cancer). A method is proposed to identify various dysplastic and nondysplastic nevi using the analysis of terahertz dielectric characteristics. It is demonstrated that terahertz pulsed spectroscopy is promising for early noninvasive diagnostics of dysplastic nevi and melanomas of the skin.

  2. Multiphoton fluorescence recovery after photobleaching: Advancements for novel in vivo applications

    NASA Astrophysics Data System (ADS)

    Sullivan, Kelley Diane

    Multiphoton fluorescence recovery after photobleaching (MP-FRAP) is a laser microscopy technique used to probe the transport properties of macromolecules in biological systems. MP-FRAP utilizes two-photon fluorescence and photobleaching to produce a three-dimensionally resolved diffusion coefficient for an ensemble of molecules in the region of the two-photon focal volume. This thesis describes two fundamental improvements to the MP-FRAP technique, which are vital steps to enable MP-FRAP to be applied to the complex in vivo environment. In Chapter 1, we lay the groundwork for our discussion of these advancements by introducing the MP-FRAP technique and the physics upon which it is based. We begin with a description of fluorescence and diffusion and discuss their importance in biomedical research. Next, we describe how two-photon fluorescence and photobleaching are applied to a diffusing system to measure the diffusion coefficient via fluorescence recovery after photobleaching (FRAP). Then, we take the reader through the evolution of FRAP, which leads to the application of two- photon fluorescence and photobleaching to produce MP-FRAP. Along the way, we highlight applications and advancements of the FRAP techniques, and introduce fluorescence correlation spectroscopy, a popular complement to FRAP. In Chapter 2, we collect the experimental methods for the studies presented in Chapters 3 and 4. We begin with an in-depth discussion of our work to build and troubleshoot our MP-FRAP apparatus, followed by a detailed description of our data analysis protocol. Next, we delve into the specific methods for producing computer generated data and fits, as well as in vitro and in vivo experimental data, for our work in Chap. 3 on improving MP-FRAP to measure diffusion in the presence of convective flow. We end with a description of the Monte Carlo algorithm we developed for our work in Chap. 4 to model diffusion and multiphoton fluorescence recovery after photobleaching in the

  3. Quantum process tomography by 2D fluorescence spectroscopy

    SciTech Connect

    Pachón, Leonardo A.; Marcus, Andrew H.; Aspuru-Guzik, Alán

    2015-06-07

    Reconstruction of the dynamics (quantum process tomography) of the single-exciton manifold in energy transfer systems is proposed here on the basis of two-dimensional fluorescence spectroscopy (2D-FS) with phase-modulation. The quantum-process-tomography protocol introduced here benefits from, e.g., the sensitivity enhancement ascribed to 2D-FS. Although the isotropically averaged spectroscopic signals depend on the quantum yield parameter Γ of the doubly excited-exciton manifold, it is shown that the reconstruction of the dynamics is insensitive to this parameter. Applications to foundational and applied problems, as well as further extensions, are discussed.

  4. Quantum process tomography by 2D fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Pachón, Leonardo A.; Marcus, Andrew H.; Aspuru-Guzik, Alán

    2015-06-01

    Reconstruction of the dynamics (quantum process tomography) of the single-exciton manifold in energy transfer systems is proposed here on the basis of two-dimensional fluorescence spectroscopy (2D-FS) with phase-modulation. The quantum-process-tomography protocol introduced here benefits from, e.g., the sensitivity enhancement ascribed to 2D-FS. Although the isotropically averaged spectroscopic signals depend on the quantum yield parameter Γ of the doubly excited-exciton manifold, it is shown that the reconstruction of the dynamics is insensitive to this parameter. Applications to foundational and applied problems, as well as further extensions, are discussed.

  5. Fluorescence-excitation and Emission Spectroscopy on Single FMO Complexes.

    PubMed

    Löhner, Alexander; Ashraf, Khuram; Cogdell, Richard J; Köhler, Jürgen

    2016-08-22

    In green-sulfur bacteria sunlight is absorbed by antenna structures termed chlorosomes, and transferred to the RC via the Fenna-Matthews-Olson (FMO) complex. FMO consists of three monomers arranged in C3 symmetry where each monomer accommodates eight Bacteriochlorophyll a (BChl a) molecules. It was the first pigment-protein complex for which the structure has been determined with high resolution and since then this complex has been the subject of numerous studies both experimentally and theoretically. Here we report about fluorescence-excitation spectroscopy as well as emission spectroscopy from individual FMO complexes at low temperatures. The individual FMO complexes are subjected to very fast spectral fluctuations smearing out any possible different information from the ensemble data that were recorded under the same experimental conditions. In other words, on the time scales that are experimentally accessible by single-molecule techniques, the FMO complex exhibits ergodic behaviour.

  6. Detectors for single-molecule fluorescence imaging and spectroscopy

    PubMed Central

    MICHALET, X.; SIEGMUND, O.H.W.; VALLERGA, J.V.; JELINSKY, P.; MILLAUD, J.E.; WEISS, S.

    2010-01-01

    Single-molecule observation, characterization and manipulation techniques have recently come to the forefront of several research domains spanning chemistry, biology and physics. Due to the exquisite sensitivity, specificity, and unmasking of ensemble averaging, single-molecule fluorescence imaging and spectroscopy have become, in a short period of time, important tools in cell biology, biochemistry and biophysics. These methods led to new ways of thinking about biological processes such as viral infection, receptor diffusion and oligomerization, cellular signaling, protein-protein or protein-nucleic acid interactions, and molecular machines. Such achievements require a combination of several factors to be met, among which detector sensitivity and bandwidth are crucial. We examine here the needed performance of photodetectors used in these types of experiments, the current state of the art for different categories of detectors, and actual and future developments of single-photon counting detectors for single-molecule imaging and spectroscopy. PMID:20157633

  7. Fluorescence-excitation and Emission Spectroscopy on Single FMO Complexes

    PubMed Central

    Löhner, Alexander; Ashraf , Khuram; Cogdell, Richard J.; Köhler, Jürgen

    2016-01-01

    In green-sulfur bacteria sunlight is absorbed by antenna structures termed chlorosomes, and transferred to the RC via the Fenna-Matthews-Olson (FMO) complex. FMO consists of three monomers arranged in C3 symmetry where each monomer accommodates eight Bacteriochlorophyll a (BChl a) molecules. It was the first pigment-protein complex for which the structure has been determined with high resolution and since then this complex has been the subject of numerous studies both experimentally and theoretically. Here we report about fluorescence-excitation spectroscopy as well as emission spectroscopy from individual FMO complexes at low temperatures. The individual FMO complexes are subjected to very fast spectral fluctuations smearing out any possible different information from the ensemble data that were recorded under the same experimental conditions. In other words, on the time scales that are experimentally accessible by single-molecule techniques, the FMO complex exhibits ergodic behaviour. PMID:27545197

  8. Identification of active fluorescence stained bacteria by Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Krause, Mario; Beyer, Beatrice; Pietsch, Christian; Radt, Benno; Harz, Michaela; Rösch, Petra; Popp, Jürgen

    2008-04-01

    Microorganisms can be found everywhere e.g. in food both as useful ingredients or harmful contaminations causing food spoilage. Therefore, a fast and easy to handle analysis method is needed to detect bacteria in different kinds of samples like meat, juice or air to decide if the sample is contaminated by harmful microorganisms. Conventional identification methods in microbiology require always cultivation and therefore are time consuming. In this contribution we present an analysis approach to identify fluorescence stained bacteria on strain level by means of Raman spectroscopy. The stained bacteria are highlighted and can be localized easier against a complex sample environment e.g. in food. The use of Raman spectroscopy in combination with chemometrical methods allows the identification of single bacteria within minutes.

  9. Identification of Atherosclerotic Plaques in Carotid Artery by Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Rocha, Rick; Villaverde, Antonio Balbin; Silveira, Landulfo; Costa, Maricília Silva; Alves, Leandro Procópio; Pasqualucci, Carlos Augusto; Brugnera, Aldo

    2008-04-01

    The aim of this work was to identify the presence of atherosclerotic plaques in carotid artery using the Fluorescence Spectroscopy. The most important pathogeny in the cardiovascular disorders is the atherosclerosis, which may affect even younger individuals. With approximately 1.2 million heart attacks and 750,000 strokes afflicting an aging American population each year, cardiovascular disease remains the number one cause of death. Carotid artery samples were obtained from the Autopsy Service at the University of São Paulo (São Paulo, SP, Brazil) taken from cadavers. After a histopathological analysis the 60 carotid artery samples were divided into two groups: normal (26) and atherosclerotic plaques (34). Samples were irradiated with the wavelength of 488 nm from an Argon laser. A 600 μm core optical fiber, coupled to the Argon laser, was used for excitation of the sample, whereas another 600 optical fiber, coupled to the spectrograph entrance slit, was used for collecting the fluorescence from the sample. Measurements were taken at different points on each sample and then averaged. Fluorescence spectra showed a single broad line centered at 549 nm. The fluorescence intensity for each sample was calculated by subtracting the intensity at the peak (550 nm) and at the bottom (510 nm) and then data were statistically analyzed, looking for differences between both groups of samples. ANOVA statistical test showed a significant difference (p<0,05) between both types of tissues, with regard to the fluorescence peak intensities. Our results indicate that this technique could be used to detect the presence of the atherosclerotic in carotid tissue.

  10. Design and evaluation of a device for fast multispectral time-resolved fluorescence spectroscopy and imaging

    PubMed Central

    Yankelevich, Diego R.; Ma, Dinglong; Liu, Jing; Sun, Yang; Sun, Yinghua; Bec, Julien; Elson, Daniel S.; Marcu, Laura

    2014-01-01

    The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8–7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence

  11. Design and evaluation of a device for fast multispectral time-resolved fluorescence spectroscopy and imaging.

    PubMed

    Yankelevich, Diego R; Ma, Dinglong; Liu, Jing; Sun, Yang; Sun, Yinghua; Bec, Julien; Elson, Daniel S; Marcu, Laura

    2014-03-01

    The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8-7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence lifetime

  12. Design and evaluation of a device for fast multispectral time-resolved fluorescence spectroscopy and imaging

    SciTech Connect

    Yankelevich, Diego R.; Ma, Dinglong; Liu, Jing; Sun, Yang; Sun, Yinghua; Bec, Julien; Marcu, Laura; Elson, Daniel S.

    2014-03-15

    The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8–7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence

  13. Visualizing Oxazine 4 nerve-specific fluorescence ex vivo in frozen tissue sections

    NASA Astrophysics Data System (ADS)

    Barth, Connor W.; Gibbs, Summer L.

    2016-03-01

    Nerve damage plagues surgical outcomes and remains a major burden for patients, surgeons, and the healthcare system. Fluorescence image-guided surgery using nerve specific small molecule fluorophores offers a solution to diminish surgical nerve damage through improved intraoperative nerve identification and visualization. Oxazine 4 has shown superior nerve specificity in initial testing in vivo, while exhibiting a red shifted excitation and emission spectra compared to other nerve-specific fluorophores. However, Oxazine 4 does not exhibit near-infrared (NIR) excitation and emission, which would be ideal to improve penetration depth and nerve signal to background ratios for in vivo imaging. Successful development of a NIR nerve-specific fluorophore will require understanding of the molecular target of fluorophore nerve specificity. While previous small molecule nerve-specific fluorophores have demonstrated excellent ex vivo nerve specificity, Oxazine 4 ex vivo nerve specific fluorescence has been difficult to visualize. In the present study, we examined each step of the ex vivo fluorescence microscopy sample preparation procedure to discover how in vivo nerve-specific fluorescence is changed during ex vivo tissue sample preparation. Through step-by-step examination we found that Oxazine 4 fluorescence was significantly diminished by washing and mounting tissue sections for microscopy. A method to preserve Oxazine 4 nerve specific fluorescence ex vivo was determined, which can be utilized for visualization by fluorescence microscopy.

  14. Classification of plum spirit drinks by synchronous fluorescence spectroscopy.

    PubMed

    Sádecká, J; Jakubíková, M; Májek, P; Kleinová, A

    2016-04-01

    Synchronous fluorescence spectroscopy was used in combination with principal component analysis (PCA) and linear discriminant analysis (LDA) for the differentiation of plum spirits according to their geographical origin. A total of 14 Czech, 12 Hungarian and 18 Slovak plum spirit samples were used. The samples were divided in two categories: colorless (22 samples) and colored (22 samples). Synchronous fluorescence spectra (SFS) obtained at a wavelength difference of 60 nm provided the best results. Considering the PCA-LDA applied to the SFS of all samples, Czech, Hungarian and Slovak colorless samples were properly classified in both the calibration and prediction sets. 100% of correct classification was also obtained for Czech and Hungarian colored samples. However, one group of Slovak colored samples was classified as belonging to the Hungarian group in the calibration set. Thus, the total correct classifications obtained were 94% and 100% for the calibration and prediction steps, respectively. The results were compared with those obtained using near-infrared (NIR) spectroscopy. Applying PCA-LDA to NIR spectra (5500-6000 cm(-1)), the total correct classifications were 91% and 92% for the calibration and prediction steps, respectively, which were slightly lower than those obtained using SFS.

  15. Fluorescence Spectroscopy and Chemometric Modeling for Bioprocess Monitoring

    PubMed Central

    Faassen, Saskia M.; Hitzmann, Bernd

    2015-01-01

    On-line sensors for the detection of crucial process parameters are desirable for the monitoring, control and automation of processes in the biotechnology, food and pharma industry. Fluorescence spectroscopy as a highly developed and non-invasive technique that enables the on-line measurements of substrate and product concentrations or the identification of characteristic process states. During a cultivation process significant changes occur in the fluorescence spectra. By means of chemometric modeling, prediction models can be calculated and applied for process supervision and control to provide increased quality and the productivity of bioprocesses. A range of applications for different microorganisms and analytes has been proposed during the last years. This contribution provides an overview of different analysis methods for the measured fluorescence spectra and the model-building chemometric methods used for various microbial cultivations. Most of these processes are observed using the BioView® Sensor, thanks to its robustness and insensitivity to adverse process conditions. Beyond that, the PLS-method is the most frequently used chemometric method for the calculation of process models and prediction of process variables. PMID:25942644

  16. Fluorescence spectroscopy and chemometric modeling for bioprocess monitoring.

    PubMed

    Faassen, Saskia M; Hitzmann, Bernd

    2015-01-01

    On-line sensors for the detection of crucial process parameters are desirable for the monitoring, control and automation of processes in the biotechnology, food and pharma industry. Fluorescence spectroscopy as a highly developed and non-invasive technique that enables the on-line measurements of substrate and product concentrations or the identification of characteristic process states. During a cultivation process significant changes occur in the fluorescence spectra. By means of chemometric modeling, prediction models can be calculated and applied for process supervision and control to provide increased quality and the productivity of bioprocesses. A range of applications for different microorganisms and analytes has been proposed during the last years. This contribution provides an overview of different analysis methods for the measured fluorescence spectra and the model-building chemometric methods used for various microbial cultivations. Most of these processes are observed using the BioView® Sensor, thanks to its robustness and insensitivity to adverse process conditions. Beyond that, the PLS-method is the most frequently used chemometric method for the calculation of process models and prediction of process variables.

  17. Fluorescence spectroscopy and chemometric modeling for bioprocess monitoring.

    PubMed

    Faassen, Saskia M; Hitzmann, Bernd

    2015-01-01

    On-line sensors for the detection of crucial process parameters are desirable for the monitoring, control and automation of processes in the biotechnology, food and pharma industry. Fluorescence spectroscopy as a highly developed and non-invasive technique that enables the on-line measurements of substrate and product concentrations or the identification of characteristic process states. During a cultivation process significant changes occur in the fluorescence spectra. By means of chemometric modeling, prediction models can be calculated and applied for process supervision and control to provide increased quality and the productivity of bioprocesses. A range of applications for different microorganisms and analytes has been proposed during the last years. This contribution provides an overview of different analysis methods for the measured fluorescence spectra and the model-building chemometric methods used for various microbial cultivations. Most of these processes are observed using the BioView® Sensor, thanks to its robustness and insensitivity to adverse process conditions. Beyond that, the PLS-method is the most frequently used chemometric method for the calculation of process models and prediction of process variables. PMID:25942644

  18. Remote excitation fluorescence correlation spectroscopy using silver nanowires

    NASA Astrophysics Data System (ADS)

    Su, Liang; Yuan, Haifeng; Lu, Gang; Hofkens, Johan; Roeffaers, Maarten; Uji-i, Hiroshi

    2014-11-01

    Fluorescence correlation spectroscopy (FCS), a powerful tool to resolve local properties, dynamical process of molecules, rotational and translational diffusion motions, relies on the fluctuations of florescence observables in the observation volume. In the case of rare transition events or small dynamical fluctuations, FCS requires few molecules or even single molecules in the observation volume at a time to minimize the background signals. Metal nanoparticle which possess unique localized surface plasmon resonance (LSPR) have been used to reduce the observation volume down to sub-diffraction limited scale while maintain at high analyst concentration up to tens of micromolar. Nevertheless, the applications of functionalized nanoparticles in living cell are limited due to the continuous diffusion after cell uptake, which makes it difficult to target the region of interests in the cell. In this work, we demonstrate the use of silver nanowires for remote excitation FCS on fluorescent molecules in solution. By using propagation surface plasmon polaritons (SPPs) which supported by the silver nanowire to excite the fluorescence, both illumination and observation volume can be reduced simultaneously. In such a way, less perturbation is induced to the target region, and this will broaden the application scope of silver nanowire as tip in single cell endoscopy.

  19. Laser-induced fluorescence and dispersed fluorescence spectroscopy of jet-cooled 1-phenylpropargyl radical

    NASA Astrophysics Data System (ADS)

    Reilly, Neil J.; Nakajima, Masakazu; Gibson, Bligh A.; Schmidt, Timothy W.; Kable, Scott H.

    2009-04-01

    The D1(A2″)-D0(A2″) electronic transition of the resonance-stabilized 1-phenylpropargyl radicalooled discharge of 3-phenyl-1-propyne, has been investigated in detail by laser-induced fluorescence excitation and dispersed single vibronic level fluorescence (SVLF) spectroscopy. The transition is dominated by the origin band at 21 007 cm-1, with weaker Franck-Condon activity observed in a' fundamentals and even overtones and combinations of a″ symmetry. Ab initio and density functional theory calculations of the D0 and D1 geometries and frequencies were performed to support and guide the experimental assignments throughout. Analysis of SVLF spectra from 16 D1 vibronic levels has led to the assignment of 15 fundamental frequencies in the excited state and 19 fundamental frequencies in the ground state; assignments for many more normal modes not probed directly by fluorescence spectroscopy are also suggested. Duschinsky mixing, in which the excited state normal modes are rotated with respect to the ground state modes, is prevalent throughout, in vibrations of both a' and a″ symmetry.

  20. Evaluation of a fiber-optic fluorescence spectroscopy system to assist neurosurgical tumor resections

    NASA Astrophysics Data System (ADS)

    Ilias, Michail A.; Richter, Johan; Westermark, Frida; Brantmark, Martin; Andersson-Engels, Stefan; Wårdell, Karin

    2007-07-01

    The highly malignant brain tumor, glioblastoma multiforme, is difficult to totally resect without aid due to its infiltrative way of growing and its morphological similarities to surrounding functioning brain under direct vision in the operating field. The need for an inexpensive and robust real-time visualizing system for resection guiding in neurosurgery has been formulated by research groups all over the world. The main goal is to develop a system that helps the neurosurgeon to make decisions during the surgical procedure. A compact fiber optic system using fluorescence spectroscopy has been developed for guiding neurosurgical resections. The system is based on a high power light emitting diode at 395 nm and a spectrometer. A fiber bundle arrangement is used to guide the excitation light and fluorescence light between the instrument and the tissue target. The system is controlled through a computer interface and software package especially developed for the application. This robust and simple instrument has been evaluated in vivo both on healthy skin but also during a neurosurgical resection procedure. Before surgery the patient received orally a low dose of 5-aminolevulinic acid, converted to the fluorescence tumor marker protoporphyrin IX in the malignant cells. Preliminary results indicate that PpIX fluorescence and brain tissue autofluorescence can be recorded with the help of the developed system intraoperatively during resection of glioblastoma multiforme.

  1. Probe pressure effects on human skin diffuse reflectance and fluorescence spectroscopy measurements.

    PubMed

    Lim, Liang; Nichols, Brandon; Rajaram, Narasimhan; Tunnell, James W

    2011-01-01

    Diffuse reflectance and fluorescence spectroscopy are popular research techniques for noninvasive disease diagnostics. Most systems include an optical fiber probe that transmits and collects optical spectra in contact with the suspected lesion. The purpose of this study is to investigate probe pressure effects on human skin spectroscopic measurements. We conduct an in-vivo experiment on human skin tissue to study the short-term (<2 s) and long-term (>30 s) effects of probe pressure on diffuse reflectance and fluorescence measurements. Short-term light probe pressure (P0<9 mN∕mm2) effects are within 0 ± 10% on all physiological properties extracted from diffuse reflectance and fluorescence measurements, and less than 0±5% for diagnostically significant physiological properties. Absorption decreases with site-specific variations due to blood being compressed out of the sampled volume. Reduced scattering coefficient variation is site specific. Intrinsic fluorescence shows a large standard error, although no specific pressure-related trend is observed. Differences in tissue structure and morphology contribute to site-specific probe pressure effects. Therefore, the effects of pressure can be minimized when the pressure is small and applied for a short amount of time; however, long-term and large pressures induce significant distortions in measured spectra. PMID:21280899

  2. Probe pressure effects on human skin diffuse reflectance and fluorescence spectroscopy measurements

    PubMed Central

    Lim, Liang; Nichols, Brandon; Rajaram, Narasimhan; Tunnell, James W.

    2011-01-01

    Diffuse reflectance and fluorescence spectroscopy are popular research techniques for noninvasive disease diagnostics. Most systems include an optical fiber probe that transmits and collects optical spectra in contact with the suspected lesion. The purpose of this study is to investigate probe pressure effects on human skin spectroscopic measurements. We conduct an in-vivo experiment on human skin tissue to study the short-term (<2 s) and long-term (>30 s) effects of probe pressure on diffuse reflectance and fluorescence measurements. Short-term light probe pressure (P0 < 9 mN∕mm2) effects are within 0 ± 10% on all physiological properties extracted from diffuse reflectance and fluorescence measurements, and less than 0 ± 5% for diagnostically significant physiological properties. Absorption decreases with site-specific variations due to blood being compressed out of the sampled volume. Reduced scattering coefficient variation is site specific. Intrinsic fluorescence shows a large standard error, although no specific pressure-related trend is observed. Differences in tissue structure and morphology contribute to site-specific probe pressure effects. Therefore, the effects of pressure can be minimized when the pressure is small and applied for a short amount of time; however, long-term and large pressures induce significant distortions in measured spectra. PMID:21280899

  3. Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein

    PubMed Central

    Guan, Yinghua; Meurer, Matthias; Raghavan, Sarada; Rebane, Aleksander; Lindquist, Jake R.; Santos, Sofia; Kats, Ilia; Davidson, Michael W.; Mazitschek, Ralph; Hughes, Thomas E.; Drobizhev, Mikhail; Knop, Michael; Shah, Jagesh V.

    2015-01-01

    We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein–protein interactions. We also use MPE-FCCS to detect drug–protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells. PMID:25877871

  4. Hazards and benefits of in-vivo Raman spectroscopy of human skin

    NASA Astrophysics Data System (ADS)

    Carter, Elizabeth A.; Williams, Adrian C.; Barry, Brian W.; Edwards, Howell G.

    1999-04-01

    The resurgence of Raman spectroscopy, in the late 1980's has led to an increase in the use of the technique for the analysis of biological tissues. Consequently, Raman spectroscopy is now regarded to be a well-established non- invasive, non-destructive technique, which is used to obtain good quality spectra from biological tissues with minimal fluorescence. What is presently of interest to our group is to develop further and establish the technique for in vivo investigations of healthy and diseased skin. This presentation discusses some potentially valuable clinical applications of the technique, and also highlights some of the experimental difficulties that were encountered when examining patients who were receiving treatment for psoriasis.

  5. Chemical analysis in vivo and in vitro by Raman spectroscopy – from single cells to humans

    PubMed Central

    Wachsmann-Hogiu, Sebastian; Weeks, Tyler

    2009-01-01

    Summary The gold standard for clinical diagnostics of tissues is immunofluorescence staining. Toxicity of many fluorescent dyes precludes their application in vivo. Raman spectroscopy, a chemically specific, label-free diagnostic technique, is rapidly gaining in acceptance as a powerful alternative. It has the ability to probe the chemical composition of biological materials in a nondestructive and mostly non-perturbing manner. We review the most recent developments in Raman spectroscopy in the life sciences, detailing advances in technology that have improved the ability to screen for diseases. Its role in the monitoring of biological function and mapping the intracellular chemical microenvironment will be discussed. Applications including endoscopy, surface-enhanced Raman scattering (SERS), and coherent Raman scattering (CRS) will be reviewed. PMID:19268566

  6. Assessing Raw and Treated Water Quality Using Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Bridgeman, J.; Baker, A.

    2006-12-01

    To date, much fluorescence spectroscopy work has focused on the use of techniques to characterize pollution in river water and to fingerprint pollutants such as, inter alia, treated and raw sewage effluent. In the face of tightening water quality standards associated with disinfection byproducts, there exists the need for a surrogate THM parameter which can be measured accurately and quickly at the water treatment works and which will give a satisfactory indication of the THM concentration leaving the water treatment works. In addition, water treatment works and distribution system managers require tools which are simple and quick, yet robust, to monitor plant and unit process performance. We extend the use of fluorescence techniques from raw water quality monitoring to (1) the monitoring of water treatment works intakes and the assessment of water treatment works performance by (2) assessing the removal of dissolved organic matter (DOM) through the unit process stages of various water treatment works treating different raw waters and (3) examining the prevalence of microbiological activity found at service reservoirs in the downstream distribution system. 16 surface water treatment works were selected in the central region of the UK and samples taken at works' intakes, downstream of each unit process, and in the distribution systems. The intakes selected abstract water from a broad range of upland and lowland water sources with varying natural and anthropogenic pollutant inputs and significantly different flows. The treatment works selected offer a range of different, but relatively standard, unit processes. The results demonstrate that raw waters exhibit more fluorescence than (partially) treated waters. However, noticeable differences between each site are observed. Furthermore, differences in unit process performance between works are also identified and quantified. Across all sites, treatment with Granular Activated Carbon is found to yield a significant

  7. Fluorescence imaging and spectroscopy of ethyl nile blue A in animal models of (pre)malignancies.

    PubMed

    van Staveren, H J; Speelman, O C; Witjes, M J; Cincotta, L; Star, W M

    2001-01-01

    Discrimination between normal and premalignant tissues by fluorescence imaging and/or spectroscopy may be enhanced by a tumor-localizing fluorescent drug. Ethyl Nile Blue A (EtNBA), a dye with no phototoxic activity, was investigated for this purpose. The pharmacokinetics and tissue-localizing properties were investigated in a rat palate model with chemically induced premalignant mucosal lesions (0.5 mg/kg EtNBA intravenous [i.v.]), a hairless mouse model with UVB-induced premalignant skin lesions (1 mg/kg EtNBA intraperitoneal) and in a rat skin-fold observation chamber model on the back of a rat with a transplanted solid tumor (2.5 mg/kg EtNBA i.v.). Fluorescence images and spectra were recorded in vivo (600 nm excitation, 665-900 nm detection) and in frozen tissue sections at several time points after EtNBA administration. In the rat palate the EtNBA fluorescence was maximum almost immediately after injection, whereas in the mouse skin and the observation chamber the fluorescence maximum was reached between 2 and 3 h after injection. EtNBA cleared from tissues after 8-24 h. EtNBA localizes in the transplantable solid tumor, but is not targeted specifically to the dysplastic location in the rat palate and mouse skin. However, in the rat palate the EtNBA fluorescence increased significantly with increasing dysplasia, apparently due to the increasing thickness of the upper keratinized layer of the epithelium where the dye was found to localize. Localization in this layer occurred both in the rat palate and in hairless mouse skin.

  8. Localized in vivo13C NMR spectroscopy of the brain

    PubMed Central

    Gruetter, Rolf; Adriany, Gregor; Choi, In-Young; Henry, Pierre-Gilles; Lei, Hongxia; Öz, Gülin

    2006-01-01

    Localized 13C NMR spectroscopy provides a new investigative tool for studying cerebral metabolism. The application of 13C NMR spectroscopy to living intact humans and animals presents the investigator with a number of unique challenges. This review provides in the first part a tutorial insight into the ingredients required for achieving a successful implementation of localized 13C NMR spectroscopy. The difficulties in establishing 13C NMR are the need for decoupling of the one-bond 13C–1H heteronuclear J coupling, the large chemical shift range, the low sensitivity and the need for localization of the signals. The methodological consequences of these technical problems are discussed, particularly with respect to (a) RF front-end considerations, (b) localization methods, (c) the low sensitivity, and (d) quantification methods. Lastly, some achievements of in vivo localized 13C NMR spectroscopy of the brain are reviewed, such as: (a) the measurement of brain glutamine synthesis and the feasibility of quantifying glutamatergic action in the brain; (b) the demonstration of significant anaplerotic fluxes in the brain; (c) the demonstration of a highly regulated malate-aspartate shuttle in brain energy metabolism and isotope flux; (d) quantification of neuronal and glial energy metabolism; and (e) brain glycogen metabolism in hypoglycemia in rats and humans. We conclude that the unique and novel insights provided by 13C NMR spectroscopy have opened many new research areas that are likely to improve the understanding of brain carbohydrate metabolism in health and disease. PMID:14679498

  9. Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Smith, Elizabeth Myhra

    The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

  10. Fluorescence correlation spectroscopy evidence for structural heterogeneity in ionic liquids

    SciTech Connect

    Guo, J C; Baker, G. A.; Hillesheim, P. C.; Dai, S.; Shaw, R. W.; Mahurin, S., M.

    2011-01-01

    In this work, we provide new experimental evidence for chain length-dependent self-aggregation in room temperature ionic liquids (RTILs) using fluorescence correlation spectroscopy (FCS). In studying a homologous series of N-alkyl-N-methylpyrrolidinium bis(trifluoromethylsulfonyl) imide, [C{sub n}MPy][Tf{sub 2}N] RTILs of varying alkyl chain length (n = 3, 4, 6, 8, and 10), biphasic rhodamine 6G solute diffusion dynamics were observed; both the fast and slow diffusion coefficients decreased with increasing alkyl chain length, with the relative contribution from slower diffusion increasing for longer-chain [C{sub n}MPy][Tf{sub 2}N]. We propose that the biphasic diffusion dynamics originate from self-aggregation of the nonpolar alkyl chains in the cationic [CnMPy]{sup +}.

  11. Shedding light on azopolymer brush dynamics by fluorescence correlation spectroscopy.

    PubMed

    Kollarigowda, R H; De Santo, I; Rianna, C; Fedele, C; Manikas, A C; Cavalli, S; Netti, P A

    2016-09-14

    Understanding the response to illumination at a molecular level as well as characterising polymer brush dynamics are key features that guide the engineering of new light-stimuli responsive materials. Here, we report on the use of a confocal microscopy technique that was exploited to discern how a single molecular event such as the photoinduced isomerisation of azobenzene can affect an entire polymeric material at a macroscopic level leading to photodriven mass-migration. For this reason, a set of polymer brushes, containing azobenzene (Disperse Red 1, DR) on the side chains of poly(methacrylic acid), was synthesised and the influence of DR on the polymer brush dynamics was investigated for the first time by Fluorescence Correlation Spectroscopy (FCS). Briefly, two dynamics were observed, a short one coming from the isomerisation of DR and a long one related to the brush main chain. Interestingly, photoinduced polymer aggregation in the confocal volume was observed. PMID:27491890

  12. Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

    SciTech Connect

    Laurence, Ted A.; Ly, Sonny; Bourguet, Feliza; Fischer, Nicholas O.; Coleman, Matthew A.

    2014-08-14

    Fluorescence correlation spectroscopy (FCS) is an important technique for studying biochemical interactions dynamically that may be used in vitro and in cell-based studies. It is generally claimed that FCS may only be used at nM concentrations. We show that this general consensus is incorrect and that the limitation to nM concentrations is not fundamental but due to detector limits as well as laser fluctuations. With a high count rate detector system and applying laser fluctuation corrections, we demonstrate FCS measurements up to 38 μM with the same signal-to-noise as at lower concentrations. Optical nanoconfinement approaches previously used to increase the concentration range of FCS are not necessary, and further increases above 38 μM may be expected using detectors and detector arrays with higher saturation rates and better laser fluctuation corrections. This approach greatly widens the possibilities of dynamic measurements of biochemical interactions using FCS at physiological concentrations.

  13. Fluorescence spectroscopy, exciton dynamics, and photochemistry of single allophycocyanin trimers

    SciTech Connect

    Ying, L.; Sie, X.S.

    1998-12-10

    The authors report a study of the allophycocyanin trimer (APC), a light-harvesting protein complex from cyanobacteria, by room-temperature single-molecule measurements of fluorescence spectra, lifetimes, intensity trajectories, and polarization modulation. Emission spectra of individual APC trimers are found to be homogeneous on the time scale of seconds. In contrast, their emission lifetimes are found to be widely distributed because of generation of long-lived exciton traps during the course of measurements. The intensity trajectories and polarization modulation experiments indicate reversible exciton trap formation within the three quasi-independent pairs of strong interacting {alpha}84 and {beta}84 chromophores in APC, as well as photobleaching of individual chromophores. Comparison experiments under continuous-wave and pulsed excitation reveal a two-photon mechanism for generating exciton traps and/or photobleaching, which involves exciton-exciton annihilation. These single-molecule experiments provide new insights into the spectroscopy, exciton dynamics, and photochemistry of light-harvesting complexes.

  14. Electrically induced microflows probed by fluorescence correlation spectroscopy.

    PubMed

    Ybert, C; Nadal, F; Salomé, R; Argoul, F; Bourdieu, L

    2005-03-01

    We report on the experimental characterisation of electrically induced flows at the micrometer scale through Fluorescence Correlation Spectroscopy (FCS) measurements. We stress the potential of FCS as a useful characterisation technique in microfluidics devices for transport properties cartography. The experimental results obtained in a model situation are in agreement with previous calculations (F. Nadal, F. Argoul, P. Kestener, B. Pouligny, C. Ybert, A. Ajdari, Eur. Phys. J. E 9, 387 (2002)) predicting the structure and electric-field dependency of the induced flow. Additionally, the present study evidences a complex behaviour of the probe nanobeads under electric field whose precise understanding might prove relevant for situations where nano-objects interact with an external electric field.

  15. Nonnegative matrix factorization: a blind spectra separation method for in vivo fluorescent optical imaging.

    PubMed

    Montcuquet, Anne-Sophie; Hervé, Lionel; Navarro, Fabrice; Dinten, Jean-Marc; Mars, Jérôme I

    2010-01-01

    Fluorescence imaging in diffusive media is an emerging imaging modality for medical applications that uses injected fluorescent markers that bind to specific targets, e.g., carcinoma. The region of interest is illuminated with near-IR light and the emitted back fluorescence is analyzed to localize the fluorescence sources. To investigate a thick medium, as the fluorescence signal decreases with the light travel distance, any disturbing signal, such as biological tissues intrinsic fluorescence (called autofluorescence) is a limiting factor. Several specific markers may also be simultaneously injected to bind to different molecules, and one may want to isolate each specific fluorescent signal from the others. To remove the unwanted fluorescence contributions or separate different specific markers, a spectroscopic approach is explored. The nonnegative matrix factorization (NMF) is the blind positive source separation method we chose. We run an original regularized NMF algorithm we developed on experimental data, and successfully obtain separated in vivo fluorescence spectra.

  16. Transcutaneous Raman Spectroscopy of Murine Bone In Vivo

    PubMed Central

    Schulmerich, Matthew V.; Cole, Jacqueline H.; Kreider, Jaclynn M.; Esmonde-White, Francis; Dooley, Kathryn A.; Goldstein, Steven A.; Morris, Michael D.

    2009-01-01

    Raman spectroscopy can provide valuable information about bone tissue composition in studies of bone development, biomechanics, and health. In order to study the Raman spectra of bone in vivo, instrumentation that enhances the recovery of subsurface spectra must be developed and validated. Five fiber-optic probe configurations were considered for transcutaneous bone Raman spectroscopy of small animals. Measurements were obtained from the tibia of sacrificed mice, and the bone Raman signal was recovered for each probe configuration. The configuration with the optimal combination of bone signal intensity, signal variance, and power distribution was then evaluated under in vivo conditions. Multiple in vivo transcutaneous measurements were obtained from the left tibia of 32 anesthetized mice. After collecting the transcutaneous Raman signal, exposed bone measurements were collected and used as a validation reference. Multivariate analysis was used to recover bone spectra from transcutaneous measurements. To assess the validity of the transcutaneous bone measurements cross-correlations were calculated between standardized spectra from the recovered bone signal and the exposed bone measurements. Additionally, the carbonate-to-phosphate height ratios of the recovered bone signals were compared to the reference exposed bone measurements. The mean cross-correlation coefficient between the recovered and exposed measurements was 0.96, and the carbonate-to-phosphate ratios did not differ significantly between the two sets of spectra (p > 0.05). During these first systematic in vivo Raman measurements, we discovered that probe alignment and animal coat color influenced the results and thus should be considered in future probe and study designs. Nevertheless, our noninvasive Raman spectroscopic probe accurately assessed bone tissue composition through the skin in live mice. PMID:19281644

  17. Imaging tumor metabolism using in vivo magnetic resonance spectroscopy.

    PubMed

    Li, Yan; Park, Ilwoo; Nelson, Sarah J

    2015-01-01

    Magnetic resonance spectroscopy (MRS) is a powerful tool for noninvasively investigating normal and abnormal metabolism. When used in combination with imaging strategies, multinuclear MRS methods provide detailed biochemical information that can be directly correlated with anatomical features. Hyperpolarized C MRS is a new technology that reflects real-time metabolic conversion and is likely to be extremely valuable in managing patients with cancer. This article reviews the use of in vivo P, H, and C MRS for assessing cancer metabolism in order to provide information for diagnosis, planning treatment, assessing response to therapy, and predicting survival for patients with cancer.

  18. Dynamic and unique nucleolar microenvironment revealed by fluorescence correlation spectroscopy.

    PubMed

    Park, Hweon; Han, Sung-Sik; Sako, Yasushi; Pack, Chan-Gi

    2015-03-01

    Organization and functions of the nucleolus is maintained by mobilities and interactions of nucleolar factors. Because the nucleolus is a densely packed structure, molecular crowding effects determined by the molecular concentrations and mobilities in the nucleolus should also be important for regulating nucleolar organization and functions. However, such molecular property of nucleolar organization is not fully understood. To understand the biophysical property of nucleolar organization, the diffusional behaviors of inert green fluorescent protein (GFP) oligomers with or without nuclear localization signals (NLSs) were analyzed under various conditions by fluorescence correlation spectroscopy. Our result demonstrates that the mobility of GFPs inside the nucleolus and the nucleoplasm can be represented by single free diffusion under normal conditions, even though the mobility in the nucleolus is considerably slower than that in the chromatin region. Moreover, the free diffusion of GFPs is found to be significantly size- and NLS-dependent only in the nucleolus. Interestingly, the mobility in the nucleolus is highly sensitive to ATP depletion, as well as actinomycin D (ActD) treatment. In contrast, the ultra-structure of the nucleolus was not significantly changed by ATP depletion but was changed by ActD treatment. These results suggest that the nucleolus behaves similarly to an open aqueous-phase medium with an increased molecular crowding effect that depends on both energy and transcription.

  19. Fluorescence Correlation Spectroscopy Close to a Fluctuating Membrane

    PubMed Central

    Fradin, Cécile; Abu-Arish, Asmahan; Granek, Rony; Elbaum, Michael

    2003-01-01

    Compartmentalization of the cytoplasm by membranes should have a strong influence on the diffusion of macromolecules inside a cell, and we have studied how this could be reflected in fluorescence correlation spectroscopy (FCS) experiments. We derived the autocorrelation function measured by FCS for fluorescent particles diffusing close to a soft membrane, and show it to be the sum of two contributions: short timescale correlations come from the diffusion of the particles (differing from free diffusion because of the presence of an obstacle), whereas long timescale correlations arise from fluctuations of the membrane itself (which create intensity fluctuations by modulating the number of detected particles). In the case of thermal fluctuations this second type of correlation depends on the elasticity of the membrane. To illustrate this calculation, we report the results of FCS experiments carried out close to a vesicle membrane. The measured autocorrelation functions display very distinctly the two expected contributions, and allow both to recover the diffusion coefficient of the fluorophore and to characterize the membrane fluctuations in term of a bending rigidity. Our results show that FCS measurements inside cells can lead to erroneous values of the diffusion coefficient if the influence of membranes is not recognized. PMID:12609903

  20. Evaluation of actinic cheilitis using fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Cosci, Alessandro; Pratavieira, Sebastião.; Takahama, Ademar; Souza Azevedo, Rebeca; Kurachi, Cristina

    2016-03-01

    Actinic cheilitis is a potentially malignant disorder that mostly affects the vermilion border of the lower lip and can lead to squamous cell carcinoma. Because of its heterogeneous clinical aspect, it is difficult to indicate representative biopsy area. Late diagnosis is a limiting factor of therapeutic possibilities available to treat oral cancer. The diagnosis of actinic cheilitis is mainly based on clinical and histopathological analysis and it is a time consuming procedure to get the results. Information about the organization and chemical composition of the tissues can be obtained using fluorescence lifetime spectroscopy techniques without the need for biopsy. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and allow a quick and non-invasive clinical investigation of injuries and to help clinicians with the early diagnosis of actinic cheilitis. This study aims to evaluate the fluorescence lifetime parameters at the discrimination of three degrees of epithelial dysplasia, the most important predictor of malignant development, described in up to 100% of actinic cheilitis cases.

  1. Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Liang, Lifang; Xing, Da; Chen, Tongshen; Pei, Yihui

    2007-11-01

    Fluorescence correlation spectroscopy (FCS) is a new kind of real-time, high-speed and single-molecule technique. It is used to detect the kinetic characteristics of fluorescent dye such as diffusion coefficient in the aqueous solution. Combined with confocal microscope optics, it has been now widely applied in cell biological research. Through a time correlation analysis of spontaneous intensity fluctuations, this technique with EGFP as a probe is capable of determining viscosity of fluids according to Stokes-Einstein equation. Nucleoplasmic viscosity is an important physical parameter to quantify the rheological characteristics of the nucleoplasm. Investigation on nucleoplasmic viscosity plays an important role in further understanding intranuclear environment. In this paper, FCS is introduced to noninvasively investigate nucleoplasmic viscosity of living cells. The results show that nucleoplasmic viscosity of lung adenocarcinoma (ASTC-a-1) cells is 2.55+/-0.61 cP and nucleoplasmic viscosity is larger than cytoplasmic viscosity at 37 °C (pH 7.4). In addition, significant changes in nucleoplasmic viscosity are detected by FCS when cells are exposed to hyper or hypotonic medium. Our study suggests that FCS can be used to detect the kinetic characteristics of biomolecules in living cells and thus helps to investigate the dynamic changes of the microenvironment in the cell.

  2. Continuous fluorescence microphotolysis and correlation spectroscopy using 4Pi microscopy.

    PubMed

    Arkhipov, Anton; Hüve, Jana; Kahms, Martin; Peters, Reiner; Schulten, Klaus

    2007-12-01

    Continuous fluorescence microphotolysis (CFM) and fluorescence correlation spectroscopy (FCS) permit measurement of molecular mobility and association reactions in single living cells. CFM and FCS complement each other ideally and can be realized using identical equipment. So far, the spatial resolution of CFM and FCS was restricted by the resolution of the light microscope to the micrometer scale. However, cellular functions generally occur on the nanometer scale. Here, we develop the theoretical and computational framework for CFM and FCS experiments using 4Pi microscopy, which features an axial resolution of approximately 100 nm. The framework, taking the actual 4Pi point spread function of the instrument into account, was validated by measurements on model systems, employing 4Pi conditions or normal confocal conditions together with either single- or two-photon excitation. In all cases experimental data could be well fitted by computed curves for expected diffusion coefficients, even when the signal/noise ratio was small due to the small number of fluorophores involved.

  3. Fluorescence correlation spectroscopy reveals strong fluorescence quenching of FITC adducts on PEGylated gold nanoparticles in water and the presence of fluorescent aggregates of desorbed thiolate ligands.

    PubMed

    Loumaigne, Matthieu; Praho, Raïssa; Nutarelli, Daniele; Werts, Martinus H V; Débarre, Anne

    2010-09-28

    Colloidal gold particles functionalised with oligoethylene-glycolated disulfide ligands and fluorescent moieties derived from fluorescein isothiocyanate (FITC) have been prepared and studied in aqueous suspension using fluorescence correlation spectroscopy (FCS). FCS probes the dynamics of the particles at the single object level, and reveals the desorption of fluorescent ligands which subsequently aggregate into larger (slower diffusing) objects. Cross-correlation spectroscopy of the FITC fluorescence and the Rayleigh-Mie scattering (RM-FCCS) of the gold cores shows that the only detectable fluorescent objects are free ligands and aggregates not associated with a gold particle. The fluorescence of bound fluorophores is quenched making their fluorescence too weak to be detected. FCS and RM-FCCS are useful tools for characterising functionalised noble metal particles in solution, under conditions similar to those used in optical bio-imaging. Desorption of thiolates from gold nanoparticles needs to be taken into account when working with these materials at low concentration.

  4. Autofluorescence spectroscopy of colorectal carcinoma: ex vivo study

    NASA Astrophysics Data System (ADS)

    Horak, Ladislav; Svec, Alexandr; Lezal, Dimitrij; Zavadil, Jiri

    2003-10-01

    Diagnosis established by means of fluorescence spectroscopy is currently used in the field of urology and bronchology. Its major advantage is that it allows the diagnosis of epithelial dysplasia or malignant proliferation even if routine diagnostic endoscopy fails to reveal any macroscopic changes. The authors present results of their observations that deal with fluorescence diagnosis of colorectal carcinoma. They examined the wet microscopic mounts of healthy colon mucosa and compared them to that prepared from colon mucosa affected by adenocarcinoma. The diagnosis of adenocarcinoma was verified by using clinical and histology means. Fluorescence spectra of tissue samples, excited by means of 488 and 514.5 nm lines of Ar ion laser and/or by He-Ne laser line 632.8 nm, have been studied. This study demonstrated differences in both the spectral shape and in the signal intensity (at unchanged spectral shape) of photoluminescence spectra emitted from tissue affected by adenocarcinoma as compared to that of healthy colon mucosa. The results encourage us to continue the study aimed at development of the diagnostic system usable in the clinical practice.

  5. Biosurfactant templated quantum sized fluorescent gold nanoclusters for in vivo bioimaging in zebrafish embryos.

    PubMed

    S, Chandirasekar; C, Chandrasekaran; T, Muthukumarasamyvel; G, Sudhandiran; N, Rajendiran

    2016-07-01

    We report the biosurfactant (sodium cholate) templated bright bluish-green emitting gold nanoclusters (AuNCs) by green chemical approach. Optical properties of the AuNCs were studied using UV-vis and luminescence spectroscopy. Lifetime of the fluorescent AuNCs was measured using time correlated single photon counting technique (TCSPC). High-resolution transmission electron microscopy (HR-TEM) and dynamic light scattering (DLS) were used to measure the sizes of the clusters. In-vivo toxicity and bioimaging studies of sodium cholate (NaC) templated AuNCs were carried out at different developmental stages of zebrafish embryos. The survival rate, hatching rate, heart rate, malformation and apoptotic gene expression experiments shows no significant toxicity in developing embryos up to 100μL/mL of AuNCs concentration and the AuNCs stained embryos exhibited green fluorescence with high intensity over the period from 4 to 96hpf (hours post fertilization) which shows that AuNCs were stable in living organisms.

  6. Heat-induced unfolding of apo-CP43 studied by fluorescence spectroscopy and CD spectroscopy.

    PubMed

    Xiao, Qing-Jie; Li, Zai-Geng; Yang, Jiao; He, Qing; Xi, Lei; Du, Lin-Fang

    2015-12-01

    CP43 is a chlorophyll-binding protein, which acts as a conduit for the excitation energy transfer. The thermal stability of apo-CP43 was studied by intrinsic fluorescence, exogenous ANS fluorescence, and circular dichroism spectroscopy. Under heat treatment, the structure of apo-CP43 changed and existed transition state occurred between 56 and 62 °C by the intrinsic, exogenous ANS fluorescence and the analysis of hydrophobicity. Besides, the isosbestic point of the sigmoidal curve was 58.10 ± 1.02 °C by calculating α-helix transition and the Tm was 56.45 ± 0.52 and 55.59 ± 0.68 °C by calculating the unfolded fraction of tryptophan and tyrosine fluorescence, respectively. During the process of unfolding, the hydrophobic structure of C-terminal segment firstly started to expose at 40 °C, and then the hydrophobic cluster adjacent to the N-terminal segment also gradually exposed to hydrophilic environment with increasing temperature. Our results indicated that heat treatment, especially above 40 °C, has an important impact on the structural stability of apo-CP43.

  7. Microneedles rollers as a potential device to increase ALA diffusion and PpIX production: evaluations by wide-field fluorescence imaging and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Gracielli Sousa, R. Phamilla; de Menezes, Priscila F. C.; Fujita, Alessandra K. L.; Requena, Michelle B.; Govone, Angelo Biassi; Escobar, André; de Nardi, Andrigo B.; Kurachi, Cristina; Bagnato, Vanderlei Salvador

    2014-03-01

    One of the limitations of topical photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) is the poor ability to penetrate biological barriers of skin and the recurrence rates in treatments. This study aimed to identify possible signs of increased diffusion of ALA-induced PpIX by fluorescence images and fluorescence spectroscopy. The research was done using in vivo porcine skin model. Before the cream application, microholes was performed with microneedles rollers in only one direction, afterward the ALA cream was applied at a 2.5cm2 area in triplicate and an occlusive dressing was placed. PpIX production was monitored using fluorescence spectroscopy collected at skin surface after 70, 100, 140, and 180 minutes of ALA incubation. About 100 fluorescence spectra of each treatment were collected, distributed by about five points for each site. Wide-field fluorescence imaging was made after 70, 90, and 170 minutes after treatment. The results obtained by imaging analysis indicated increase of the PpIX diffusion in the skin surface using the microneedles rollers (MNs) before ALA application. Circular regions of red fluorescence around the microholes were observed. In addition, the fluorescence spectra showed a greater intensity (2 times as many) in groups microneedles rollers associated. In conclusion, our data shown greater homogeneity and PpIX production in the groups pre-treated with microneedles indicating that the technique can be used to greater uniformity of PpIX production throughout the area to be treated reducing the chances of recurrent tumor as well as has potential for decreasing the time of therapy. (FUNDING SUPPORT:CAPES, CNPq and FAPESP)

  8. Analysis of green fluorescent protein bioluminescence in vivo and in vitro using a glow discharge

    NASA Astrophysics Data System (ADS)

    Hernández, L.; Mandujano, L. A.; Cuevas, J.; Reyes, P. G.; Osorio-González, D.

    2015-03-01

    The discovery of fluorescent proteins has been a revolution in cell biology and related sciences because of their many applications, mainly emphasizing their use as cellular markers. The green fluorescent protein (GFP) is one of the most used as it requires no cofactors to generate fluorescence and retains this property into any organism when it is expressed by recombinant DNA techniques, which is a great advantage. In this work, we analyze the emission spectra of recombinant green fluorescent protein in vivo and in vitro exposed to a glow discharge plasma of nitrogen in order to relate electron temperature to fluorescence intensity.

  9. In vivo EPR spectroscopy of free radicals in the heart.

    PubMed Central

    Zweier, J L; Kuppusamy, P

    1994-01-01

    Electron paramagnetic resonance (EPR) spectroscopy can be applied to directly measure free radicals; however, it has not been possible to measure important biologic radicals in situ because conventional spectrometer designs are not suitable for the performance of measurements on large aqueous structures such as whole organs or tissues. We describe the design, construction, and application of instrumentation developed in an effort to obtain optimum performance in measuring free radicals in intact biologic organs or tissues. This spectrometer consists of a 1- to 2-GHz microwave bridge with the source locked to the resonant frequency of a specially designed recessed gap, loop-gap resonator. The principles of resonator design and construction are analyzed and described. Using this spectrometer radical concentrations as low as 0.4 microM in aqueous solutions could be measured. Studies of isolated beating hearts involving simultaneous real time measurements of free radicals and cardiac contractile function are performed. This in vivo EPR technique is applied to study the kinetics of free radical uptake and metabolism in normally perfused and globally ischemic hearts. In addition, it is demonstrated that this technique can be used to noninvasively measure tissue oxygen consumption. Thus, low frequency EPR spectroscopy offers great promise in the study of in vivo free radical generation and the effects of this radical generation on whole biologic tissues. PMID:7705304

  10. Molecular aggregation characterized by high order autocorrelation in fluorescence correlation spectroscopy.

    PubMed Central

    Palmer, A G; Thompson, N L

    1987-01-01

    The use of high order autocorrelation in fluorescence correlation spectroscopy for investigating aggregation in a sample that contains fluorescent molecules is described. Theoretical expressions for the fluorescence fluctuation autocorrelation functions defined by gm,n(tau) = [(delta fm(t + tau)delta fm(t] - (delta Fm(t] (delta Fn(t

  11. Fluorescence Spectroscopy: An Adjunct Diagnostic Tool to Image-Guided Core Needle Biopsy of the Breast

    PubMed Central

    Zhu, Changfang; Burnside, Elizabeth S.; Sisney, Gale A.; Salkowski, Lonie R.; Harter, Josephine M.; Yu, Bing

    2009-01-01

    We explored the use of a fiber-optic probe for in vivo fluorescence spectroscopy of breast tissues during percutaneous image-guided breast biopsy. A total of 121 biopsy samples with accompanying histological diagnosis were obtained clinically and investigated in this study. The tissue spectra were analyzed using partial least-squares analysis and represented using a set of principal components (PCs) with dramatically reduced data dimension. For nonmalignant tissue samples, a set of PCs that account for the largest amount of variance in the spectra displayed correlation with the percent tissue composition. For all tissue samples, a set of PCs was identified using a Wilcoxon rank-sum test as showing statistically significant differences between: 1) malignant and fibrous/benign; 2) malignant and adipose; and 3) malignant and nonmalignant breast samples. These PCs were used to distinguish malignant from other nonmalignant tissue types using a binary classification scheme based on both linear and nonlinear support vector machine (SVM) and logistic regression (LR). For the sample set investigated in this study, the SVM classifier provided a cross-validated sensitivity and specificity of up to 81% and 87%, respectively, for discrimination between malignant and fibrous/benign samples, and up to 81% and 81%, respectively, for discriminating between malignant and adipose samples. Classification based on LR was used to generate receiver operator curves with an area under the curve (AUC) of 0.87 for discriminating malignant versus fibrous/benign tissues, and an AUC of 0.84 for discriminating malignant from adipose tissue samples. This study demonstrates the feasibility of performing fluorescence spectroscopy during clinical core needle breast biopsy, and the potential of this technique for identifying breast malignancy in vivo. PMID:19272976

  12. In vivo two-dimensional NMR correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Kraft, Robert A.

    1999-10-01

    The poor resolution of in-vivo one- dimensional nuclear magnetic resonance spectroscopy (NMR) has limited its clinical potential. Currently, only the large singlet methyl resonances arising from N-acetyl aspartate (NAA), choline, and creatine are quantitated in a clinical setting. Other metabolites such as myo- inositol, glutamine, glutamate, lactate, and γ- amino butyric acid (GABA) are of clinical interest but quantitation is difficult due to the overlapping resonances and limited spectral resolution. To improve the spectral resolution and distinguish between overlapping resonances, a series of two- dimensional chemical shift correlation spectroscopy experiments were developed for a 1.5 Tesla clinical imaging magnet. Two-dimensional methods are attractive for in vivo spectroscopy due to their ability to unravel overlapping resonances with the second dimension, simplifying the interpretation and quantitation of low field NMR spectra. Two-dimensional experiments acquired with mix-mode line shape negate the advantages of the second dimension. For this reason, a new experiment, REVOLT, was developed to achieve absorptive mode line shape in both dimensions. Absorptive mode experiments were compared to mixed mode experiments with respect to sensitivity, resolution, and water suppression. Detailed theoretical and experimental calculations of the optimum spin lock and radio frequency power deposition were performed. Two-dimensional spectra were acquired from human bone marrow and human brain tissue. The human brain tissue spectra clearly reveal correlations among the coupled spins of NAA, glutamine, glutamate, lactate, GABA, aspartate and myo-inositol obtained from a single experiment of 23 minutes from a volume of 59 mL. (Copies available exclusively from MIT Libraries, Rm. 14-0551, Cambridge, MA 02139-4307. Ph. 617-253-5668; Fax 617-253-1690.)

  13. Fluorescence spectroscopy of fulvic acids from fen peatlands

    NASA Astrophysics Data System (ADS)

    Maryganova, Victoria; Wojciech Szajdak, Lech

    2010-05-01

    Intensive cultivation and agricultural use of peatlands lead to the degradation and mineralization of peat. Fulvic acids (FA) as the most mobile part of peat organic matter can be considered as an early indicator of its changes. One of the most sensitive and simple methods for studying the structural chemistry of humic substances is fluorescence spectroscopy. The objective of this study was to analyze comparatively the fluorescence properties of FA from low-moor peats of different genesis and decomposition degree with respect to the peculiarities of their chemical structure. FA were isolated from 4 peat samples collected from different fen peatlands of Belarus. Fluorescence spectra were obtained on water solutions of FA at a concentration of 50 mg/L after adjustment to pH=2, 6 and 13 on a MSL-4800 spectrofluorimeter (Perkin Elmer, USA.) at 20 ± 2 oC. Emission spectra were obtained using an excitation wavelength of 365 nm. Excitation spectra were recorded by varying the excitation wavelength from 260 to 520 nm and measuring the fluorescence emission at a fixed wavelength of 520 nm. Elemental composition of FA and optical density at 465 nm (D465) of FA solutions in 0.1 N NaOH were determined. Emission spectra of FA are characterized by a broad featureless band of the maximum wavelengths at λ=460-475 nm. Excitation spectra of FA have three peaks localized in different wavelength regions. The maximum wavelengths and intensities of the excitation peaks depend on the pH values. The highest intensities are observed at pH=6. FA exhibit a main excitation peak at λ=355-370 nm, a minor peak at λ=395-400 nm, and a weak band at λ=430-440 nm. At pH=2, all the peaks decrease in intensity. With increasing the pH to 13, the excitation maximum at λ=355-370 nm shifts from 10 to 20 nm towards longer wavelengths compared to acidic solutions. A general decrease in fluorescence intensity is observed, the intensity decline of the peak at λ=355-370 nm being more marked than of the

  14. In-vivo concentration ratio estimation of two fluorescent probes for early detection of Alzheimer's Disease

    NASA Astrophysics Data System (ADS)

    Harbater, Osnat; Gannot, Israel

    2015-03-01

    In-vivo measurement of the concentrations of biological compounds using fluorescence is one of the challenging biophotonic fields. These measurements are useful in diagnostic and treatment monitoring applications that use fluorescent probes which may bond to specific proteins and drugs. In some cases the relative concentration of two compounds is a sufficient biological indicator. For instance, it has been shown that the ratio between Amyloid-Beta and tau protein in the Cerebrospinal fluid (CSF) may predict the development of Alzheimer's disease (AD) several years before current diagnosis. We have previously suggested a system that could measure the concentration ratio of these two proteins in-vivo without the need to collect CSF samples. This system uses a miniature needle with an optical fiber which is coupled to a laser source and a detector. The fiber excites fluorescent probes which were injected and bond to the proteins in the CSF, and collects the fluorescence emission. Using the fluorescence intensity ratio, the concentration ratio between the proteins is estimated, and AD may be diagnosed. In this work we present the results of an in-vivo trial performed on mice. Miniature tubes containing two fluorescent probes in several concentration ratios were inserted into the mice in two locations: subcutaneously, and deeper in the abdomen. The fluorescent probes were excited and the fluorescence intensity was measured. The concentration ratios were extracted from the fluorescence intensities using a simple calibration curve. The extracted ratios are compared to the true ratios and the system's accuracy is estimated.

  15. In-vivo imaging of murine tumors using complete-angle projection fluorescence molecular tomography

    NASA Astrophysics Data System (ADS)

    Deliolanis, Nikolaos C.; Dunham, Joshua; Wurdinger, Thomas; Figueiredo, Jose-Luiz; Bakhos, Tannous; Ntziachristos, Vasilis

    2009-05-01

    We interrogate the ability of free-space fluorescence tomography to image small animals in vivo using charge-coupled device (CCD) camera measurements over 360-deg noncontact projections. We demonstrate the performance of normalized dual-wavelength measurements that are essential for in-vivo use, as they account for the heterogeneous distribution of photons in tissue. In-vivo imaging is then showcased on mouse lung and brain tumors cross-validated by x-ray microcomputed tomography and histology.

  16. DNA binding and oligomerization of NtrC studied by fluorescence anisotropy and fluorescence correlation spectroscopy.

    PubMed Central

    Sevenich, F W; Langowski, J; Weiss, V; Rippe, K

    1998-01-01

    Fluorescence anisotropy and fluorescence correlation spectroscopy measurements of rhodamine-labeled DNA oligonucleotide duplexes have been used to determine equilibrium binding constants for DNA binding of the prokaryotic transcription activator protein NtrC. Measurements were made with wild-type NtrC from Escherichia coli and the constitutively active mutant NtrCS160Ffrom Salmonella using DNA duplexes with one or two binding sites. The following results were obtained: (i) the dissociation constant K d for binding of one NtrC dimer to a single binding site was the same for the wild-type and mutant proteins within the error of measurement. (ii) The value of K d decreased from 1.4 +/- 0.7 x 10(-11) M at 15 mM K acetate to 5.8 +/- 2.6 x 10(-9) M at 600 mM K acetate. From the salt dependence of the dissociation constant we calculated that two ion pairs form upon binding of one dimeric protein to the DNA. (iii) Binding of two NtrC dimers to the DNA duplex with two binding sites occured with essentially no cooperativity. Titration curves of NtrCS160Fbinding to the same duplex demonstrated that more than two protein dimers of the mutant protein could bind to the DNA. PMID:9490780

  17. Tubulin equilibrium unfolding followed by time-resolved fluorescence and fluorescence correlation spectroscopy

    PubMed Central

    Sánchez, Susana A.; Brunet, Juan E.; Jameson, David M.; Lagos, Rosalba; Monasterio, Octavio

    2004-01-01

    The pathway for the in vitro equilibrium unfolding of the tubulin heterodimer by guanidinium chloride (GdmCl) has been studied using several spectroscopic techniques, specifically circular dichroism (CD), two-photon Fluorescence Correlation Spectroscopy (FCS), and time-resolved fluorescence, including lifetime and dynamic polarization. The results show that tubulin unfolding is characterized by distinct processes that occur in different GdmCl concentration ranges. From 0 to 0.5 M GdmCl, a slight alteration of the tubulin heterodimer occurs, as evidenced by a small, but reproducible increase in the rotational correlation time of the protein and a sharp decrease in the secondary structure monitored by CD. In the range 0.5–1.5 M GdmCl, significant decreases in the steady-state anisotropy and average lifetime of the intrinsic tryptophan fluorescence occur, as well as a decrease in the rotational correlation time, from 48 to 26 nsec. In the same GdmCl range, the number of protein molecules (labeled with Alexa 488), as determined by two-photon FCS measurements, increases by a factor of two, indicating dissociation of the tubulin dimer into monomers. From 1.5 to 4 M GdmCl, these monomers unfold, as evidenced by the continual decrease in the tryptophan steady-state anisotropy, average lifetime, and rotational correlation time, concomitant with secondary structural changes. These results help to elucidate the unfolding pathway of the tubulin heterodimer and demonstrate the value of FCS measurements in studies on oligomeric protein systems. PMID:14691224

  18. Fluorescence spectroscopy and confocal microscopy of the mycotoxin citrinin in condensed phase and hydrogel films.

    PubMed

    Lauer, Milena H; Gehlen, Marcelo H; de Jesus, Karen; Berlinck, Roberto G S

    2014-05-01

    The emission spectra, quantum yields and fluorescence lifetimes of citrinin in organic solvents and hydrogel films have been determined. Citrinin shows complex fluorescence decays due to the presence of two tautomers in solution and interconversion from excited-state double proton transfer (ESDPT) process. The fluorescence decay times associated with the two tautomers have values near 1 and 5 ns depending on the medium. In hydrogel films of agarose and alginate, fluorescence imaging showed that citrinin is not homogeneously dispersed and highly emissive micrometer spots may be formed. Fluorescence spectrum and decay analysis are used to recognize the presence of citrinin in hydrogel films using confocal fluorescence microscopy and spectroscopy.

  19. Long-term in vivo glucose monitoring using fluorescent hydrogel fibers

    PubMed Central

    Heo, Yun Jung; Shibata, Hideaki; Okitsu, Teru; Kawanishi, Tetsuro; Takeuchi, Shoji

    2011-01-01

    The use of fluorescence-based sensors holds great promise for continuous glucose monitoring (CGM) in vivo, allowing wireless transdermal transmission and long-lasting functionality in vivo. The ability to monitor glucose concentrations in vivo over the long term enables the sensors to be implanted and replaced less often, thereby bringing CGM closer to practical implementation. However, the full potential of long-term in vivo glucose monitoring has yet to be realized because current fluorescence-based sensors cannot remain at an implantation site and respond to blood glucose concentrations over an extended period. Here, we present a long-term in vivo glucose monitoring method using glucose-responsive fluorescent hydrogel fibers. We fabricated glucose-responsive fluorescent hydrogels in a fibrous structure because this structure enables the sensors to remain at the implantation site for a long period. Moreover, these fibers allow easy control of the amount of fluorescent sensors implanted, simply by cutting the fibers to the desired length, and facilitate sensor removal from the implantation site after use. We found that the polyethylene glycol (PEG)-bonded polyacrylamide (PAM) hydrogel fibers reduced inflammation compared with PAM hydrogel fibers, transdermally glowed, and continuously responded to blood glucose concentration changes for up to 140 days, showing their potential application for long-term in vivo continuous glucose monitoring. PMID:21808049

  20. Long-term in vivo glucose monitoring using fluorescent hydrogel fibers.

    PubMed

    Heo, Yun Jung; Shibata, Hideaki; Okitsu, Teru; Kawanishi, Tetsuro; Takeuchi, Shoji

    2011-08-16

    The use of fluorescence-based sensors holds great promise for continuous glucose monitoring (CGM) in vivo, allowing wireless transdermal transmission and long-lasting functionality in vivo. The ability to monitor glucose concentrations in vivo over the long term enables the sensors to be implanted and replaced less often, thereby bringing CGM closer to practical implementation. However, the full potential of long-term in vivo glucose monitoring has yet to be realized because current fluorescence-based sensors cannot remain at an implantation site and respond to blood glucose concentrations over an extended period. Here, we present a long-term in vivo glucose monitoring method using glucose-responsive fluorescent hydrogel fibers. We fabricated glucose-responsive fluorescent hydrogels in a fibrous structure because this structure enables the sensors to remain at the implantation site for a long period. Moreover, these fibers allow easy control of the amount of fluorescent sensors implanted, simply by cutting the fibers to the desired length, and facilitate sensor removal from the implantation site after use. We found that the polyethylene glycol (PEG)-bonded polyacrylamide (PAM) hydrogel fibers reduced inflammation compared with PAM hydrogel fibers, transdermally glowed, and continuously responded to blood glucose concentration changes for up to 140 days, showing their potential application for long-term in vivo continuous glucose monitoring. PMID:21808049

  1. In Vivo and Ex Vivo Transcutaneous Glucose Detection Using Surface-Enhanced Raman Spectroscopy

    NASA Astrophysics Data System (ADS)

    Ma, Ke

    Diabetes mellitus is widely acknowledged as a large and growing health concern. The lack of practical methods for continuously monitoring glucose levels causes significant difficulties in successful diabetes management. Extensive validation work has been carried out using surface-enhanced Raman spectroscopy (SERS) for in vivo glucose sensing. This dissertation details progress made towards a Raman-based glucose sensor for in vivo, transcutaneous glucose detection. The first presented study combines spatially offset Raman spectroscopy (SORS) with SERS (SESORS) to explore the possibility of in vivo, transcutaneous glucose sensing. A SERS-based glucose sensor was implanted subcutaneously in Sprague-Dawley rats. SERS spectra were acquired transcutaneously and analyzed using partial least-squares (PLS). Highly accurate and consistent results were obtained, especially in the hypoglycemic range. Additionally, the sensor demonstrated functionality at least17 days after implantation. A subsequent study further extends the application of SESORS to the possibility of in vivo detection of glucose in brain through skull. Specifically, SERS nanoantennas were buried in an ovine tissue behind a bone with 8 mm thickness and detected by using SESORS. In addition, quantitative detection through bones by using SESORS was also demonstrated. A device that could measure glucose continuously as well as noninvasively would be of great use to patients with diabetes. The inherent limitation of the SESORS approach may prevent this technique from becoming a noninvasive method. Therefore, the prospect of using normal Raman spectroscopy for glucose detection was re-examined. Quantitative detection of glucose and lactate in the clinically relevant range was demonstrated by using normal Raman spectroscopy with low power and short acquisition time. Finally, a nonlinear calibration method called least-squares support vector machine regression (LS-SVR) was investigated for analyzing spectroscopic

  2. The Intermediate Scattering Function in Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Guerra, Rodrigo; Andrews, Ballard; Sen, Pabitra

    2006-03-01

    We formulate the autocorrelation function for Fluorescence Correlation Spectroscopy (FCS) GD(τ) in reciprocal space in terms of the of the Intermediate Scattering Function ISF(k,t) and the fourier transform of the Optical Response Function ORF(k). In this way we may extend the use of FCS to processes that have been studied using NMR, DLS, and neutron scattering. This formalism is useful for the complicated propagators involved in confined systems and in the study of diffusion in cells: where diffusion is either restricted or permeation through membrane is important. Calculations in k-space produce approximate expressions for the ORF using cumulant expansions that are accurate for small wavevectors. This provides descriptions for longer timescales better suited for studying time-dependent diffusion ISF(k,t)->exp[-tD(t)k^2] and provides a natural separation of contributions from system dynamics and from optical artifacts and aberrations. We will show an explicit derivation of a semi-analytical fit function for free diffusion based on standard electromagnetic analysis of a confocal optical apparatus. This fit function is then used to analyze a representative data set and has no free fit parameters other than the diffusion constant.

  3. Resonance fluorescence spectroscopy in laser-induced cavitation bubbles.

    PubMed

    Koch, Sandra; Garen, Walter; Neu, Walter; Reuter, Rainer

    2006-05-01

    Laser-induced breakdown spectroscopy (LIBS) in liquids using a double-pulse Q-switched Nd:YAG laser system has provided reliable results that give trace detection limits in water. Resonant laser excitation has been added to enhance detection sensitivity. A primary laser pulse (at 532 nm), transmitted via an optical fiber, induces a cavitation bubble and shockwave at a target immersed in a 10 mg l(-1)-100 mg l(-1) indium (In) water suspension. The low-pressure rear of the shockwave induces bubble expansion and a resulting reduction in cavity pressure as it extends away from the target. Shortly before the maximum diameter is expected, a secondary laser pulse (also at 532 nm) is fed into the bubble in order to reduce quenching processes. The plasma field generated is then resonantly excited by a fiber-guided dye laser beam to increase detection selectivity. The resulting resonance fluorescence emission is optically detected and processed by an intensified optical multichannel analyzer system.

  4. Fluorescence Correlation Spectroscopy Evidence for Structural Heterogeneity in Ionic Liquids

    SciTech Connect

    Guo, Jianchang; Baker, Gary A; Hillesheim, Patrick C; Dai, Sheng; Shaw, Robert W; Mahurin, Shannon Mark

    2011-01-01

    Self-aggregation in room temperature ionic liquids (RTILs) has been a subject of intense interest in recent years. In this work, we provide new experimental evidence for chain length-dependent self-aggregation in RTILs using fluorescence correlation spectroscopy (FCS). In studying a homologous series of N-alkyl-N-methylpyrrolidinium bis(trifluoromethylsulfonyl) imide, [CnMPy][Tf2N] RTILs of varying alkyl chain length (n = 3, 4, 6, 8, and 10), biphasic rhodamine 6G solute diffusion dynamics were observed; both the fast and slow diffusion coefficients decrease with increasing alkyl chain length, with the relative contribution from slower diffusion increasing for longer-chained [CnMPy][Tf2N]. We propose that the biphasic diffusion dynamics originate from self-aggregation of the nonpolar alkyl chains in the cationic [CnMPy]+. The presence of this local liquid structuring provides important insight into the behavior of RTILs relevant to their application in photovoltaics, fuel cells, and batteries.

  5. DOM transformations in stream biofilms shown by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Burns, N. R.; Rosentreter, J. A.; Bengtsson, M. M.; Wagner, K.; Herberg, E. R.; Battin, T. J.

    2012-04-01

    Alpine streams are hotspots of biogeochemical activity, where dissolved organic matter (DOM) is mineralised and transformed by heterotrophic microorganisms while travelling downstream. The chemical composition of DOM strongly affects the rate and type of transformations that occur, and a portion of the DOM is thought to be chemically resistant to decomposition by biofilm microorganisms. In soil studies, interactions between decomposition rates of recalcitrant soil organic matter (SOM) and labile rhizodeposits have often been described as 'priming effects'. Labile substrate additions have been observed both to stimulate and to suppress mineralisation of recalcitrant substrates under different conditions, due to substrate co-metabolism or microbial community dynamics. Although the same principles are likely to apply to decomposition of recalcitrant DOM and labile algal exudates, few studies so far have investigated priming effects in an aquatic context. In this presentation, we describe results from a microcosm experiment. Streamwater with added recalcitrant DOM was passed through bioreactors mimicking streambed heterotrophic biofilms. Three potential priming treatments were applied; glucose (G), glucose with nitrate and phosphate (GNP) or an algal extract with nitrate and phosphate (ANP). We used fluorescence emission-excitation matrices (EEM) and UV spectroscopy on the DOM input to and output from the bioreactors to unravel potential interactions between recalcitrant and labile DOM during priming in biofilms.

  6. Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

    DOE PAGES

    Laurence, Ted A.; Ly, Sonny; Bourguet, Feliza; Fischer, Nicholas O.; Coleman, Matthew A.

    2014-08-14

    Fluorescence correlation spectroscopy (FCS) is an important technique for studying biochemical interactions dynamically that may be used in vitro and in cell-based studies. It is generally claimed that FCS may only be used at nM concentrations. We show that this general consensus is incorrect and that the limitation to nM concentrations is not fundamental but due to detector limits as well as laser fluctuations. With a high count rate detector system and applying laser fluctuation corrections, we demonstrate FCS measurements up to 38 μM with the same signal-to-noise as at lower concentrations. Optical nanoconfinement approaches previously used to increase themore » concentration range of FCS are not necessary, and further increases above 38 μM may be expected using detectors and detector arrays with higher saturation rates and better laser fluctuation corrections. This approach greatly widens the possibilities of dynamic measurements of biochemical interactions using FCS at physiological concentrations.« less

  7. Fluorescence Correlation Spectroscopy and Nonlinear Stochastic Reaction-Diffusion

    SciTech Connect

    Del Razo, Mauricio; Pan, Wenxiao; Qian, Hong; Lin, Guang

    2014-05-30

    The currently existing theory of fluorescence correlation spectroscopy (FCS) is based on the linear fluctuation theory originally developed by Einstein, Onsager, Lax, and others as a phenomenological approach to equilibrium fluctuations in bulk solutions. For mesoscopic reaction-diffusion systems with nonlinear chemical reactions among a small number of molecules, a situation often encountered in single-cell biochemistry, it is expected that FCS time correlation functions of a reaction-diffusion system can deviate from the classic results of Elson and Magde [Biopolymers (1974) 13:1-27]. We first discuss this nonlinear effect for reaction systems without diffusion. For nonlinear stochastic reaction-diffusion systems there are no closed solutions; therefore, stochastic Monte-Carlo simulations are carried out. We show that the deviation is small for a simple bimolecular reaction; the most significant deviations occur when the number of molecules is small and of the same order. Extending Delbrück-Gillespie’s theory for stochastic nonlinear reactions with rapidly stirring to reaction-diffusion systems provides a mesoscopic model for chemical and biochemical reactions at nanometric and mesoscopic level such as a single biological cell.

  8. Noninvasive determination of cell nucleoplasmic viscosity by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Liang, Lifang; Wang, Xichao; Xing, Da; Chen, Tongsheng; Chen, Wei R.

    2009-03-01

    Noninvasive and reliable quantification of rheological characteristics in the nucleus is extremely useful for fundamental research and practical applications in medicine and biology. This study examines the use of fluorescence correlation spectroscopy (FCS) to noninvasively determine nucleoplasmic viscosity (ɛnu), an important parameter of nucleoplasmic rheology. Our FCS analyses show that ɛnu of lung adenocarcinoma (ASTC-a-1) and HeLa cells are 1.77+/-0.42 cP and 1.40+/-0.27 cP, respectively, about three to four times larger than the water viscosity at 37 °C. ɛnu was reduced by 31 to 36% upon hypotonic exposure and increased by 28 to 52% from 37 to 24 °C. In addition, we found that ɛnu of HeLa cells reached the lowest value in the S phase and that there was no significant difference of ɛnu between in the G1 and G2 phases. Last, nucleoplasmic viscosity was found to be larger than cytoplasmic viscosity in both HeLa and ASTC-a-1 cells. These results indicate that FCS can be used as a noninvasive tool to investigate the microenvironment of living cells. This is the first report on the measurement of ɛnu in living cells synchronized in the G1, S, and G2 phases.

  9. A Rapid and Convenient Method for in Vivo Fluorescent Imaging of Protoscolices of Echinococcus multilocularis

    PubMed Central

    Yang, Tao; Wang, Sibo; Zhang, Xuyong; Xia, Jie; Guo, Jun; Hou, Jixue; Zhang, Hongwei; Chen, Xueling; Wu, Xiangwei

    2016-01-01

    Human and animal alveolar echinococcosis (AE) are important helminth infections endemic in wide areas of the Northern hemisphere. Monitoring Echinococcus multilocularis viability and spread using real-time fluorescent imaging in vivo provides a fast method to evaluate the load of parasite. Here, we generated a kind of fluorescent protoscolices in vivo imaging model and utilized this model to assess the activity against E. multilocularis protoscolices of metformin (Met). Results indicated that JC-1 tagged E. multilocularis can be reliably and confidently used to monitor protoscolices in vitro and in vivo. The availability of this transient in vivo fluorescent imaging of E. multilocularis protoscolices constitutes an important step toward the long term bio-imaging research of the AE-infected mouse models. In addition, this will be of great interest for further research on infection strategies and development of drugs and vaccines against E. multilocularis and other cestodes. PMID:27180584

  10. A Rapid and Convenient Method for in Vivo Fluorescent Imaging of Protoscolices of Echinococcus multilocularis.

    PubMed

    Yang, Tao; Wang, Sibo; Zhang, Xuyong; Xia, Jie; Guo, Jun; Hou, Jixue; Zhang, Hongwei; Chen, Xueling; Wu, Xiangwei

    2016-04-01

    Human and animal alveolar echinococcosis (AE) are important helminth infections endemic in wide areas of the Northern hemisphere. Monitoring Echinococcus multilocularis viability and spread using real-time fluorescent imaging in vivo provides a fast method to evaluate the load of parasite. Here, we generated a kind of fluorescent protoscolices in vivo imaging model and utilized this model to assess the activity against E. multilocularis protoscolices of metformin (Met). Results indicated that JC-1 tagged E. multilocularis can be reliably and confidently used to monitor protoscolices in vitro and in vivo. The availability of this transient in vivo fluorescent imaging of E. multilocularis protoscolices constitutes an important step toward the long term bio-imaging research of the AE-infected mouse models. In addition, this will be of great interest for further research on infection strategies and development of drugs and vaccines against E. multilocularis and other cestodes. PMID:27180584

  11. Intrinsic fluorescence spectroscopy in turbid media: disentangling effects of scattering and absorption.

    PubMed

    Müller, M G; Georgakoudi, I; Zhang, Q; Wu, J; Feld, M S

    2001-09-01

    The fluorescence from a turbid medium such as biologic tissue contains information about scattering and absorption, as well as the intrinsic fluorescence, i.e., the fluorescence from an optically thin sample of pure fluorophores. The interplay of scattering and absorption can result in severe distortion of the intrinsic spectral features. These distortions can be removed by use of a photon-migration-based picture and information from simultaneously acquired fluorescence and reflectance spectra. We present experimental evidence demonstrating the validity of such an approach for extracting the intrinsic fluorescence for a wide range of scatterer and absorber concentrations in tissue models, ex vivo and in vivo tissues. We show that variations in line shape and intensity in intrinsic tissue fluorescence are significantly reduced compared with the corresponding measured fluorescence.

  12. Spectral fluorescent properties of tissues in vivo with excitation in the red wavelength range

    NASA Astrophysics Data System (ADS)

    Stratonnikov, Alexander A.; Loschenov, Victor B.; Klimov, D. V.; Edinac, N. E.; Wolnukhin, V. A.; Strashkevich, I. A.

    1997-12-01

    The spectral fluorescence analysis is a promising method for differential tissue diagnostic. Usually the UV and visible light is used for fluorescence excitation with emission registration in the visible wavelength range. The light penetration length in this wavelength range is very small allowing one to analyze only the surface region of the tissue. Here we present the tissue fluorescent spectra in vivo excited in the red wavelength region. As excitation light source we used compact He-Ne laser (632.8 nm) and observed the fluorescence in 650 - 800 nm spectral range. The various tissues including normal skin, psoriasis, tumors, necrosis as well as photosensitized tissues have been measured.

  13. Analyses of the Dynamic Properties of Nuclear Lamins by Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS).

    PubMed

    Takeshi, Shimi; Pack, Chan-Gi; Goldman, Robert D

    2016-01-01

    The major structural components of the nuclear lamina are the A- and B-type nuclear lamin proteins which are also present in the nucleoplasm. Studies of molecular movements of the lamins in both the lamina and nucleoplasm of living cell nuclei have provided insights into their roles in maintaining nuclear architecture. In this chapter, we present protocols for quantitatively measuring the mobilities of lamin proteins by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) in mammalian cell nuclei. PMID:27147036

  14. In-vivo optical imaging and spectroscopy of cerebral hemodynamics

    NASA Astrophysics Data System (ADS)

    Zhou, Chao

    Functional optical imaging techniques, such as diffuse optical imaging and spectroscopy and laser speckle imaging (LSI), were used in research and clinical settings to measure cerebral hemodynamics. In this thesis, theoretical and experimental developments of the techniques and their in-vivo applications ranging from small animals to adult humans are demonstrated. Near infrared diffuse optical techniques non-invasively measure hemoglobin concentrations, blood oxygen saturation (diffuse reflectance spectroscopy, DRS) and blood flow (diffuse correlation spectroscopy, DCS) in deep tissues, e.g. brain. A noise model was derived for DCS measurements. Cerebral blood flow (CBF) measured with DCS was validated with arterial-spin-labeling MRI. Three-dimensional CBF tomography was obtained during cortical spreading depression from a rat using the optimized diffuse correlation tomographic method. Cerebral hemodynamics in newborn piglets after traumatic brain injury were continuously monitored optically for six hours to demonstrate the feasibility of using diffuse optical techniques as bedside patient monitors. Cerebral autoregulation in piglets and human stroke patients was demonstrated to be non-invasively assessable via the continuous DCS measurement. Significant differences of CBF responses to head-of-bead maneuvers were observed between the peri- and contra-infarct hemispheres in human stroke patients. A significant portion of patient population showed paradoxical CBF responses, indicating the importance of individualized stroke management. The development of a speckle noise model revealed the source of noise for LSI. LSI was then applied to study the acute functional recovery of the rat brain following transient brain ischemia. The spatial and temporal cerebral blood flow responses to functional stimulation were statistically quantified. The area of activation, and the temporal response to stimulation were found significantly altered by the ischemic insult, while the

  15. Localized phosphorus spectroscopy in vivo: Quantitation of metabolite concentrations

    NASA Astrophysics Data System (ADS)

    Wylezinska-Arridge, Marzena Malgorzata

    This project was dedicated to the investigation of the factors that may affect absolute quantitation in localized 31P MRS and if possible to the improvement of the accuracy of both localization and quantification. Three aspects have been looked at: 1) the acquisition /localization technique used; 2) the strategy used for conversion of signal amplitude/peak areas into concentrations; and 3) methods for MRS signal processing and analysis. With respect to the first aspect, image selected in vivo spectroscopy (ISIS) and point resolved spectroscopy (PRESS), were considered. Aspects of ISIS localization, including relaxation effects during inversion and excitation adiabatic pulses, and uniformity of spin excitation across the "in vivo" 31P spectral range, were investigated using simulation. In order to reduce the chemical shift displacement error in ISIS, a new adiabatic pulse for spin inversion, has been designed and experimentally verified. For PRESS, the performance of the selective 90[degrees] and 180[degrees] pulses was investigated experimentally and using simulations. The consequences of nonideal flip angles on T1 measurements based on two PRESS experiments were analyzed. Effects of amplitude and phase modulation of the ATP signal during the PRESS sequence were analyzed using product-operator formalism for an AMX system. A tissue substitute material, with known metabolite concentrations and simulating the 31P spectrum obtained from neonatal brain, has been developed for testing quantitation accuracy. The manufacture, physical properties and chemical stability of a material has been presented. The following calibration protocols have been experimentally verified: use of water as an internal concentration reference (ICR), and use of a standard phantom as an external concentration reference (ECR). A modified ECR protocol using the tissue substitute material as a reference, has been suggested to deal with problems related to off-resonance effects. This protocol has

  16. Fluorescent Pluronic nanodots for in vivo two-photon imaging

    NASA Astrophysics Data System (ADS)

    Maurin, Mathieu; Vurth, Laeticia; Vial, Jean-Claude; Baldeck, Patrice; Marder, Seth R.; Sanden, Boudewijn Van der; Stephan, Olivier

    2009-06-01

    We report the synthesis of new nanosized fluorescent probes based on bio-compatible polyethylene-polypropylene glycol (Pluronic) materials. In aqueous solution, mini-emulsification of Pluronic with a high fluorescent di-stryl benzene-modified derivative, exhibiting a two-photon absorption cross section as high as 2500 Goeppert-Mayer units at 800 nm, leads to nanoparticles exhibiting a hydrodynamic radius below 100 nm. We have demonstrated that these new probes with luminescence located in the spectral region of interest for bio-imaging (the yellow part of the visible spectrum) allow deep (500 µm) bio-imaging of the mice brain vasculature. The dose injected during our experiments is ten times lower when compared to the classical commercial rhodamine-B isothicyanate-Dextran system but gives similar results to homogeneous blood plasma staining. The mean fluorescent signal intensity stayed constant during more than 1 h.

  17. Fluorescent Pluronic nanodots for in vivo two-photon imaging.

    PubMed

    Maurin, Mathieu; Vurth, Laeticia; Vial, Jean-Claude; Baldeck, Patrice; Marder, Seth R; Van der Sanden, Boudewijn; Stephan, Olivier

    2009-06-10

    We report the synthesis of new nanosized fluorescent probes based on bio-compatible polyethylene-polypropylene glycol (Pluronic) materials. In aqueous solution, mini-emulsification of Pluronic with a high fluorescent di-stryl benzene-modified derivative, exhibiting a two-photon absorption cross section as high as 2500 Goeppert-Mayer units at 800 nm, leads to nanoparticles exhibiting a hydrodynamic radius below 100 nm. We have demonstrated that these new probes with luminescence located in the spectral region of interest for bio-imaging (the yellow part of the visible spectrum) allow deep (500 microm) bio-imaging of the mice brain vasculature. The dose injected during our experiments is ten times lower when compared to the classical commercial rhodamine-B isothicyanate-Dextran system but gives similar results to homogeneous blood plasma staining. The mean fluorescent signal intensity stayed constant during more than 1 h.

  18. Fluorescent Pluronic nanodots for in vivo two-photon imaging.

    PubMed

    Maurin, Mathieu; Vurth, Laeticia; Vial, Jean-Claude; Baldeck, Patrice; Marder, Seth R; Van der Sanden, Boudewijn; Stephan, Olivier

    2009-06-10

    We report the synthesis of new nanosized fluorescent probes based on bio-compatible polyethylene-polypropylene glycol (Pluronic) materials. In aqueous solution, mini-emulsification of Pluronic with a high fluorescent di-stryl benzene-modified derivative, exhibiting a two-photon absorption cross section as high as 2500 Goeppert-Mayer units at 800 nm, leads to nanoparticles exhibiting a hydrodynamic radius below 100 nm. We have demonstrated that these new probes with luminescence located in the spectral region of interest for bio-imaging (the yellow part of the visible spectrum) allow deep (500 microm) bio-imaging of the mice brain vasculature. The dose injected during our experiments is ten times lower when compared to the classical commercial rhodamine-B isothicyanate-Dextran system but gives similar results to homogeneous blood plasma staining. The mean fluorescent signal intensity stayed constant during more than 1 h. PMID:19448291

  19. Influence of Autofluorescence Derived From Living Body on In Vivo Fluorescence Imaging Using Quantum Dots.

    PubMed

    Yukawa, Hiroshi; Watanabe, Masaki; Kaji, Noritada; Baba, Yoshinobu

    2015-02-01

    Quantum dots (QDs) are thought to be a novel inorganic probe for in vivo fluorescence imaging because of their excellent fluorescence properties. Autofluorescence is generally known to be produced from various living bodies including humans, rats, and mice. However, the influence of the autofluorescence on in vivo fluorescence imaging using QDs remains poorly understood. In this article, we assessed the autofluorescence derived from a mouse body and the influence of the autofluorescence on in vivo fluorescence imaging using QDs. The dorsal and ventral autofluorescence derived from a mouse from which the hair was removed were detected under all kinds of excitation/fluorescence filter settings (blue, green, yellow, red, deep red, and NIR) using the Maestro™ in vivo imaging system. The degree of autofluorescence was found to be extremely high in the red filter condition, but transplanted ASCs labeled with QDs on the back of a mouse could be detected in the red filter condition. Moreover, the ASCs labeled with QDs could be traced for at least 5 days. We suggest that fluorescence imaging using QDs can be useful for the detection of transplanted cells. PMID:26858896

  20. Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging.

    PubMed

    Slaughter, Brian D; Schwartz, Joel W; Li, Rong

    2007-12-18

    Fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and photon counting histograms (PCH) are fluctuation methods that emerged recently as potentially useful tools for obtaining parameters of molecular dynamics, interactions, and oligomerization in vivo. Here, we report the successful implementation of FCS, FCCS, and PCH in live yeast cells using fluorescent protein-tagged proteins expressed from their native chromosomal loci, examining cytosolic dynamics and interactions among components of the mitogen activated protein kinase (MAPK) cascade, a widely occurring signaling motif, in response to mating pheromone. FCS analysis detailed the diffusion characteristics and mobile concentrations of MAPK proteins. FCCS analysis using EGFP and mCherry-tagged protein pairs observed the interactions of Ste7 (MAPK kinase) with the MAPKs, Fus3 or Kss1, and of the scaffold protein, Ste5, with Ste7 and Ste11 (MAPK kinase kinase) in the cytosol, providing in vivo constants of their binding equilibrium. The interaction of Ste5 with Fus3 in the cytosol was below the limit of detection, suggesting a weak interaction, if it exists, with K(d) >400-500 nM. Using PCH, we show that cytosolic Ste5 were mostly monomers. Artificial dimerization of Ste5, as confirmed by PCH, using a dimerizing tag, stimulated the interaction between Ste5 and Fus3. Native Ste5 was found to bind Fus3 preferentially at the cortex in pheromone-treated cells, as detected by fluorescence resonance energy transfer (FRET). These results provide a quantitative spatial map of MAPK complexes in vivo and directly support the model that membrane association and regulation of the Ste5 scaffold are critical steps in MAPK activation. PMID:18077328

  1. Image-selected in Vivo spectroscopy (ISIS). A new technique for spatially selective nmr spectroscopy

    NASA Astrophysics Data System (ADS)

    Ordidge, R. J.; Connelly, A.; Lohman, J. A. B.

    A method of spatial localization is described which is particularly suitable for the in vivo spectroscopic investigation of biological and medical samples. The technique overcomes most of the technical problems associated with localized NMR spectroscopy and allows the spectrum to be investigated from a cube which can be positioned by reference to an NMR image. The cube can be reduced or enlarged, and can be rapidly moved in space to investigate further volumes of interest within the sample. The first experimental results from a phantom and the human leg are presented.

  2. Fluorescence lifetime spectroscopy for breast cancer margins assessment

    NASA Astrophysics Data System (ADS)

    Gorpas, Dimitris; Fatakdawala, Hussain; Zhang, Yanhong; Bold, Richard; Marcu, Laura

    2015-03-01

    During breast conserving surgery (BCS), which is the preferred approach to treat most early stage breast cancers, the surgeon attempts to excise the tumor volume, surrounded by thin margin of normal tissue. The intra-operative assessment of cancerous areas is a challenging procedure, with the surgeon usually relying on visual or tactile guidance. This study evaluates whether time-resolved fluorescence spectroscopy (TRFS) presents the potential to address this problem. Point TRFS measurements were obtained from 19 fresh tissue slices (7 patients) and parameters that characterize the transient signals were quantified via constrained least squares deconvolution scheme. Fibrotic tissue (FT, n=69), adipose tissue (AT, n=76), and invasive ductal carcinoma (IDC, n=27) were identified in histology and univariate statistical analysis, followed by multi-comparison test, was applied to the corresponding lifetime data. Significant differentiation between the three tissue types exists at 390 nm and 500 nm bands. The average lifetime is 3.23+/-0.74 ns for AT, 4.21+/-0.83 ns for FT and 4.71+/-0.35 ns (p<0.05) for IDC at 390 nm. Due to the smaller contribution of collagen in AT the average lifetime value is different from FT and IDC. Additionally, although intensity measurements do not show difference between FT and IDC, lifetime can distinguish them. Similarly, in 500 nm these values are 7.01+/-1.08 ns, 5.43+/-1.05 ns and 4.39+/-0.88 ns correspondingly (p<0.05) and this contrast is due to differentiation in retinol or flavins relative concentration, mostly contributing to AT. Results demonstrate the potential of TRFS to intra-operatively characterize BCS breast excised tissue in real-time and assess tumor margins.

  3. Differential Laser-Induced Perturbation Spectroscopy for Analysis of Mixtures of the Fluorophores l-Phenylalanine, l-Tyrosine and l-Tryptophan Using a Fluorescence Probe.

    PubMed

    Oztekin, Erman K; Hahn, David W

    2016-09-01

    Quantitative detection of common endogenous fluorophores is accomplished using differential laser-induced perturbation spectroscopy (DLIPS) with a 193-nm UV fluorescence probe and various UV perturbation wavelengths. In this study, DLIPS is explored as an alternative to traditional fluorescence spectroscopy alone, with a goal of exploring natural fluorophores pursuant to biological samples and tissue analysis. To this end, aromatic amino acids, namely, l-phenylalanine, l-tyrosine and l-tryptophan are mixed with differing mass ratios and then classified with various DLIPS schemes. Classification with a traditional fluorescence probe is used as a benchmark. The results show a 20% improvement in classification performance of the DLIPS method over the traditional fluorescence method using partial least squares (PLS) analysis. Additional multivariate analyses are explored, and the relevant photochemistry is elucidated in the context of perturbation wavelengths. We conclude that DLIPS is a promising biosensing approach with potential for in vivo analysis given the current findings with fluorophores relevant to biological tissues.

  4. Multimodal Raman-fluorescence spectroscopy of formalin fixed samples is able to discriminate brain tumors from dysplastic tissue

    NASA Astrophysics Data System (ADS)

    Anand, Suresh; Cicchi, Riccardo; Giordano, Flavio; Buccoliero, Anna Maria; Pavone, Francesco Saverio

    2014-05-01

    In the recent years, there has been a considerable surge in the application of spectroscopy for disease diagnosis. Raman and fluorescence spectra provide characteristic spectral profile related to biochemical and morphological changes when tissues progress from normal state towards malignancy. Spectroscopic techniques offer the advantage of being minimally invasive compared to traditional histopathology, real time and quantitative. In biomedical optical diagnostics, freshly excised specimens are preferred for making ex-vivo spectroscopic measurements. With regard to fresh tissues, if the lab is located far away from the clinic it could pose a problem as spectral measurements have to be performed immediately after dissection. Tissue samples are usually placed in a fixative agent such as 4% formaldehyde to preserve the samples before processing them for routine histopathological studies. Fixation prevents the tissues from decomposition by arresting autolysis. In the present study, we intend to investigate the possibility of using formalin fixed samples for discrimination of brain tumours from dysplastic tissue using Raman and fluorescence spectroscopy. Formalin fixed samples were washed with phosphate buffered saline for about 5 minutes in order to remove the effects of formalin during spectroscopic measurements. In case of fluorescence spectroscopy, changes in spectral profile have been observed in the region between 550-670 nm between dysplastic and tumor samples. For Raman measurements, we found significant differences in the spectral profiles between dysplasia and tumor. In conclusion, formalin fixed samples can be potentially used for the spectroscopic discrimination of tumor against dysplastic tissue in brain samples.

  5. Real time monitoring of superoxide dynamics in vivo through fluorescent proteins using a sensitive fiber probe

    NASA Astrophysics Data System (ADS)

    Chang, Yu-Chung; Ken, Chuian-Fu; Hsu, Che-Wei; Liu, Ya-Ging

    2014-03-01

    Superoxide anion is the primary oxygen free radical generated in mitochondria that causes intracellular oxidative stress. The lack of a method to directly monitor superoxide concentration in vivo in real time has severely hindered our understanding on its pathophysiology. We made transgenic zebrafish to specifically express fluorescent proteins, which are recently developed as reversible superoxide-specific indicators, in the liver. A fiber-optic fluorescent probe was used to noninvasively monitor superoxide generation in the liver in real time. The fish were placed in microfluidic channels for manipulation and reagents administration. Several superoxide-inducing and scavenging reagents were administrated onto the fish to investigate their effects on superoxide anion balancing. The biochemical dynamics of superoxide due to the application reagents were revealed in the transient behaviors of fluorescence time courses. With the ability to monitor superoxide dynamics in vivo in real time, this method can be used as an in vivo pharmaceutical screening platform.

  6. Cutaneous tumors in vivo investigations using fluorescence and diffuse reflectance techniques

    NASA Astrophysics Data System (ADS)

    Borisova, E.; Troyanova, P.; Nikolova, E.; Avramov, L.

    2008-06-01

    In the recent years, there has been growing interest in the common use of laser-induced autofluorescence (LIAF) and reflectance spectroscopy (RS) to differentiate disease from normal surrounding tissue - so called optical biopsy method. Painless, instant diagnoses from optical biopsies will soon be a reality. These forms of optical diagnoses are preferable to the removal of several square millimeters of tissue surface - common in traditional biopsies - followed by delays while samples are sent for clinical analysis. The goal of this work was investigation of cutaneous benign and malignant lesions by the methods of LIAFS and RS. A nitrogen laser at 337 nm was applied for the needs of autofluorescence excitation. Broad-spectrum halogen lamp (from 400 to 900 nm) was applied for diffuse reflectance measurements. An associated microspectrometer detected in vivo the fluorescence and reflectance signals from human skin. The main spectral features of benign lesions - compound nevus, dysplastic nevi, heamangioma and basal cell papilloma and malignant lesions - pigmented, amelanotic and secondary malignant melanoma, as well as basal cell carcinoma are discussed and their possible origins are indicated. Spectra from healthy skin areas near to the lesion were detected to be used posteriori to reveal changes between healthy and lesion skin spectra. Influence of the main skin pigments on the spectra detected is discussed and evaluation of possibilities for differentiation between malignant and benign lesions is made based on their spectral properties. This research shows that non-invasive and high-sensitive in vivo detection by means of appropriate light sources and detectors should be possible, related to real-time determination of existing pathological conditions.

  7. Modeling in vivo fluorescence of small animals using TracePro software

    NASA Astrophysics Data System (ADS)

    Leavesley, Silas; Rajwa, Bartek; Freniere, Edward R.; Smith, Linda; Hassler, Richard; Robinson, J. Paul

    2007-02-01

    The theoretical modeling of fluorescence excitation, emission, and propagation within living tissue has been a limiting factor in the development and calibration of in vivo small animal fluorescence imagers. To date, no definitive calibration standard, or phantom, has been developed for use with small animal fluorescence imagers. Our work in the theoretical modeling of fluorescence in small animals using solid modeling software is useful in optimizing the design of small animal imaging systems, and in predicting their response to a theoretical model. In this respect, it is also valuable in the design of a fluorescence phantom for use in in vivo small animal imaging. The use of phantoms is a critical step in the testing and calibration of most diagnostic medical imaging systems. Despite this, a realistic, reproducible, and informative phantom has yet to be produced for use in small animal fluorescence imaging. By modeling the theoretical response of various types of phantoms, it is possible to determine which parameters are necessary for accurately modeling fluorescence within inhomogenous scattering media such as tissue. Here, we present the model that has been developed, the challenges and limitations associated with developing such a model, and the applicability of this model to experimental results obtained in a commercial small animal fluorescence imager.

  8. Multiple stimulated emission fluorescence photoacoustic sensing and spectroscopy

    NASA Astrophysics Data System (ADS)

    Li, Gaoming; Gao, Fei; Qiu, Yishen; Feng, Xiaohua; Zheng, Yuanjin

    2016-07-01

    Multiple stimulated emission fluorescence photoacoustic (MSEF-PA) phenomenon is demonstrated in this letter. Under simultaneous illumination of pumping light and stimulated emission light, the fluorescence emission process is speeded up by the stimulated emission effect. This leads to nonlinear enhancement of photoacoustic signal while the quantity of absorbed photons is more than that of fluorescent molecules illuminated by pumping light. The electronic states' specificity of fluorescent molecular can also be labelled by the MSEF-PA signals, which can potentially be used to obtain fluorescence excitation spectrum in deep scattering tissue with nonlinearly enhanced photoacoustic detection. In this preliminary study, the fluorescence excitation spectrum is reconstructed by MSEF-PA signals through sweeping the wavelength of exciting light, which confirms the theoretical derivation well.

  9. Fluorescent spectroscopy, a technique for characterizing surface films

    USGS Publications Warehouse

    Goldberg, Marvin C.; Devonald, David H.

    1973-01-01

    A relationship is established between fluorescent spectra obtained by using a light path through the liquid solution and the fluorescent spectra obtained by a direct reading of surface reflection (remote sensing). A brief review of quantum fundamentals provides the necessary information to conclude that "see-through" and reflectance spectra are identical in wavelength response. Many floating films contain fluorescent materials; thus fluorescent spectroscopic techniques were used to obtain the fluorescent spectra of lube oil, crude oil, andlignosulfonie acid in an effort to detect, identify, and quantify these representative fluorescent materials in water solutions. For each material tested the emission maximum was established. The emission maximum was then held constant while the absorption spectrum was recorded and the absorption maximum established. The complete spectral curves are presented.

  10. High volume confinement in two-photon fluorescence correlation spectroscopy with radially polarized light

    NASA Astrophysics Data System (ADS)

    Ivanov, D.; Shcheslavskiy, V.; Märki, I.; Leutenegger, M.; Lasser, T.

    2009-02-01

    We present the results on two-photon total-internal-reflection fluorescence correlation spectroscopy. The combination of liquid crystal spatial light modulator, providing radial polarization, with ultrafast laser (picosecond Nd:GdVO4 laser) allowed us to take the advantage of nonlinear optical contrast mechanisms to suppress the side-lobe energy specific for radial polarization and reduce the effective excited volume twice compared to one-photon evanescent wave excitation in fluorescence correlation spectroscopy.

  11. High volume confinement in two-photon total-internal-reflection fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Ivanov, D.; Shcheslavskiy, V.; Märki, I.; Leutenegger, M.; Lasser, T.

    2009-02-01

    We report results on two-photon total-internal-reflection fluorescence correlation spectroscopy with radially polarized light. The combination of liquid crystal spatial light modulator, providing radial polarization with ultrafast laser (picosecond Nd:GdVO4 laser), allowed us to take the advantage of nonlinear optical contrast mechanisms to suppress the side-lobe energy specific for radial polarization and reduce the effective excited volume twice compared to one-photon evanescent wave excitation in fluorescence correlation spectroscopy.

  12. Fluorescence dynamics of human epidermis (ex vivo) and skin (in vivo)

    NASA Astrophysics Data System (ADS)

    Salomatina, Elena V.; Pravdin, Alexander B.

    2003-10-01

    The temporal behavior of autofluorescence of human skin and epidermis under continuous UV-irradiation has been studied. Fluorescence spectra and kinetic curves of fluorescence intensity have been obtained. The fluorescence intensity recovery after dark period also has been examined. The vitiligo skin and epidermis were used for comparing their spectra with reflectance and fluorescence spectra of healthy skin. The epidermal samples were prepared using surface epidermis stripping technique. It has been concluded that fluorophores being undergone the UVA photobleaching are actually present in epidermal layer, and immediate pigment darkening does contribute, no less than a half of magnitude, to the autofluorescence decrease under continuous UVA irradiation.

  13. Biocompatible fluorescent nanoparticles for in vivo stem cell tracking

    NASA Astrophysics Data System (ADS)

    Cova, Lidia; Bigini, Paolo; Diana, Valentina; Sitia, Leopoldo; Ferrari, Raffaele; Pesce, Ruggiero Maria; Khalaf, Rushd; Bossolasco, Patrizia; Ubezio, Paolo; Lupi, Monica; Tortarolo, Massimo; Colombo, Laura; Giardino, Daniela; Silani, Vincenzo; Morbidelli, Massimo; Salmona, Mario; Moscatelli, Davide

    2013-06-01

    Efficient application of stem cells to the treatment of neurodegenerative diseases requires safe cell tracking to follow stem cell fate over time in the host environment after transplantation. In this work, for the first time, fluorescent and biocompatible methyl methacrylate (MMA)-based nanoparticles (fluoNPs) were synthesized through a free-radical co-polymerization process with a fluorescent macromonomer obtained by linking Rhodamine B and hydroxyethyl methacrylate. We demonstrate that the fluoNPs produced by polymerization of MMA-Rhodamine complexes (1) were efficient for the labeling and tracking of multipotent human amniotic fluid cells (hAFCs); (2) did not alter the main biological features of hAFCs (such as viability, cell growth and metabolic activity); (3) enabled us to determine the longitudinal bio-distribution of hAFCs in different brain areas after graft in the brain ventricles of healthy mice by a direct fluorescence-based technique. The reliability of our approach was furthermore confirmed by magnetic resonance imaging analyses, carried out by incubating hAFCs with both superparamagnetic iron oxide nanoparticles and fluoNPs. Our data suggest that these finely tunable and biocompatible fluoNPs can be exploited for the longitudinal tracking of stem cells.

  14. Tryptophan content for monitoring breast cancer cell aggressiveness by native fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Lin; Pu, Yang; Xue, Jianpeng; Pratavieira, Sebastião.; Xu, Baogang; Achilefu, Samuel; Alfano, R. R.

    2014-03-01

    This study shows tryptophan as the key native marker in cells to determine the level of aggressive cancer in breast cell lines using native fluorescence spectroscopy. An algorithm based on the ratio of tryptophan fluorescence intensity at 340 nm to intensity at 460 nm is associated with aggressiveness of the cancer cells. The higher the ratio is, the more aggressive the tumor towards metastasis.

  15. Lipidots: competitive organic alternative to quantum dots for in vivo fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Gravier, Julien; Navarro, Fabrice P.; Delmas, Thomas; Mittler, Frédérique; Couffin, Anne-Claude; Vinet, Françoise; Texier, Isabelle

    2011-09-01

    The use of fluorescent nanostructures can bring several benefits on the signal to background ratio for in vitro microscopy, in vivo small animal imaging, and image-guided surgery. Fluorescent quantum dots (QDs) display outstanding optical properties, with high brightness and low photobleaching rate. However, because of their toxic element core composition and their potential long term retention in reticulo-endothelial organs such as liver, their in vivo human applications seem compromised. The development of new dye-loaded (DiO, DiI, DiD, DiR, and Indocyanine Green (ICG)) lipid nanoparticles for fluorescence imaging (lipidots) is described here. Lipidot optical properties quantitatively compete with those of commercial QDs (QTracker®705). Multichannel in vivo imaging of lymph nodes in mice is demonstrated for doses as low as 2 pmols of particles. Along with their optical properties, fluorescent lipidots display very low cytotoxicity (IC50 > 75 nM), which make them suitable tools for in vitro, and especially in vivo, fluorescence imaging applications.

  16. In vivo imaging of cerebral energy metabolism with two-photon fluorescence lifetime microscopy of NADH

    PubMed Central

    Yaseen, Mohammad A.; Sakadžić, Sava; Wu, Weicheng; Becker, Wolfgang; Kasischke, Karl A.; Boas, David A.

    2013-01-01

    Minimally invasive, specific measurement of cellular energy metabolism is crucial for understanding cerebral pathophysiology. Here, we present high-resolution, in vivo observations of autofluorescence lifetime as a biomarker of cerebral energy metabolism in exposed rat cortices. We describe a customized two-photon imaging system with time correlated single photon counting detection and specialized software for modeling multiple-component fits of fluorescence decay and monitoring their transient behaviors. In vivo cerebral NADH fluorescence suggests the presence of four distinct components, which respond differently to brief periods of anoxia and likely indicate different enzymatic formulations. Individual components show potential as indicators of specific molecular pathways involved in oxidative metabolism. PMID:23412419

  17. In vivo fluorescent imaging of the mouse retina using adaptive optics

    PubMed Central

    Biss, David P.; Sumorok, Daniel; Burns, Stephen A.; Webb, Robert H.; Zhou, Yaopeng; Bifano, Thomas G.; Côté, Daniel; Veilleux, Israel; Zamiri, Parisa; Lin, Charles P.

    2009-01-01

    In vivo imaging of the mouse retina using visible and near infrared wavelengths does not achieve diffraction-limited resolution due to wavefront aberrations induced by the eye. Considering the pupil size and axial dimension of the eye, it is expected that unaberrated imaging of the retina would have a transverse resolution of 2 μm. Higher-order aberrations in retinal imaging of human can be compensated for by using adaptive optics. We demonstrate an adaptive optics system for in vivo imaging of fluorescent structures in the retina of a mouse, using a microelectromechanical system membrane mirror and a Shack–Hartmann wavefront sensor that detects fluorescent wavefront. PMID:17308593

  18. Monitoring the biodegradation of dendritic near-infrared nanoprobes by in vivo fluorescence imaging.

    PubMed

    Almutairi, Adah; Akers, Walter J; Berezin, Mikhail Y; Achilefu, Samuel; Fréchet, Jean M J

    2008-01-01

    Synthetic polymers and dendrimers have been widely used by the medical community to overcome biological barriers and enhance in vivo biomedical applications. Despite the widespread use of biomaterials it has been generally extremely difficult to monitor noninvasively their fate in vivo. Here we report multilayered nanoprobes, consisting of a near-infrared core, nanoencapsulated in a biodegradable dendrimer, and surrounded by a shell of polyethylene oxide. Covalent encapsulation of the near-infrared fluorophores in the dendritic scaffold conferred enhanced stability to the nanoprobe with added resistance to enzymatic oxidation and prolonged blood residence time. Insight into the time course of biodegradation of the dendritic aliphatic polyester nanoprobe was gained using noninvasive whole body in vivo fluorescence lifetime imaging. As the dendritic shell biodegrades the NIR probe becomes exposed, enabling monitoring of fluorescence lifetime changes in vivo. PMID:19434857

  19. In Vivo Two-Photon Fluorescence Kinetics of Primate Rods and Cones

    PubMed Central

    Sharma, Robin; Schwarz, Christina; Williams, David R.; Palczewska, Grazyna; Palczewski, Krzysztof; Hunter, Jennifer J.

    2016-01-01

    Purpose The retinoid cycle maintains vision by regenerating bleached visual pigment through metabolic events, the kinetics of which have been difficult to characterize in vivo. Two-photon fluorescence excitation has been used previously to track autofluorescence directly from retinoids and pyridines in the visual cycle in mouse and frog retinas, but the mechanisms of the retinoid cycle are not well understood in primates. Methods We developed a two-photon fluorescence adaptive optics scanning light ophthalmoscope dedicated to in vivo imaging in anesthetized macaques. Using pulsed light at 730 nm, two-photon fluorescence was captured from rods and cones during light and dark adaptation through the eye's pupil. Results The fluorescence from rods and cones increased with light exposure but at different rates. During dark adaptation, autofluorescence declined, with cone autofluorescence decreasing approximately 4 times faster than from rods. Rates of autofluorescence decrease in rods and cones were approximately 4 times faster than their respective rates of photopigment regeneration. Also, subsets of sparsely distributed cones were less fluorescent than their neighbors immediately following bleach at 565 nm and they were comparable with the S cone mosaic in density and distribution. Conclusions Although other molecules could be contributing, we posit that these fluorescence changes are mediated by products of the retinoid cycle. In vivo two-photon ophthalmoscopy provides a way to monitor noninvasively stages of the retinoid cycle that were previously inaccessible in the living primate eye. This can be used to assess objectively photoreceptor function in normal and diseased retinas. PMID:26903225

  20. A fluorescence spectroscopy study of traditional Chinese medicine Angelica

    NASA Astrophysics Data System (ADS)

    Zhao, Hongyan; Song, Feng; Liu, Shujing; Chen, Guiyang; Wei, Chen; Liu, Yanling; Liu, Jiadong

    2013-10-01

    By measuring the fluorescence spectra of Chinese medicine (CM) Angelica water solutions with different concentrations from 0.025 to 2.5 mg/mL, results showed that the fluorescence intensity was proportional to the concentration. Through fluorescence spectra of Angelica solution under different pH values, results indicated coumarin compounds were the active ingredients of Angelica. We also observed fluorescence quenching of the Angelica solution in the presence of spherical silver nanoparticles with radius of 12 nm. Keeping a certain value for the volume of the silver nanoparticles, the fluorescence intensity at 402 nm was linearly proportional to the Angelica in the range of 1-3 mg/mL.

  1. Portable instrument that integrates irradiation with fluorescence and reflectance spectroscopies during clinical photodynamic therapy of cutaneous disease

    NASA Astrophysics Data System (ADS)

    Cottrell, W. J.; Oseroff, A. R.; Foster, T. H.

    2006-06-01

    We report a portable clinical instrument for delivering photodynamic therapy (PDT) while performing noninvasive spectroscopic monitoring in vivo. Using an off-surface probe, the instrument delivers the treatment beam to a user-defined field on the skin and performs reflectance and fluorescence spectroscopies at two regions within this field. The instrument is being used to monitor photosensitizer fluorescence photobleaching, fluorescent photoproduct kinetics, blood volume, and hemoglobin oxygen saturation during a pilot clinical trial of 5-aminolevulinic acid-PDT treatment of superficial basal cell carcinoma (BCC). Protoporphyrin IX and photoproduct fluorescence excited by the 633nm PDT treatment laser is collected between 655 and 800nm. During a series of brief treatment interruptions at programable time points, white light reflectance spectra between 475 and 800nm are acquired. Fluorescence spectra are corrected for the effects of absorption and scattering, informed by the reflectance measurements, and then decomposed into known fluorophore contributions in real time using a robust singular value decomposition fitting routine. Reflectance spectra additionally provide information on blood volume and hemoglobin oxygen saturation. Monitoring blood oxygenation and implicit dose metrics such as photosensitizer photobleaching during PDT allows the improved interpretation of clinical results and is helping to guide the treatment protocol for an anticipated low-irradiance PDT clinical trial of BCC.

  2. In vivo diffuse correlation spectroscopy investigation of the ocular fundus

    NASA Astrophysics Data System (ADS)

    Cattini, Stefano; Staurenghi, Giovanni; Gatti, Antonietta; Rovati, Luigi

    2013-05-01

    Diffuse correlation spectroscopy (DCS) measurements in vivo recorded from rabbits' ocular fundus are presented. Despite the complexity of these ocular tissues, we provide a clear and simple demonstration of the DCS abilities to analyze variations in physiological quantities of clinical interest. Indeed, the reported experimental activities demonstrate that DCS can reveal both choroidal-flow and temperature variations and detect nano- and micro-aggregates in ocular fundus circulation. Such abilities can be of great interest both in fundamental research and practical clinical applications. The proposed measuring system can be useful in: (a) monitoring choroidal blood flow variations, (b) determining the end-point for photo-dynamic therapy and transpupillary thermo therapy and, (c) managing the dye injection and determining an end-point for dye-enhanced photothrombosis. Moreover, it could allow both diagnoses when the presence of nano- and micro-aggregates is related to specific diseases and verifying the effects of nanoparticle injection in nanomedicine. Even though the reported results demonstrate the applicability of DCS to investigate ocular fundus, a detailed and accurate investigation of the limits of detection is beyond the scope of this article.

  3. In vivo diffuse correlation spectroscopy investigation of the ocular fundus.

    PubMed

    Cattini, Stefano; Staurenghi, Giovanni; Gatti, Antonietta; Rovati, Luigi

    2013-05-01

    Diffuse correlation spectroscopy (DCS) measurements in vivo recorded from rabbits' ocular fundus are presented. Despite the complexity of these ocular tissues, we provide a clear and simple demonstration of the DCS abilities to analyze variations in physiological quantities of clinical interest. Indeed, the reported experimental activities demonstrate that DCS can reveal both choroidal-flow and temperature variations and detect nano- and micro-aggregates in ocular fundus circulation. Such abilities can be of great interest both in fundamental research and practical clinical applications. The proposed measuring system can be useful in: (a) monitoring choroidal blood flow variations, (b) determining the end-point for photo-dynamic therapy and transpupillary thermo therapy and, (c) managing the dye injection and determining an end-point for dye-enhanced photothrombosis. Moreover, it could allow both diagnoses when the presence of nano- and micro-aggregates is related to specific diseases and verifying the effects of nanoparticle injection in nanomedicine. Even though the reported results demonstrate the applicability of DCS to investigate ocular fundus, a detailed and accurate investigation of the limits of detection is beyond the scope of this article.

  4. In vivo impedance spectroscopy of deep brain stimulation electrodes

    NASA Astrophysics Data System (ADS)

    Lempka, Scott F.; Miocinovic, Svjetlana; Johnson, Matthew D.; Vitek, Jerrold L.; McIntyre, Cameron C.

    2009-08-01

    Deep brain stimulation (DBS) represents a powerful clinical technology, but a systematic characterization of the electrical interactions between the electrode and the brain is lacking. The goal of this study was to examine the in vivo changes in the DBS electrode impedance that occur after implantation and during clinically relevant stimulation. Clinical DBS devices typically apply high-frequency voltage-controlled stimulation, and as a result, the injected current is directly regulated by the impedance of the electrode-tissue interface. We monitored the impedance of scaled-down clinical DBS electrodes implanted in the thalamus and subthalamic nucleus of a rhesus macaque using electrode impedance spectroscopy (EIS) measurements ranging from 0.5 Hz to 10 kHz. To further characterize our measurements, equivalent circuit models of the electrode-tissue interface were used to quantify the role of various interface components in producing the observed electrode impedance. Following implantation, the DBS electrode impedance increased and a semicircular arc was observed in the high-frequency range of the EIS measurements, commonly referred to as the tissue component of the impedance. Clinically relevant stimulation produced a rapid decrease in electrode impedance with extensive changes in the tissue component. These post-operative and stimulation-induced changes in impedance could play an important role in the observed functional effects of voltage-controlled DBS and should be considered during clinical stimulation parameter selection and chronic animal research studies.

  5. Fluorescence imaging of experimental rheumatoid arthritis in vivo using a fast flying-spot scanner

    NASA Astrophysics Data System (ADS)

    Berger, J.; Voigt, J.; Seifert, F.; Ebert, B.; Macdonald, R.; Gemeinhardt, I.; Gemeinhardt, O.; Schnorr, J.; Taupitz, M.; Vater, A.; Vollmer, S.; Licha, K.; Schirner, M.

    2007-07-01

    We have developed a flying-spot scanner for fluorescence imaging of rheumatoid arthritis in the near infrared (NIR) spectral range following intravenous administration of contrast agents. The new imaging system has been characterized with respect to linearity, dynamic range and spatial resolution with the help of fluorescent phantoms. In vivo experiments were performed on an animal model of rheumatoid arthritis. Finally, NIR-fluorescence images of early stages of joint inflammation have been compared with findings from contrast enhanced MR imaging and histology.

  6. In vivo simultaneous multispectral fluorescence imaging with spectral multiplexed volume holographic imaging system

    NASA Astrophysics Data System (ADS)

    Lv, Yanlu; Zhang, Jiulou; Zhang, Dong; Cai, Wenjuan; Chen, Nanguang; Luo, Jianwen

    2016-06-01

    A simultaneous multispectral fluorescence imaging system incorporating multiplexed volume holographic grating (VHG) is developed to acquire multispectral images of an object in one shot. With the multiplexed VHG, the imaging system can provide the distribution and spectral characteristics of multiple fluorophores in the scene. The implementation and performance of the simultaneous multispectral imaging system are presented. Further, the system's capability in simultaneously obtaining multispectral fluorescence measurements is demonstrated with in vivo experiments on a mouse. The demonstrated imaging system has the potential to obtain multispectral images fluorescence simultaneously.

  7. Construction of In Vivo Fluorescent Imaging of Echinococcus granulosus in a Mouse Model.

    PubMed

    Wang, Sibo; Yang, Tao; Zhang, Xuyong; Xia, Jie; Guo, Jun; Wang, Xiaoyi; Hou, Jixue; Zhang, Hongwei; Chen, Xueling; Wu, Xiangwei

    2016-06-01

    Human hydatid disease (cystic echinococcosis, CE) is a chronic parasitic infection caused by the larval stage of the cestode Echinococcus granulosus. As the disease mainly affects the liver, approximately 70% of all identified CE cases are detected in this organ. Optical molecular imaging (OMI), a noninvasive imaging technique, has never been used in vivo with the specific molecular markers of CE. Thus, we aimed to construct an in vivo fluorescent imaging mouse model of CE to locate and quantify the presence of the parasites within the liver noninvasively. Drug-treated protoscolices were monitored after marking by JC-1 dye in in vitro and in vivo studies. This work describes for the first time the successful construction of an in vivo model of E. granulosus in a small living experimental animal to achieve dynamic monitoring and observation of multiple time points of the infection course. Using this model, we quantified and analyzed labeled protoscolices based on the intensities of their red and green fluorescence. Interestingly, the ratio of red to green fluorescence intensity not only revealed the location of protoscolices but also determined the viability of the parasites in vivo and in vivo tests. The noninvasive imaging model proposed in this work will be further studied for long-term detection and observation and may potentially be widely utilized in susceptibility testing and therapeutic effect evaluation. PMID:27417083

  8. Construction of In Vivo Fluorescent Imaging of Echinococcus granulosus in a Mouse Model.

    PubMed

    Wang, Sibo; Yang, Tao; Zhang, Xuyong; Xia, Jie; Guo, Jun; Wang, Xiaoyi; Hou, Jixue; Zhang, Hongwei; Chen, Xueling; Wu, Xiangwei

    2016-06-01

    Human hydatid disease (cystic echinococcosis, CE) is a chronic parasitic infection caused by the larval stage of the cestode Echinococcus granulosus. As the disease mainly affects the liver, approximately 70% of all identified CE cases are detected in this organ. Optical molecular imaging (OMI), a noninvasive imaging technique, has never been used in vivo with the specific molecular markers of CE. Thus, we aimed to construct an in vivo fluorescent imaging mouse model of CE to locate and quantify the presence of the parasites within the liver noninvasively. Drug-treated protoscolices were monitored after marking by JC-1 dye in in vitro and in vivo studies. This work describes for the first time the successful construction of an in vivo model of E. granulosus in a small living experimental animal to achieve dynamic monitoring and observation of multiple time points of the infection course. Using this model, we quantified and analyzed labeled protoscolices based on the intensities of their red and green fluorescence. Interestingly, the ratio of red to green fluorescence intensity not only revealed the location of protoscolices but also determined the viability of the parasites in vivo and in vivo tests. The noninvasive imaging model proposed in this work will be further studied for long-term detection and observation and may potentially be widely utilized in susceptibility testing and therapeutic effect evaluation.

  9. Construction of In Vivo Fluorescent Imaging of Echinococcus granulosus in a Mouse Model

    PubMed Central

    Wang, Sibo; Yang, Tao; Zhang, Xuyong; Xia, Jie; Guo, Jun; Wang, Xiaoyi; Hou, Jixue; Zhang, Hongwei; Chen, Xueling; Wu, Xiangwei

    2016-01-01

    Human hydatid disease (cystic echinococcosis, CE) is a chronic parasitic infection caused by the larval stage of the cestode Echinococcus granulosus. As the disease mainly affects the liver, approximately 70% of all identified CE cases are detected in this organ. Optical molecular imaging (OMI), a noninvasive imaging technique, has never been used in vivo with the specific molecular markers of CE. Thus, we aimed to construct an in vivo fluorescent imaging mouse model of CE to locate and quantify the presence of the parasites within the liver noninvasively. Drug-treated protoscolices were monitored after marking by JC-1 dye in in vitro and in vivo studies. This work describes for the first time the successful construction of an in vivo model of E. granulosus in a small living experimental animal to achieve dynamic monitoring and observation of multiple time points of the infection course. Using this model, we quantified and analyzed labeled protoscolices based on the intensities of their red and green fluorescence. Interestingly, the ratio of red to green fluorescence intensity not only revealed the location of protoscolices but also determined the viability of the parasites in vivo and in vivo tests. The noninvasive imaging model proposed in this work will be further studied for long-term detection and observation and may potentially be widely utilized in susceptibility testing and therapeutic effect evaluation. PMID:27417083

  10. A 32-channel photon counting module with embedded auto/cross-correlators for real-time parallel fluorescence correlation spectroscopy

    SciTech Connect

    Gong, S.; Labanca, I.; Rech, I.; Ghioni, M.

    2014-10-15

    Fluorescence correlation spectroscopy (FCS) is a well-established technique to study binding interactions or the diffusion of fluorescently labeled biomolecules in vitro and in vivo. Fast FCS experiments require parallel data acquisition and analysis which can be achieved by exploiting a multi-channel Single Photon Avalanche Diode (SPAD) array and a corresponding multi-input correlator. This paper reports a 32-channel FPGA based correlator able to perform 32 auto/cross-correlations simultaneously over a lag-time ranging from 10 ns up to 150 ms. The correlator is included in a 32 × 1 SPAD array module, providing a compact and flexible instrument for high throughput FCS experiments. However, some inherent features of SPAD arrays, namely afterpulsing and optical crosstalk effects, may introduce distortions in the measurement of auto- and cross-correlation functions. We investigated these limitations to assess their impact on the module and evaluate possible workarounds.

  11. A 32-channel photon counting module with embedded auto/cross-correlators for real-time parallel fluorescence correlation spectroscopy

    PubMed Central

    Gong, S.; Labanca, I.; Rech, I.; Ghioni, M.

    2014-01-01

    Fluorescence correlation spectroscopy (FCS) is a well-established technique to study binding interactions or the diffusion of fluorescently labeled biomolecules in vitro and in vivo. Fast FCS experiments require parallel data acquisition and analysis which can be achieved by exploiting a multi-channel Single Photon Avalanche Diode (SPAD) array and a corresponding multi-input correlator. This paper reports a 32-channel FPGA based correlator able to perform 32 auto/cross-correlations simultaneously over a lag-time ranging from 10 ns up to 150 ms. The correlator is included in a 32 × 1 SPAD array module, providing a compact and flexible instrument for high throughput FCS experiments. However, some inherent features of SPAD arrays, namely afterpulsing and optical crosstalk effects, may introduce distortions in the measurement of auto- and cross-correlation functions. We investigated these limitations to assess their impact on the module and evaluate possible workarounds. PMID:25362365

  12. Biosynthesis of fluorescent gold nanoclusters for in vitro and in vivo tumor imaging

    NASA Astrophysics Data System (ADS)

    Li, Linlin; Liu, Xi; Fu, Changhui; Tan, Longfei; Liu, Huiyu

    2015-11-01

    Recently, fluorescent metallic nanoclusters with sizes in the few-nanometer range have showed great potentials in biomedical applications for their stable and tailorable fluorescence. Although many studies have focused on fabricating these kinds of materials with chemical methods, there has been little focus on the biosynthesis of gold nanoclusters with green and facile methods. In this study, a facile, scalable, cost-effective and environmentally benign biosynthesis approach was developed to produce fluorescent gold nanoclusters (AuNCs). Biomasses including egg white, egg yolk and serums were used as both capping agents and reductants in the biosynthesis of AuNCs. As a new kind of fluorescent imaging agent, they were used for in vitro and in vivo tumor imaging that can efficiently track cancer cells with excellent biocompatibility. This work provides new insight into green biosynthesis and biomedical applications of fluorescent metallic nanoclusters.

  13. In vivo fluorescence spectra unmixing and autofluorescence removal by sparse nonnegative matrix factorization.

    PubMed

    Montcuquet, Anne-Sophie; Hervé, Lionel; Navarro, Fabrice; Dinten, Jean-Marc; Mars, Jérôme I

    2011-09-01

    Fluorescence imaging locates fluorescent markers that specifically bind to targets; like tumors, markers are injected to a patient, optimally excited with near-infrared light, and located thanks to backward-emitted fluorescence analysis. To investigate thick and diffusive media, as the fluorescence signal decreases exponentially with the light travel distance, the autofluorescence of biological tissues comes to be a limiting factor. To remove autofluorescence and isolate specific fluorescence, a spectroscopic approach, based on nonnegative matrix factorization (NMF), is explored. To improve results on spatially sparse markers detection, we suggest a new constrained NMF algorithm that takes sparsity constraints into account. A comparative study between both algorithms is proposed on simulated and in vivo data.

  14. Soft nanomaterial-based targeting polymersomes for near-infrared fluorescence multispectral in vivo imaging.

    PubMed

    Li, Zuhong; Wu, Liyuan; Hu, Peiran; Han, Sihai; Zhang, Tao; Fan, Hongliang; Jin, Wei; Jin, Qinhan; Mu, Ying

    2012-11-21

    We report here the soft nanomaterial-based targeting polymersomes for near-infrared (NIR) fluorescence imaging to carry out in vivo tumor detection. Two polymersome-based NIR fluorescent probes were prepared through the self-assembly of amphiphilic block copolymers, poly(butadiene-b-ethylene oxide) (PEO-b-PBD). Each of them was encapsulated with distinct hydrophobic near-infrared dyes (DiD and DiR) and modified with different targeting ligands (anti-CEA antibody and anti-EGFR antibody), respectively. After simultaneous injection of these two probes into the tumor-bearing mice via tail vein, multispectral near-infrared fluorescence images were obtained. The results indicate that both probes are successfully directed to the tumor foci, where two distinguishable fluorescent signals were detected through the unmixed fluorescence images. By taking advantage of two targeting polymersome-based probes with distinct fluorescent features, the proposed multispectral near-infrared fluorescence imaging method can greatly improve the specificity and accuracy for in vivo tumor detection.

  15. Near-Infrared Squaraine Dye Encapsulated Micelles for in Vivo Fluorescence and Photoacoustic Bimodal Imaging.

    PubMed

    Sreejith, Sivaramapanicker; Joseph, James; Lin, Manjing; Menon, Nishanth Venugopal; Borah, Parijat; Ng, Hao Jun; Loong, Yun Xian; Kang, Yuejun; Yu, Sidney Wing-Kwong; Zhao, Yanli

    2015-06-23

    Combined near-infrared (NIR) fluorescence and photoacoustic imaging techniques present promising capabilities for noninvasive visualization of biological structures. Development of bimodal noninvasive optical imaging approaches by combining NIR fluorescence and photoacoustic tomography demands suitable NIR-active exogenous contrast agents. If the aggregation and photobleaching are prevented, squaraine dyes are ideal candidates for fluorescence and photoacoustic imaging. Herein, we report rational selection, preparation, and micelle encapsulation of an NIR-absorbing squaraine dye (D1) for in vivo fluorescence and photoacoustic bimodal imaging. D1 was encapsulated inside micelles constructed from a biocompatible nonionic surfactant (Pluoronic F-127) to obtain D1-encapsulated micelles (D1(micelle)) in aqueous conditions. The micelle encapsulation retains both the photophysical features and chemical stability of D1. D1(micelle) exhibits high photostability and low cytotoxicity in biological conditions. Unique properties of D1(micelle) in the NIR window of 800-900 nm enable the development of a squaraine-based exogenous contrast agent for fluorescence and photoacoustic bimodal imaging above 820 nm. In vivo imaging using D1(micelle), as demonstrated by fluorescence and photoacoustic tomography experiments in live mice, shows contrast-enhanced deep tissue imaging capability. The usage of D1(micelle) proven by preclinical experiments in rodents reveals its excellent applicability for NIR fluorescence and photoacoustic bimodal imaging.

  16. Soft nanomaterial-based targeting polymersomes for near-infrared fluorescence multispectral in vivo imaging

    NASA Astrophysics Data System (ADS)

    Li, Zuhong; Wu, Liyuan; Hu, Peiran; Han, Sihai; Zhang, Tao; Fan, Hongliang; Jin, Wei; Jin, Qinhan; Mu, Ying

    2012-10-01

    We report here the soft nanomaterial-based targeting polymersomes for near-infrared (NIR) fluorescence imaging to carry out in vivo tumor detection. Two polymersome-based NIR fluorescent probes were prepared through the self-assembly of amphiphilic block copolymers, poly(butadiene-b-ethylene oxide) (PEO-b-PBD). Each of them was encapsulated with distinct hydrophobic near-infrared dyes (DiD and DiR) and modified with different targeting ligands (anti-CEA antibody and anti-EGFR antibody), respectively. After simultaneous injection of these two probes into the tumor-bearing mice via tail vein, multispectral near-infrared fluorescence images were obtained. The results indicate that both probes are successfully directed to the tumor foci, where two distinguishable fluorescent signals were detected through the unmixed fluorescence images. By taking advantage of two targeting polymersome-based probes with distinct fluorescent features, the proposed multispectral near-infrared fluorescence imaging method can greatly improve the specificity and accuracy for in vivo tumor detection.

  17. Single gold nanoparticles to enhance the detection of single fluorescent molecules at micromolar concentration using fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Punj, Deep; Rigneault, Hervé; Wenger, Jérôme

    2014-05-01

    Single nanoparticles made of noble metals are strongly appealing to develop practical applications to detect fluorescent molecules in solution. Here, we detail the use of a single gold nanoparticle of 100 nm diameter to enhance the detection of single Alex Fluor 647 fluorescent molecules at high concentrations of several micromolar. We discuss the implementation of fluorescence correlation spectroscopy, and provide a new method to reliably extract the enhanced fluorescence signal stemming from the nanoparticle near-field from the background generated in the confocal volume. The applicability of our method is checked by reporting the invariance of the single molecule results as function of the molecular concentration, and the experimental data is found in good agreement with numerical simulations.

  18. Visualization of in vivo degradation of aliphatic polyesters by a fluorescent dendritic star macromolecule.

    PubMed

    Duan, Shun; Ma, Shiqing; Huang, Zhaohui; Zhang, Xu; Yang, Xiaoping; Gao, Ping; Yin, Meizhen; Cai, Qing

    2015-12-01

    In tissue engineering, most polymeric scaffolds should degrade along with the formation of the new tissues. Therefore, it is necessary to look into the in vivo degradation of scaffolds. In this study, a fluorescent perylenediimide-cored (PDI-cored) dendritic star macromolecule bearing multiple amines (d-p48) was incorporated into biodegradable polyester nanofibrous scaffolds by eletrospinning as an indicator. The polyester/d-p48 blend nanofibers could emit strong red fluorescence when they were irradiated under exciting light. Initially, using slowly degradable polyester, poly(L-lactide) (PLLA)/d-p48 nanofibers were soaked in phosphate buffered saline for various lengths of time to determine the possible diffusing release of d-p48 macromolecule from nanofibers. The PLLA/d-p48 nanofibers were then implanted subcutaneously into mice and left for up to 2 weeks. In both cases, no undesirable release of the incorporated d-p48 macromolecule was detected, and the nanofibers were clearly visualized in vivo by fluorescence microscopy. Using a fast degradable polyester, poly(lactide-co-glycolide) (PLGA)/d-p48 nanofibers were electrospun and implanted subcutaneously to determine the possibility of monitoring in vivo degradation by fluorescence during 12 weeks. The results showed that the location and the contour of PLGA/d-p48 nanofibrous scaffolds could be clearly visualized using an animal fluorescent imaging system. The fluorescent intensities decreased gradually with the degradation of the scaffolds. No side effects on liver and kidney were found during the detection. This study indicates that the fluorescent PDI-cored dendritic star macromolecule can be used as a stable bioimaging indicator for biodegradable aliphatic polyesters in vivo. PMID:26526346

  19. Optical spectroscopy of a highly fluorescent aggregate of bacteriochlorophyll c

    NASA Technical Reports Server (NTRS)

    Causgrove, T. P.; Cheng, P.; Brune, D. C.; Blankenship, R. E.

    1993-01-01

    Bacteriochlorophyll (BChl) c and a similar model compound, Mg-methyl bacteriopheophorbide d, form several types of aggregates in nonpolar solvents. One of these aggregates is highly fluorescent, with a quantum yield higher than that of the monomer. This aggregate is also unusual in that it shows a rise time in its fluorescence emission decay at certain wavelengths, which is ascribed to a change in conformation of the aggregate. An analysis of fluorescence depolarization data is consistent with either a linear aggregate of four or five monomers or preferably a cyclic arrangement of three dimers.

  20. Laser-induced fluorescence spectroscopy of the secondary cataract

    NASA Astrophysics Data System (ADS)

    Maslov, N. A.; Larionov, P. M.; Rozhin, I. A.; Druzhinin, I. B.; Chernykh, V. V.

    2016-06-01

    Excitation-emission matrices of laser-induced fluorescence of lens capsule epithelium, the lens nucleus, and the lens capsule are investigated. A solid-state laser in combination with an optical parametric generator tunable in the range from 210 to 350 nm was used for excitation of fluorescence. The spectra of fluorescence of all three types of tissues exhibit typical features that are specific to them and drastically differ from one another. This effect can be used for intrasurgical control of presence of residual lens capsule epithelium cells in the capsular bag after surgical treatment of a cataract.

  1. Detection of mechanical and disease stresses in citrus plants by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Belasque, J., Jr.; Gasparoto, M. C. G.; Marcassa, L. G.

    2008-04-01

    We have investigated the detection of mechanical and disease stresses in citrus plants (Citrus limonia [L.] Osbeck) using laser-induced fluorescence spectroscopy. Due to its economic importance we have chosen to investigate the citrus canker disease, which is caused by the Xanthomonas axonopodis pv. citri bacteria. Mechanical stress was also studied because it plays an important role in the plant's infection by such bacteria. A laser-induced fluorescence spectroscopy system, composed of a spectrometer and a 532 nm10 mW excitation laser was used to perform fluorescence spectroscopy. The ratio of two chlorophyll fluorescence bands allows us to detect and discriminate between mechanical and disease stresses. This ability to discriminate may have an important application in the field to detect citrus canker infected trees.

  2. Vectorized data acquisition and fast triple-correlation integrals for Fluorescence Triple Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Ridgeway, William K.; Millar, David P.; Williamson, James R.

    2013-04-01

    Fluorescence Correlation Spectroscopy (FCS) is widely used to quantify reaction rates and concentrations of molecules in vitro and in vivo. We recently reported Fluorescence Triple Correlation Spectroscopy (F3CS), which correlates three signals together instead of two. F3CS can analyze the stoichiometries of complex mixtures and detect irreversible processes by identifying time-reversal asymmetries. Here we report the computational developments that were required for the realization of F3CS and present the results as the Triple Correlation Toolbox suite of programs. Triple Correlation Toolbox is a complete data analysis pipeline capable of acquiring, correlating and fitting large data sets. Each segment of the pipeline handles error estimates for accurate error-weighted global fitting. Data acquisition was accelerated with a combination of off-the-shelf counter-timer chips and vectorized operations on 128-bit registers. This allows desktop computers with inexpensive data acquisition cards to acquire hours of multiple-channel data with sub-microsecond time resolution. Off-line correlation integrals were implemented as a two delay time multiple-tau scheme that scales efficiently with multiple processors and provides an unprecedented view of linked dynamics. Global fitting routines are provided to fit FCS and F3CS data to models containing up to ten species. Triple Correlation Toolbox is a complete package that enables F3CS to be performed on existing microscopes. Catalogue identifier: AEOP_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEOP_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 50189 No. of bytes in distributed program, including test data, etc.: 6135283 Distribution format: tar.gz Programming language: C/Assembly. Computer: Any with GCC and

  3. Anabaena cell ageing monitored with confocal fluorescence spectroscopy.

    PubMed

    Ke, Shan; Bindokas, Vytas; Haselkorn, Robert

    2015-01-01

    Cyanobacteria use a sophisticated system of pigments to collect light energy across the visible spectrum for photosynthesis. The pigments are assembled in structures called phycobilisomes, composed of phycoerythrocyanin, phycocyanin and allophycocyanin, which absorb energy and transfer it to chlorophyll in photosystem II reaction centres. All of the components of this system are fluorescent, allowing sensitive measurements of energy transfer using single cell confocal fluorescence microscopy. The native pigments can be interrogated without the use of reporters. Here, we use confocal fluorescence microscopy to monitor changes in the efficiency of energy transfer as single cells age, between the time they are born at cell division until they are ready to divide again. Alteration of fluorescence was demonstrated to change with the age of the cyanobacterial cell.

  4. Excitation emission and time-resolved fluorescence spectroscopy of selected varnishes used in historical musical instruments.

    PubMed

    Nevin, Austin; Echard, Jean-Philippe; Thoury, Mathieu; Comelli, Daniela; Valentini, Gianluca; Cubeddu, Rinaldo

    2009-11-15

    The analysis of various varnishes from different origins, which are commonly found on historical musical instruments was carried out for the first time with both fluorescence excitation emission spectroscopy and laser-induced time-resolved fluorescence spectroscopy. Samples studied include varnishes prepared using shellac, and selected diterpenoid and triterpenoid resins from plants, and mixtures of these materials. Fluorescence excitation emission spectra have been collected from films of naturally aged varnishes. In parallel, time-resolved fluorescence spectroscopy of varnishes provides means for discriminating between short- (less than 2.0 ns) and long-lived (greater than 7.5 ns) fluorescence emissions in each of these complex materials. Results suggest that complementary use of the two non destructive techniques allows a better understanding of the main fluorophores responsible for the emission in shellac, and further provides means for distinguishing the main classes of other varnishes based on differences in fluorescence lifetime behaviour. Spectrofluorimetric data and time resolved spectra presented here may form the basis for the interpretation of results from future in situ fluorescence examination and time resolved fluorescence imaging of varnished musical instruments.

  5. Comparative studies on the interaction of cefixime with bovine serum albumin by fluorescence quenching spectroscopy and synchronous fluorescence spectroscopy.

    PubMed

    Zhang, Lihui; Liu, Baosheng; Li, Zhiyun; Guo, Ying

    2015-08-01

    Under simulated physiological conditions, the reaction mechanism between cefixime and bovine serum albumin at different temperatures (293, 303 and 310 K) was investigated using a fluorescence quenching method and synchronous fluorescence method, respectively. The results indicated that the fluorescence intensity and synchronous fluorescence intensity of bovine serum albumin decreased regularly on the addition of cefixime. In addition, the quenching mechanism, binding constants, number of binding sites, type of interaction force and energy-transfer parameters of cefixime with bovine serum albumin obtained from two methods using the same equation were consistent. The results indicated that the synchronous fluorescence spectrometry could be used to study the binding mechanism between drug and protein, and was a useful supplement to the conventional method.

  6. Two-photon fluorescence correlation spectroscopy with high count rates and low background using dielectric microspheres

    PubMed Central

    Aouani, Heykel; Schön, Peter; Brasselet, Sophie; Rigneault, Hervé; Wenger, Jérôme

    2010-01-01

    Two-photon excitation fluorescence is a powerful technique commonly used for biological imaging. However, the low absorption cross section of this non-linear process is a critical issue for performing biomolecular spectroscopy at the single molecule level. Enhancing the two-photon fluorescence signal would greatly improve the effectiveness of this technique, yet current methods struggle with medium enhancement factors and/or high background noise. Here, we show that the two-photon fluorescence signal from single Alexa Fluor 488 molecules can be enhanced up to 10 times by using a 3 µm diameter latex sphere while adding almost no photoluminescence background. We report a full characterization of the two-photon fluorescence enhancement by a single microsphere using fluorescence correlation spectroscopy. This opens new routes to enhance non-linear optical signals and extend biophotonic applications. PMID:21258531

  7. Pin-Hole Array Correlation Imaging: Highly Parallel Fluorescence Correlation Spectroscopy

    PubMed Central

    Needleman, Daniel J.; Xu, Yangqing; Mitchison, Timothy J.

    2009-01-01

    Abstract In this work, we describe pin-hole array correlation imaging, a multipoint version of fluorescence correlation spectroscopy, based upon a stationary Nipkow disk and a high-speed electron multiplying charged coupled detector. We characterize the system and test its performance on a variety of samples, including 40 nm colloids, a fluorescent protein complex, a membrane dye, and a fluorescence fusion protein. Our results demonstrate that pin-hole array correlation imaging is capable of simultaneously performing tens or hundreds of fluorescence correlation spectroscopy-style measurements in cells, with sufficient sensitivity and temporal resolution to study the behaviors of membrane-bound and soluble molecules labeled with conventional chemical dyes or fluorescent proteins. PMID:19527665

  8. Ultrafast fluorescence spectroscopy via upconversion applications to biophysics.

    PubMed

    Xu, Jianhua; Knutson, Jay R

    2008-01-01

    This chapter reviews basic concepts of nonlinear fluorescence upconversion, a technique whose temporal resolution is essentially limited only by the pulse width of the ultrafast laser. Design aspects for upconversion spectrophotofluorometers are discussed, and a recently developed system is described. We discuss applications in biophysics, particularly the measurement of time-resolved fluorescence spectra of proteins (with subpicosecond time resolution). Application of this technique to biophysical problems such as dynamics of tryptophan, peptides, proteins, and nucleic acids is reviewed.

  9. Instrument for fluorescence sensing of circulating cells with diffuse light in mice in vivo

    PubMed Central

    Zettergren, Eric; Vickers, Dwayne; Runnels, Judith; Murthy, Shashi K.; Lin, Charles P.

    2012-01-01

    Abstract. Accurate quantification of circulating cell populations in mice is important in many areas of preclinical biomedical research. Normally, this is done either by extraction and analysis of small blood samples or, more recently, by using microscopy-based in vivo fluorescence flow cytometry. We describe a new technological approach to this problem using detection of diffuse fluorescent light from relatively large blood vessels in vivo. The diffuse fluorescence flow cytometer (DFFC) uses a laser to illuminate a mouse limb and an array of optical fibers coupled to a high-sensitivity photomultiplier tube array operating in photon counting mode to detect weak fluorescence signals from cells. We first demonstrate that the DFFC instrument is capable of detecting fluorescent microspheres and Vybrant-DiD-labeled cells in a custom-made optical flow phantom with similar size, optical properties, linear flow rates, and autofluorescence as a mouse limb. We also present preliminary data demonstrating that the DFFC is capable of detecting circulating cells in nude mice in vivo. In principle, this device would allow interrogation of the whole blood volume of a mouse in minutes, with sensitivity improvement by several orders of magnitude compared to current approaches. PMID:22502573

  10. In Vivo Visualization of Tumor Antigen-containing Microparticles Generated in Fluorescent-protein-elicited Immunity

    PubMed Central

    Yang, Fei; Liu, Shun; Liu, Xiuli; Liu, Lei; Luo, Meijie; Qi, Shuhong; Xu, Guoqiang; Qiao, Sha; Lv, Xiaohua; Li, Xiangning; Fu, Ling; Luo, Qingming; Zhang, Zhihong

    2016-01-01

    In vivo optical spatio-temporal imaging of the tumor microenvironment is useful to explain how tumor immunotherapies work. However, the lack of fluorescent antigens with strong immunogenicity makes it difficult to study the dynamics of how tumors are eliminated by any given immune response. Here, we develop an effective fluorescent model antigen based on the tetrameric far-red fluorescent protein KatushkaS158A (tfRFP), which elicits both humoral and cellular immunity. We use this fluorescent antigen to visualize the dynamic behavior of immunocytes as they attack and selectively eliminate tfRFP-expressing tumors in vivo; swarms of immunocytes rush toward tumors with high motility, clusters of immunocytes form quickly, and numerous antigen-antibody complexes in the form of tfRFP+ microparticles are generated in the tumor areas and ingested by macrophages in the tumor microenvironment. Therefore, tfRFP, as both a model antigen and fluorescent reporter, is a useful tool to visualize specific immune responses in vivo. PMID:27375792

  11. In-vivo validation of fluorescence lifetime imaging (FLIm) of coronary arteries in swine

    NASA Astrophysics Data System (ADS)

    Bec, Julien; Ma, Dinglong; Yankelevich, Diego R.; Gorpas, Dimitris S.; Ferrier, William T.; Southard, Jeffrey; Marcu, Laura

    2015-02-01

    We report a scanning imaging system that enables high speed multispectral fluorescence lifetime imaging (FLIm) of coronary arteries. This system combines a custom low profile (3 Fr) imaging catheter using a 200 μm core side viewing UV-grade silica fiber optic, an acquisition system able to measure fluorescence decays over four spectral bands at 20 kHz and a fast data analysis and display module. In vivo use of the system has been optimized, with particular emphasis on clearing blood from the optical pathway. A short acquisition time (5 seconds for a 20 mm long coronary segment) enabled data acquisition during a bolus saline solution injection through the 7 Fr catheter guide. The injection parameters were precisely controlled using a power injector and optimized to provide good image quality while limiting the bolus injection duration and volume (12 cc/s, 80 cc total volume). The ability of the system to acquire data in vivo was validated in healthy swine by imaging different sections of the left anterior descending (LAD) coronary. A stent coated with fluorescent markers was placed in the LAD and imaged, demonstrating the ability of the system to discriminate in vivo different fluorescent features and structures from the vessel background fluorescence using spectral and lifetime information. Intensity en face images over the four bands of the instrument were available within seconds whereas lifetime images were computed in 2 minutes, providing efficient feedback during the procedure. This successful demonstration of FLIm in coronaries enables future study of atherosclerotic cardiovascular diseases.

  12. Fluorescence spectroscopy of anisole at elevated temperatures and pressures

    NASA Astrophysics Data System (ADS)

    Tran, K. H.; Morin, C.; Kühni, M.; Guibert, P.

    2014-06-01

    Laser-induced fluorescence of anisole as tracer of isooctane at an excitation wavelength of 266 nm was investigated for conditions relevant to rapid compression machine studies and for more general application of internal combustion engines regarding temperature, pressure, and ambient gas composition. An optically accessible high pressure and high temperature chamber was operated by using different ambient gases (Ar, N2, CO2, air, and gas mixtures). Fluorescence experiments were investigated at a large range of pressure and temperature (0.2-4 MPa and 473-823 K). Anisole fluorescence quantum yield decreases strongly with temperature for every considered ambient gas, due to efficient radiative mechanisms of intersystem crossing. Concerning the pressure effect, the fluorescence signal decreases with increasing pressure, because increasing the collisional rate leads to more important non-radiative collisional relaxation. The quenching effect is strongly efficient in oxygen, with a fluorescence evolution described by Stern-Volmer relation. The dependence of anisole fluorescence versus thermodynamic parameters suggests the use of this tracer for temperature imaging in specific conditions detailed in this paper. The calibration procedure for temperature measurements is established for the single-excitation wavelength and two-color detection technique.

  13. Time-resolved fluorescence spectroscopy of spinach chloroplast.

    PubMed

    Yu, W; Pellegrino, F; Alfano, R R

    1977-04-11

    Picosecond fluorescent kinetics and time-resolved spectra of spinach chloroplast were measured at room temperature and low temperatures. The measurement is conducted with 530 nm excitation at an average intensity of 2-10(14) photons/cm2, pluse and at a pulse separation of 6 ns for the 100 pulses used. The 685 nm fluorescent kinetics was found to decay with two components, a fast component with a 56 ps lifetime, and a slow component with a 220 ps lifetime. The 730 nm fluorescent kinetics at room temperature is a single exponential decay with a 100 ps lifetime. The 730 nm fluorescence lifetime was found to increase by a factor of 6 when the temperature was lowered from room temperature to 90 K, while the 685 and 695 nm fluorescent kinetics were unchanged. The time-resolved spectra data obtained within 10 ps after excitation is consistent with the kinetic data reported here. A two-level fluorescence scheme is proposed to explain the kinetics. The effect of excitation with high light intensity and multiple pulses is discussed.

  14. Infrared Fluorescent Imaging as a Potent Tool for In Vitro, Ex Vivo and In Vivo Models of Visceral Leishmaniasis

    PubMed Central

    Calvo-Álvarez, Estefanía; Stamatakis, Kostantinos; Punzón, Carmen; Álvarez-Velilla, Raquel; Tejería, Ana; Escudero-Martínez, José Miguel; Pérez-Pertejo, Yolanda; Fresno, Manuel; Balaña-Fouce, Rafael; Reguera, Rosa M.

    2015-01-01

    Background Visceral leishmaniasis (VL) is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years. Methodology/Principal findings We generated two infrared fluorescent L. infantum strains, which stably overexpress the IFP 1.4 and iRFP reporter genes and performed comparative studies of their biophotonic properties at both promastigote and amastigote stages. To improve the fluorescence emission of the selected reporter in intracellular amastigotes, we engineered distinct constructs by introducing regulatory sequences of differentially-expressed genes (A2, AMASTIN and HSP70 II). The final strain that carries the iRFP gene under the control of the L. infantum HSP70 II downstream region (DSR), was employed to perform a phenotypic screening of a collection of small molecules by using ex vivo splenocytes from infrared-infected BALB/c mice. In order to further investigate the usefulness of this infrared strain, we monitored an in vivo infection by imaging BALB/c mice in a time-course study of 20 weeks. Conclusions/Significance The near-infrared fluorescent L. infantum strain represents an important step forward in bioimaging research of VL, providing a robust model of phenotypic screening suitable for HTS of small molecule collections in the mammalian parasite stage. Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL. This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program

  15. Fluorescence imaging of tryptophan and collagen cross-links to evaluate wound closure ex vivo

    NASA Astrophysics Data System (ADS)

    Wang, Ying; Ortega-Martinez, Antonio; Farinelli, Bill; Anderson, R. R.; Franco, Walfre

    2016-02-01

    Wound size is a key parameter in monitoring healing. Current methods to measure wound size are often subjective, time-consuming and marginally invasive. Recently, we developed a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds. This method exploits the fluorescence of native molecules to tissue as functional and structural markers. The objective of the present study is to demonstrate the feasibility of using variations in the fluorescence intensity of tryptophan and cross-links of collagen to evaluate proliferation of keratinocyte cells and quantitate size of wound during healing, respectively. Circular dermal wounds were created in ex vivo human skin and cultured in different media. Two serial fluorescence images of tryptophan and collagen cross-links were acquired every two days. Histology and immunohistology were used to validate correlation between fluorescence and epithelialization. Images of collagen cross-links show fluorescence of the exposed dermis and, hence, are a measure of wound area. Images of tryptophan show higher fluorescence intensity of proliferating keratinocytes forming new epithelium, as compared to surrounding keratinocytes not involved in epithelialization. These images are complementary since collagen cross-links report on structure while tryptophan reports on function. HE and immunohistology show that tryptophan fluorescence correlates with newly formed epidermis. We have established a fluorescence imaging method for studying epithelialization processes during wound healing in a skin organ culture model, our approach has the potential to provide a non-invasive, non-contact, quick, objective and direct method for quantitative measurements in wound healing in vivo.

  16. Comparing Compositions of Modern Cast Bronze Sculptures: Optical Emission Spectroscopy Versus x-Ray Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Young, M. L.; Dunand, D. C.

    2015-07-01

    Bulk elemental compositions of 74 modern cast bronze sculptures from the collection at the Art Institute of Chicago, the Philadelphia Museum of Art, and the Rodin Museum (Philadelphia, PA) were determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES) and a handheld x-ray fluorescence (XRF) spectrometer. The elemental compositions of the cast sculptures as measured previously by ICP-OES and presently by XRF are compared: A good match is found between the two methods for the base metal (Cu) and the two majority alloying elements (Zn and Sn). For both ICP-OES and XRF data, when the Zn composition is plotted versus the Sn composition, three discernable clusters are found that are related to the artist, foundry, casting date, and casting method; they consist of (A) high-zinc brass, (B) low-zinc, low-tin brass, and (C) low-zinc, tin bronze. Thus, our study confirms that the relatively fast, nondestructive XRF spectrometry can be used effectively over slower and invasive, but more accurate, ICP-OES to help determine a sculpture's artist, foundry, date of creation, date of casting, and casting method.

  17. In vivo time-gated fluorescence imaging with biodegradable luminescent porous silicon nanoparticles.

    PubMed

    Gu, Luo; Hall, David J; Qin, Zhengtao; Anglin, Emily; Joo, Jinmyoung; Mooney, David J; Howell, Stephen B; Sailor, Michael J

    2013-01-01

    Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (<10 ns) emission signals from organic chromophores or tissue autofluorescence. Here using a conventional animal imaging system not optimized for such long-lived excited states, we demonstrate improvement of signal to background contrast ratio by >50-fold in vitro and by >20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed.

  18. Fluorescent risedronate analogues reveal bisphosphonate uptake by bone marrow monocytes and localization around osteocytes in vivo.

    PubMed

    Roelofs, Anke J; Coxon, Fraser P; Ebetino, Frank H; Lundy, Mark W; Henneman, Zachary J; Nancollas, George H; Sun, Shuting; Blazewska, Katarzyna M; Bala, Joy Lynn F; Kashemirov, Boris A; Khalid, Aysha B; McKenna, Charles E; Rogers, Michael J

    2010-03-01

    Bisphosphonates are effective antiresorptive agents owing to their bone-targeting property and ability to inhibit osteoclasts. It remains unclear, however, whether any non-osteoclast cells are directly affected by these drugs in vivo. Two fluorescent risedronate analogues, carboxyfluorescein-labeled risedronate (FAM-RIS) and Alexa Fluor 647-labeled risedronate (AF647-RIS), were used to address this question. Twenty-four hours after injection into 3-month-old mice, fluorescent risedronate analogues were bound to bone surfaces. More detailed analysis revealed labeling of vascular channel walls within cortical bone. Furthermore, fluorescent risedronate analogues were present in osteocytic lacunae in close proximity to vascular channels and localized to the lacunae of newly embedded osteocytes close to the bone surface. Following injection into newborn rabbits, intracellular uptake of fluorescently labeled risedronate was detected in osteoclasts, and the active analogue FAM-RIS caused accumulation of unprenylated Rap1A in these cells. In addition, CD14(high) bone marrow monocytes showed relatively high levels of uptake of fluorescently labeled risedronate, which correlated with selective accumulation of unprenylated Rap1A in CD14(+) cells, as well as osteoclasts, following treatment with risedronate in vivo. Similar results were obtained when either rabbit or human bone marrow cells were treated with fluorescent risedronate analogues in vitro. These findings suggest that the capacity of different cell types to endocytose bisphosphonate is a major determinant for the degree of cellular drug uptake in vitro as well as in vivo. In conclusion, this study shows that in addition to bone-resorbing osteoclasts, bisphosphonates may exert direct effects on bone marrow monocytes in vivo.

  19. Fluorescent Risedronate Analogues Reveal Bisphosphonate Uptake by Bone Marrow Monocytes and Localization Around Osteocytes In Vivo

    PubMed Central

    Roelofs, Anke J; Coxon, Fraser P; Ebetino, Frank H; Lundy, Mark W; Henneman, Zachary J; Nancollas, George H; Sun, Shuting; Blazewska, Katarzyna M; Bala, Joy Lynn F; Kashemirov, Boris A; Khalid, Aysha B; McKenna, Charles E; Rogers, Michael J

    2010-01-01

    Bisphosphonates are effective antiresorptive agents owing to their bone-targeting property and ability to inhibit osteoclasts. It remains unclear, however, whether any non-osteoclast cells are directly affected by these drugs in vivo. Two fluorescent risedronate analogues, carboxyfluorescein-labeled risedronate (FAM-RIS) and Alexa Fluor 647–labeled risedronate (AF647-RIS), were used to address this question. Twenty-four hours after injection into 3-month-old mice, fluorescent risedronate analogues were bound to bone surfaces. More detailed analysis revealed labeling of vascular channel walls within cortical bone. Furthermore, fluorescent risedronate analogues were present in osteocytic lacunae in close proximity to vascular channels and localized to the lacunae of newly embedded osteocytes close to the bone surface. Following injection into newborn rabbits, intracellular uptake of fluorescently labeled risedronate was detected in osteoclasts, and the active analogue FAM-RIS caused accumulation of unprenylated Rap1A in these cells. In addition, CD14high bone marrow monocytes showed relatively high levels of uptake of fluorescently labeled risedronate, which correlated with selective accumulation of unprenylated Rap1A in CD14+ cells, as well as osteoclasts, following treatment with risedronate in vivo. Similar results were obtained when either rabbit or human bone marrow cells were treated with fluorescent risedronate analogues in vitro. These findings suggest that the capacity of different cell types to endocytose bisphosphonate is a major determinant for the degree of cellular drug uptake in vitro as well as in vivo. In conclusion, this study shows that in addition to bone-resorbing osteoclasts, bisphosphonates may exert direct effects on bone marrow monocytes in vivo. © 2010 American Society for Bone and Mineral Research PMID:20422624

  20. Intracellular distribution of fluorescent copper and zinc bis(thiosemicarbazonato) complexes measured with fluorescence lifetime spectroscopy.

    PubMed

    Hickey, James L; James, Janine L; Henderson, Clare A; Price, Katherine A; Mot, Alexandra I; Buncic, Gojko; Crouch, Peter J; White, Jonathan M; White, Anthony R; Smith, Trevor A; Donnelly, Paul S

    2015-10-01

    The intracellular distribution of fluorescently labeled copper and zinc bis(thiosemicarbazonato) complexes was investigated in M17 neuroblastoma cells and primary cortical neurons with a view to providing insights into the neuroprotective activity of a copper bis(thiosemicarbazonato) complex known as Cu(II)(atsm). Time-resolved fluorescence measurements allowed the identification of the Cu(II) and Zn(II) complexes as well as the free ligand inside the cells by virtue of the distinct fluorescence lifetime of each species. Confocal fluorescent microscopy of cells treated with the fluorescent copper(II)bis(thiosemicarbazonato) complex revealed significant fluorescence associated with cytoplasmic puncta that were identified to be lysosomes in primary cortical neurons and both lipid droplets and lysosomes in M17 neuroblastoma cells. Fluorescence lifetime imaging microscopy confirmed that the fluorescence signal emanating from the lipid droplets could be attributed to the copper(II) complex but also that some degree of loss of the metal ion led to diffuse cytosolic fluorescence that could be attributed to the metal-free ligand. The accumulation of the copper(II) complex in lipid droplets could be relevant to the neuroprotective activity of Cu(II)(atsm) in models of amyotrophic lateral sclerosis and Parkinson's disease.

  1. Intracellular distribution of fluorescent copper and zinc bis(thiosemicarbazonato) complexes measured with fluorescence lifetime spectroscopy.

    PubMed

    Hickey, James L; James, Janine L; Henderson, Clare A; Price, Katherine A; Mot, Alexandra I; Buncic, Gojko; Crouch, Peter J; White, Jonathan M; White, Anthony R; Smith, Trevor A; Donnelly, Paul S

    2015-10-01

    The intracellular distribution of fluorescently labeled copper and zinc bis(thiosemicarbazonato) complexes was investigated in M17 neuroblastoma cells and primary cortical neurons with a view to providing insights into the neuroprotective activity of a copper bis(thiosemicarbazonato) complex known as Cu(II)(atsm). Time-resolved fluorescence measurements allowed the identification of the Cu(II) and Zn(II) complexes as well as the free ligand inside the cells by virtue of the distinct fluorescence lifetime of each species. Confocal fluorescent microscopy of cells treated with the fluorescent copper(II)bis(thiosemicarbazonato) complex revealed significant fluorescence associated with cytoplasmic puncta that were identified to be lysosomes in primary cortical neurons and both lipid droplets and lysosomes in M17 neuroblastoma cells. Fluorescence lifetime imaging microscopy confirmed that the fluorescence signal emanating from the lipid droplets could be attributed to the copper(II) complex but also that some degree of loss of the metal ion led to diffuse cytosolic fluorescence that could be attributed to the metal-free ligand. The accumulation of the copper(II) complex in lipid droplets could be relevant to the neuroprotective activity of Cu(II)(atsm) in models of amyotrophic lateral sclerosis and Parkinson's disease. PMID:26397162

  2. In vivo and ex vivo measurements: noninvasive assessment of alcoholic fatty liver using 1H-MR spectroscopy

    PubMed Central

    Keese, Daniel; Korkusuz, Hüdayi; Huebner, Frank; Namgaladze, Dmitry; Raschidi, Bahram; Vogl, Thomas J.

    2016-01-01

    PURPOSE We aimed to evaluate the ability of 1H-magnetic resonance spectroscopy (1H-MRS) to detect and quantify hepatic fat content in vivo and ex vivo in an experimental rat model of alcoholic fatty liver using histopathology, biochemistry, and laboratory analyses as reference. METHODS Alcoholic fatty liver was induced within 48 hours in 20 Lewis rats; 10 rats served as control. Intrahepatic fat content determined by 1H-MRS was expressed as the percent ratio of the lipid and water peaks and was correlated with intrahepatic fat content determined histologically and biochemically. Liver enzymes were measured in serum. RESULTS Fatty liver could be detected in vivo as well as ex vivo using 1H-MRS, in all 20 animals. Histologic analysis showed a fatty liver in 16 of 20 animals. Histology and 1H-MRS results were highly correlated (in vivo, r=0.93, P = 0.0005; ex vivo, r=0.92, P = 0.0006). Also a strong correlation was noted between in vivo 1H-MRS measurements and the fat content determined biochemically (r=0.96, P = 0.0003). Ex vivo results showed a similarly strong correlation between 1H-MRS and biochemistry (r=0.89, P = 0.0011). CONCLUSION 1H-MRS can be carried out in ex vivo models, as well as in vivo, to detect and quantify intrahepatic fat content in the acute fatty liver. PMID:26627137

  3. Research of the interaction between kangai injection and human serum albumin by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Ye, Changbin; Lin, Xiaogang; Zhu, Hao; Li, Wenchao; Wu, Jie

    2015-10-01

    The interaction between drugs and serum albumin is the theoretical basis of pharmacology research. Kangai injection with invigorating Qi, enhancing the immune function, is widely used for a variety of malignant tumor treatment. Fluorescence spectroscopy was adopted due to its high sensitivity and other advantages. The interaction between kangai injection and human serum albumin (HSA) in physiological buffer (pH 7.4) was investigated by fluorescence spectroscopy and UV-Vis absorption spectroscopy. The results of fluorescence spectrum at three temperature (296K, 303K and 310K) showed the degree of binding at 310K is the highest. Also, the maximum emission peak has a slight blue shift, which indicates that the interaction between kangai injection and HSA has an effect on the conformation of HSA. That is, the microenvironment of tryptophan increase hydrophobic due to the increase of the concentration of kangai injection. Results obtained from analysis of fluorescence spectrum and fluorescence intensity indicated that kangai injection has a strong ability to quench the intrinsic fluorescence of HSA. And according to the Stern-Volume equation, the quenching mechanism is static quenching, which is further proved by the UV-Vis absorption spectroscopy.

  4. Fluorescence polarization standard for near infrared spectroscopy and microscopy.

    PubMed

    Luchowski, Rafal; Sarkar, Pabak; Bharill, Shashank; Laczko, Gabor; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, Ignacy

    2008-11-20

    We present studies of polarized absorption [linear dichroism (LD)] and fluorescence polarization of the styryl derivative (LDS 798) embedded in oriented poly(vinyl alcohol) (PVA) films. These films were oriented by progressive stretching up to eight folds. Both vertical and horizontal components of absorptions and fluorescence were measured and dichroic ratios were determined for different film stretching ratios. The dichroic ratio and fluorescence anisotropy values were analyzed as a function of PVA film stretching ratio by fitting according to the previously developed theory. For maximum stretching ratios, exceptionally high anisotropy (approximately 0.8) and polarization (approximately 0.9) values have been measured. The stretched films have high polarization values also for isotropic excitation in a wide spectral range (500-700 nm). Such films can be conveniently used as high polarization standards and we envision they will also have applications in near infrared (NIR) imaging microscopy, where they can be used for correcting an instrumental factor in polarization measurements.

  5. Compact point-detection fluorescence spectroscopy system for quantifying intrinsic fluorescence redox ratio in brain cancer diagnostics

    NASA Astrophysics Data System (ADS)

    Liu, Quan; Grant, Gerald; Li, Jianjun; Zhang, Yan; Hu, Fangyao; Li, Shuqin; Wilson, Christy; Chen, Kui; Bigner, Darell; Vo-Dinh, Tuan

    2011-03-01

    We report the development of a compact point-detection fluorescence spectroscopy system and two data analysis methods to quantify the intrinsic fluorescence redox ratio and diagnose brain cancer in an orthotopic brain tumor rat model. Our system employs one compact cw diode laser (407 nm) to excite two primary endogenous fluorophores, reduced nicotinamide adenine dinucleotide, and flavin adenine dinucleotide. The spectra were first analyzed using a spectral filtering modulation method developed previously to derive the intrinsic fluorescence redox ratio, which has the advantages of insensitivty to optical coupling and rapid data acquisition and analysis. This method represents a convenient and rapid alternative for achieving intrinsic fluorescence-based redox measurements as compared to those complicated model-based methods. It is worth noting that the method can also extract total hemoglobin concentration at the same time but only if the emission path length of fluorescence light, which depends on the illumination and collection geometry of the optical probe, is long enough so that the effect of absorption on fluorescence intensity due to hemoglobin is significant. Then a multivariate method was used to statistically classify normal tissues and tumors. Although the first method offers quantitative tissue metabolism information, the second method provides high overall classification accuracy. The two methods provide complementary capabilities for understanding cancer development and noninvasively diagnosing brain cancer. The results of our study suggest that this portable system can be potentially used to demarcate the elusive boundary between a brain tumor and the surrounding normal tissue during surgical resection.

  6. Time-resolved and steady-state fluorescence spectroscopy for the assessment of skin photoaging process

    NASA Astrophysics Data System (ADS)

    D´Almeida, Camila de Paula; Campos, Carolina; Saito Nogueira, Marcelo; Pratavieira, Sebastião.; Kurachi, Cristina

    2015-06-01

    pathology. The optical properties of these intrinsic fluorophores respond to the microenvironment and the metabolic status, thus making fluorescence spectroscopy a valuable tool to study the conditions of biological tissues. The purpose of this study is to investigate the hairless mice skin metabolic changes during the photoaging process through lifetime and fluorescence measurements targeting NADH and FAD. Two lasers centered at 378 nm and 445 nm, respectively, perform excitation of NADH and FAD. The fluorescence acquisition is carried out at mice dorsal and ventral regions throughout the photoaging protocol and aging process. Differences in fluorescence and lifetime data between young and photoaged mice measurements were observed. The endogenous fluorescence spectrum of photoaged dorsal skin showed an increase compared to young and aged skin. Lifetime of bound NADH and free FAD presented an increase in the first week that continued until the end of the protocol. Aging process is being investigated to complement the information obtained from fluorescence data and lifetime of photoaging process.

  7. Real-time background-free selective imaging of fluorescent nanodiamonds in vivo.

    PubMed

    Igarashi, Ryuji; Yoshinari, Yohsuke; Yokota, Hiroaki; Sugi, Takuma; Sugihara, Fuminori; Ikeda, Kazuhiro; Sumiya, Hitoshi; Tsuji, Shigenori; Mori, Ikue; Tochio, Hidehito; Harada, Yoshie; Shirakawa, Masahiro

    2012-11-14

    Recent developments of imaging techniques have enabled fluorescence microscopy to investigate the localization and dynamics of intracellular substances of interest even at the single-molecule level. However, such sensitive detection is often hampered by autofluorescence arising from endogenous molecules. Those unwanted signals are generally reduced by utilizing differences in either wavelength or fluorescence lifetime; nevertheless, extraction of the signal of interest is often insufficient, particularly for in vivo imaging. Here, we describe a potential method for the selective imaging of nitrogen-vacancy centers (NVCs) in nanodiamonds. This method is based on the property of NVCs that the fluorescence intensity sensitively depends on the ground state spin configuration which can be regulated by electron spin magnetic resonance. Because the NVC fluorescence exhibits neither photobleaching nor photoblinking, this protocol allowed us to conduct long-term tracking of a single nanodiamond in both Caenorhabditis elegans and mice, with excellent imaging contrast even in the presence of strong background autofluorescence.

  8. Variation of fluorescence spectroscopy during the menstrual cycle

    NASA Astrophysics Data System (ADS)

    Macaulay, Calum; Richards-Kortum, Rebecca; Utzinger, Urs; Fedyk, Amanda; Neely Atkinson, E.; Cox, Dennis; Follen, Michele

    2002-06-01

    Cervical autofluorescence has been demonstrated to have potential for real-time diagnosis. Inter-patient and intra-patient variations in fluorescence intensity have been measured. Inter-patient measurements may vary by a factor of ten, while intra-patient measurements may vary by a factor of two. Age and menopausal status have been demonstrated to account for some of the variations, while race and smoking have not. In order to explore in detail the role of the menstrual cycle in intra-patient variation, a study was designed to measure fluorescence excitation emission matrices (EEMs) in patients daily throughout one cycle. Ten patients with a history of normal menstrual cycles and normal Papanicolaou smears underwent daily measurements of fluorescence EEMs from three colposcopically normal sites throughout one menstrual cycle. Changes in signals from porphyrin, NADH, and FAD fluorescence and blood absorption were noted when the data was viewed in a graphical format. Visually interpreted features of the EEMs in this graphical format did not appear to correlate with the day of the menstrual cycle with the exception that blood absorption features were more prominent during the menstrual phase (during which bleeding occurs), suggesting that measurements during the menstrual phase should be avoided. Variations in cycle date likely do not account for inter- or intra-patient variations.

  9. Luminescence and fluorescence of essential oils. Fluorescence imaging in vivo of wild chamomile oil

    PubMed Central

    Boschi, F.; Fontanella, M.; Calderan, L.; Sbarbati, A.

    2011-01-01

    Essential oils are currently of great importance to pharmaceutical companies, cosmetics producers and manufacturers of veterinary products. They are found in perfumes, creams, bath products, and household cleaning substances, and are used for flavouring food and drinks. It is well known that some of them act on the respiratory apparatus. The increasing interest in optical imaging techniques and the development of related technologies have made possible the investigation of the optical properties of several compounds. Luminescent properties of essential oils have not been extensively investigated. We evaluated the luminescent and fluorescent emissions of several essential oils, in order to detect them in living organisms by exploiting their optical properties. Some fluorescent emission data were high enough to be detected in dermal treatments. Consequently, we demonstrated how the fluorescent signal can be monitored for at least three hours on the skin of living mice treated with wild chamomile oil. The results encourage development of this technique to investigate the properties of drugs and cosmetics containing essential oils. PMID:22193298

  10. Plasmonic antennas and zero-mode waveguides to enhance single molecule fluorescence detection and fluorescence correlation spectroscopy toward physiological concentrations.

    PubMed

    Punj, Deep; Ghenuche, Petru; Moparthi, Satish Babu; de Torres, Juan; Grigoriev, Victor; Rigneault, Hervé; Wenger, Jérôme

    2014-01-01

    Single-molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as Förster fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) are limited by diffraction to the nanomolar concentration range, far below the physiological micromolar concentration range where most biological reaction occur. To breach the diffraction limit, zero-mode waveguides (ZMW) and plasmonic antennas exploit the surface plasmon resonances to confine and enhance light down to the nanometer scale. The ability of plasmonics to achieve extreme light concentration unlocks an enormous potential to enhance fluorescence detection, FRET, and FCS. Single molecule spectroscopy techniques greatly benefit from ZMW and plasmonic antennas to enter a new dimension of molecular concentration reaching physiological conditions. The application of nano-optics to biological problems with FRET and FCS is an emerging and exciting field, and is promising to reveal new insights on biological functions and dynamics.

  11. Performance of computer vision in vivo flow cytometry with low fluorescence contrast

    NASA Astrophysics Data System (ADS)

    Markovic, Stacey; Li, Siyuan; Niedre, Mark

    2015-03-01

    Detection and enumeration of circulating cells in the bloodstream of small animals are important in many areas of preclinical biomedical research, including cancer metastasis, immunology, and reproductive medicine. Optical in vivo flow cytometry (IVFC) represents a class of technologies that allow noninvasive and continuous enumeration of circulating cells without drawing blood samples. We recently developed a technique termed computer vision in vivo flow cytometry (CV-IVFC) that uses a high-sensitivity fluorescence camera and an automated computer vision algorithm to interrogate relatively large circulating blood volumes in the ear of a mouse. We detected circulating cells at concentrations as low as 20 cells/mL. In the present work, we characterized the performance of CV-IVFC with low-contrast imaging conditions with (1) weak cell fluorescent labeling using cell-simulating fluorescent microspheres with varying brightness and (2) high background tissue autofluorescence by varying autofluorescence properties of optical phantoms. Our analysis indicates that CV-IVFC can robustly track and enumerate circulating cells with at least 50% sensitivity even in conditions with two orders of magnitude degraded contrast than our previous in vivo work. These results support the significant potential utility of CV-IVFC in a wide range of in vivo biological models.

  12. Optical spectroscopy of the bladder washout fluid to optimize fluorescence cystoscopy with Hexvix®

    NASA Astrophysics Data System (ADS)

    Martoccia, Carla; Zellweger, Matthieu; Lovisa, Blaise; Jichlinski, Patrice; van den Bergh, Hubert; Wagnières, Georges

    2014-09-01

    Fluorescence cystoscopy enhances detection of early bladder cancer. Water used to inflate the bladder during the procedure rapidly contains urine, which may contain fluorochromes. This frequently degrades fluorescence images. Samples of bladder washout fluid (BWF) or urine were collected (15 subjects). We studied their fluorescence properties and assessed changes induced by pH (4 to 9) and temperature (15°C to 41°C). A typical fluorescence spectrum of BWF features a main peak (excitation/emission: 320/420 nm, FWHM=50/100 nm) and a weaker (5% to 20% of main peak intensity), secondary peak (excitation/emission: 455/525 nm, FWHM=80/50 nm). Interpatient fluctuations of fluorescence intensity are observed. Fluorescence intensity decreases when temperature increases (max 30%) or pH values vary (max 25%). Neither approach is compatible with clinical settings. Fluorescence lifetime measurements suggest that 4-pyridoxic acid/riboflavin is the most likely molecule responsible for urine's main/secondary fluorescence peak. Our measurements give an insight into the spectroscopy of the detrimental background fluorescence. This should be included in the optical design of fluorescence cystoscopes. We estimate that restricting the excitation range from 370-430 nm to 395-415 nm would reduce the BWF background by a factor 2.

  13. Ex Vivo Sentinel Node Mapping in Colon Cancer Combining Blue Dye Staining and Fluorescence Imaging

    PubMed Central

    Schaafsma, Boudewijn E.; Verbeek, Floris P.R.; van der Vorst, Joost R.; Hutteman, Merlijn; Kuppen, Peter J.K.; Frangioni, John V.; van de Velde, Cornelis J.H.; Vahrmeijer, Alexander L.

    2013-01-01

    Background The sentinel lymph node procedure has been proposed to improve nodal staging in colon cancer patients. The aim of this study was to assess the added value of near-infrared fluorescence imaging to conventional blue dye staining for ex vivo sentinel lymph node mapping. Materials and Methods Twenty-two consecutive patients undergoing surgery for colon cancer were included. After tumor resection, a premixed cocktail of the near-infrared lymphatic tracer HSA800 and blue dye was submucosally injected around the tumor for detection of sentinel lymph nodes. The Mini-FLARE imaging system was used for fluorescence imaging. Results In 95% of the patients, at least one sentinel lymph node was identified. Overall, a total of 77 sentinel lymph nodes were identified, of which 77 were fluorescent (100%) and 70 (91%) were blue. Sentinel lymph nodes that were located deeper in the mesenteric fat could easily be located by NIR fluorescence. In 4 out of 5 patients with lymph node metastases, tumor cells were present in at least 1 of the sentinel lymph nodes. Conclusions This study shows the successful use and added value of the near-infrared fluorescence tracer HSA800 to conventional blue dye for the ex vivo sentinel lymph node procedure in colon cancer. PMID:23391167

  14. Changes and characteristics of dissolved organic matter in a constructed wetland system using fluorescence spectroscopy.

    PubMed

    Yao, Yuan; Li, Yun-Zhen; Guo, Xu-Jing; Huang, Tao; Gao, Ping-Ping; Zhang, Ying-Pei; Yuan, Feng

    2016-06-01

    Domestic wastewater was treated by five constructed wetland beds in series. Dissolved organic matter (DOM) collected from influent and effluent samples from the constructed wetland was investigated using fluorescence spectroscopy combined with fluorescence regional integration (FRI), parallel factor (PARAFAC) analysis, and two-dimensional correlation spectroscopy (2D-COS). This study evaluates the capability of these methods in detecting the spectral characteristics of fluorescent DOM fractions and their changes in constructed wetlands. Fluorescence excitation-emission matrix (EEM) combined with FRI analysis showed that protein-like materials displayed a higher removal ratio compared to humic-like substances. The PARAFAC analysis of wastewater DOM indicated that six fluorescent components, i.e., two protein-like substances (C1 and C6), three humic-like substances (C2, C3 and C5), and one non-humic component (C4), could be identified. Tryptophan-like C1 was the dominant component in the influent DOM. The removal ratios of six fluorescent components (C1-C6) were 56.21, 32.05, 49.19, 39.90, 29.60, and 45.87 %, respectively, after the constructed wetland treatment. Furthermore, 2D-COS demonstrated that the sequencing of spectral changes for fluorescent DOM followed the order 298 nm → 403 nm → 283 nm (310-360 nm) in the constructed wetland, suggesting that the peak at 298 nm is associated with preferential tryptophan fluorescence removal. Variation of the fluorescence index (FI) and the ratio of fluorescence components indicated that the constructed wetland treatment resulted in the decrease of fluorescent organic pollutant with increasing the humification and chemical stability of the DOM. PMID:26976008

  15. Time-resolved fluorescence spectroscopy for chemical sensors

    NASA Astrophysics Data System (ADS)

    Draxler, Sonja; Lippitsch, Max E.

    1996-07-01

    A family of sensors is presented with fluorescence decay-time measurements used as the sensing technique. The concept is to take a single fluorophore with a suitably long fluorescence decay time as the basic building block for numerous different sensors. Analyte recognition can be performed by different functional groups that are necessary for selective interaction with the analyte. To achieve this, the principle of excited-state electron transfer is applied with pyrene as the fluorophore. Therefore the same instrumentation based on a small, ambient air-nitrogen laser and solid-state electronics can be used to measure different analytes, for example, oxygen, pH, carbon dioxide, potassium, ammonium, lead, cadmium, zinc, and phosphate.

  16. A scanning fluorescence spectroscopy of decorin under high pressure

    NASA Astrophysics Data System (ADS)

    Komoda, Takahito; Kim, Yun-Jung; Suzuki, Atsushi; Nishiumi, Tadayuki

    2013-06-01

    High pressure processing is able to tenderize not only meat but also intramuscular connective tissue, which is mainly composed of collagen. Decorin, one of the proteoglycans, binds to and stabilizes collagen fibrils. It has been suggested that structural weakening of intramuscular connective tissue may result from the disappearance of the decorin-collagen interaction. In this study, the fluorescence spectra and the surface hydrophobicity of decorin molecules were measured under high pressure in order to examine the resulting change in the tertiary structure. The fluorescence intensity and the surface hydrophobicity of decorin molecules both decreased with increasing applied pressure and with applied time at the constant applied pressure, respectively. The observations indicate that the native structure of decorin is maintained during 200 MPa pressurization for less than 30 min.

  17. Surface modified single molecules free-diffusion evidenced by fluorescence correlation spectroscopy.

    PubMed

    Boutin, Céline; Jaffiol, Rodolphe; Plain, Jérôme; Royer, Pascal

    2008-11-01

    We report on the free diffusion of single molecule near an interface studied using fluorescence correlation spectroscopy. In particular, we show that the chemical nature of the substrate may modify the free diffusion of a widely used molecule (rhodamine 6G), thus inducing unexpected effects in fluorescence correlation spectroscopy measurements. Our results show a strong influence, up to a few micrometer from the interface, of the surface polarity. This effect is assessed through the relative weight of the two dimensions diffusion process observed close to the surface. Our results are discussed in terms of competition between surface-solvent, solvent-molecule and molecule-surface specific interactions.

  18. Quantitative frequency-domain fluorescence spectroscopy in tissues and tissue-like media

    NASA Astrophysics Data System (ADS)

    Cerussi, Albert Edward

    1999-09-01

    In the never-ending quest for improved medical technology at lower cost, modern near-infrared optical spectroscopy offers the possibility of inexpensive technology for quantitative and non-invasive diagnoses. Hemoglobin is the dominant chromophore in the 700-900 nm spectral region and as such it allows for the optical assessment of hemoglobin concentration and tissue oxygenation by absorption spectroscopy. However, there are many other important physiologically relevant compounds or physiological states that cannot be effectively sensed via optical methods because of poor optical contrast. In such cases, contrast enhancements are required. Fluorescence spectroscopy is an attractive component of optical tissue spectroscopy. Exogenous fluorophores, as well as some endogenous ones, may furnish the desperately needed sensitivity and specificity that is lacking in near-infrared optical tissue spectroscopy. The main focus of this thesis was to investigate the generation and propagation of fluorescence photons inside tissues and tissue-like media (i.e., scattering dominated media). The standard concepts of fluorescence spectroscopy have been incorporated into a diffusion-based picture that is sometimes referred to as photon migration. The novelty of this work lies in the successful quantitative recovery of fluorescence lifetimes, absolute fluorescence quantum yields, fluorophore concentrations, emission spectra, and both scattering and absorption coefficients at the emission wavelength from a tissue-like medium. All of these parameters are sensitive to the fluorophore local environment and hence are indicators of the tissue's physiological state. One application demonstrating the capabilities of frequency-domain lifetime spectroscopy in tissue-like media is a study of the binding of ethidium bromide to bovine leukocytes in fresh milk. Ethidium bromide is a fluorescent dye that is commonly used to label DNA, and hence visualize chromosomes in cells. The lifetime of

  19. Impurity studies in fusion devices using laser-fluorescence-spectroscopy

    SciTech Connect

    Husinsky, W.R.

    1980-08-01

    Resonance fluorescence excitation of neutral atoms using tunable radiation from dye lasers offers a number of unique advantages for impurity studies in fusion devices. Using this technique, it is possible to perform local, time-resolved measurements of the densities and velocity distributions of metallic impurities in fusion devices without disturbing the plasma. Velocities are measured by monitoring the fluorescence intensity while tuning narrow bandwidth laser radiation through the Doppler - broadened absorbtion spectrum of the transition. The knowledge of the velocity distribution of neutral impurities is particularly useful for the determination of impurity introduction mechanisms. The laser fluorescence technique will be described in terms of its application to metallic impurities in fusion devices and related laboratory experiments. Particular attention will be given to recent results from the ISX-B tokamak using pulsed dye lasers where detection sensitivities for neutral Fe of 10/sup 6/ atoms/cm/sup 3/ with a velocity resolution of 600 m/sec (0.1 eV) have been achieved. Techniques for exciting plasma particles (H,D) will also be discussed.

  20. Design optimization of fiber optic probes for remote fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Bhowmick, G. K.; Gautam, Nutan; Gantayet, L. M.

    2009-07-01

    Fiber optic probes are designed, developed and fabricated in the laboratories for remote fluorescence spectroscopic studies in various fields such as investigation of tissues, environmental monitoring, and analysis of samples in hostile environment. Optimized probe design is very much important for efficient transport and collection of photons, which ultimately helps in quantifying resultant emission and understanding light-matter interaction. Instead of the conventional ray optics, Monte Carlo technique has been used to optimize the design of fiber optic probes, comprising only of flat tipped fibers with and without focusing lenses, for remote fluorescence measurement in three different types of target media having different optical properties. Typical probe geometry consists of one excitation fiber surrounded by a ring of collection fibers. The effects of fiber parameters like fiber diameter, numerical aperture, core-clad ratio, arrangement of collection fibers around the excitation fiber and dead space between them, and optical properties of the medium on the performance of probes have been analysed and compared with the results of previous observations, wherever the data are available. The results show a significant difference between the collected emission with and without consideration of dead space, which plays a very significant role in probe design and is dependent on the number of collection fibers in the geometry, relative dimension of collection and excitation fibers and separation between the two. Introduction of a convex lens in the probe increases the amount of fluorescence signal for a given probe arrangement.

  1. In vivo stepwise multi-photon activation fluorescence imaging of melanin in human skin

    NASA Astrophysics Data System (ADS)

    Lai, Zhenhua; Gu, Zetong; Abbas, Saleh; Lowe, Jared; Sierra, Heidy; Rajadhyaksha, Milind; DiMarzio, Charles

    2014-03-01

    The stepwise multi-photon activated fluorescence (SMPAF) of melanin is a low cost and reliable method of detecting melanin because the activation and excitation can be a continuous-wave (CW) mode near infrared (NIR) laser. Our previous work has demonstrated the melanin SMPAF images in sepia melanin, mouse hair, and mouse skin. In this study, we show the feasibility of using SMPAF to detect melanin in vivo. in vivo melanin SMPAF images of normal skin and benign nevus are demonstrated. SMPAF images add specificity for melanin detection than MPFM images and CRM images. Melanin SMPAF is a promising technology to enable early detection of melanoma for dermatologists.

  2. Fluorescence spectroscopy of kerosene vapour at high temperatures and pressures: potential for gas turbines measurements

    NASA Astrophysics Data System (ADS)

    Orain, M.; Baranger, P.; Ledier, C.; Apeloig, J.; Grisch, F.

    2014-09-01

    Laser-induced fluorescence spectroscopy of kerosene vapour was performed in a heated test cell operating between 450 and 900 K, at pressure from 0.1 to 3.0 MPa, for oxygen molar fraction between 0 and 21 %, with different laser excitation wavelengths (248, 266, 282 and 308 nm). Results show that, depending on the laser excitation scheme, kerosene fluorescence spectrum exhibits one or two fluorescence bands in the UV-visible range (attributed to aromatics naturally present in kerosene fuel). Fluorescence intensity of these bands decreases with increasing temperature, pressure and oxygen molar fraction. Different imaging strategies were derived from spectroscopic findings to simultaneously measure temperature and equivalence ratio fields in kerosene/air sprays, or flame structure and fuel spatial distribution in kerosene/air aeronautical combustors, by means of planar laser-induced fluorescence on kerosene vapour (K-PLIF).

  3. Wavefront sensorless adaptive optics fluorescence biomicroscope for in vivo retinal imaging in mice.

    PubMed

    Wahl, Daniel J; Jian, Yifan; Bonora, Stefano; Zawadzki, Robert J; Sarunic, Marinko V

    2016-01-01

    Cellular-resolution in vivo fluorescence imaging is a valuable tool for longitudinal studies of retinal function in vision research. Wavefront sensorless adaptive optics (WSAO) is a developing technology that enables high-resolution imaging of the mouse retina. In place of the conventional method of using a Shack-Hartmann wavefront sensor to measure the aberrations directly, WSAO uses an image quality metric and a search algorithm to drive the shape of the adaptive element (i.e. deformable mirror). WSAO is a robust approach to AO and it is compatible with a compact, low-cost lens-based system. In this report, we demonstrated a hill-climbing algorithm for WSAO with a variable focus lens and deformable mirror for non-invasive in vivo imaging of EGFP (enhanced green fluorescent protein) labelled ganglion cells and microglia cells in the mouse retina.

  4. Wavefront sensorless adaptive optics fluorescence biomicroscope for in vivo retinal imaging in mice

    PubMed Central

    Wahl, Daniel J.; Jian, Yifan; Bonora, Stefano; Zawadzki, Robert J.; Sarunic, Marinko V.

    2015-01-01

    Cellular-resolution in vivo fluorescence imaging is a valuable tool for longitudinal studies of retinal function in vision research. Wavefront sensorless adaptive optics (WSAO) is a developing technology that enables high-resolution imaging of the mouse retina. In place of the conventional method of using a Shack-Hartmann wavefront sensor to measure the aberrations directly, WSAO uses an image quality metric and a search algorithm to drive the shape of the adaptive element (i.e. deformable mirror). WSAO is a robust approach to AO and it is compatible with a compact, low-cost lens-based system. In this report, we demonstrated a hill-climbing algorithm for WSAO with a variable focus lens and deformable mirror for non-invasive in vivo imaging of EGFP (enhanced green fluorescent protein) labelled ganglion cells and microglia cells in the mouse retina. PMID:26819812

  5. In Vivo Photoacoustic and Fluorescence Cystography Using Clinically Relevant Dual Modal Indocyanine Green

    PubMed Central

    Park, Sungjo; Kim, Jeesu; Jeon, Mansik; Song, Jaewon; Kim, Chulhong

    2014-01-01

    Conventional X-ray-based cystography uses radio-opaque materials, but this method uses harmful ionizing radiation and is not sensitive. In this study, we demonstrate nonionizing and noninvasive photoacoustic (PA) and fluorescence (FL) cystography using clinically relevant indocyanine green (ICG) in vivo. After transurethral injection of ICG into rats through a catheter, their bladders were photoacoustically and fluorescently visualized. A deeply positioned bladder below the skin surface (i.e., ∼1.5–5 mm) was clearly visible in the PA and FL image using a laser pulse energy of less than 2 mJ/cm2 (1/15 of the safety limit). Then, the in vivo imaging results were validated through in situ studies. Our results suggest that dual modal cystography can provide a nonionizing and noninvasive imaging tool for bladder mapping. PMID:25337743

  6. Determination of dissolved organic matter removal efficiency in wastewater treatment works using fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Carstea, Elfrida M.; Bridgeman, John

    2015-04-01

    Fluorescence spectroscopy was used to investigate the removal efficiency of dissolved organic matter (DOM) in several wastewater treatment works, at different processing stages. The correlation between fluorescence values and biochemical oxygen demand (BOD), chemical oxygen demand (COD) and total organic carbon (TOC) has been examined. Fluorescence was measured for unfiltered and filtered (0.45 and 0.20 μm) samples of crude, settled and secondary treated wastewater (activated sludge), and final effluent. Moreover, the potential of using portable fluorimeters has been explored in a laboratory scale activated sludge process. Good correlations were observed for filtered and unfiltered wastewater samples between protein-like fluorescence intensity (excitation 280 nm, emission 350 nm) and BOD (r = 0.78), COD (r = 0.90) and TOC (r = 0.79). BOD displayed a higher correlation at the 0.20 μm filtered samples compared to COD and TOC. Slightly better relation was seen between fluorescence and conventional parameters at the portable fluorimeters compared to laboratory-based instruments. The results indicated that fluorescence spectroscopy, in particular protein-like fluorescence, could be used for continuous, real-time assessment of DOM removal efficiency in wastewater treatment works.

  7. The application of anti-ESAT-6 monoclonal antibody fluorescent probe in ex vivo near-infrared fluorescence imaging in mice with pulmonary tuberculosis.

    PubMed

    Feng, Feng; Zhang, Haoling; Zhu, Zhaoqin; Li, Cong; Shi, Yuxin; Zhang, Zhiyong

    2014-09-01

    Here, we aimed to assess the feasibility of anti-ESAT-6 monoclonal antibody (mAb) coupling with IR783 and rhodamine fluorescent probe in the detection of ESAT-6 expression in tuberculosis tissue of mice using near-infrared fluorescence imaging. IR783 and rhodamine were conjugated to the anti-ESAT-6 mAb or IgG. Mice in the experimental group were injected with fluorescence-labeled mAb probe, and mice in the control group were injected with fluorescence-labeled non-specific IgG antibody. Twenty-four hours later, the lung tissue of mice was examined using ex vivo near-infrared fluorescence imaging. In addition, the contrast-to-noise ratio (CNR) was calculated by measuring the signal intensities of the pulmonary lesions, normal lung tissue and background noise. The frozen lung tissue section was examined under fluorescence microscopy and compared with hemoxylin and eosin (HE) staining. The ex vivo near-infrared fluorescence imaging showed that the fluorescence signal in the lung tuberculosis lesions in the experimental group was significantly enhanced, whereas there was only a weak fluorescence signal or even no fluorescence signal in the control group. CNR values were 64.40 ± 7.02 (n = 6) and 8.75 ± 3.87 (n = 6), respectively (t = 17.01, p < 0.001). The fluorescence accumulation distribution detected under fluorescence microscopy was consistent with HE staining of the tuberculosis region. In conclusion, anti-ESAT-6 mAb fluorescent probe could target and be applied in specific ex vivo imaging of mice tuberculosis, and may be of further use in tuberculosis in living mice.

  8. Near-infrared-excited confocal Raman spectroscopy advances in vivo diagnosis of cervical precancer

    NASA Astrophysics Data System (ADS)

    Duraipandian, Shiyamala; Zheng, Wei; Ng, Joseph; Low, Jeffrey J. H.; Ilancheran, Arunachalam; Huang, Zhiwei

    2013-06-01

    Raman spectroscopy is a unique optical technique that can probe the changes of vibrational modes of biomolecules associated with tissue premalignant transformation. This study evaluates the clinical utility of confocal Raman spectroscopy over near-infrared (NIR) autofluorescence (AF) spectroscopy and composite NIR AF/Raman spectroscopy for improving early diagnosis of cervical precancer in vivo at colposcopy. A rapid NIR Raman system coupled with a ball-lens fiber-optic confocal Raman probe was utilized for in vivo NIR AF/Raman spectral measurements of the cervix. A total of 1240 in vivo Raman spectra [normal (n=993), dysplasia (n=247)] were acquired from 84 cervical patients. Principal components analysis (PCA) and linear discriminant analysis (LDA) together with a leave-one-patient-out, cross-validation method were used to extract the diagnostic information associated with distinctive spectroscopic modalities. The diagnostic ability of confocal Raman spectroscopy was evaluated using the PCA-LDA model developed from the significant principal components (PCs) [i.e., PC4, 0.0023% PC5, 0.00095% PC8, 0.00022%, (p<0.05)], representing the primary tissue Raman features (e.g., 854, 937, 1095, 1253, 1311, 1445, and 1654 cm-1). Confocal Raman spectroscopy coupled with PCA-LDA modeling yielded the diagnostic accuracy of 84.1% (a sensitivity of 81.0% and a specificity of 87.1%) for in vivo discrimination of dysplastic cervix. The receiver operating characteristic curves further confirmed that the best classification was achieved using confocal Raman spectroscopy compared to the composite NIR AF/Raman spectroscopy or NIR AF spectroscopy alone. This study illustrates that confocal Raman spectroscopy has great potential to improve early diagnosis of cervical precancer in vivo during clinical colposcopy.

  9. In Vivo Fluorescence Imaging and Tracking of Circulating Cells and Therapeutic Nanoparticles

    NASA Astrophysics Data System (ADS)

    Markovic, Stacey

    Noninvasive enumeration of rare circulating cells in small animals is of great importance in many areas of biomedical research, but most existing enumeration techniques involve drawing and enriching blood which is known to be problematic. Recently, small animal "in vivo flow cytometry" (IVFC) techniques have been developed, where cells flowing through small arterioles are counted continuously and noninvasively in vivo. However, higher sensitivity IVFC techniques are needed for studying low-abundance (<100/mL) circulating cells. To this end, we developed a macroscopic fluorescence imaging system and automated computer vision algorithm that allows in vivo detection, enumeration and tracking of circulating fluorescently labeled cells from multiple large blood vessels in the ear of a mouse. This technique ---"computer vision IVFC" (CV-IVFC) --- allows cell detection and enumeration at concentrations of 20 cells/mL. Performance of CV-IVFC was also characterized for low-contrast imaging scenarios, representing conditions of weak cell fluorescent labeling or high background tissue autofluorescence, and showed efficient tracking and enumeration of circulating cells with 50% sensitivity in contrast conditions degraded 2 orders of magnitude compared to in vivo testing supporting the potential utility of CV-IVFC in a range of biological models. Refinement of prior work in our lab of a separate rare-cell detection platform - "diffuse fluorescence flow cytometry" (DFFC) --- implemented a "frequency encoding" scheme by modulating two excitation lasers. Fluorescent light from both lasers can be simultaneously detected and split by frequency allowing for better discrimination of noise, sensitivity, and cell localization. The system design is described in detail and preliminary data is shown. Last, we developed a broad-field transmission fluorescence imaging system to observe nanoparticle (NP) diffusion in bulk biological tissue. Novel, implantable NP spacers allow controlled, long

  10. Fluorescence diagnosis of the status of the human lens in vivo

    NASA Astrophysics Data System (ADS)

    Vladimirova, E. S.; Salmin, V. V.; Salmina, A. B.; Oskirko, S. A.; Lazarenko, V. I.; Provorov, A. S.

    2012-03-01

    We have studied fluorescence spectra of the human lens in vivo for healthy eyes and in different stages of senile cataract development. We propose a spectral criterion, the lens opacity index, allowing us to differentiate between stages of cataract development. We show a high correlation between the stage of cataract development and the opacity index. We propose an empirical expression for determining the stage of senile cataract development from the value of the lens opacity index. The technique has been clinically tested.

  11. In vivo and in situ tracking cancer chemotherapy by highly photostable NIR fluorescent theranostic prodrug.

    PubMed

    Wu, Xumeng; Sun, Xuanrong; Guo, Zhiqian; Tang, Jianbin; Shen, Youqing; James, Tony D; Tian, He; Zhu, Weihong

    2014-03-01

    In vivo monitoring of the biodistribution and activation of prodrugs is urgently required. Near infrared (NIR) fluorescence-active fluorophores with excellent photostability are preferable for tracking drug release in vivo. Herein, we describe a NIR prodrug DCM-S-CPT and its polyethylene glycol-polylactic acid (PEG-PLA) loaded nanoparticles as a potent cancer therapy. We have conjugated a dicyanomethylene-4H-pyran derivative as the NIR fluorophore with camptothecin (CPT) as the anticancer drug using a disulfide linker. In vitro experiments verify that the high intracellular glutathione (GSH) concentrations in tumor cells cause cleavage of the disulfide linker, resulting in concomitantly the active drug CPT release and significant NIR fluorescence turn-on with large Stokes shift (200 nm). The NIR fluorescence of DCM-S-CPT at 665 nm with fast response to GSH can act as a direct off-on signal reporter for the GSH-activatable prodrug. Particularly, DCM-S-CPT possesses much better photostability than ICG, which is highly desirable for in situ fluorescence-tracking of cancer chemotherapy. DCM-S-CPT has been successfully utilized for in vivo and in situ tracking of drug release and cancer therapeutic efficacy in living animals by NIR fluorescence. DCM-S-CPT exhibits excellent tumor-activatable performance when intravenously injected into tumor-bearing nude mice, as well as specific cancer therapy with few side effects. DCM-S-CPT loaded in PEG-PLA nanoparticles shows even higher antitumor activity than free CPT, and is also retained longer in the plasma. The tumor-targeting ability and the specific drug release in tumors make DCM-S-CPT as a promising prodrug, providing significant advances toward deeper understanding and exploration of theranostic drug-delivery systems.

  12. Excitation-emission matrices (EEMs) and synchronous fluorescence spectroscopy (SFS) investigations of gastrointestinal tissues

    NASA Astrophysics Data System (ADS)

    Genova, Ts.; Borisova, E.; Zhelyazkova, Al.; Semyachkina-Glushkovskaya, O.; Penkov, N.; Keremedchiev, M.; Vladimirov, B.; Avramov, L.

    2015-01-01

    In this report we will present our recent investigations of the fluorescence properties of lower part gastrointestinal tissues using excitation-emission matrix and synchronous fluorescence spectroscopy measurement modalities. The spectral peculiarities observed will be discussed and the endogenous sources of the fluorescence signal will be addressed. For these fluorescence spectroscopy measurements the FluoroLog 3 system (HORIBA Jobin Yvon, France) was used. It consists of a Xe lamp (300 W, 200-650 nm), a double mono-chromators, and a PMT detector with a work region at 220- 850 nm. Autofluorescence signals were detected in the form of excitation-emission matrices for the samples of normal mucosa, dysphasia and colon carcinoma and specific spectral features for each tissue were found. Autofluorescence signals from the same samples are observed through synchronous fluorescence spectroscopy, which is a novel promising modality for fluorescence spectroscopy measurements of bio-samples. It is one of the most powerful techniques for multicomponent analysis, because of its sensitivity. In the SFS regime, the fluorescence signal is recorded while both excitation λexc and emission wavelengths λem are simultaneously scanned. A constant wavelength interval is maintained between the λexc and λem wavelengths throughout the spectrum. The resulted fluorescence spectrum shows narrower peak widths, in comparison with EEMs, which are easier for identification and minimizes the chance for false determinations or pretermission of specific spectral feature. This modality is also faster, than EEMs, a much smaller number of data points are required.1 In our measurements we use constant wavelength interval Δλ in the region of 10-200 nm. Measurements are carried out in the terms of finding Δλ, which results in a spectrum with most specific spectral features for comparison with spectral characteristics observed in EEMs. Implementing synchronous fluorescence spectroscopy in optical

  13. In Vivo Imaging of Retinal Oxidative Stress Using a Reactive Oxygen Species–Activated Fluorescent Probe

    PubMed Central

    Prunty, Megan C.; Aung, Moe H.; Hanif, Adam M.; Allen, Rachael S.; Chrenek, Micah A.; Boatright, Jeffrey H.; Thule, Peter M.; Kundu, Kousik; Murthy, Niren; Pardue, Machelle T.

    2015-01-01

    Purpose In vivo methods for detecting oxidative stress in the eye would improve screening and monitoring of the leading causes of blindness: diabetic retinopathy, glaucoma, and age-related macular degeneration. Methods To develop an in vivo biomarker for oxidative stress in the eye, we tested the efficacy of a reactive oxygen species (ROS)–activated, near-infrared hydrocyanine-800CW (H-800CW) fluorescent probe in light-induced retinal degeneration (LIRD) mouse models. After intravitreal delivery in LIRD rats, fluorescent microscopy was used to confirm that the oxidized H-800CW appeared in the same retinal layers as an established ROS marker (dichlorofluorescein). Results Dose–response curves of increasing concentrations of intravenously injected H-800CW demonstrated linear increases in both intensity and total area of fundus hyperfluorescence in LIRD mice, as detected by scanning laser ophthalmoscopy. Fundus hyperfluorescence also correlated with the duration of light damage and functional deficits in vision after LIRD. In LIRD rats with intravitreal injections of H-800CW, fluorescent labeling was localized to photoreceptor inner segments, similar to dichlorofluorescein. Conclusions Hydrocyanine-800CW detects retinal ROS in vivo and shows potential as a novel biomarker for ROS levels in ophthalmic diseases. PMID:26348635

  14. In vivo target bio-imaging of Alzheimer's disease by fluorescent zinc oxide nanoclusters.

    PubMed

    Lai, Lanmei; Zhao, Chunqiu; Su, Meina; Li, Xiaoqi; Liu, Xiaoli; Jiang, Hui; Amatore, Christian; Wang, Xuemei

    2016-07-21

    Alzheimer's disease (AD) is an irreversible neurodegenerative disease which is difficult to cure. When Alzheimer's disease occurs, the level of zinc ions in the brain changes, and the relevant amount of zinc ions continue decreasing in the cerebrospinal fluid and plasma of Alzheimer's patients with disease exacerbation. In view of these considerations, we have explored a new strategy for the in vivo rapid fluorescence imaging of Alzheimer's disease through target bio-labeling of zinc oxide nanoclusters which were biosynthesized in vivo in the Alzheimer's brain via intravenous injection of zinc gluconate solution. By using three-month-old and six-month-old Alzheimer's model mice as models, our observations demonstrate that biocompatible zinc ions could pass through the blood-brain barrier of the Alzheimer's disease mice and generate fluorescent zinc oxide nanoclusters (ZnO NCs) through biosynthesis, and then the bio-synthesized ZnO NCs could readily accumulate in situ on the hippocampus specific region for the in vivo fluorescent labeling of the affected sites. This study provides a new way for the rapid diagnosis of Alzheimer's disease and may have promising prospects in the effective diagnosis of Alzheimer's disease.

  15. In vivo optical imaging of brain tumors and arthritis using fluorescent SapC-DOPS nanovesicles.

    PubMed

    Chu, Zhengtao; LaSance, Kathleen; Blanco, Victor; Kwon, Chang-Hyuk; Kaur, Balveen; Frederick, Malinda; Thornton, Sherry; Lemen, Lisa; Qi, Xiaoyang

    2014-01-01

    We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10° steps over 380°. A rectangular region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models. PMID:24837630

  16. Photoacoustic imaging of the near-infrared fluorescent protein iRFP in vivo

    NASA Astrophysics Data System (ADS)

    Krumholz, Arie; Filonov, Grigory S.; Xia, Jun; Yao, Junjie; Verkhusha, Vladislav V.; Wang, Lihong V.

    2012-02-01

    Genetically encoded probes powerfully and non-invasively target specific tissues, cells, and subcellular locations. iRFP, a novel near-infrared fluorescent protein with low quantum yield whose absorption and fluorescence maxima are located at wavelengths longer than the Q-band of hemoglobin absorption, is ideal for PAT. Here, we report on an in vitro comparison of iRFP with other far-red fluorescent proteins, and its use in imaging a mouse tumor xenograft model. In an in vivo experiment, we stably transfected iRFP into MTLn3 adenocarcinoma cells and injected them into the mammary fat pad of female SCID/NCr mice, then imaged the resulting tumors two and three weeks post injection. The contrast increase from the protein expression was high enough to clearly separate the tumor region from the rest of the animal.

  17. In vivo detecting matrix metalloproteinase (MMP) activity by a genetically engineered fluorescent probe

    NASA Astrophysics Data System (ADS)

    Yang, Jie; Zhang, Zhihong; Su, Ting; Luo, Qingming

    2007-02-01

    Degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) enhances tumor invasion and metastasis. To monitor MMP activity, we constructed plasmid that encoded a fluorescent sensor DC, in which an MMP substrate site (MSS) is sandwiched between DsRed2 and ECFP. MMPs are secretory proteins, only acting on the outside of cells; hence, an expressing vector was used that displayed the fluorescent sensor on the cellular surface. The DC was expressed in cells with high secretory MMP, so MSS was cleaved by MMP. Also, GM6001, an MMP inhibitor, causes DsRed2 signals to increase in living cells and on the chick embryo chorioallantoic membrane (CAM). Thus, this fluorescent sensor was able to sensitively monitor MMP activation in vivo. Potential applications for this sensor include high-throughput screening for MMP inhibitors for anti-cancer research, and detailed analysis of the effects of MMP inhibitors.

  18. Dendrimer-Based Fluorescent Indicators: In Vitro and In Vivo Applications

    PubMed Central

    Albertazzi, Lorenzo; Brondi, Marco; Pavan, Giovanni M.; Sato, Sebastian Sulis; Signore, Giovanni; Storti, Barbara; Ratto, Gian Michele; Beltram, Fabio

    2011-01-01

    Background The development of fluorescent proteins and synthetic molecules whose fluorescence properties are controlled by the environment makes it possible to monitor physiological and pathological events in living systems with minimal perturbation. A large number of small organic dyes are available and routinely used to measure biologically relevant parameters. Unfortunately their application is hindered by a number of limitations stemming from the use of these small molecules in the biological environment. Principal Findings We present a novel dendrimer-based architecture leading to multifunctional sensing elements that can overcome many of these problems. Applications in vitro, in living cells and in vivo are reported. In particular, we image for the first time extracellular pH in the brain in a mouse epilepsy model. Conclusion We believe that the proposed architecture can represent a useful and novel tool in fluorescence imaging that can be widely applied in conjunction with a broad range of sensing dyes and experimental setups. PMID:22163303

  19. Europium Uptake and Partitioning in Oat (Avena sativa) Roots as studied By Laser-Induced Fluorescence Spectroscopy and Confocal Microscopy Profiling Technique

    SciTech Connect

    Fellows, Robert J.; Wang, Zheming; Ainsworth, Calvin C.

    2003-11-15

    The uptake of Eu3+ by elongating oat plant roots was studied by fluorescence spectroscopy, fluorescence lifetime measurement, as well as laser excitation time-resolved confocal fluorescence profiling technique. The results of this work indicated that the initial uptake of Eu(III) by oat root was most evident within the apical meristem of the root just proximal to the root cap. Distribution of assimilated Eu(III) within the roots differentiation and elongation zone was non-uniform. Higher concentrations were observed within the vascular cylinder, specifically in the phloem and developing xylem parenchyma. Elevated levels of the metal were also observed in the root hairs of the mature root. The concentration of assimilated Eu3+ dropped sharply from the apical meristem to the differentiation and elongation zone and then gradually decreased as the distance from the root cap increased. Fluorescence spectroscopic characteristics of the assimilated Eu3+ suggested that the Eu3+ exists a s inner-sphere mononuclear complexes inside the root. This work has also demonstrated the effectiveness of a time-resolved Eu3+ fluorescence spectroscopy and confocal fluorescence profiling techniques for the in vivo, real-time study of metal[Eu3+] accumulation by a functioning intact plant root. This approach can prove valuable for basic and applied studies in plant nutrition and environmental uptake of actinide radionuclides.

  20. Picosecond time-resolved fluorescence spectroscopy of phytochrome and stentorin

    NASA Astrophysics Data System (ADS)

    Song, Pill-Soon

    1991-05-01

    Phytochrome is a tetrapyrrole chromoprotein. It serves as a sensitive photosensor for red lightmediated gene expression and other developmental/morphological responses in plants. In this paper photochemical dynamics of the phytochrome molecule have been described in terms of photoisomerization of the tetrapyrrole chromophore in its singlet excited state and subsequent thermal processes in the Pr Pfr phototransformation of phytochrome. Stentorin acts as the photosensor molecule in the ciliate Stentor coeruleus. This unicellular protozoan is most sensitive to red light (610-620 urn). Stentor also senses the direction of light propagation as evidenced by their light-avoiding and negative phototactic swimming behaviors. This aneural photosensory phenomenon is triggered by the photoreceptor stentorin. The possible involvement of a light-induced transient proton release from the photoreceptor as the primary mechanism of light-signal processing has been discussed on the basis of picosecond fluorescence decays and time-resolved fluorescence spectra of stentorin in solution. An initial sensory signal generated by the primary photoprocess of stentorin then triggers subsequent transduction steps that include calcium ion influx from the extracellular medium. Calcium ion influx from the extracellular medium to the cytosol causes the Stentor cell to reverse its ciliary beating and subsequently steer away from the light trap. II.

  1. Novel In Vivo Model for Combinatorial Fluorescence Labeling in Mouse Prostate

    PubMed Central

    Fang, Xiaolan; Gyabaah, Kenneth; Nickkholgh, Bita; Cline, J. Mark; Balaji, K.C.

    2015-01-01

    BACKGROUND The epithelial layer of prostate glands contains several types of cells, including luminal and basal cells. Yet there is paucity of animal models to study the cellular origin of normal or neoplastic development in the prostate to facilitate the treatment of heterogenous prostate diseases by targeting individual cell lineages. METHODS We developed a mouse model that expresses different types of fluorescent proteins (XFPs) specifically in prostatic cells. Using an in vivo stochastic fluorescent protein combinatorial strategy, XFP signals were expressed specifically in prostate of Protein Kinase D1 (PKD1) knock-out, K-RasG12D knock-in, and Phosphatase and tensin homolog (PTEN) and PKD1 double knock-out mice under the control of PB-Cre promoter. RESULTS In vivo XFP signals were observed in prostate of PKD1 knock-out, K-RasG12D knock-in, and PTEN PKD1 double knock-out mice, which developed normal, hyperplastic, and neoplastic prostate, respectively. The patchy expression pattern of XFPs in neoplasia tissue indicated the clonal origin of cancer cells in the prostate. CONCLUSIONS The transgenic mouse models demonstrate combinatorial fluorescent protein expression in normal and cancerous prostatic tissues. This novel prostate-specific fluorescent labeled mouse model, which we named Prorainbow, could be useful in studying benign and malignant pathology of prostate. PMID:25753731

  2. Redox-responsive branched-bottlebrush polymers for in vivo MRI and fluorescence imaging

    PubMed Central

    Sowers, Molly A.; McCombs, Jessica R.; Wang, Ying; Paletta, Joseph T.; Morton, Stephen W.; Dreaden, Erik C.; Boska, Michael D.; Ottaviani, M. Francesca; Hammond, Paula T.; Rajca, Andrzej; Johnson, Jeremiah A.

    2014-01-01

    Stimuli-responsive multimodality imaging agents have broad potential in medical diagnostics. Herein, we report the development of a new class of branched-bottlebrush polymer dual-modality organic radical contrast agents—ORCAFluors—for combined magnetic resonance and near-infrared fluorescence imaging in vivo. These nitroxide radical-based nanostructures have longitudinal and transverse relaxation times that are on par with commonly used heavy-metal-based magnetic resonance imaging (MRI) contrast agents. Furthermore, these materials display a unique compensatory redox response: fluorescence is partially quenched by surrounding nitroxides in the native state; exposure to ascorbate or ascorbate/glutathione leads to nitroxide reduction and a concomitant 2- to 3.5-fold increase in fluorescence emission. This behaviour enables correlation of MRI contrast, fluorescence intensity and spin concentration with tissues known to possess high concentrations of ascorbate in mice. Our in vitro and in vivo results, along with our modular synthetic approach, make ORCAFluors a promising new platform for multimodality molecular imaging. PMID:25403521

  3. From the shape of the vertical profile of in vivo fluorescence to Chlorophyll-a concentration

    NASA Astrophysics Data System (ADS)

    Mignot, A.; Claustre, H.; D'Ortenzio, F.; Xing, X.; Poteau, A.; Ras, J.

    2011-04-01

    In vivo fluorescence of Chlorophyll-a (Chl-a) is a potentially useful property to study the vertical distribution of phytoplankton biomass. However the technique is presently not fully exploited as it should be, essentially because of the difficulties in converting the fluorescence signal into an accurate Chl-a concentration. These difficulties arise noticeably from natural variations in the Chl-a fluorescence relationship, which is under the control of community composition as well as of their nutrient and light status. As a consequence although vertical profiles of fluorescence are likely the most recorded biological property in the open ocean, the corresponding large databases are underexploited. Here with the aim to convert a fluorescence profile into a Chl-a concentration profile, we test the hypothesis that the Chl-a concentration can be gathered from the sole knowledge of the shape of the fluorescence profile. We analyze a large dataset from 18 oceanographic cruises conducted in case-1 waters from the highly stratified hyperoligotrophic waters (surface Chl-a = 0.02 mg m-3) of the South Pacific Gyre to the eutrophic waters of the Benguela upwelling (surface Chl-a = 32 mg m-3) and including the very deep mixed waters in the North Atlantic (Mixed Layer Depth = 690 m). This dataset encompasses more than 700 vertical profiles of Chl-a fluorescence as well as accurate estimations of Chl-a by High Performance Liquid Chromatography (HPLC). Two typical fluorescence profiles are identified, the uniform profile, characterized by a homogeneous layer roughly corresponding to the mixed layer, and the non-uniform profile, characterized by the presence of a Deep Chlorophyll Maximum. Using appropriate mathematical parameterizations, a fluorescence profile is subsequently represented by 3 or 5 shape parameters for uniform or non-uniform profiles, respectively. For both situations, an empirical model is developed to predict the "true" Chl-a concentration from these shape

  4. From the shape of the vertical profile of in vivo fluorescence to Chlorophyll-a concentration

    NASA Astrophysics Data System (ADS)

    Mignot, A.; Claustre, H.; D'Ortenzio, F.; Xing, X.; Poteau, A.; Ras, J.

    2011-08-01

    In vivo fluorescence of Chlorophyll-a (Chl-a) is a potentially useful property to study the vertical distribution of phytoplankton biomass. However the technique is presently not fully exploited as it should be, essentially because of the difficulties in converting the fluorescence signal into an accurate Chl-a concentration. These difficulties arise noticeably from natural variations in the Chl-a fluorescence relationship, which is under the control of community composition as well as of their nutrient and light status. As a consequence, although vertical profiles of fluorescence are likely the most recorded biological property in the open ocean, the corresponding large databases are underexploited. Here with the aim to convert a fluorescence profile into a Chl-a concentration profile, we test the hypothesis that the Chl-a concentration can be gathered from the sole knowledge of the shape of the fluorescence profile. We analyze a large dataset from 18 oceanographic cruises conducted in case-1 waters from the highly stratified hyperoligotrophic waters (surface Chl-a = 0.02 mg m-3) of the South Pacific Gyre to the eutrophic waters of the Benguela upwelling (surface Chl-a = 32 mg m-3) and including the very deep mixed waters in the North Atlantic (Mixed Layer Depth = 690 m). This dataset encompasses more than 700 vertical profiles of Chl-a fluorescence as well as accurate estimations of Chl-a by High Performance Liquid Chromatography (HPLC). Two typical fluorescence profiles are identified, the uniform profile, characterized by a homogeneous layer roughly corresponding to the mixed layer, and the non-uniform profile, characterized by the presence of a Deep Chlorophyll Maximum. Using appropriate mathematical parameterizations, a fluorescence profile is subsequently represented by 3 or 5 shape parameters for uniform or non-uniform profiles, respectively. For both situations, an empirical model is developed to predict the "true" Chl-a concentration from these shape

  5. The Quality of In Vivo Upconversion Fluorescence Signals Inside Different Anatomic Structures.

    PubMed

    Wang, Lijiang; Draz, Mohamed Shehata; Wang, Wei; Liao, Guodong; Xu, Yuhong

    2015-02-01

    Fluorescence imaging is a broadly interesting and rapidly growing strategy for non-invasive clinical applications. However, because of interference from light scattering, absorbance, and tissue autofluorescence, the images can exhibit low sensitivity and poor quality. Upconversion fluorescence imaging, which is based on the use of near-infrared (NIR) light for excitation, has recently been introduced as an improved approach to minimize the effects of light scattering and tissue autofluorescence. This strategy is promising for ultrasensitive and deep tissue imaging applications. However, the emitted upconversion fluorescence signals are primarily in the visible range and are likely to be absorbed and scattered by tissues. Therefore, different anatomic structures could impose various effects on the quality of the images. In this study, we used upconversion-core/silica-shell nanoprobes to evaluate the quality of upconversion fluorescence at different anatomic locations in athymic nude mice. The nanoprobe contained an upconversion core, which was green (β-NaYF4:Yb3+/Ho3+) or red (β-NaYF4:Yb3+/Er3+), and a nonporous silica shell to allow for multicolor imaging. High-quality upconversion fluorescence signals were detected with signal-to-noise ratios of up to 170 at tissue depths of up to - 1.0 cm when a 980 nm laser excitation source and a bandpass emission filter were used. The presence of dense tissue structures along the imaging path reduced the signal intensity and imaging quality, and nanoprobes with longer-wavelength emission spectra were therefore preferable. This study offers a detailed analysis of the quality of upconversion signals in vivo inside different anatomic structures. Such information could be essential for the analysis of upconversion fluorescence images in any in vivo biodiagnostic and microbial tracking applications.

  6. In vivo cell biology of cancer cells visualized with fluorescent proteins.

    PubMed

    Hoffman, Robert M

    2005-01-01

    This chapter describes a new cell biology where the behavior of individual cells can be visualized in the living animal. Previously it has been demonstrated that fluorescent proteins can be used for whole-body imaging of metastatic tumor growth, bacterial infection, and gene expression. An example of the new cell biology is dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP)-expressing transgenic mice. These models show with great clarity the details of tumor-stroma interactions and especially tumor-induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts, and macrophages. Another example is the color coding of cells with RFP or GFP such that both cell types can be simultaneously visualized in vivo. Stem cells can also be visualized and tracked in vivo. Mice in which the regulatory elements of the stem cell marker nestin drive GFP expression enable nascent vasculature to be visualized interacting with transplanted RFP-expressing cancer cells. Nestin-driven GFP expression can also be used to visualize hair follicle stem cells. Dual-color cells expressing GFP in the nucleus and RFP in the cytoplasm enable real-time visualization of nuclear-cytoplasm dynamics including cell cycle events and apoptosis. Highly elongated cancer cells in capillaries in living mice were observed within skin flaps. The migration velocities of the cancer cells in the capillaries were measured by capturing images of the dual-color fluorescent cells over time. The cells in the capillaries elongated to fit the width of these vessels. The use of the dual-color cancer cells differentially labeled in the cytoplasm and nucleus and associated fluorescent imaging provide a powerful tool to understand the mechanism of cancer cell migration and deformation in small vessels.

  7. Classification of aortic atherosclerotic lesions with time-resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Maarek, Jean-Michel I.; Marcu, Laura; Grundfest, Warren S.; Fishbein, Michael C.

    1999-07-01

    In this study, we examine the possibility of differentiating between classes of atherosclerotic lesions based on time- resolved fluorescence spectroscopy and we compare the performance of classification schemes that use either the time-resolved spectra or only the intensity spectra. Transient fluorescence emissions induced by pulsed nitrogen laser excitation was measured on 87 excised samples of human aorta. The samples were classified histologically using the AHA classification Predictor variables derived from the time-resolved spectra included the spectral intensities at 360-510 nm and parameters of a biexponential fit of the fluorescence impulse response function. Stepwise discriminant analysis using these predict variables showed that a few predictor variables sufficed to correctly classify 89 percent of the samples. Excluding the time- dependent decay and using only the spectral intensities, the percentage of correctly classified cases was significantly lower: 51 percent. These results establish that time- resolved fluorescence spectroscopy markedly improved on the performance of steady-state fluorescence spectroscopy for fine classification of atherosclerotic lesions.

  8. 2D fluorescence spectroscopy for monitoring ion-exchange membrane based technologies - Reverse electrodialysis (RED).

    PubMed

    Pawlowski, Sylwin; Galinha, Claudia F; Crespo, João G; Velizarov, Svetlozar

    2016-01-01

    Reverse electrodialysis (RED) is one of the emerging, membrane-based technologies for harvesting salinity gradient energy. In RED process, fouling is an undesirable operation constraint since it leads to a decrease of the obtainable net power density due to increasing stack electric resistance and pressure drop. Therefore, early fouling detection is one of the main challenges for successful RED technology implementation. In the present study, two-dimensional (2D) fluorescence spectroscopy was used, for the first time, as a tool for fouling monitoring in RED. Fluorescence excitation-emission matrices (EEMs) of ion-exchange membrane surfaces and of natural aqueous streams were acquired during one month of a RED stack operation. Fouling evolvement on the ion-exchange membrane surfaces was successfully followed by 2D fluorescence spectroscopy and quantified using principal components analysis (PCA). Additionally, the efficiency of cleaning strategy was assessed by measuring the membrane fluorescence emission intensity before and after cleaning. The anion-exchange membrane (AEM) surface in contact with river water showed to be significantly affected due to fouling by humic compounds, which were found to cross through the membrane from the lower salinity (river water) to higher salinity (sea water) stream. The results obtained show that the combined approach of using 2D fluorescence spectroscopy and PCA has a high potential for studying fouling development and membrane cleaning efficiency in ion exchange membrane processes.

  9. Studies of multifrequency phase-resolved fluorescence spectroscopy for spectral fingerprinting

    SciTech Connect

    McGown, L.B.

    1990-01-01

    During the past two project periods (7/1/88--12/31/90), we have made significant advances towards our goal of characterizing samples in terms of their dynamic spectral characteristics through the use of phase-resolved fluorescence spectroscopy. Specific achievements are discussed, each of which describes a particular area of focus in our studies.

  10. En route to traceable reference standards for surface group quantifications by XPS, NMR and fluorescence spectroscopy.

    PubMed

    Hennig, Andreas; Dietrich, Paul M; Hemmann, Felix; Thiele, Thomas; Borcherding, Heike; Hoffmann, Angelika; Schedler, Uwe; Jäger, Christian; Resch-Genger, Ute; Unger, Wolfgang E S

    2015-03-21

    The fluorine content of polymer particles labelled with 2,2,2-trifluoroethylamine was reliably quantified with overlapping sensitivity ranges by XPS and solid-state NMR. This provides a first step towards reference materials for the metrological traceability of surface group quantifications. The extension of this concept to fluorescence spectroscopy is illustrated.

  11. Fluorescence excitation and multiphoton ionization spectroscopy of 3-methylindole in a supersonic jet

    NASA Astrophysics Data System (ADS)

    Hays, T. R.; Henke, W. E.; Selzle, H. L.; Schlag, E. W.

    1983-05-01

    The fluorescence excitation and multiphoton ionization spectroscopy of 3-methylindole (skatole) is reported. One electronic origin ( 1L b) is assigned at 34875 cm -1, the second ( 1L a) suspected at 35483 cm -1. Some 1L b vibrational assignments are also made. Complex formation between skatole and some small molecules is indicated but not directly observed.

  12. Organ transplant tissue rejection: detection and staging by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    MacAulay, Calum E.; Whitehead, Peter D.; McManus, Bruce; Zeng, Haishan; Wilson-McManus, Janet; MacKinnon, Nick; Morgan, David C.; Dong, Chunming; Gerla, Paul; Kenyon, Jennifer

    1998-07-01

    Patients receiving heart or other organ transplants usually require some level of anti-rejection drug therapy, most commonly cyclosporine. The rejection status of the organ must be monitored to determine the optimal anti-rejection drug therapy. The current method for monitoring post-transplant rejection status of heart transplant patients consists of taking biopsies from the right ventricle. In this work we have developed a system employing optical and signal-processing techniques that will allow a cardiologist to measure spectral changes associated with tissue rejection using an optical catheter probe. The system employs time gated illumination and detection systems to deal with the dynamic signal acquisition problems associated with in vivo measurements of a beating heart. Spectral data processing software evaluates and processes the data to produce a simple numerical score. Results of measurements made on 100 excised transplanted isograft and allograft rat hearts have demonstrated the ability of the system to detect the presence of rejection and to accurately correlate the spectroscopic results with the ISHLT (International Society for Heart and Lung Transplantation) stage of rejection determined by histopathology. In vivo measurements using a pig transplant model are now in process.

  13. Fluorescence kinetics of Trp-Trp dipeptide and its derivatives in water via ultrafast fluorescence spectroscopy.

    PubMed

    Jia, Menghui; Yi, Hua; Chang, Mengfang; Cao, Xiaodan; Li, Lei; Zhou, Zhongneng; Pan, Haifeng; Chen, Yan; Zhang, Sanjun; Xu, Jianhua

    2015-08-01

    Ultrafast fluorescence dynamics of Tryptophan-Tryptophan (Trp-Trp/Trp2) dipeptide and its derivatives in water have been investigated using a picosecond resolved time correlated single photon counting (TCSPC) apparatus together with a femtosecond resolved upconversion spectrophotofluorometer. The fluorescence decay profiles at multiple wavelengths were fitted by a global analysis technique. Nanosecond fluorescence kinetics of Trp2, N-tert-butyl carbonyl oxygen-N'-aldehyde group-l-tryptophan-l-tryptophan (NBTrp2), l-tryptophan-l-tryptophan methyl ester (Trp2Me), and N-acetyl-l-tryptophan-l-tryptophan methyl ester (NATrp2Me) exhibit multi-exponential decays with the average lifetimes of 1.99, 3.04, 0.72 and 1.22ns, respectively. Due to the intramolecular interaction between two Trp residues, the "water relaxation" lifetime was observed around 4ps, and it is noticed that Trp2 and its derivatives also exhibit a new decay with a lifetime of ∼100ps, while single-Trp fluorescence decay in dipeptides/proteins shows 20-30ps. The intramolecular interaction lifetime constants of Trp2, NBTrp2, Trp2Me and NATrp2Me were then calculated to be 3.64, 0.93, 11.52 and 2.40ns, respectively. Candidate mechanisms (including heterogeneity, solvent relaxation, quasi static self-quenching or ET/PT quenching) have been discussed.

  14. Doppler-free Yb spectroscopy with the fluorescence spot technique

    SciTech Connect

    Nizamani, Altaf H.; McLoughlin, James J.; Hensinger, Winfried K.

    2010-10-15

    We demonstrate a simple technique to measure the resonant frequency of the 398.9-nm {sup 1}S{sub 0}{leftrightarrow}{sup 1}P{sub 1} transition for the different Yb isotopes. The technique, which works by observing and aligning fluorescence spots, has enabled us to measure transition frequencies and isotope shifts with an accuracy of 60 MHz. We provide wavelength measurements for the transition that differ from previously published work. Our technique also allows for the determination of Doppler-shifted transition frequencies for photoionization experiments when the atomic beam and the laser beam are not perpendicular and furthermore allows us to determine the average velocity of the atoms along the direction of the atomic beam.

  15. Gold nephropathy: tissue analysis by X-ray fluorescent spectroscopy.

    PubMed

    Viol, G W; Minielly, J A; Bistricki, T

    1977-12-01

    Three patients developed proteinuria following gold therapy for rheumatoid arthritis. The clinical syndrome was a self-limiting proteinuria with normal renal function. By light and electron microscopic appearances the renal lesion was an epimembranous deposit form of membranous glomerulopathy. Immunofluorescent study showed granular deposits of IgG and C3 complement along glomerular basement membranes. By X-ray fluorescent spectroscopic examination, gold was seen to be present within the proximal convoluted tubular cells but was not identified in the glomerular subepithelial deposits. These findings are consistent with an immune-complex form of glomerulopathy in which gold is neither the antigen nor a hapten in the glomerular deposits, and they suggest the hypothesis that antibodies to tubular epithelial antigens induced by gold therapy may be a causative factor in the renal disease associated with gold therapy in rheumatoid arthritis. PMID:412488

  16. Hyperspectral Imaging and Spectroscopy of Fluorescently Coupled Acyl-CoA: Cholesterol Acyltransferase in Insect Cells

    NASA Technical Reports Server (NTRS)

    Malak, H.; Mahtani, H.; Herman, P.; Vecer, J.; Lu, X.; Chang, T. Y.; Richmond, Robert C.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    A high-performance hyperspectral imaging module with high throughput of light suitable for low-intensity fluorescence microscopic imaging and subsequent analysis, including single-pixel-defined emission spectroscopy, was tested on Sf21 insect cells expressing green fluorescence associated with recombinant green fluorescent protein linked or not with the membrane protein acyl-CoA:cholesterol acyltransferase. The imager utilized the phenomenon of optical activity as a new technique providing information over a spectral range of 220-1400 nm, and was inserted between the microscope and an 8-bit CCD video-rate camera. The resulting fluorescence image did not introduce observable image aberrations. The images provided parallel acquisition of well resolved concurrent spatial and spectral information such that fluorescence associated with green fluorescent protein alone was demonstrated to be diffuse within the Sf21 insect cell, and that green fluorescence associated with the membrane protein was shown to be specifically concentrated within regions of the cell cytoplasm. Emission spectra analyzed from different regions of the fluorescence image showed blue shift specific for the regions of concentration associated with the membrane protein.

  17. Nonlinear Theory of Anomalous Diffusion and Application to Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Boon, Jean Pierre; Lutsko, James F.

    2015-12-01

    The nonlinear theory of anomalous diffusion is based on particle interactions giving an explicit microscopic description of diffusive processes leading to sub-, normal, or super-diffusion as a result of competitive effects between attractive and repulsive interactions. We present the explicit analytical solution to the nonlinear diffusion equation which we then use to compute the correlation function which is experimentally measured by correlation spectroscopy. The theoretical results are applicable in particular to the analysis of fluorescence correlation spectroscopy of marked molecules in biological systems. More specifically we consider the cases of fluorescently labeled lipids in the plasma membrane and of fluorescent apoferritin (a spherically shaped oligomer) in a crowded dextran solution and we find that the nonlinear correlation spectra reproduce very well the experimental data indicating sub-diffusive molecular motion.

  18. Fluorescence excitation-emission matrix spectroscopy as a tool for determining quality of sparkling wines.

    PubMed

    Elcoroaristizabal, Saioa; Callejón, Raquel M; Amigo, Jose M; Ocaña-González, Juan A; Morales, M Lourdes; Ubeda, Cristina

    2016-09-01

    Browning in sparkling wines was assessed by the use of excitation-emission fluorescence spectroscopy combined with PARAllel FACtor analysis (PARAFAC). Four different cava sparkling wines were monitored during an accelerated browning process and subsequently storage. Fluorescence changes observed during the accelerated browning process were monitored and compared with other conventional parameters: absorbance at 420nm (A420) and the content of 5-hydroxymethyl-2-furfural (5-HMF). A high similarity of the spectral profiles for all sparkling wines analyzed was observed, being explained by a four component PARAFAC model. A high correlation between the third PARAFAC factor (465/530nm) and the commonly used non-enzymatic browning indicators was observed. The fourth PARAFAC factor (280/380nm) gives us also information about the browning process following a first order kinetic reaction. Hence, excitation-emission fluorescence spectroscopy, together with PARAFAC, provides a faster alternative for browning monitoring to conventional methods, as well as useful key indicators for quality control.

  19. Fluorescence spectroscopy and molecular weight distribution of extracellular polymers from full-scale activated sludge biomass.

    PubMed

    Esparza-Soto, M; Westerhoff, P K

    2001-01-01

    Two fractions of extracellular polymer substances (EPSs), soluble and readily extractable (RE), were characterised in terms of their molecular weight distributions (MWD) and 3-D excitation-emission-matrix (EEM) fluorescence spectroscopy signatures. The EPS fractions were different: the soluble EPSs were composed mainly of high molecular weight compounds, while the RE EPSs were composed of small molecular weight compounds. Contrary to previous thought, EPS may not be considered only as macromolecular because most organic matter present in both fractions had low molecular weight. Three different fluorophore peaks were identified in the EEM fluorescence spectra. Two peaks were attributed to protein-like fluorophores, and the third to a humic-like fluorophore. Fluorescence signatures were different from other previously published signatures for marine and riverine environments. EEM spectroscopy proved to be a suitable method that may be used to characterise and trace organic matter of bacterial origin in wastewater treatment operations.

  20. High-throughput single-molecule fluorescence spectroscopy using parallel detection

    PubMed Central

    Michalet, X.; Colyer, R. A.; Scalia, G.; Kim, T.; Levi, Moran; Aharoni, Daniel; Cheng, Adrian; Guerrieri, F.; Arisaka, Katsushi; Millaud, Jacques; Rech, I.; Resnati, D.; Marangoni, S.; Gulinatti, A.; Ghioni, M.; Tisa, S.; Zappa, F.; Cova, S.; Weiss, S.

    2011-01-01

    Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. The basic concept of this technique is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements obtained with different novel multipixel single-photon counting detectors. PMID:21625288

  1. Fabrication of folate bioconjugated near-infrared fluorescent silver nanoclusters for targeted in vitro and in vivo bioimaging.

    PubMed

    Wang, Yong; Dai, Cong; Yan, Xiu-Ping

    2014-11-28

    Thiolpolyethyleneimine stabilized silver nanoclusters (SH-PEI-AgNCs) with intense NIR fluorescence and chemical stability were fabricated in aqueous solution. The SH-PEI-AgNCs were subsequently bioconjugated with folate for targeted in vitro and in vivo bioimaging.

  2. Characterising organic matter in recirculating aquaculture systems with fluorescence EEM spectroscopy.

    PubMed

    Hambly, A C; Arvin, E; Pedersen, L-F; Pedersen, P B; Seredyńska-Sobecka, B; Stedmon, C A

    2015-10-15

    The potential of recirculating aquaculture systems (RAS) in the aquaculture industry is increasingly being acknowledged. Along with intensified application, the need to better characterise and understand the accumulated dissolved organic matter (DOM) within these systems increases. Mature RASs, stocked with rainbow trout and operated at steady state at four feed loadings, were analysed by dissolved organic carbon (DOC) analysis and fluorescence excitation-emission matrix (EEM) spectroscopy. The fluorescence dataset was then decomposed by PARAFAC analysis using the drEEM toolbox. This revealed that the fluorescence character of the RAS water could be represented by five components, of which four have previously been identified in fresh water, coastal marine water, wetlands and drinking water. The fluorescence components as well as the DOC showed positive correlations with feed loading, however there was considerable variation between the five fluorescence components with respect to the degree of accumulation with feed loading. The five components were found to originate from three sources: the feed; the influent tap water (groundwater); and processes related to the fish and the water treatment system. This paper details the first application of fluorescence EEM spectroscopy to assess DOM in RAS, and highlights the potential applications of this technique within future RAS management strategies.

  3. Determination of the in vivo redox potential using roGFP and fluorescence spectra obtained from one-wavelength excitation

    NASA Astrophysics Data System (ADS)

    Wierer, S.; Elgass, K.; Bieker, S.; Zentgraf, U.; Meixner, A. J.; Schleifenbaum, F.

    2011-02-01

    The analysis of molecular processes in living (plant) cells such as signal transduction, DNA replication, carbon metabolism and senescence has been revolutionized by the use of green fluorescent protein (GFP) and its variants as specific cellular markers. Many cell biological processes are accompanied by changes in the intracellular redox potential. To monitor the redox potential, a redox-sensitive mutant of GFP (roGFP) was created, which shows changes in its optical properties in response to changes in the redox state of its surrounding medium. For a quantitative analysis in living systems, it is essential to know the optical properties of roGFP in vitro. Therefore, we applied spectrally resolved fluorescence spectroscopy on purified roGFP exposed to different redox potentials to determine shifts in both the absorption and the emission spectra of roGFP. Based on these in vitro findings, we introduce a new approach using one-wavelength excitation to use roGFP for the in vivo analysis of cell biological processes. We demonstrate the ability this technique by investigating chloroplast-located Grx1-roGFP2 expressing Arabidopsis thaliana cells as example for dynamically moving intracellular compartments. This is not possible with the two-wavelength excitation technique established so far, which hampers a quantitative analysis of highly mobile samples due to the time delay between the two measurements and the consequential displacement of the investigated area.

  4. Noise and artifact characterization of in vivo Raman spectroscopy skin measurements.

    PubMed

    Ramírez-Elías, Miguel G; Alda, Javier; González, Francisco J

    2012-06-01

    In this work principal component analysis (PCA), a multivariate pattern recognition technique, is used to characterize the noise contribution of the experimental apparatus and two commonly used methods for fluorescence removal used in biomedical Raman spectroscopy measurements. These two methods are a fifth degree polynomial fitting and an iterative variation of it commonly known as the Vancouver method. The results show that the noise in Raman spectroscopy measurements is related to the spectral resolution of the measurement equipment, the intrinsic variability of the biological measurements, and the fluorescence removal algorithm used.

  5. Fluorescence spectroscopy incorporating a ratiometric approach for the diagnosis and classification of urothelial carcinoma

    NASA Astrophysics Data System (ADS)

    Anand, Suresh; Cicchi, Riccardo; Crisci, Alfonso; Nesi, Gabriella; Carini, Marco; Pavone, Francesco S.

    2016-02-01

    The current most popular clinical method for the screening of urothelial carcinoma is white light cystoscopy. This method has inherent disadvantages making a strong genesis towards developing more powerful diagnostic techniques. Laser induced intrinsic fluorescence spectroscopy has been studied as an adjunct to current methods for the detection of tumors. This technique allows real time results based on the changes in spectral profile between normal and tumor tissues. We conducted a pilot study based on fluorescence spectroscopy at two wavelengths 378 and 445 nm excitation for the differentiation of urothelial carcinoma. At both the excitation wavelengths, the measured fluorescence signal showed an increased intensity at wavelengths greater than 520 nm. In addition, the emission profile showed modulation at 580 nm which is due to the reabsorption of emitted fluo- rescence due to hemoglobin. Additionally, we developed a tissue characterizing algorithm, based on fluorescence intensity ratios, F510/F600 and F520/F580 at 378 and 445 nm excitation wavelengths respectively. Further, the results were correlated with the pathologists assessment of urothelial carcinoma. This ratiometric classification algorithm yielded 81% sensitivity and 83% specificity at 378 nm and while at 445 nm excitation we achieved a sensitivity and specificity of 85% and 86% for classifying normal and tumor bladder tissues. In this study we have demonstrated the potential of a simple ratiometric algorithm based on fluorescence spectroscopy could be an alternative tool to tissue biopsy. Furthermore, this technique based fiber-based fluorescence spectroscopy could be integrated into an endoscopy system for use in the operating room.

  6. Wavefront sensorless approaches to adaptive optics for in vivo fluorescence imaging of mouse retina

    NASA Astrophysics Data System (ADS)

    Wahl, Daniel J.; Bonora, Stefano; Mata, Oscar S.; Haunerland, Bengt K.; Zawadzki, Robert J.; Sarunic, Marinko V.; Jian, Yifan

    2016-03-01

    Adaptive optics (AO) is necessary to correct aberrations when imaging the mouse eye with high numerical aperture. In order to obtain cellular resolution, we have implemented wavefront sensorless adaptive optics for in vivo fluorescence imaging of mouse retina. Our approach includes a lens-based system and MEMS deformable mirror for aberration correction. The AO system was constructed with a reflectance channel for structural images and fluorescence channel for functional images. The structural imaging was used in real-time for navigation on the retina using landmarks such as blood vessels. We have also implemented a tunable liquid lens to select the retinal layer of interest at which to perform the optimization. At the desired location on the mouse retina, the optimization algorithm used the fluorescence image data to drive a modal hill-climbing algorithm using an intensity or sharpness image quality metric. The optimization requires ~30 seconds to complete a search up to the 20th Zernike mode. In this report, we have demonstrated the AO performance for high-resolution images of the capillaries in a fluorescence angiography. We have also made progress on an approach to AO with pupil segmentation as a possible sensorless technique suitable for small animal retinal imaging. Pupil segmentation AO was implemented on the same ophthalmic system and imaging performance was demonstrated on fluorescent beads with induced aberrations.

  7. Sensitive β-galactosidase-targeting fluorescence probe for visualizing small peritoneal metastatic tumours in vivo.

    PubMed

    Asanuma, Daisuke; Sakabe, Masayo; Kamiya, Mako; Yamamoto, Kyoko; Hiratake, Jun; Ogawa, Mikako; Kosaka, Nobuyuki; Choyke, Peter L; Nagano, Tetsuo; Kobayashi, Hisataka; Urano, Yasuteru

    2015-01-01

    Fluorescence-guided diagnostics is one of the most promising approaches for facile detection of cancer in situ. Here we focus on β-galactosidase, which is overexpressed in primary ovarian cancers, as a molecular target for visualizing peritoneal metastases from ovarian cancers. As existing fluorescence probes are unsuitable, we have designed membrane-permeable HMRef-βGal, in which the optimized intramolecular spirocyclic function affords >1,400-fold fluorescence enhancement on activation. We confirm that HMRef-βGal sensitively detects intracellular β-galactosidase activity in several ovarian cancer lines. In vivo, this probe visualizes metastases as small as <1 mm in diameter in seven mouse models of disseminated human peritoneal ovarian cancer (SHIN3, SKOV3, OVK18, OVCAR3, OVCAR4, OVCAR5 and OVCAR8). Because of its high brightness, real-time detection of metastases with the naked eye is possible. Endoscopic fluorescence detection of metastases is also demonstrated. The results clearly indicate preclinical potential value of the probe for fluorescence-guided diagnosis of peritoneal metastases from ovarian cancers.

  8. Measurement of diffusion of fluorescent compounds and autofluorescence in skin in vivo using a confocal instrument

    NASA Astrophysics Data System (ADS)

    Buttenschoen, K. K.; Sutton, E. E.; Daly, D.; Girkin, J. M.

    2016-02-01

    Using compact and affordable instrumentation based upon fluorescent confocal imaging we have tracked the movement of autofluorescent compounds through skin in near real time with high temporal and spatial resolution and sensitivity. The ability to measure the diffusion of compounds through skin with such resolution plays an important role for applications such as monitoring the penetration of pharmaceuticals applied to skin and assessing the integrity of the skin barrier. Several measurement methods exist, but they suffer from a number of problems such as being slow, expensive, non-portable and lacking sensitivity. To address these issues, we adapted a technique that we previously developed for tracking fluorescent compounds in the eye to measure the autofluorescence and the diffusion of externally applied fluorescent compounds in skin in vivo. Results are presented that show the change in autofluorescence of the volar forearm over the course of a week. We furthermore demonstrate the ability of the instrument to measure the diffusion speed and depth of externally applied fluorescent compounds both in healthy skin and after the skin barrier function has been perturbed. The instrument is currently being developed further for increased sensitivity and multi-wavelength excitation. We believe that the presented instrument is suitable for a large number of applications in fields such as assessment of damage to the skin barrier, development of topical and systemic medication and tracking the diffusion of fluorescent compounds through skin constructs as well as monitoring effects of skin products and general consumer products which may come into contact with the skin.

  9. Fluorescence spectra of blood plasma treated with ultraviolet irradiation in vivo

    NASA Astrophysics Data System (ADS)

    Zalesskaya, G. A.; Maslova, T. O.

    2010-09-01

    We have studied the fluorescence spectra of blood plasma from patients with acute coronary syndrome, and also the effect of therapeutic doses of in vivo ultraviolet blood irradiation (UBI) on the spectra. We have established that the maxima in the fluorescence spectra of the original plasma samples, obtained from unirradiated blood, are located in the wavelength interval 330-340 nm, characteristic for the fluorescence of tryptophan residues. In extracorporeal UBI ( λ = 254 nm), we observed changes in the shape and also both a blue and a red shift in the maxima of the fluorescence spectra, differing in magnitude for blood plasma samples from different patients in the test group. We show that UBI-initiated changes in the fluorescence spectra of the plasma depend on the original pathological disturbances of metabolite levels, and also on the change in the oxygen-transport function of the blood and the acid-base balance, affecting the oxidative stability of the plasma. We have concluded that UV irradiation, activating buffer systems in the blood, has an effect on the universal and specific interactions of the tryptophan residue with the amino acid residues and water surrounding it.

  10. Human in vivo fluorescence microimaging of the alveolar ducts and sacs during bronchoscopy.

    PubMed

    Thiberville, L; Salaün, M; Lachkar, S; Dominique, S; Moreno-Swirc, S; Vever-Bizet, C; Bourg-Heckly, G

    2009-05-01

    The aim of the present study was to assess fibred confocal fluorescence microscopy (FCFM) as a tool for imaging the alveolar respiratory system in vivo during bronchoscopy. A 488-nm excitation wavelength FCFM device was used in 41 healthy subjects including 17 active smokers. After topical anaesthesia, the 1.4-mm miniprobe was introduced into the bronchoscope working channel and advanced distally to the alveoli. Morphometric and cellular analyses were performed on selected frames harbouring a minimal compression effect. In vivo acinar microimaging was obtained from each lung segment except for the apical and posterior segments of both upper lobes. Reproducible patterns, corresponding to the elastic framework of the axial and peripheral interstitial systems, were recorded from 192 separate acini. The mean+/-sd thickness of the acinar elastic fibres was 10+/-2.7 microm. Alveolar mouth diameters (mean+/-sd 278+/-53 microm) were normally distributed but appeared smaller in the right upper lobe and right medial basal segment. Lobular microvessels (median diameter 90 microm) were equally distributed throughout the lungs. Alveolar macrophages were not detectable in nonsmokers, whereas a specific tobacco-tar-induced fluorescence was observed in smoking subjects, providing fine details of the alveolar walls and macrophages. A strong correlation was found between the number of cigarettes smoked per day and the amount of large and mobile macrophages observed in vivo, as well as with the intensity of the macrophage alveolitis. Fibred confocal fluorescence microscopy enables accurate exploration of the peripheral lung in vivo in both smokers and nonsmokers. PMID:19213792

  11. Improved Diffuse Fluorescence Flow Cytometer Prototype for High Sensitivity Detection of Rare Circulating Cells In Vivo

    NASA Astrophysics Data System (ADS)

    Pestana, Noah Benjamin

    Accurate quantification of circulating cell populations is important in many areas of pre-clinical and clinical biomedical research, for example, in the study of cancer metastasis or the immune response following tissue and organ transplants. Normally this is done "ex-vivo" by drawing and purifying a small volume of blood and then analyzing it with flow cytometry, hemocytometry or microfludic devices, but the sensitivity of these techniques are poor and the process of handling samples has been shown to affect cell viability and behavior. More recently "in vivo flow cytometry" (IVFC) techniques have been developed where fluorescently-labeled cells flowing in a small blood vessel in the ear or retina are analyzed, but the sensitivity is generally poor due to the small sampling volume. To address this, our group recently developed a method known as "Diffuse Fluorescence Flow Cytometry" (DFFC) that allows detection and counting of rare circulating cells with diffuse photons, offering extremely high single cell counting sensitivity. In this thesis, an improved DFFC prototype was designed and validated. The chief improvements were three-fold, i) improved optical collection efficiency, ii) improved detection electronics, and iii) development of a method to mitigate motion artifacts during in vivo measurements. In combination, these improvements yielded an overall instrument detection sensitivity better than 1 cell/mL in vivo, which is the most sensitive IVFC system reported to date. Second, development and validation of a low-cost microfluidic device reader for analysis of ocular fluids is described. We demonstrate that this device has equivalent or better sensitivity and accuracy compared a fluorescence microscope, but at an order-of-magnitude reduced cost with simplified operation. Future improvements to both instruments are also discussed.

  12. Multicolored two-photon fluorescent microscopy and localized two-photon fluorescent spectroscopy in living cells

    NASA Astrophysics Data System (ADS)

    Bergey, Earl J.; Wang, Xiaopeng; Krebs, Linda J.; Pudavar, Haridas E.; Kapoor, Rakesh; Friend, Christopher S.; Liebow, Charles; Prasad, Paras N.

    2001-04-01

    Imaging in biological systems has become one of the most relied upon tools in the study of human disease. Two-photon excitation methodology in laser scanning microscopy has resulted in 3D-imaging capability not easily achieved in one- photon systems. Our Institute, in conjunction with Andrew Schally (Noble Laureate, Tulane University), has used two- photon laser scanning microscopy (TPLSM) to understand the real time cellular transport of the chemotherapeutic agent, Luteinizing Hormone-Releasing Hormone-Doxorubicin (AN152) covalently coupled to a novel two-photon fluorophore (C625). At the Institute, new and highly efficient two-photon fluorophores that fluoresce at different wavelengths have been developed. The coupling of LH-RH and AN152 with two-photon fluorophores having different spectroscopic profiles allows for the simultaneous determination of their cellular compartmentalization. Coupled with the two-photon microspectrofluorometer, we acquired localized fluorescence spectra from the inside of living cells to differentiate the cytoplasmic and nuclear localization of the LH-RH and AN152 respectively. The ability of these new dyes to fluoresce at different wavelengths using the same excitation wavelength provides a major advantage over single photon dyes. This technology has great promise in imaging the dynamic changes or events occurring in living cells over short periods of time. Another approach to bioimaging at the Institute is the integration of two-photon and nanosized technologies. Nanoclinics (20 - 30 nm silica bubbles) can be fabricated to contain these two photon fluorophores and the surface functionalized with biological agents which can target specific cells. These highly fluorescent nanoclinics are sufficiently small in size to allow for tissue penetration, allowing for the multiple probing for different cellular functions in normal and cancerous tissues.

  13. Probing the extracellular diffusion of antibodies in brain using in vivo integrative optical imaging and ex vivo fluorescence imaging.

    PubMed

    Wolak, Daniel J; Pizzo, Michelle E; Thorne, Robert G

    2015-01-10

    Antibody-based therapeutics exhibit great promise in the treatment of central nervous system (CNS) disorders given their unique customizable properties. Although several clinical trials have evaluated therapeutic antibodies for treatment of CNS disorders, success to date has likely been limited in part due to complex issues associated with antibody delivery to the brain and antibody distribution within the CNS compartment. Major obstacles to effective CNS delivery of full length immunoglobulin G (IgG) antibodies include transport across the blood-brain and blood-cerebrospinal fluid barriers. IgG diffusion within brain extracellular space (ECS) may also play a role in limiting central antibody distribution; however, IgG transport in brain ECS has not yet been explored using established in vivo methods. Here, we used real-time integrative optical imaging to measure the diffusion properties of fluorescently labeled, non-targeted IgG after pressure injection in both free solution and in adult rat neocortex in vivo, revealing IgG diffusion in free medium is ~10-fold greater than in brain ECS. The pronounced hindered diffusion of IgG in brain ECS is likely due to a number of general factors associated with the brain microenvironment (e.g. ECS volume fraction and geometry/width) but also molecule-specific factors such as IgG size, shape, charge and specific binding interactions with ECS components. Co-injection of labeled IgG with an excess of unlabeled Fc fragment yielded a small yet significant increase in the IgG effective diffusion coefficient in brain, suggesting that binding between the IgG Fc domain and endogenous Fc-specific receptors may contribute to the hindered mobility of IgG in brain ECS. Importantly, local IgG diffusion coefficients from integrative optical imaging were similar to those obtained from ex vivo fluorescence imaging of transport gradients across the pial brain surface following controlled intracisternal infusions in anesthetized animals. Taken

  14. Laser-induced fluorescence-cued, laser-induced breakdown spectroscopy biological-agent detection

    SciTech Connect

    Hybl, John D.; Tysk, Shane M.; Berry, Shaun R.; Jordan, Michael P

    2006-12-01

    Methods for accurately characterizing aerosols are required for detecting biological warfare agents. Currently, fluorescence-based biological agent sensors provide adequate detection sensitivity but suffer from high false-alarm rates. Combining single-particle fluorescence analysis with laser-induced breakdown spectroscopy (LIBS) provides additional discrimination and potentially reduces false-alarm rates. A transportable UV laser-induced fluorescence-cued LIBS test bed has been developed and used to evaluate the utility of LIBS for biological-agent detection. Analysis of these data indicates that LIBS adds discrimination capability to fluorescence-based biological-agent detectors.However, the data also show that LIBS signatures of biological agent simulants are affected by washing. This may limit the specificity of LIBS and narrow the scope of its applicability in biological-agent detection.

  15. Laser-induced fluorescence-cued, laser-induced breakdown spectroscopy biological-agent detection.

    PubMed

    Hybl, John D; Tysk, Shane M; Berry, Shaun R; Jordan, Michael P

    2006-12-01

    Methods for accurately characterizing aerosols are required for detecting biological warfare agents. Currently, fluorescence-based biological agent sensors provide adequate detection sensitivity but suffer from high false-alarm rates. Combining single-particle fluorescence analysis with laser-induced breakdown spectroscopy (LIBS) provides additional discrimination and potentially reduces false-alarm rates. A transportable UV laser-induced fluorescence-cued LIBS test bed has been developed and used to evaluate the utility of LIBS for biological-agent detection. Analysis of these data indicates that LIBS adds discrimination capability to fluorescence-based biological-agent detectors. However, the data also show that LIBS signatures of biological agent simulants are affected by washing. This may limit the specificity of LIBS and narrow the scope of its applicability in biological-agent detection.

  16. In vivo targeting of colonic dysplasia on fluorescence endoscopy with near-infrared octapeptide

    PubMed Central

    Liu, Zhongyao; Miller, Sharon J.; Joshi, Bishnu P.; Wang, Thomas D.

    2012-01-01

    Objective To demonstrate a near-infrared peptide that is highly specific for colonic adenomas on fluorescence endoscopy in vivo. Design A 3 mm diameter endoscope was adapted to deliver 671 nm illumination and collect NIR fluorescence (696–736 nm). Target (QPIHPNNM) and control (YTTNKH) peptides were labeled with Cy5.5, a near-infrared dye, and characterized by mass spectra. The peptides were topically administered separately (100 µM) through the endoscope’s instrument channel into the distal colon of CPC;Apc mice, genetically-engineered to spontaneously develop adenomas. After 5 minutes for incubation, the unbound peptides were rinsed off, and images were collected at a rate of 10 per second. Regions-of-interest were identified around the adenoma and adjacent normal-appearing mucosa on white light. Intensity measurements were made from these same regions on fluorescence, and the target-to-background ratio (TBR) was calculated. Results We achieved an image resolution of 9.8 µm and field-of-view of 3.6 mm at a distance of 2.5 mm between the distal end of the instrument and the tissue surface. On mass spectra, the experimental mass-to-charge ratio for the Cy5.5-labeled target and control peptides agreed with expected values. The near-infrared fluorescence images of adenomas revealed individual dysplastic crypts with distorted morphology. By comparison, only amorphous surface features could be visualized from reflected near-infrared light. The average TBR for adenomas was found to be 3.42±1.30 and 1.88±0.38 for the target and control peptides, respectively, p=0.007. Conclusion We demonstrate a near-infrared peptide that is highly specific for colonic adenomas on fluorescence endoscopy in vivo and achieved sub-cellular resolution images. PMID:22427239

  17. Rapid screening test for porphyria diagnosis using fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Lang, A.; Stepp, H.; Homann, C.; Hennig, G.; Brittenham, G. M.; Vogeser, M.

    2015-07-01

    Porphyrias are rare genetic metabolic disorders, which result from deficiencies of enzymes in the heme biosynthesis pathway. Depending on the enzyme defect, different types of porphyrins and heme precursors accumulate for the different porphyria diseases in erythrocytes, liver, blood plasma, urine and stool. Patients with acute hepatic porphyrias can suffer from acute neuropathic attacks, which can lead to death when undiagnosed, but show only unspecific clinical symptoms such as abdominal pain. Therefore, in addition to chromatographic methods, a rapid screening test is required to allow for immediate identification and treatment of these patients. In this study, fluorescence spectroscopic measurements were conducted on blood plasma and phantom material, mimicking the composition of blood plasma of porphyria patients. Hydrochloric acid was used to differentiate the occurring porphyrins (uroporphyrin-III and coproporphyrin-III) spectroscopically despite their initially overlapping excitation spectra. Plasma phantom mixtures were measured using dual wavelength excitation and the corresponding concentrations of uroporphyrin-III and coproporphyrin-III were determined. Additionally, three plasma samples of porphyria patients were examined and traces of coproporphyrin-III and uroporphyrin-III were identified. This study may therefore help to establish a rapid screening test method with spectroscopic differentiation of the occurring porphyrins, which consequently allows for the distinction of different porphyrias. This may be a valuable tool for clinical porphyria diagnosis and rapid or immediate treatment.

  18. Longitudinal in vivo imaging of bone formation and resorption using fluorescence molecular tomography.

    PubMed

    Lambers, F M; Stuker, F; Weigt, C; Kuhn, G; Koch, K; Schulte, F A; Ripoll, J; Rudin, M; Müller, R

    2013-02-01

    Bone research often focuses on anatomical imaging of the bone microstructure, but in order to gain better understanding in how bone remodeling is modulated through interventions also bone formation and resorption processes should be investigated. With this in mind, the purpose of this study was to establish a longitudinal in vivo imaging approach of bone formation and resorption using fluorescence molecular tomography (FMT). In this study the reproducibility, accuracy and sensitivity of FMT for bone imaging were assessed by performing longitudinal measurements with FMT and comparing it to in vivo micro-computed tomography on a set of control mice, and mice in which load-adaptation was induced in the sixth caudal vertebra. The precision error for FMT measurements, expressed as coefficient of variation, was smaller than 16%, indicating acceptable reproducibility. A correlation was found between bone resorption measured with FMT and bone resorption rate measured with in vivo micro-computed tomography only over the first 14days (R=0.81, p<0.01), but not between bone formation measured with FMT and bone formation rate measured with in vivo micro-CT. Bone formation measured by FMT was 89-109% greater (p<0.05) for mice subjected to mechanical loading than control mice. Bone resorption was 5-8% lower, but did not reach a significant difference between groups, indicating moderate sensitivity for FMT. In conclusion, in vivo FMT in mouse tail bones is feasible but needs to be optimized for monitoring load adaptation in living mice.

  19. An (125)I-labeled octavalent peptide fluorescent nanoprobe for tumor-homing imaging in vivo.

    PubMed

    Luo, Haiming; Shi, Jiyun; Jin, Honglin; Fan, Di; Lu, Lisen; Wang, Fan; Zhang, Zhihong

    2012-06-01

    Targeting radiopeptides are promising agents for radio-theranostics. However, in vivo evaluation of their targeting specificity is often obscured by their short biologic half-lives and low binding affinities. Here, we report an approach to efficiently examine targeting radiopeptides with a new class of octavalent peptide fluorescent nanoprobe (Octa-FNP) platform, which is composed of candidate targeting peptides and a tetrameric far-red fluorescent protein (tfRFP) scaffold. To shed light on this process, (125)I-Octa-FNP, (125)I-tfRFP and (125)I-peptide were synthesized, and their targeting functionalities were compared. Both fluorescence imaging and radioactive quantification results confirmed that (125)I-Octa-FNP had a significantly higher cellular binding capability than (125)I-tfRFP. In vivo biodistribution studies show that at 6 h post-injection, (125)I-Octa-FNP had 2-fold and 30-fold higher tumor uptake than that of (125)I-tfRFP and (125)I-peptide, respectively. Moreover, γ-imaging at 24 h post-injection revealed a remarkable accumulation of (125)I-Octa-FNP in the tumor while maintaining an extremely low background contrast, which was further confirmed by immunofluorescence analysis. These data suggested that, as an engineered and multivalent platform, Octa-FNP could enhance the tumor targeting of a designed peptide and provide excellent contrast radioimaging, making it a valuable tool for the evaluation of the targeting ability of specifically designed radiopeptides for cancer theranostics.

  20. Probing Ternary Complex Equilibria of Crown Ether Ligands by Time-Resolved Fluorescence Spectroscopy

    PubMed Central

    2015-01-01

    Ternary complex formation with solvent molecules and other adventitious ligands may compromise the performance of metal-ion-selective fluorescent probes. As Ca(II) can accommodate more than 6 donors in the first coordination sphere, commonly used crown ether ligands are prone to ternary complex formation with this cation. The steric strain imposed by auxiliary ligands, however, may result in an ensemble of rapidly equilibrating coordination species with varying degrees of interaction between the cation and the specific donor atoms mediating the fluorescence response, thus diminishing the change in fluorescence properties upon Ca(II) binding. To explore the influence of ligand architecture on these equilibria, we tethered two structurally distinct aza-15-crown-5 ligands to pyrazoline fluorophores as reporters. Due to ultrafast photoinduced electron-transfer (PET) quenching of the fluorophore by the ligand moiety, the fluorescence decay profile directly reflects the species composition in the ground state. By adjusting the PET driving force through electronic tuning of the pyrazoline fluorophores, we were able to differentiate between species with only subtle variations in PET donor abilities. Concluding from a global analysis of the corresponding fluorescence decay profiles, the coordination species composition was indeed strongly dependent on the ligand architecture. Altogether, the combination of time-resolved fluorescence spectroscopy with selective tuning of the PET driving force represents an effective analytical tool to study dynamic coordination equilibria and thus to optimize ligand architectures for the design of high-contrast cation-responsive fluorescence switches. PMID:25313708

  1. Fluorescence lifetime spectroscopy in multiple-scattering environments: an application to biotechnology

    NASA Astrophysics Data System (ADS)

    Cerussi, Albert E.; Gratton, Enrico; Fantini, Sergio

    1999-07-01

    Over the past few years, there has been significant research activity devoted to the application of fluorescence spectroscopy to strongly scattering media, where photons propagate diffusely. Much of this activity focused on fluorescence as a source of contrast enhancement in optical tomography. Our efforts have emphasized the quantitative recovery of fluorescence parameters for spectroscopy. Using a frequency-domain diffusion-based model, we have successfully recovered the lifetime, the absolute quantum yield, the fluorophore concentration, and the emission spectrum of the fluorophore, as well as the absorption and the reduced scattering coefficients at the emission wavelength of the medium in different measurements. In this contribution, we present a sensitive monitor of the binding between ethidium bromide and bovine cells in fresh milk. The spectroscopic contrast was the approximately tenfold increase in the ethidium bromide lifetime upon binding to DNA. The measurement clearly demonstrated that we could quantitatively measure the density of cells in the milk, which is an application vital to the tremendous economic burden of bovine subclinical mastitis detection. Furthermore, we may in principle use the spirit of this technique as a quantitative monitor of the binding of fluorescent drugs inside tissues. This is a first step towards lifetime spectroscopy in tissues.

  2. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds.

    PubMed

    Holt, David; Parthasarathy, Ashwin B; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G; Singhal, Sunil

    2015-07-01

    Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ∼16 . Ten animals showed no residual tumor cells in the wound bed (mean SBR<2 , P<0.001 ). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15 , and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

  3. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds

    NASA Astrophysics Data System (ADS)

    Holt, David; Parthasarathy, Ashwin B.; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G.; Singhal, Sunil

    2015-07-01

    Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ˜16. Ten animals showed no residual tumor cells in the wound bed (mean SBR<2, P<0.001). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

  4. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds

    PubMed Central

    Holt, David; Parthasarathy, Ashwin B.; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G.; Singhal, Sunil

    2015-01-01

    Abstract. Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ∼16. Ten animals showed no residual tumor cells in the wound bed (mean SBR<2, P<0.001). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations. PMID:26160347

  5. The Monte Carlo modelling of in vivo x-ray fluorescence measurement of lead in tissue.

    PubMed

    Wallace, J D

    1994-10-01

    A Monte Carlo model has been developed, using the EGS4 code, to model the in vivo x-ray fluorescence (XRF) measurement of Pb in non-superficial bone/tissue. Unlike previous work in this field the current model incorporates a correction for Doppler broadening of the Compton scatter peak due to the electron momentum distribution of the medium (tissue/water) in which the photons are Compton scattered by convolving the Compton peak of the Monte Carlo generated spectrum with a modified Compton profile for water. This correction improves the agreement between the measured spectral shape obtained using an experimental in vivo x-ray fluorescence Pb analyser with a 109Cd/180 degrees source/geometry combination, measuring a bone phantom at depth in water and the generated spectral shape obtained from the equivalent Monte Carlo model. The model enables improved estimates to be made of the spectral background beneath the Pb Kalpha1 and Kalpha2 x-ray peaks compared with estimates based on simpler models that assume that Compton interactions are with 'free' electrons and hence permits better optimization of in vivo analyser system design. PMID:15551542

  6. Near-infrared fluorescence imaging platform for quantifying in vivo nanoparticle diffusion from drug loaded implants

    PubMed Central

    Markovic, Stacey; Belz, Jodi; Kumar, Rajiv; Cormack, Robert A; Sridhar, Srinivas; Niedre, Mark

    2016-01-01

    Drug loaded implants are a new, versatile technology platform to deliver a localized payload of drugs for various disease models. One example is the implantable nanoplatform for chemo-radiation therapy where inert brachytherapy spacers are replaced by spacers doped with nanoparticles (NPs) loaded with chemotherapeutics and placed directly at the disease site for long-term localized drug delivery. However, it is difficult to directly validate and optimize the diffusion of these doped NPs in in vivo systems. To better study this drug release and diffusion, we developed a custom macroscopic fluorescence imaging system to visualize and quantify fluorescent NP diffusion from spacers in vivo. To validate the platform, we studied the release of free fluorophores, and 30 nm and 200 nm NPs conjugated with the same fluorophores as a model drug, in agar gel phantoms in vitro and in mice in vivo. Our data verified that the diffusion volume was NP size-dependent in all cases. Our near-infrared imaging system provides a method by which NP diffusion from implantable nanoplatform for chemo-radiation therapy spacers can be systematically optimized (eg, particle size or charge) thereby improving treatment efficacy of the platform. PMID:27069363

  7. Assessing the photoaging process at sun exposed and non-exposed skin using fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Kurachi, Cristina

    2016-03-01

    Photoaging is the skin premature aging due to exposure to ultraviolet light, which damage the collagen, elastin and can induce alterations on the skin cells DNA, and, then, it may evolve to precancerous lesions, which are widely investigated by fluorescence spectroscopy and lifetime. The fluorescence spectra and fluorescence lifetime analysis has been presented as a technique of great potential for biological tissue characterization at optical diagnostics. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and may contribute to a non-invasive clinical investigation of injuries such as skin lesions. These lesions and the possible areas where they may develop can be interrogated using fluorescence lifetime spectroscopy taking into account the variability of skin phototypes and the changes related to melanin, collagen and elastin, endogenous fluorophores which have emissions that spectrally overlap to the NADH and FAD emission. The objective of this study is to assess the variation on fluorescence lifetimes of normal skin at sun exposed and non-exposed areas and associate this variation to the photoaging process.

  8. Towards in situ fluorescence spectroscopy and microscopy investigations of asphaltene precipitation kinetics.

    PubMed

    Franco, Juliana C; Gonçalves, Grasiele; Souza, Monique S; Rosa, Samantha B C; Thiegue, Larissa M; Atvars, Teresa D Z; Rosa, Paulo T V; Nome, René A

    2013-12-16

    We perform a spectroscopic analysis of asphaltene in solution and in crude oil with the goal of designing an optical probe of asphaltene precipitation inside high-pressure cells. Quantitative analysis of steady-state spectroscopic data is employed to identify fluorescence and Raman contributions to the observed signals. Time-resolved fluorescence spectroscopy indicates that fluorescence lifetime can be used as a spectroscopic probe of asphaltene in crude oil. Quantitative confocal laser-scanning microscopy studies of asphaltene in n-heptane are used to calculate particle-size distributions as a function of time, both at the sample surface and asphaltene interior. The resulting precipitation kinetics is well described by stochastic numerical simulations of diffusion-limited aggregation. Based on these results, we present the design and construction of an apparatus to optically probe the in situ precipitation of asphaltene suitable for studies inside high pressure cells. Design considerations include the use of a spatial light modulator for aberration correction in microscopy measurements, together with the design of epi-fluorescence spectrometer, both fiber-based and for remote sensing fluorescence spectroscopy. PMID:24514660

  9. Applicability of Fluorescence and Absorbance Spectroscopy to Estimate Organic Pollution in Rivers

    PubMed Central

    Knapik, Heloise Garcia; Fernandes, Cristovão Vicente Scapulatempo; de Azevedo, Júlio Cesar Rodrigues; do Amaral Porto, Monica Ferreira

    2014-01-01

    Abstract This article explores the applicability of fluorescence and absorbance spectroscopy for estimating organic pollution in polluted rivers. The relationship between absorbance, fluorescence intensity, dissolved organic carbon, biochemical oxygen demand (BOD), chemical oxygen demand (COD), and other water quality parameters were used to characterize and identify the origin and the spatial variability of the organic pollution in a highly polluted watershed. Analyses were performed for the Iguassu River, located in southern Brazil, with area about 2,700 km2 and ∼3 million inhabitants. Samples were collect at six monitoring sites covering 107 km of the main river. BOD, COD, nitrogen, and phosphorus concentration indicates a high input of sewage to the river. Specific absorbance at 254 and 285 nm (SUVA254 and A285/COD) did not show significant variation between sites monitored, indicating the presence of both dissolved compounds found in domestic effluents and humic and fulvic compounds derived from allochthonous organic matter. Correlations between BOD and tryptophan-like fluorescence peak (peak T2, r=0.7560, and peak T1, r=0.6949) and tyrosine-like fluorescence peak (peak B, r=0.7321) indicated the presence of labile organic matter and thus confirmed the presence of sewage in the river. Results showed that fluorescence and absorbance spectroscopy provide useful information on pollution in rivers from critical watersheds and together are a robust method that is simpler and more rapid than traditional methods employed by regulatory agencies. PMID:25469076

  10. Optical fluorescence spectroscopy to detect hepatic necrosis after normothermic ischemia: animal model

    NASA Astrophysics Data System (ADS)

    Romano, Renan A.; Vollet-Filho, Jose D.; Pratavieira, Sebastião.; Fernandez, Jorge L.; Kurachi, Cristina; Bagnato, Vanderlei S.; Castro-e-Silva, Orlando; Sankarankutty, Ajith K.

    2015-06-01

    Liver transplantation is a well-established treatment for liver failure. However, the success of the transplantation procedure depends on liver graft conditions. The tissue function evaluation during the several transplantation stages is relevant, in particular during the organ harvesting, when a decision is made concerning the viability of the graft. Optical fluorescence spectroscopy is a good option because it is a noninvasive and fast technique. A partial normothermic hepatic ischemia was performed in rat livers, with a vascular occlusion of both median and left lateral lobes, allowing circulation only for the right lateral lobe and the caudate lobe. Fluorescence spectra under excitation at 532 nm (doubled frequency Nd:YAG laser) were collected using a portable spectrometer (USB2000, Ocean Optics, USA). The fluorescence emission was collected before vascular occlusion, after ischemia, and 24 hours after reperfusion. A morphometric histology analysis was performed as the gold standard evaluation - liver samples were analyzed, and the percentage of necrotic tissue was obtained. The results showed that changes in the fluorescence emission after ischemia can be correlated with the amount of necrosis evaluated by a morphometric analysis, the Pearson correlation coefficient of the generated model was 0.90 and the root mean square error was around 20%. In this context, the laser-induced fluorescence spectroscopy technique after normothermic ischemia showed to be a fast and efficient method to differentiate ischemic injury from viable tissues.

  11. [Study on determination of carbaryl content in duck meat based on synchronous fluorescence spectroscopy].

    PubMed

    Xiao, Hai-Bin; Liu, Mu-Hua; Yuan, Hai-Chao; Xu, Jiang; Zhao, Jin-Hui

    2012-11-01

    For the rapid detection of carbaryl residue in duck meat, synchronous fluorescence spectroscopy was used, and GA combined with SVR was used to establish regression forecasting mode for the application of forecasting carbaryl residue in duck meat. Firstly, fluorescence spectrophotometer was used to get the 3D synchronous fluorescence spectra of carbaryl hydrolysate and duck solution containing carbaryl, and 140 nm was selected as the optimum wavelength difference delta lambda; Secondly, some concentration quenching was analysed. Finally, GA was used to optimize and choose the 3D synchronous fluorescence spectra. According to the root mean square error of cross-validation (RMSECV) 21 characteristic wavelengths were chosen, then the full wavelength and 21 characteristic wavelengths were used as input characteristic variables of SVR regression forecasting model respectively. At last the results showed that characteristic wavelengths chosen by GA can get better forecasting results, and the correlation coefficient of the prediction samples set and the root mean squared error (RMSEP) were 0.976 4 and 12.232 2, respectively. The results of experiments showed that the synchronous fluorescence spectroscopy could be used to detect carbaryl residue in duck meat efficiently and rapidly when combined with GA-SVR.

  12. In vivo soft tissue differentiation by diffuse reflectance spectroscopy: preliminary results

    NASA Astrophysics Data System (ADS)

    Zam, Azhar; Stelzle, Florian; Tangermann-Gerk, Katja; Adler, Werner; Nkenke, Emeka; Neukam, Friedrich Wilhelm; Schmidt, Michael; Douplik, Alexandre

    Remote laser surgery does not provide haptic feedback to operate layer by layer and preserve vulnerable anatomical structures like nerve tissue or blood vessels. The aim of this study is identification of soft tissue in vivo by diffuse reflectance spectroscopy to set the base for a feedback control system to enhance nerve preservation in oral and maxillofacial laser surgery. Various soft tissues can be identified by diffuse reflectance spectroscopy in vivo. The results may set the base for a feedback system to prevent nerve damage during oral and maxillofacial laser surgery.

  13. In vivo fluorescence and photodynamic activity of zinc phthalocyanine administered in liposomes.

    PubMed

    van Leengoed, H L; Cuomo, V; Versteeg, A A; van der Veen, N; Jori, G; Star, W M

    1994-05-01

    Zinc(II) phthalocyanine, a hydrophobic photosensitiser, was incorporated in unilamellar liposomes and studied in vivo for fluorescence kinetics and photodynamic activity. An observation chamber mounted in a dorsal skinfold of female WAG/Rij rats was used as a model system. In the chamber, an isogeneic mammary carcinoma was transplanted in the subcutaneous tissue. Phthalocyanine fluorescence was excited at 610 nm with a power density of 0.25 mW cm-2 and was detected above 665 nm through a high-pass filter using a two-stage image intensifier coupled to a charge-coupled device (CCD) camera. Following i.v. administration of 0.14 mg kg-1 of the drug, the fluorescence pharmacokinetics of the dye in vasculature, normal tissue and tumour tissue was determined as a function of time. Tumour fluorescence increased slowly to a maximum about 3 h post injection (p.i.), and remained well above the normal tissue fluorescence till 24 h p.i. Fluorescence in the circulation was always stronger than in the tissues. A treatment light dose at a wavelength of 675 nm was delivered 24 h p.i. One group of six animals received a total light dose of 150 J cm-2 (100 mW cm-2). A second group of six animals received a total light dose of 450 J cm-2 at the same dose rate. Vascular damage resulting from treatment was observed only at the final stages of the irradiation, despite the relatively high levels of fluorescence in the circulation. Immediate post-treatment (re)transplantation of the content of the chamber into the flank always resulted in tumour regrowth, confirming the presence of viable tumour cells following photodynamic therapy (PDT). When the chamber was left intact, the light dose of 450 J cm-2 yielded complete tissue necrosis. The role of the dye-carrier complex in shielding the vascular surrounding from photoproducts was studied in a third group of animals. The presence of peroxides was demonstrated in the serum of these animals after PDT with zinc phthalocyanine in liposomes (Zn

  14. In vivo macroscopic HPD fluorescence reflectance imaging on small animals bearing surface ARO/NPA tumor

    NASA Astrophysics Data System (ADS)

    Autiero, Maddalena; Celentano, Luigi; Laccetti, Paolo; Marotta, Marcello; Mettivier, Giovanni; Montesi, Maria C.; Riccio, Patrizia; Russo, Paolo; Roberti, Giuseppe

    2005-08-01

    Recently multimodal imaging systems have been devised because the combination of different imaging modalities results in the complementarity and integration of the techniques and in a consequent improvement of the diagnostic capabilities of the multimodal system with respect to each separate imaging modality. We developed a simple and reliable HematoPorphyrin (HP) mediated Fluorescence Reflectance Imaging (FRI) system that allows for in vivo real time imaging of surface tumors with a large field of view. The tumor cells are anaplastic human thyroid carcinoma-derived ARO cells, or human papillary thyroid carcinoma-derived NPA cells. Our measurements show that the optical contrast of the tumor region image is increased by a simple digital subtraction of the background fluorescence and that HP fluorescence emissivity of ARO tumors is about 2 times greater than that of NPA tumors, and about 4 times greater than that of healthy tissues. This is also confirmed by spectroscopic measurements on histological sections of tumor and healthy tissues. It was shown also the capability of this system to distinguish the tumor type on the basis of the different intensity of the fluorescence emission, probably related to the malignancy degree. The features of this system are complementary with those ones of a pixel radionuclide detection system, which allows for relatively time expensive, narrow field of view measurements, and applicability to tumors also deeply imbedded in tissues. The fluorescence detection could be used as a large scale and quick analysis tool and could be followed by narrow field, higher resolution radionuclide measurements on previously determined highly fluorescent regions.

  15. Precise quantification of cellular uptake of cell-penetrating peptides using fluorescence-activated cell sorting and fluorescence correlation spectroscopy.

    PubMed

    Rezgui, Rachid; Blumer, Katy; Yeoh-Tan, Gilbert; Trexler, Adam J; Magzoub, Mazin

    2016-07-01

    Cell-penetrating peptides (CPPs) have emerged as a potentially powerful tool for drug delivery due to their ability to efficiently transport a whole host of biologically active cargoes into cells. Although concerted efforts have shed some light on the cellular internalization pathways of CPPs, quantification of CPP uptake has proved problematic. Here we describe an experimental approach that combines two powerful biophysical techniques, fluorescence-activated cell sorting (FACS) and fluorescence correlation spectroscopy (FCS), to directly, accurately and precisely measure the cellular uptake of fluorescently-labeled molecules. This rapid and technically simple approach is highly versatile and can readily be applied to characterize all major CPP properties that normally require multiple assays, including amount taken up by cells (in moles/cell), uptake efficiency, internalization pathways, intracellular distribution, intracellular degradation and toxicity threshold. The FACS-FCS approach provides a means for quantifying any intracellular biochemical entity, whether expressed in the cell or introduced exogenously and transported across the plasma membrane. PMID:27033412

  16. Strengths and Weaknesses of Recently Engineered Red Fluorescent Proteins Evaluated in Live Cells Using Fluorescence Correlation Spectroscopy

    PubMed Central

    Siegel, Amanda P.; Baird, Michelle A.; Davidson, Michael W.; Day, Richard N.

    2013-01-01

    The scientific community is still looking for a bright, stable red fluorescent protein (FP) as functional as the current best derivatives of green fluorescent protein (GFP). The red FPs exploit the reduced background of cells imaged in the red region of the visible spectrum, but photophysical short comings have limited their use for some spectroscopic approaches. Introduced nearly a decade ago, mCherry remains the most often used red FP for fluorescence correlation spectroscopy (FCS) and other single molecule techniques, despite the advent of many newer red FPs. All red FPs suffer from complex photophysics involving reversible conversions to a dark state (flickering), a property that results in fairly low red FP quantum yields and potential interference with spectroscopic analyses including FCS. The current report describes assays developed to determine the best working conditions for, and to uncover the shortcoming of, four recently engineered red FPs for use in FCS and other diffusion and spectroscopic studies. All five red FPs assayed had potential shortcomings leading to the conclusion that the current best red FP for FCS is still mCherry. The assays developed here aim to enable the rapid evaluation of new red FPs and their smooth adaptation to live cell spectroscopic microscopy and nanoscopy. PMID:24129172

  17. Cellular characterization of adenylate kinase and its isoform: two-photon excitation fluorescence imaging and fluorescence correlation spectroscopy.

    PubMed Central

    Ruan, Qiaoqiao; Chen, Yan; Gratton, Enrico; Glaser, Michael; Mantulin, William W

    2002-01-01

    Adenylate kinase (AK) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. AK1beta (long form) from murine cells shares the same protein sequence as AK1 (short form) except for the addition of 18 amino acid residues at its N-terminus. It is hypothesized that these residues serve as a signal for protein lipid modification and targeting of the protein to the plasma membrane. To better understand the cellular function of these AK isoforms, we have used several modern fluorescence techniques to characterize these two isoforms of AK enzyme. We fused cytosolic adenylate kinase (AK1) and its isoform (AK1beta) with enhanced green fluorescence protein (EGFP) and expressed the chimera proteins in HeLa cells. Using two-photon excitation scanning fluorescence imaging, we were able to directly visualize the localization of AK1-EGFP and AK1beta-EGFP in live cells. AK1beta-EGFP mainly localized on the plasma membrane, whereas AK1-EGFP distributed throughout the cell except for trace amounts in the nuclear membrane and some vesicles. We performed fluorescence correlation spectroscopy measurements and photon-counting histogram analysis in specific domains of live cells. For AK1-EGFP, we observed only one diffusion component in the cytoplasm. For AK1beta-EGFP, we observed two distinct diffusion components on the plasma membrane. One corresponded to the free diffusing protein, whereas the other represented the membrane-bound AK1beta-EGFP. The diffusion rate of AK1-EGFP was slowed by a factor of 1.8 with respect to that of EGFP, which was 50% more than what we would expect for a free diffusing AK1-EGFP. To rule out the possibility of oligomer formation, we performed photon-counting histogram analysis to direct analyze the brightness difference between AK1-EGFP and EGFP. From our analysis, we concluded that cytoplasmic AK1-EGFP is monomeric. fluorescence correlation spectroscopy proved to be a powerful technique for quantitatively studying the

  18. In vivo terahertz pulsed spectroscopy of dysplastic and non-dysplastic skin nevi

    NASA Astrophysics Data System (ADS)

    Zaytsev, Kirill I.; Chernomyrdin, Nikita V.; Kudrin, Konstantin G.; Gavdush, Arseniy A.; Nosov, Pavel A.; Yurchenko, Stanislav O.; Reshetov, Igor V.

    2016-08-01

    The results of the in vivo terahertz (THz) pulsed spectroscopy (TPS) of pigmentary skin nevi are reported. Observed THz dielectric permittivity of healthy skin and dysplastic and non-dysplastic skin nevi exhibits significant contrast in THz frequency range. Dysplastic skin nevus is a precursor of melanoma, which is reportedly the most dangerous cancer of the skin. Therefore, the THz dielectric spectroscopy is potentially an effective tool for non-invasive early diagnosis of melanomas of the skin.

  19. Portable fluorescence spectroscopy platform for Huanglongbing (HLB) citrus disease in situ detection

    NASA Astrophysics Data System (ADS)

    Mota, Alessandro D.; Rossi, Giuliano; de Castro, Guilherme Cunha; Ortega, Tiago A.; de Castro N., Jarbas C.

    2014-02-01

    In this work, the development of a portable fluorescence spectroscopy platform for Huanglongbing (HLB) citrus disease in situ detection is presented. The equipment consists of an excitation blue LED light source, a commercial miniature spectrometer and embedded software. Measurements of healthy, HLB-symptomatic and HLB-asymptomatic citrus leafs were performed. Leafs were excited with the blue LED and their fluorescence spectra collected. Embedded electronics and software were responsible for the spectrum processing and classification via partial least squares regression. Global success rates above 80% and 100% distinction of healthy and HLB-symptomatic leafs were obtained.

  20. Two-photon-excited fluorescence spectroscopy of atomic fluorine at 170 nm

    NASA Technical Reports Server (NTRS)

    Herring, G. C.; Dyer, Mark J.; Jusinski, Leonard E.; Bischel, William K.

    1988-01-01

    Two-photon-excited fluorescence spectroscopy of atomic fluorine is reported. A doubled dye laser at 286-nm is Raman shifted in H2 to 170 nm (sixth anti-Stokes order) to excite ground-state 2P(0)J fluorine atoms to the 2D(0)J level. The fluorine atoms are detected by one of two methods: observing the fluorescence decay to the 2PJ level or observing F(+) production through the absorption of an additional photon by the excited atoms. Relative two-photon absorption cross sections to and the radiative lifetimes of the 2D(0)J states are measured.

  1. Rapid detection of authenticity and adulteration of walnut oil by FTIR and fluorescence spectroscopy: a comparative study.

    PubMed

    Li, Bingning; Wang, Haixia; Zhao, Qiaojiao; Ouyang, Jie; Wu, Yanwen

    2015-08-15

    Fourier transform infrared (FTIR) and fluorescence spectroscopy combined with soft independent modeling of class analogies (SIMCA) and partial least square (PLS) were used to detect the authenticity of walnut oil and adulteration amount of soybean oil in walnut oil. A SIMCA model of FTIR spectra could differentiate walnut oil and other oils into separate categories; the classification limit of soybean oil in walnut oil was 10%. Fluorescence spectroscopy could differentiate oil composition by the peak position and intensity of emission spectrum without multivariate analysis. The classification limit of soybean oil adulterated in walnut oil by fluorescence spectroscopy was below 5%. The deviation of the prediction model for fluorescence spectra was lower than that for FTIR spectra. Fluorescence spectroscopy was more applicable than FTIR in the adulteration detection of walnut oil, both from the determination limit and prediction deviation.

  2. Tomographic sensing and localization of fluorescently labeled circulating cells in mice in vivo

    PubMed Central

    Zettergren, Eric; Swamy, Tushar; Runnels, Judith; Lin, Charles P; Niedre, Mark

    2013-01-01

    Sensing and enumeration of specific types of circulating cells in small animals is an important problem in many areas of biomedical research. Microscopy-based fluorescence in vivo flow cytometry methods have been developed previously, but these are typically limited to sampling of very small blood volumes, so that very rare circulating cells may escape detection. Recently, we described the development of a ‘diffuse fluorescence flow cytometer’ (DFFC) that allows sampling of much larger blood vessels and therefore circulating blood volumes in the hindlimb, forelimb or tail of a mouse. In this work, we extend this concept by developing and validating a method to tomographically localize circulating fluorescently labeled cells in the cross section of a tissue simulating optical flow phantom and mouse limb. This was achieved using two modulated light sources and an array of six fiber-coupled detectors that allowed rapid, high-sensitivity acquisition of full tomographic data sets at 10 Hz. These were reconstructed into two-dimensional cross-sectional images using Monte Carlo models of light propagation and the randomized algebraic reconstruction technique. We were able to obtain continuous images of moving cells in the sample cross section with 0.5 mm accuracy or better. We first demonstrated this concept in limb-mimicking optical flow photons with up to four flow channels, and then in the tails of mice with fluorescently labeled multiple myeloma cells. This approach increases the overall diagnostic utility of our DFFC instrument. PMID:22750660

  3. Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

    SciTech Connect

    Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

    2014-10-31

    Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.

  4. Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

    DOE PAGES

    Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

    2014-10-31

    Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip windowmore » surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.« less

  5. Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

    PubMed Central

    Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

    2014-01-01

    Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria. PMID:25358460

  6. Tomographic sensing and localization of fluorescently labeled circulating cells in mice in vivo

    NASA Astrophysics Data System (ADS)

    Zettergren, Eric; Swamy, Tushar; Runnels, Judith; Lin, Charles P.; Niedre, Mark

    2012-07-01

    Sensing and enumeration of specific types of circulating cells in small animals is an important problem in many areas of biomedical research. Microscopy-based fluorescence in vivo flow cytometry methods have been developed previously, but these are typically limited to sampling of very small blood volumes, so that very rare circulating cells may escape detection. Recently, we described the development of a ‘diffuse fluorescence flow cytometer’ (DFFC) that allows sampling of much larger blood vessels and therefore circulating blood volumes in the hindlimb, forelimb or tail of a mouse. In this work, we extend this concept by developing and validating a method to tomographically localize circulating fluorescently labeled cells in the cross section of a tissue simulating optical flow phantom and mouse limb. This was achieved using two modulated light sources and an array of six fiber-coupled detectors that allowed rapid, high-sensitivity acquisition of full tomographic data sets at 10 Hz. These were reconstructed into two-dimensional cross-sectional images using Monte Carlo models of light propagation and the randomized algebraic reconstruction technique. We were able to obtain continuous images of moving cells in the sample cross section with 0.5 mm accuracy or better. We first demonstrated this concept in limb-mimicking optical flow photons with up to four flow channels, and then in the tails of mice with fluorescently labeled multiple myeloma cells. This approach increases the overall diagnostic utility of our DFFC instrument.

  7. Label-free in vivo imaging of human leukocytes using two-photon excited endogenous fluorescence

    NASA Astrophysics Data System (ADS)

    Zeng, Yan; Yan, Bo; Sun, Qiqi; Teh, Seng Khoon; Zhang, Wei; Wen, Zilong; Qu, Jianan Y.

    2013-04-01

    We demonstrate that two-photon excited endogenous fluorescence enables label-free morphological and functional imaging of various human blood cells. Specifically, we achieved distinctive morphological contrast to visualize morphology of important leukocytes, such as polymorphonuclear structure of granulocyte and mononuclear feature of agranulocyte, through the employment of the reduced nicotinamide adenine dinucleotide (NADH) fluorescence signals. In addition, NADH fluorescence images clearly reveal the morphological transformation process of neutrophils during disease-causing bacterial infection. Our findings also show that time-resolved NADH fluorescence can be potentially used for functional imaging of the phagocytosis of pathogens by leukocytes (neutrophils) in vivo. In particular, we found that free-to-bound NADH ratios measured in infected neutrophils increased significantly, which is consistent with a previous study that the energy consumed in the phagocytosis of neutrophils is mainly generated through the glycolysis pathway that leads to the accumulation of free NADH. Future work will focus on further developing and applying label-free imaging technology to investigate leukocyte-related diseases and disorders.

  8. Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

    NASA Astrophysics Data System (ADS)

    Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

    2014-10-01

    Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.

  9. Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo

    NASA Astrophysics Data System (ADS)

    Razansky, Daniel; Distel, Martin; Vinegoni, Claudio; Ma, Rui; Perrimon, Norbert; Köster, Reinhard W.; Ntziachristos, Vasilis

    2009-07-01

    Fluorescent proteins have become essential reporter molecules for studying life at the cellular and sub-cellular level, re-defining the ways in which we investigate biology. However, because of intense light scattering, most organisms and tissues remain inaccessible to current fluorescence microscopy techniques at depths beyond several hundred micrometres. We describe a multispectral opto-acoustic tomography technique capable of high-resolution visualization of fluorescent proteins deep within highly light-scattering living organisms. The method uses multiwavelength illumination over multiple projections combined with selective-plane opto-acoustic detection for artifact-free data collection. Accurate image reconstruction is enabled by making use of wavelength-dependent light propagation models in tissue. By performing whole-body imaging of two biologically important and optically diffuse model organisms, Drosophila melanogaster pupae and adult zebrafish, we demonstrate the facility to resolve tissue-specific expression of eGFP and mCherrry fluorescent proteins for precise morphological and functional observations in vivo.

  10. Using Fluorescence Imaging to Track Drug Delivery and Guide Treatment Planning In Vivo.

    PubMed

    Lin, Qiaoya; Huang, Huang; Chen, Juan; Zheng, Gang

    2016-01-01

    Imaging has become an indispensable tool in both clinical medicine and preclinical sciences. It enables doctors to locate sites of cancer/disease, track drug delivery, and guide operative planning, thus enhancing the treatment efficacy. Recently, we developed a multimodal theranostic lipid nanoparticles, named HPPS(NIR)-chol-siRNA with its built-in near-infrared (NIR) fluorescent probe core as a useful surrogate for tracking small interfering RNA (siRNA) delivery. By using the image co-registration of computed tomography (CT) and fluorescence molecular tomography (FMT), we achieved noninvasive assessment and treatment planning of siRNA delivery into the orthotopic tumor, thus enabling efficacious RNA interference (RNAi) therapy. In this chapter, we introduce this method to illustrate the use of CT-FMT co-registration for tracking drug delivery and guiding treatment planning in vivo. PMID:27283425

  11. Fluorescence lifetime measurement with confocal endomicroscopy for direct analysis of tissue biochemistry in vivo.

    PubMed

    Won, Youngjae; Park, Byungjun; Kim, Inwook; Lee, Seungrag

    2016-08-01

    Confocal endomicroscopy is a powerful tool for in vivo real-time imaging at cellular resolution inside a living body without tissue resection. Microscopic fluorescence lifetime measurement can provide information about localized biochemical conditions such as pH and the concentrations of oxygen and calcium. We hypothesized that combining these techniques could assist accurate cancer discrimination by providing both biochemical and morphological information. We designed a dual-mode experimental setup for confocal endomicroscopic imaging and fluorescence lifetime measurement and applied it to a mouse xenograft model of activated human pancreatic cancer generated by subcutaneous injection of AsPC-1 tumor cells. Using this method with pH-sensitive sodium fluorescein injection, we demonstrated discrimination between normal and cancerous tissues in a living mouse. With further development, this method may be useful for clinical cancer detection. PMID:27547835

  12. Laser-induced fluorescence spectroscopy of benign and malignant cutaneous lesions

    NASA Astrophysics Data System (ADS)

    Borisova, Ekaterina G.; Troyanova, P. P.; Stoyanova, V. P.; Avramov, Lachezar A.

    2005-04-01

    The goals of this work were investigation of pigmented skin lesions by the method of laser-induced fluorescence spectroscopy. Fluorescence spectra were obtained from malignant and benign skin lesions after excitation with nitrogen laser at 337 nm, namely: benign nevi, dysplastic nevi, malignant melanoma (MM), keratopapilloma, base-cell papilloma and base-cell carcinoma, as well as from healthy skin areas near to the lesion that were used posteriori to reveal changes between healthy and lesion skin spectra. Initially lesions were classified by ABCD-dermatscopic method. All suspicious lesions were excised and were investigated histologically. Spectrum of healthy skin consists of one main maximum at 470-500 nm spectral region and secondary maxima at in the regions round 400 and 440 nm. In the cases of nevi and melanoma significant decrease of fluorescence intensity, which correlated with the type of pigment lesion was observed. This reduction of the signal is related to the accumulation of melanin in the lesions that re-absorb strongly the fluorescence from native skin fluorophores in whole visible spectral region. In cases of papilloma and base-cell carcinoma an intensity decrease was also observed, related to accumulation of pigments in these cutaneous lesions. An relative increase of the fluorescence peak at 440 nm were registered in the case of base-cell carcinoma, and appearance of green fluorescence, related to increase of keratin content in benign papilloma lesions were detected. The results, obtained in this investigation of the different pigment lesions could be used for better comprehension of the skin optical properties. The fluorescence spectroscopy of the human skin are very prominent for early diagnosis and differentiation of cutaneous diseases and gives a wide range of possibilities related to real-time determination of existing pathological condition.

  13. In Vivo Optical Imaging of Acute Cell Death Using a Near-Infrared Fluorescent Zinc-Dipicolylamine Probe

    PubMed Central

    Smith, Bryan A.; Gammon, Seth T.; Xiao, Shuzhang; Wang, Wei; Chapman, Sarah; McDermott, Ryan; Suckow, Mark A.; Johnson, James R.; Piwnica-Worms, David; Gokel, George W.; Smith, Bradley D.; Leevy, W. Matthew

    2013-01-01

    Cell death is a fundamental biological process that is present in numerous disease pathologies. Fluorescent probes that detect cell death have been developed for a myriad of research applications ranging from microscopy to in vivo imaging. Here we describe a synthetic near infrared conjugate of zinc(II)-dipicolylamine (Zn2+-DPA) for in vivo imaging of cell death. Chemically induced in vivo models of myopathy were established using an ionphore, ethanol, or ketamine as chemical cytotoxins. The Zn2+-DPA fluorescent probe or corresponding control was subsequently injected and whole animal fluorescence imaging demonstrated probe uptake at the site of muscle damage, which was confirmed by ex vivo and histological analyses. Further, a comparative study with a near-infrared fluorescent conjugate Annexin V showed less intense uptake at the site of muscle damage and high accumulation in the bladder. The results indicate that the fluorescent Zn2+-DPA conjugate is an effective probe for in vivo cell death detection and in some cases may be an appropriate alternative to fluorescent Annexin V conjugates. PMID:21323375

  14. In vivo optical imaging of acute cell death using a near-infrared fluorescent zinc-dipicolylamine probe.

    PubMed

    Smith, Bryan A; Gammon, Seth T; Xiao, Shuzhang; Wang, Wei; Chapman, Sarah; McDermott, Ryan; Suckow, Mark A; Johnson, James R; Piwnica-Worms, David; Gokel, George W; Smith, Bradley D; Leevy, W Matthew

    2011-04-01

    Cell death is a fundamental biological process that is present in numerous disease pathologies. Fluorescent probes that detect cell death have been developed for a myriad of research applications ranging from microscopy to in vivo imaging. Here we describe a synthetic near-infrared (NIR) conjugate of zinc(II)-dipicolylamine (Zn²+-DPA) for in vivo imaging of cell death. Chemically induced in vivo models of myopathy were established using an ionphore, ethanol, or ketamine as cytotoxins. The Zn²+-DPA fluorescent probe or corresponding control was subsequently injected, and whole animal fluorescence imaging demonstrated probe uptake at the site of muscle damage, which was confirmed by ex vivo and histological analyses. Further, a comparative study with a NIR fluorescent conjugate Annexin V showed less intense uptake at the site of muscle damage and high accumulation in the bladder. The results indicate that the fluorescent Zn²+-DPA conjugate is an effective probe for in vivo cell death detection and in some cases may be an appropriate alternative to fluorescent Annexin V conjugates.

  15. Multimodal in vivo imaging of oral cancer using fluorescence lifetime, photoacoustic and ultrasound techniques

    PubMed Central

    Fatakdawala, Hussain; Poti, Shannon; Zhou, Feifei; Sun, Yang; Bec, Julien; Liu, Jing; Yankelevich, Diego R.; Tinling, Steven P.; Gandour-Edwards, Regina F.; Farwell, D. Gregory; Marcu, Laura

    2013-01-01

    This work reports a multimodal system for label-free tissue diagnosis combining fluorescence lifetime imaging (FLIm), ultrasound backscatter microscopy (UBM), and photoacoustic imaging (PAI). This system provides complementary biochemical, structural and functional features allowing for enhanced in vivo detection of oral carcinoma. Results from a hamster oral carcinoma model (normal, precancer and carcinoma) are presented demonstrating the ability of FLIm to delineate biochemical composition at the tissue surface, UBM and related radiofrequency parameters to identify disruptions in the tissue microarchitecture and PAI to map optical absorption associated with specific tissue morphology and physiology. PMID:24049693

  16. Spectral editing for in vivo 13C magnetic resonance spectroscopy

    NASA Astrophysics Data System (ADS)

    Xiang, Yun; Shen, Jun

    2012-01-01

    In vivo detection of carboxylic/amide carbons is a promising technique for studying cerebral metabolism and neurotransmission due to the very low RF power required for proton decoupling. In the carboxylic/amide region, however, there is severe spectral overlap between acetate C1 and glutamate C5, complicating studies that use acetate as an astroglia-specific substrate. There are no known in vivo MRS techniques that can spectrally resolve acetate C1 and glutamate C5 singlets. In this study, we propose to spectrally separate acetate C1 and glutamate C5 by a two-step J-editing technique after introducing homonuclear 13C- 13C scalar coupling between carboxylic/amide carbons and aliphatic carbons. By infusing [1,2- 13C 2]acetate instead of [1- 13C]acetate the acetate doublet can be spectrally edited because of the large separation between acetate C2 and glutamate C4 in the aliphatic region. This technique can be applied to studying acetate transport and metabolism in brain in the carboxylic/amide region without spectral interference.

  17. In vivo monitoring of toxic metals: assessment of neutron activation and x-ray fluorescence techniques

    SciTech Connect

    Ellis, K.J.

    1986-01-01

    To date, cadmium, lead, aluminum, and mercury have been measured in vivo in humans. The possibilities of monitoring other toxic metals have also been demonstrated, but no human studies have been performed. Neutron activation analysis appears to be most suitable for Cd and Al measurements, while x-ray fluorescence is ideally suited for measurement of lead in superficial bone. Filtered neutron beams and polarized x-ray sources are being developed which will improve in vivo detection limits. Even so, several of the current facilities are already suitable for use in epidemiological studies of selected populations with suspected long-term low-level ''environmental'' exposures. Evaluation and diagnosis of patients presenting with general clinical symptoms attributable to possible toxic metal exposure may be assisted by in vivo examination. Continued in vivo monitoring of industrial workers, especially follow-up measurements, will provide the first direct assessment of changes in body burden and a direct measure of the biological life-times of these metals in humans. 50 refs., 4 figs., 2 tabs.

  18. Interactions of indocyanine green and lipid in enhancing near-infrared fluorescence properties: the basis for near-infrared imaging in vivo.

    PubMed

    Kraft, John C; Ho, Rodney J Y

    2014-03-01

    Indocyanine green (ICG) is a near-infrared (NIR) contrast agent commonly used for in vivo cardiovascular and eye imaging. For medical diagnosis, ICG is limited by its aqueous instability, concentration-dependent aggregation, and rapid degradation. To overcome these limitations, scientists have formulated ICG in various liposomes, which are spherical lipid membrane vesicles with an aqueous core. Some encapsulate ICG, while others mix it with liposomes. There is no clear understanding of lipid-ICG interactions. Therefore, we investigated lipid-ICG interactions by fluorescence and photon correlation spectroscopy. These data were used to design stable and maximally fluorescent liposomal ICG nanoparticles for NIR optical imaging of the lymphatic system. We found that ICG binds to and is incorporated completely and stably into the lipid membrane. At a lipid:ICG molar ratio of 250:1, the maximal fluorescence intensity was detected. ICG incorporated into liposomes enhanced the fluorescence intensity that could be detected across 1.5 cm of muscle tissue, while free ICG only allowed 0.5 cm detection. When administered subcutaneously in mice, lipid-bound ICG in liposomes exhibited a higher intensity, NIR image resolution, and enhanced lymph node and lymphatic vessel visualization. It also reduced the level of fluorescence quenching due to light exposure and degradation in storage. Lipid-bound ICG could provide additional medical diagnostic value with NIR optical imaging for early intervention in cases of lymphatic abnormalities.

  19. A fast, flexible algorithm for calculating correlations in Fluorescence Correlation Spectroscopy

    SciTech Connect

    Laurence, T; Fore, S; Huser, T

    2005-10-13

    A new algorithm is introduced for computing correlations of photon arrival time data acquired in single-molecule fluorescence spectroscopy and fluorescence correlation spectroscopy (FCS). The correlation is first rewritten as a counting operation on photon pairs. For each photon, the contribution to the correlation function for each subsequent photon is calculated for arbitrary bin spacings of the correlation time lag. By retaining the bin positions in the photon sequence after each photon, the correlation can be performed efficiently. Example correlations for simulations of FCS experiments are shown, with comparable execution speed to the commonly used multiple-tau correlation technique. Also, wide bin spacings are possible that allow for real-time software calculation of correlations even for high count rates ({approx}350 kHz). The flexibility and broad applicability of the algorithm is demonstrated using results from single molecule photon antibunching experiments.

  20. Spot Variation Fluorescence Correlation Spectroscopy Allows for Superresolution Chronoscopy of Confinement Times in Membranes

    PubMed Central

    Ruprecht, Verena; Wieser, Stefan; Marguet, Didier; Schütz, Gerhard J.

    2011-01-01

    Resolving the dynamical interplay of proteins and lipids in the live-cell plasma membrane represents a central goal in current cell biology. Superresolution concepts have introduced a means of capturing spatial heterogeneity at a nanoscopic length scale. Similar concepts for detecting dynamical transitions (superresolution chronoscopy) are still lacking. Here, we show that recently introduced spot-variation fluorescence correlation spectroscopy allows for sensing transient confinement times of membrane constituents at dramatically improved resolution. Using standard diffraction-limited optics, spot-variation fluorescence correlation spectroscopy captures signatures of single retardation events far below the transit time of the tracer through the focal spot. We provide an analytical description of special cases of transient binding of a tracer to pointlike traps, or association of a tracer with nanodomains. The influence of trap mobility and the underlying binding kinetics are quantified. Experimental approaches are suggested that allow for gaining quantitative mechanistic insights into the interaction processes of membrane constituents. PMID:21641330

  1. Dynamic disorder in horseradish peroxidase observed with total internal reflection fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Hassler, Kai; Rigler, Per; Blom, Hans; Rigler, Rudolf; Widengren, Jerker; Lasser, Theo

    2007-04-01

    This paper discusses the application of objective-type total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to the study of the kinetics of immobilized horseradish peroxidase on a single molecule level. Objective-type TIR-FCS combines the advantages of FCS with TIRF microscopy in a way that allows for simultaneous ultra-sensitive spectroscopic measurements using a single-point detector and convenient localization of single molecules on a surface by means of parallel imaging.

  2. TOTAL INTERNAL REFLECTION WITH FLUORESCENCE CORRELATION SPECTROSCOPY: APPLICATIONS TO SUBSTRATE-SUPPORTED PLANAR MEMBRANES

    PubMed Central

    Thompson, Nancy L.; Wang, Xiang; Navaratnarajah, Punya

    2009-01-01

    In this review paper, the conceptual basis and experimental design of total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) is described. The few applications to date of TIR-FCS to supported membranes are discussed, in addition to a variety of applications not directly involving supported membranes. Methods related, but not technically equivalent, to TIR-FCS are also summarized. Future directions for TIR-FCS are outlined. PMID:19269331

  3. New energy levels of atomic niobium by laser induced fluorescence spectroscopy in the near infrared

    NASA Astrophysics Data System (ADS)

    Öztürk, I. K.; Başar, Gö; Er, A.; Güzelçimen, F.; Başar, Gü; Kröger, S.

    2015-01-01

    Laser-induced fluorescence spectroscopy was applied in order to find new energy levels of the niobium atom. A continuous wave tuneable titanium-sapphire laser in the wavelength range from 750 to 865 nm and a hollow-cathode lamp were used. We discovered four energy levels of even parity, three lying levels below 19 000 cm-1 and one at much higher energy. Additionally hyperfine structure data of six levels of odd parity were determined.

  4. Fluorescence Lifetime Imaging and Spectroscopy as Tools for Nondestructive Analysis of Works of Art

    NASA Astrophysics Data System (ADS)

    Comelli, Daniela; D'Andrea, Cosimo; Valentini, Gianluca; Cubeddu, Rinaldo; Colombo, Chiara; Toniolo, Lucia

    2004-04-01

    A system for advanced fluorescence investigation of works of art has been assembled and integrated in a characterization procedure that allows one to localize and identify organic compounds that are present in artworks. At the beginning of the investigation, fluorescence lifetime imaging and spectroscopy address a selective microsampling of the artwork. Then analytical measurements of microsamples identify the chemical composition of the materials under investigation. Finally, on the basis of fluorescence lifetime and amplitude maps, analytical data are extended to the whole artwork. In such a way, information on the spatial distribution of organic materials can be inferred. These concepts have been successfully applied in an extensive campaign for analysis of Renaissance fresco paintings in Castiglione Olona, Italy. Residue of various types of glue and stucco left from a restoration carried out in the early 1970s was localized and classified. Insight into the technique used by the painter to make gilded reliefs was also obtained.

  5. Polarization-dependent fluorescence correlation spectroscopy for studying structural properties of proteins in living cell

    PubMed Central

    Oura, Makoto; Yamamoto, Johtaro; Ishikawa, Hideto; Mikuni, Shintaro; Fukushima, Ryousuke; Kinjo, Masataka

    2016-01-01

    Rotational diffusion measurement is predicted as an important method in cell biology because the rotational properties directly reflect molecular interactions and environment in the cell. To prove this concept, polarization-dependent fluorescence correlation spectroscopy (pol-FCS) measurements of purified fluorescent proteins were conducted in viscous solution. With the comparison between the translational and rotational diffusion coefficients obtained from pol-FCS measurements, the hydrodynamic radius of an enhanced green fluorescent protein (EGFP) was estimated as a control measurement. The orientation of oligomer EGFP in living cells was also estimated by pol-FCS and compared with Monte Carlo simulations. The results of this pol-FCS experiment indicate that this method allows an estimation of the molecular orientation using the characteristics of rotational diffusion. Further, it can be applied to analyze the degree of molecular orientation and multimerization or detection of tiny aggregation of aggregate-prone proteins. PMID:27489044

  6. Fluorescence spectroscopy for assessment of liver transplantation grafts concerning graft viability and patient survival

    NASA Astrophysics Data System (ADS)

    Vollet Filho, José D.; da Silveira, Marina R.; Castro-e-Silva, Orlando; Bagnato, Vanderlei S.; Kurachi, Cristina

    2015-06-01

    Evaluating transplantation grafts at harvest is essential for its success. Laser-induced fluorescence spectroscopy (LIFS) can help monitoring changes in metabolic/structural conditions of tissue during transplantation. The aim of the present study is to correlate LIFSobtained spectra of human hepatic grafts during liver transplantation with post-operative patients' mortality rate and biochemical parameters, establishing a method to exclude nonviable grafts before implantation. Orthotopic liver transplantation, piggyback technique was performed in 15 patients. LIFS was performed under 408nm excitation. Collection was performed immediately after opening donor's abdominal cavity, after cold perfusion, end of back-table period, and 5 min and 1 h after warm perfusion at recipient. Fluorescence information was compared to lactate, creatinine, bilirubin and INR levels and to survival status. LIFS was sensitive to liver changes during transplantation stages. Study-in-progress; initial results indicate correlation between fluorescence and life/death status of patients.

  7. Revealing the photophysics of gold-nanobeacons via time-resolved fluorescence spectroscopy.

    PubMed

    Wei, Guoke; Simionesie, Dorin; Sefcik, Jan; Sutter, Jens U; Xue, Qingjiang; Yu, Jun; Wang, Jinliang; Birch, David J S; Chen, Yu

    2015-12-15

    We demonstrate that time-resolved fluorescence spectroscopy is a powerful tool to investigate the conformation states of hairpin DNA on the surface of gold nanoparticles (AuNPs) and energy transfer processes in Au-nanobeacons. Long-range fluorescence quenching of Cy5 by AuNPs has been found to be in good agreement with electrodynamics modeling. Moreover, time-correlated single-photon counting (TCSPC) is shown to be promising for real-time monitoring of the hybridization kinetics of Au-nanobeacons, with up to 60% increase in decay time component and 300% increase in component fluorescence fraction observed. Our results also indicate the importance of the stem and spacer designs for the performance of Au-nanobeacons. PMID:26670500

  8. Site-specific interaction of thrombin and inhibitors observed by fluorescence correlation spectroscopy.

    PubMed Central

    Klingler, J; Friedrich, T

    1997-01-01

    We report on the application of fluorescence correlation spectroscopy (FCS) to observe the interaction between thrombin and thrombin inhibitors. Two site-specific fluorescent labels were used to distinguish between inhibitors directed to the active site, the exosite, or both binding sites of thrombin. For several well-known inhibitors of thrombin, the binding sites observed by FCS correspond to previous studies. The interaction of the recently discovered thrombin inhibitor ornithodorin from the tick Ornithodorus moubata with thrombin was investigated. It was found that this inhibitor, like hirudin and rhodniin, binds to both the active site and exosite of thrombin simultaneously. This study shows the feasibility of FCS as a sensitive and selective method for observing protein-ligand interactions. As an additional technique, simultaneous labeling with both fluorescent labels was successfully demonstrated. Images FIGURE 1 PMID:9336216

  9. X-ray microprobe for micro x-ray fluorescence and absorption spectroscopies at GSECARS

    NASA Astrophysics Data System (ADS)

    Newville, M.; Sutton, S.; Rivers, M.

    2002-12-01

    The hard x-ray microprobe for x-ray fluorescence and absorption spectroscopy at GeoSoilEnviroCARS is presented. Using focused synchrotron radiation from an undulator beamline at the Advanced Photon Source at Argonne National Lab, the x-ray microprobe provides bright, monochromatic x-rays with typical spot sizes down to 1x1 μm for x-ray fluorescence and absorption spectroscopies. Quantitative x-ray fluorescence (XRF) analysis gives precise elemental composition and correlations, while x-ray absorption spectroscopy (XAS) gives the chemical state and local atomic coordination for a selected atomic species. These two techniques can be used in conjunction with one another on a wide range of samples, including minerals, glasses, fluid inclusions, soils, sediments, and plant tissue. This x-ray microprobe is part of the GeoSoilEnviroCARS user facility, available for use in all areas geological, soil, and environmental sciences, and selected examples from these fields will be given.

  10. Native fluorescence spectroscopy of cervical tissues: classification by different statistical methods

    NASA Astrophysics Data System (ADS)

    Ganesan, Singaravelu; Vengadesan, Nammalver; Anbupalam, Thalaimuthu; Hemamalini, Srinivasan; Aruna, Prakasa R.; Karkuzhali, P.

    2002-05-01

    Optical Spectroscopy in the diagnosis of diseases has attracted the medical community due to their minimally invasive nature. Among various optical spectroscopic techniques, native fluorescence spectroscopy has emerged as a potential tool in diagnostic oncology. However, still the reasons for the altered spectral signatures between normal and cancer tissues not yet completely understood. Recently, data reported that emission due to the alteration of some proteins is responsible for the transformation of normal in to malignant one. In this regard, the present study is aimed to characterize the native fluorescence spectroscopy of abnormal and normal cervical tissues, at 280nm excitation. From the study, it is observed that the normal and pathologically diseased cervical tissues have their peak emission around 339 and 336nm respectively with a secondary peak around 440nm. The FWHM value of emission spectra of abnormal tissues is lower than that of normal tissues. The fluorescence spectra of normal and various pathological conditions of cancerous tissues were analyzed by various empirical and statistical methods. Among various type of discriminant analysis, combination of ratio values and linear discrimination method provides better discrimination of normal from pre-malignant and malignant tissues.

  11. Laser-induced fluorescence and pure rotational spectroscopy of the CH2CHS (vinylthio) radical.

    PubMed

    Nakajima, Masakazu; Miyoshi, Akira; Sumiyoshi, Yoshihiro; Endo, Yasuki

    2007-01-28

    Laser-induced fluorescence (LIF) excitation spectra of the B-X (2)A(") electronic transition of the CH(2)CHS radical, which is the sulfur analog of the vinoxy (CH(2)CHO) radical, were observed under room temperature and jet-cooled conditions. The LIF excitation spectra show very poor vibronic structures, since the fluorescence quantum yields of the upper vibronic levels are too small to detect fluorescence, except for the vibrationless level in the B state. A dispersed fluorescence spectrum of jet-cooled CH(2)CHS from the vibrationless level of the B state was also observed, and vibrational frequencies in the X state were determined. Precise rotational and spin-rotation constants in the ground vibronic level of the radical were determined from pure rotational spectroscopy using a Fourier-transform microwave (FTMW) spectrometer and a FTMW-millimeter wave double-resonance technique [Y. Sumiyoshi et al., J. Chem. Phys. 123, 054324 (2005)]. The rotationally resolved LIF excitation spectrum for the vibronic origin band of the jet-cooled CH(2)CHS radical was analyzed using the ground state molecular constants determined from pure rotational spectroscopy. Determined molecular constants for the upper and lower electronic states agree well with results of ab initio calculations.

  12. Spoilage of foods monitored by native fluorescence spectroscopy with selective excitation wavelength

    NASA Astrophysics Data System (ADS)

    Pu, Yang; Wang, Wubao; Alfano, Robert R.

    2015-03-01

    The modern food processing and storage environments require the real-time monitoring and rapid microbiological testing. Optical spectroscopy with selective excitation wavelengths can be the basis of a novel, rapid, reagent less, noncontact and non-destructive technique for monitoring the food spoilage. The native fluorescence spectra of muscle foods stored at 2-4°C (in refrigerator) and 20-24°C (in room temperature) were measured as a function of time with a selective excitation wavelength of 340nm. The contributions of the principal molecular components to the native fluorescence spectra of meat were measured spectra of each fluorophore: collagen, reduced nicotinamide adenine dinucleotide (NADH), and flavin. The responsible components were extracted using a method namely Multivariate Curve Resolution with Alternating Least-Squares (MCR-ALS). The native fluorescence combined with MCR-ALS can be used directly on the surface of meat to produce biochemically interpretable "fingerprints", which reflects the microbial spoilage of foods involved with the metabolic processes. The results show that with time elapse, the emission from NADH in meat stored at 24°C increases much faster than that at 4°C. This is because multiplying of microorganisms and catabolism are accompanied by the generation of NADH. This study presents changes of relative content of NADH may be used as criterion for detection of spoilage degree of meat using native fluorescence spectroscopy.

  13. New photon-counting detectors for single-molecule fluorescence spectroscopy and imaging

    PubMed Central

    Michalet, X.; Colyer, R. A.; Scalia, G.; Weiss, S.; Siegmund, Oswald H. W.; Tremsin, Anton S.; Vallerga, John V.; Villa, F.; Guerrieri, F.; Rech, I.; Gulinatti, A.; Tisa, S.; Zappa, F.; Ghioni, M.; Cova, S.

    2013-01-01

    Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. Two typical geometries can be used for these experiments: point-like and widefield excitation and detection. In point-like geometries, the basic concept is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements. In widefield geometries, the same issues of background reduction and single-molecule concentration apply, but the duration of the experiment is fixed by the time scale of the process studied and the survival time of the fluorescent probe. Temporal resolution on the other hand, is limited by signal-to-noise and/or detector resolution, which calls for new detector concepts. We will briefly present our recent results in this domain. PMID:24729836

  14. Determination of Dissociation Constants in Living Zebrafish Embryos with Single Wavelength Fluorescence Cross-Correlation Spectroscopy

    PubMed Central

    Shi, Xianke; Foo, Yong Hwee; Sudhaharan, Thankiah; Chong, Shang-Wei; Korzh, Vladimir; Ahmed, Sohail; Wohland, Thorsten

    2009-01-01

    Abstract The quantification of biological interactions is very important in life sciences. Here we report for the first time, to our knowledge, the determination of a biomolecular dissociation constant (KD) in living zebrafish embryos at physiological protein expression levels. For that purpose, we extend the application of single wavelength fluorescence cross-correlation spectroscopy into small organisms and measure the interaction of Cdc42, a small Rho-GTPase, and IQGAP1, an actin-binding scaffolding protein. Cdc42 and IQGAP1 were labeled with monomeric red fluorescent protein and enhanced green fluorescent protein, respectively. Both fluorophores were excited at a single wavelength of 514 nm, simplifying the fluorescence spectroscopy measurements and allowing quantification. For the determination of the interaction, we used two Cdc42 mutants, the constitutively active Cdc42G12V which is in a predominantly GTP-bound form and the dominant-negative GDP-bound Cdc42T17N. While Cdc42G12V binds to IQGAP1 with an apparent KD of ∼100 nM, Cdc42T17N has at least a one-order-of-magnitude lower affinity for the same protein. As a comparison, we measure the same protein-protein interactions in Chinese hamster ovary cell cultures but observe significant differences in protein mobility and KD from the zebrafish measurements, supporting the notion that bimolecular interactions depend on the biological system under investigation and are best performed under physiologically relevant conditions. PMID:19619483

  15. Applications of Fluorescence Spectroscopy for dissolved organic matter characterization in wastewater treatment plants

    NASA Astrophysics Data System (ADS)

    Goffin, Angélique; Guérin, Sabrina; Rocher, Vincent; Varrault, Gilles

    2016-04-01

    Dissolved organic matter (DOM) influences wastewater treatment plants efficiency (WTTP): variations in its quality and quantity can induce a foaming phenomenon and a fouling event inside biofiltration processes. Moreover, in order to manage denitrification step (control and optimization of the nitrate recirculation), it is important to be able to estimate biodegradable organic matter quantity before biological treatment. But the current methods used to characterize organic matter quality, like biological oxygen demand are laborious, time consuming and sometimes not applicable to directly monitor organic matter in situ. In the context of MOCOPEE research program (www.mocopee.com), this study aims to assess the use of optical techniques, such as UV-Visible absorbance and more specifically fluorescence spectroscopy in order to monitor and to optimize process efficiency in WWTP. Fluorescence excitation-emission matrix (EEM) spectroscopy was employed to prospect the possibility of using this technology online and in real time to characterize dissolved organic matter in different effluents of the WWTP Seine Centre (240,000 m3/day) in Paris, France. 35 sewage water influent samples were collected on 10 days at different hours. Data treatment were performed by two methods: peak picking and parallel factor analysis (PARAFAC). An evolution of DOM quality (position of excitation - emission peaks) and quantity (intensity of fluorescence) was observed between the different treatment steps (influent, primary treatment, biological treatment, effluent). Correlations were found between fluorescence indicators and different water quality key parameters in the sewage influents. We developed different multivariate linear regression models in order to predict a variety of water quality parameters by fluorescence intensity at specific excitation-emission wavelengths. For example dissolved biological oxygen demand (r2=0,900; p<0,0001) and ammonium concentration (r2=0,898; p<0

  16. Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Krumholz, Arie; Shcherbakova, Daria M.; Xia, Jun; Wang, Lihong V.; Verkhusha, Vladislav V.

    2014-02-01

    Non-invasive imaging of biological processes in vivo is invaluable in advancing biology. Photoacoustic tomography is a scalable imaging technique that provides higher resolution at greater depths in tissue than achievable by purely optical methods. Here we report the application of two spectrally distinct near-infrared fluorescent proteins, iRFP670 and iRFP720, engineered from bacterial phytochromes, as photoacoustic contrast agents. iRFPs provide tissue-specific contrast without the need for delivery of any additional substances. Compared to conventional GFP-like red-shifted fluorescent proteins, iRFP670 and iRFP720 demonstrate stronger photoacoustic signals at longer wavelengths, and can be spectrally resolved from each other and hemoglobin. We simultaneously visualized two differently labeled tumors, one with iRFP670 and the other with iRFP720, as well as blood vessels. We acquired images of a mouse as 2D sections of a whole animal, and as localized 3D volumetric images with high contrast and sub-millimeter resolution at depths up to 8 mm. Our results suggest iRFPs are genetically-encoded probes of choice for simultaneous photoacoustic imaging of several tissues or processes in vivo.

  17. Ultrafast fluorescence imaging in vivo with conjugated polymer fluorophores in the second near-infrared window

    NASA Astrophysics Data System (ADS)

    Hong, Guosong; Zou, Yingping; Antaris, Alexander L.; Diao, Shuo; Wu, Di; Cheng, Kai; Zhang, Xiaodong; Chen, Changxin; Liu, Bo; He, Yuehui; Wu, Justin Z.; Yuan, Jun; Zhang, Bo; Tao, Zhimin; Fukunaga, Chihiro; Dai, Hongjie

    2014-06-01

    In vivo fluorescence imaging in the second near-infrared window (1.0-1.7 μm) can afford deep tissue penetration and high spatial resolution, owing to the reduced scattering of long-wavelength photons. Here we synthesize a series of low-bandgap donor/acceptor copolymers with tunable emission wavelengths of 1,050-1,350 nm in this window. Non-covalent functionalization with phospholipid-polyethylene glycol results in water-soluble and biocompatible polymeric nanoparticles, allowing for live cell molecular imaging at >1,000 nm with polymer fluorophores for the first time. Importantly, the high quantum yield of the polymer allows for in vivo, deep-tissue and ultrafast imaging of mouse arterial blood flow with an unprecedented frame rate of >25 frames per second. The high time-resolution results in spatially and time resolved imaging of the blood flow pattern in cardiogram waveform over a single cardiac cycle (~200 ms) of a mouse, which has not been observed with fluorescence imaging in this window before.

  18. In vivo fluorescence confocal microscopy: indocyanine green enhances the contrast of epidermal and dermal structures

    NASA Astrophysics Data System (ADS)

    Skvara, Hans; Kittler, Harald; Schmid, Johannes A.; Plut, Ulrike; Jonak, Constanze

    2011-09-01

    In recent years, in vivo skin imaging devices have been successfully implemented in skin research as well as in clinical routine. Of particular importance is the use of reflectance confocal microscopy (RCM) and fluorescence confocal microscopy (FCM) that enable visualization of the tissue with a resolution comparable to histology. A newly developed commercially available multi-laser device in which both technologies are integrated now offers the possibility to directly compare RCM with FCM. The fluorophore indocyanine green (ICG) was intradermally injected into healthy forearm skin of 10 volunteers followed by in vivo imaging at various time points. In the epidermis, accurate assessment of cell morphology with FCM was supplemented by identification of pigmented cells and structures with RCM. In dermal layers, only with FCM connective tissue fibers were clearly contoured down to a depth of more than 100 μm. The fluorescent signal still provided a favorable image contrast 24 and 48 hours after injection. Subsequently, ICG was applied to different types of skin diseases (basal cell carcinoma, actinic keratosis, seborrhoeic keratosis, and psoriasis) in order to demonstrate the diagnostic benefit of FCM when directly compared with RCM. Our data suggest a great impact of FCM in combination with ICG on clinical and experimental dermatology in the future.

  19. Dynamic dual-tracer MRI-guided fluorescence tomography to quantify receptor density in vivo

    PubMed Central

    Davis, Scott C.; Samkoe, Kimberley S.; Tichauer, Kenneth M.; Sexton, Kristian J.; Gunn, Jason R.; Deharvengt, Sophie J.; Hasan, Tayyaba; Pogue, Brian W.

    2013-01-01

    The up-regulation of cell surface receptors has become a central focus in personalized cancer treatment; however, because of the complex nature of contrast agent pharmacokinetics in tumor tissue, methods to quantify receptor binding in vivo remain elusive. Here, we present a dual-tracer optical technique for noninvasive estimation of specific receptor binding in cancer. A multispectral MRI-coupled fluorescence molecular tomography system was used to image the uptake kinetics of two fluorescent tracers injected simultaneously, one tracer targeted to the receptor of interest and the other tracer a nontargeted reference. These dynamic tracer data were then fit to a dual-tracer compartmental model to estimate the density of receptors available for binding in the tissue. Applying this approach to mice with deep-seated gliomas that overexpress the EGF receptor produced an estimate of available receptor density of 2.3 ± 0.5 nM (n = 5), consistent with values estimated in comparative invasive imaging and ex vivo studies. PMID:23671066

  20. Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins.

    PubMed

    Krumholz, Arie; Shcherbakova, Daria M; Xia, Jun; Wang, Lihong V; Verkhusha, Vladislav V

    2014-01-01

    Non-invasive imaging of biological processes in vivo is invaluable in advancing biology. Photoacoustic tomography is a scalable imaging technique that provides higher resolution at greater depths in tissue than achievable by purely optical methods. Here we report the application of two spectrally distinct near-infrared fluorescent proteins, iRFP670 and iRFP720, engineered from bacterial phytochromes, as photoacoustic contrast agents. iRFPs provide tissue-specific contrast without the need for delivery of any additional substances. Compared to conventional GFP-like red-shifted fluorescent proteins, iRFP670 and iRFP720 demonstrate stronger photoacoustic signals at longer wavelengths, and can be spectrally resolved from each other and hemoglobin. We simultaneously visualized two differently labeled tumors, one with iRFP670 and the other with iRFP720, as well as blood vessels. We acquired images of a mouse as 2D sections of a whole animal, and as localized 3D volumetric images with high contrast and sub-millimeter resolution at depths up to 8 mm. Our results suggest iRFPs are genetically-encoded probes of choice for simultaneous photoacoustic imaging of several tissues or processes in vivo. PMID:24487319

  1. Functional surface engineering of C-dots for fluorescent biosensing and in vivo bioimaging.

    PubMed

    Ding, Changqin; Zhu, Anwei; Tian, Yang

    2014-01-21

    Nanoparticles are promising scaffolds for applications such as imaging, chemical sensors and biosensors, diagnostics, drug delivery, catalysis, energy, photonics, medicine, and more. Surface functionalization of nanoparticles introduces an additional dimension in controlling nanoparticle interfacial properties and provides an effective bridge to connect nanoparticles to biological systems. With fascinating photoluminescence properties, carbon dots (C-dots), carbon-containing nanoparticles that are attracting considerable attention as a new type of quantum dot, are becoming both an important class of imaging probes and a versatile platform for engineering multifunctional nanosensors. In order to transfer C-dots from proof-of-concept studies toward real world applications such as in vivo bioimaging and biosensing, careful design and engineering of C-dot probes is becoming increasingly important. A comprehensive knowledge of how C-dot surfaces with various properties behave is essential for engineering C-dots with useful imaging properties such as high quantum yield, stability, and low toxicity, and with desirable biosensing properties such as high selectivity, sensitivity, and accuracy. Several reviews in recent years have reported preparation methods and properties of C-dots and described their application in biosensors, catalysis, photovoltatic cells, and more. However, no one has yet systematically summarized the surface engineering of C-dots, nor the use of C-dots as fluorescent nanosensors or probes for in vivo imaging in cells, tissues, and living organisms. In this Account, we discuss the major design principles and criteria for engineering the surface functionality of C-dots for biological applications. These criteria include brightness, long-term stability, and good biocompatibility. We review recent developments in designing C-dot surfaces with various functionalities for use as nanosensors or as fluorescent probes with fascinating analytical performance

  2. In vivo validation of high frequency ultrasound-guided fluorescence tomography system to improve delivery of photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Paliwal, Akshat; Torosean, Sason; Gruber, Josiah; O'Hara, Julia; Hasan, Tayyaba; Pogue, Brian; Maytin, Edward V.

    2011-02-01

    Photodynamic therapy (PDT) for skin cancer is sometimes only partially effective, due to inadequate levels of the fluorescent drug (photosensitizer, PS) and due to heterogeneous distribution of PS within the tissue. To image the PS distribution within skin tumors, we have developed a fluorescence tomography system (FTS) that combines a fluorescence detection array with a high frequency ultrasound (HFUS) transducer. In this paper we describe in vitro and in vivo validation of this new system. The target fluorophore for detection was Protoporphyrin IX (PPIX). Validation experiments were performed in vivo using a subcutaneous tumor model in which A431 tumor-bearing mice were treated with 5-aminolevulinic acid to induce production of PPIX. FTS reconstructions were compared with standard histology and with data from bulk tumor slices imaged ex vivo on a fluorescence scanner. Reconstructed images obtained from the FTS were correlated with the histology and the ex vivo scans, confirming several-fold increases in PPIX fluorescence in the skin and in the tumor relative to the surrounding tissues. Our data demonstrate the feasibility of using the FTS for subsurface imaging of PPIX in skin carcinoma in vivo. Future aims are to use this device for individualized treatment planning, in order to improve overall patient responses to PDT.

  3. In vivo spatial frequency domain spectroscopy of two layer media

    NASA Astrophysics Data System (ADS)

    Yudovsky, Dmitry; Nguyen, John Quan M.; Durkin, Anthony J.

    2012-10-01

    Monitoring of tissue blood volume and local oxygen saturation can inform the assessment of tissue health, healing, and dysfunction. These quantities can be estimated from the contribution of oxyhemoglobin and deoxyhemoglobin to the absorption spectrum of the dermis. However, estimation of blood related absorption in skin can be confounded by the strong absorption of melanin in the epidermis and epidermal thickness and pigmentation varies with anatomic location, race, gender, and degree of disease progression. Therefore, a method is desired that decouples the effect of melanin absorption in the epidermis from blood absorption in the dermis for a large range of skin types and thicknesses. A previously developed inverse method based on a neural network forward model was applied to simulated spatial frequency domain reflectance of skin for multiple wavelengths in the near infrared. It is demonstrated that the optical thickness of the epidermis and absorption and reduced scattering coefficients of the dermis can be determined independently and with minimal coupling. Then, the same inverse method was applied to reflectance measurements from a tissue simulating phantom and in vivo human skin. Oxygen saturation and total hemoglobin concentrations were estimated from the volar forearms of weakly and strongly pigmented subjects using a standard homogeneous model and the present two layer model.

  4. Whole Cell-SELEX Aptamers for Highly Specific Fluorescence Molecular Imaging of Carcinomas In Vivo

    PubMed Central

    He, Xiaoxiao; Guo, Qiuping; Wang, Kemin; Ye, Xiaosheng; Tang, Jinlu

    2013-01-01

    Background Carcinomas make up the majority of cancers. Their accurate and specific diagnoses are of great significance for the improvement of patients' curability. Methodology/Principal Findings In this paper, we report an effectual example of the in vivo fluorescence molecular imaging of carcinomas with extremely high specificity based on whole cell-SELEX aptamers. Firstly, S6, an aptamer against A549 lung carcinoma cells, was adopted and labeled with Cy5 to serve as a molecular imaging probe. Flow cytometry assays revealed that Cy5-S6 could not only specifically label in vitro cultured A549 cells in buffer, but also successfully achieve the detection of ex vivo cultured target cells in serum. When applied to in vivo imaging, Cy5-S6 was demonstrated to possess high specificity in identifying A549 carcinoma through a systematic comparison investigation. Particularly, after Cy5-S6 was intravenously injected into nude mice which were simultaneously grafted with A549 lung carcinoma and Tca8113 tongue carcinoma, a much longer retention time of Cy5-S6 in A549 tumor was observed and a clear targeted cancer imaging result was presented. On this basis, to further promote the application to imaging other carcinomas, LS2 and ZY8, which are two aptamers selected by our group against Bel-7404 and SMMC-7721 liver carcinoma cells respectively, were tested in a similar way, both in vitro and in vivo. Results showed that these aptamers were even effective in differentiating liver carcinomas of different subtypes in the same body. Conclusions/Significance This work might greatly advance the application of whole cell-SELEX aptamers to carcinomas-related in vivo researches. PMID:23950940

  5. P2A-Fluorophore Tagging of BRAF Tightly Links Expression to Fluorescence In Vivo

    PubMed Central

    McMahon, Martin

    2016-01-01

    The Braf proto-oncogene is a key component of the mitogen-activated protein kinase signaling cascade and is a critical regulator of both normal development and tumorigenesis in a variety of tissues. In order to elucidate BRAF’s differing roles in varying cell types, it is important to understand both the pattern and timing of BRAF expression. Here we report the production of a mouse model that links the expression of Braf with the bright red fluorescent protein, tdTomato. We have utilized a P2A knock-in strategy, ensuring that BRAF and the fluorophore are expressed from the same endogenous promoter and from the same bicistronic mRNA transcript. This mouse model (BrafTOM) shows bright red fluorescence in organs and cell types known to be sensitive to BRAF perturbation. We further show that on a cell-by-cell basis, fluorescence correlates with BRAF protein levels. Finally, we extend the utility of this mouse by demonstrating that the remnant P2A fragment attached to BRAF acts as a suitable epitope for immunoprecipitation and biochemical characterization of BRAF in vivo. PMID:27348307

  6. Development of in-vivo fluorescence imaging with the Matrix-Free method

    NASA Astrophysics Data System (ADS)

    Zacharopoulos, Athanasios; Garofalakis, Anikitos; Ripoll, Jorge; Arridge, Simon

    2010-11-01

    Non-contact Fluorescence Molecular Tomography is an emerging technique for imaging of fluorescent probes or proteins in live animals. One of the main characteristics of the non-contact acquisition systems in comparison to the usual fibre-based systems is the much denser boundary data sets that are created. When model-based reconstruction methods are used that rely on the inversion of a derivative operator, the large number of measurements poses a challenge since the explicit formulation and storage of the Jacobian matrix could be in general not feasible. In this paper we test a matrix-free method that addresses the problems of large data sets and reduces the computational cost and memory requirements for the reconstruction. More specifically we challenged the Matrix-Free method with in-vivo measurements from mice where fluorescence tubes of different but controlled concentrations are inserted, to assess the quantification performance of the method. We extended the test with simulations, using realistic geometries extracted from a mouse-atlas and including prior known information about the optical properties of tissue into the forward model.

  7. Fiber optic-based fluorescence detection system for in vivo studies of exogenous chromophore pharmacokinetics

    NASA Astrophysics Data System (ADS)

    Doiron, Daniel R.; Dunn, J. B.; Mitchell, W. L.; Dalton, Brian K.; Garbo, Greta M.; Warner, Jon A.

    1995-05-01

    The detection and quantification of the concentration of exogenous chromophores in-vivo by their fluorescence is complicated by many physical and geometrical parameters. Measurement of such signals is advantageous in determining the pharmacokinetics of photosensitizers such as those used in photodynamic therapy (PDT) or to assist in the diagnosis of tissue histological state. To overcome these difficulties a ratio based fiber optic contact fluorometer has been developed. This fluorescence detection system (FDS) uses the ratio of the fluorescence emission peak of the exogenous chromophore to that of endogenous chromophores, i.e. autofluorescence, to correct for a variety of parameters affecting the magnitude of the measured signals. By doing so it also minimizes the range of baseline measurements prior to exogenous drug injection, for various tissue types. Design of the FDS and results of its testing in animals and patients using the second generation photosensitizer Tin ethyletiopurpurin (SnET2) are presented. These results support the feasibility and usefulness of the Ratio FDS system.

  8. Compensation of optical heterogeneity-induced artifacts in fluorescence molecular tomography: theory and in vivo validation

    NASA Astrophysics Data System (ADS)

    Mohajerani, Pouyan; Adibi, Ali; Kempner, Joshua; Yared, Wael

    2009-05-01

    We present a method for reduction of image artifacts induced by the optical heterogeneities of tissue in fluorescence molecular tomography (FMT) through identification and compensation of image regions that evidence propagation of emission light through thin or low-absorption tunnels in tissue. The light tunneled as such contributes to the emission image as spurious components that might substantially overwhelm the desirable fluorescence emanating from the targeted lesions. The proposed method makes use of the strong spatial correlation between the emission and excitation images to estimate the tunneled components and yield a residual image that mainly consists of the signal due to the desirable fluorescence. This residual image is further refined using a coincidence mask constructed for each excitation-emission image pair. The coincidence mask is essentially a map of the ``hot spots'' that occur in both excitation and emission images, as such areas are often associated with tunneled emission. In vivo studies are performed on a human colon adenocarcinoma xenograft tumor model with subcutaneous tumors and a murine breast adenocarcinoma model with aggressive tumor cell metastasis and growth in the lungs. Results demonstrate significant improvements in the reconstructions achieved by the proposed method.

  9. Clinical approved fluorescent dyes coupled to endomicroscopy for in vivo diagnostic of peritoneal carcinomatosis

    NASA Astrophysics Data System (ADS)

    Abbaci, Muriel; Dartigues, Peggy; Soufan, Ranya; De Leeuw, Frederic; Fabre, Monique; Laplace-Builhé, Corinne

    2015-03-01

    Peritoneal carcinomatosis is metastatic stage aggravating digestive, gynecological or bladder cancer dissemination and the preoperative evaluation of lesions remains difficult. There is therefore a need for minimal invasive innovative techniques to establish a precise preoperative assessment of cancer peritoneal cavity. Probe-based confocal laser endomicroscopy (pCLE) provides dynamic images of the microarchitecture of tissues during an endoscopy. The PERSEE project proposes new developments in robotics and pCLE for the exploration of the peritoneal cavity during laparoscopy. Two fluorescent dyes, Patent blue V and Indocyanine green have been evaluated on human ex vivo samples to improve the contrast of pCLE images. For a future implementation in clinical study, two topically staining protocols operable in vivo have been validated on 70 specimens from 25 patients with a peritoneal carcinomatosis. The specimens were then imaged by pCLE with an optical probe designed for the application. A histo-morphological correlative study was performed on 350 pCLE images and 70 standard histological preparations. All images were interpreted in a random way by two pathologists. Differential histological diagnostics such as normal peritoneum or pseudomyxoma could be recognized on fluorescence images. The statistical analysis of the correlative study is underway. These dyes already approved for human use are interesting for pCLE imaging because some micromorphological criteria look like to conventional histology and are readable by pathologist. Thus pCLE images using both dyes do not require a specific semiology unlike to what is described in the literature, for pCLE associated with fluorescein for the in vivo imaging of pancreatic cysts.

  10. Identifying the origins of microbially derived aquatic DOM using fluorescence spectroscopy.

    NASA Astrophysics Data System (ADS)

    Fox, Bethany; Thorn, Robin; Anesio, Alexandre; Reynolds, Darren

    2016-04-01

    Dissolved organic matter (DOM) in aquatic systems is an essential support of the microbial population and, therefore, of the entire aquatic ecosystem. Aquatic DOM is also key for global biogeochemical cycling of nutrients and connects land processes to the marine environment via hydrological transportation. There have been multiple advances in technological assessments of the characteristics of aquatic DOM, with spectroscopy becoming widely used. The extensive use of benchtop spectroscopic instruments has led to the development of in situ sensors, improving the spatiotemporal scale of data acquisition. Whilst this has greatly improved understanding of DOM characteristics and patterns, there are still unknown variables, parameters and interactions of DOM within the aquatic environment. In particular, the interactions of aquatic DOM with the microbial population is still mostly unidentified. It is generally accepted that certain DOM fluorescence regions are autochthonous and microbially derived, such as "peak T" fluorescence. However, the origins and metabolic pathways involved in the production and release of these fluorescent molecules is, as yet, not definitively known. Our work focuses on the identification of these metabolic pathways from whence this microbially derived DOM originates. The most recent stage of the research has utilised traditional microbiological techniques, such as growth curves and chemostat experiments, alongside DOM fluorescence spectroscopic analysis and flow cytometry. The information gained regarding the microbial production and processing of DOM is central for the development of novel in situ fluorescence technology, the ultimate aim of this project.

  11. Evaluating aggregation of gold nanoparticles and humic substances using fluorescence spectroscopy.

    PubMed

    Pallem, Vasanta L; Stretz, Holly A; Wells, Martha J M

    2009-10-01

    The fate and transport of diagnostic gold nanoparticles in surface waters would significantly depend on their interactions with humic substances, which are ubiquitously found in natural aquatic systems. The current study employs UV-visible absorbance and fluorescence spectroscopy to investigate the interactions of commercial humic acid (HA) with gold nanoparticles having a core size of 5 nm and coated with two different stabilizers, beta-D-glucose and citrate. Humic substances (HS) are fluorescent in nature, providing a unique probe of nanometer-scale morphological changes for interactions between these natural polyelectrolytes and water-soluble gold nanoparticles. Quenching of fluorescence intensity was observed with beta-D-glucose-coated gold nanoparticles, whereas an enhancement effect was noticed with the citrate-coated particles when mixed with HA having concentrations of 2 and 8 ppm (surface waters typically may contain approximately 10 ppm HS). Examining the quenching and enhancement of fluorescence provides insight into the structural changes taking place at the coated gold nanoparticle-HA interface. The quenching behavior suggested ligand exchange due to nanometer-scale contact between the HA and beta-D-glucose-coated gold nanoparticles, whereas the enhancement effect with citrate particles would indicate overcoating, leading to increased transfer distances for fluorescence resonance energy transfer. PMID:19848172

  12. Applications of fluorescence spectroscopy for predicting percent wastewater in an urban stream

    USGS Publications Warehouse

    Goldman, Jami H.; Rounds, Stewart A.; Needoba, Joseph A.

    2012-01-01

    Dissolved organic carbon (DOC) is a significant organic carbon reservoir in many ecosystems, and its characteristics and sources determine many aspects of ecosystem health and water quality. Fluorescence spectroscopy methods can quantify and characterize the subset of the DOC pool that can absorb and re-emit electromagnetic energy as fluorescence and thus provide a rapid technique for environmental monitoring of DOC in lakes and rivers. Using high resolution fluorescence techniques, we characterized DOC in the Tualatin River watershed near Portland, Oregon, and identified fluorescence parameters associated with effluent from two wastewater treatment plants and samples from sites within and outside the urban region. Using a variety of statistical approaches, we developed and validated a multivariate linear regression model to predict the amount of wastewater in the river as a function of the relative abundance of specific fluorescence excitation/emission pairs. The model was tested with independent data and predicts the percentage of wastewater in a sample within 80% confidence. Model results can be used to develop in situ instrumentation, inform monitoring programs, and develop additional water quality indicators for aquatic systems.

  13. Time-resolved fluorescence polarization spectroscopy of visible and near infrared dyes in picosecond dynamics

    NASA Astrophysics Data System (ADS)

    Pu, Yang; Alfano, Robert R.

    2015-03-01

    Near-infrared (NIR) dyes absorb and emit light within the range from 700 to 900 nm have several benefits in biological studies for one- and/or two-photon excitation for deeper penetration of tissues. These molecules undergo vibrational and rotational motion in the relaxation of the excited electronic states, Due to the less than ideal anisotropy behavior of NIR dyes stemming from the fluorophores elongated structures and short fluorescence lifetime in picosecond range, no significant efforts have been made to recognize the theory of these dyes in time-resolved polarization dynamics. In this study, the depolarization of the fluorescence due to emission from rotational deactivation in solution will be measured with the excitation of a linearly polarized femtosecond laser pulse and a streak camera. The theory, experiment and application of the ultrafast fluorescence polarization dynamics and anisotropy are illustrated with examples of two of the most important medical based dyes. One is NIR dye, namely Indocyanine Green (ICG) and is compared with Fluorescein which is in visible range with much longer lifetime. A set of first-order linear differential equations was developed to model fluorescence polarization dynamics of NIR dye in picosecond range. Using this model, the important parameters of ultrafast polarization spectroscopy were identified: risetime, initial time, fluorescence lifetime, and rotation times.

  14. On-chip integrated lensless fluorescence microscopy/spectroscopy module for cell-based sensors

    NASA Astrophysics Data System (ADS)

    Li, Wei; Knoll, Thorsten; Sossalla, Adam; Bueth, Heiko; Thielecke, Hagen

    2011-03-01

    The integration of a fluorescence microscopy/spectroscopy module in cell-based lab-on-a-chip systems is of high interest for applications in cell-based diagnostics and substance evaluation in situ. We present an on-chip integrated lensless fluorescence imaging module applying the principle of contact/proximate optical lithography. The pixel resolution is comparable with a 4 x objective microscope. The module can be used for morphology and fluorescence imaging of mammalian cells (15 - 20 μm) as well as for testing the concentration of a fluorescent substance. The biological samples or solutions are sustained in disposable sterilized microfluidic chips with 1 μm thick silicon nitride (Si3N4) membranes. These chips are assembled on the surface of a 5 megapixel colored CMOS image sensor array with 1.75 μm pixel size, which is coated with an additional interference filter. Each culturing chip consists of a MEMS cavity chip and a PDMS microfluidic interface. The surface of the CMOS image sensor is smoothened using SU-8 photoresist spin-coating for a commercial grade interference filter (optical density >= 5) coating by Plasma-Ion Assisted Deposition thereafter. The function is demonstrated by primary imaging results of the non-/fluorescent mammalian cells/microspheres as well as by differentiating different concentrations of FITC solutions.

  15. Construction, figures of merit, and testing of a single-cell fluorescence excitation spectroscopy system

    PubMed Central

    Hill, Laura S.; Richardson, Tammi L.; Profeta, Luisa T. M.; Shaw, Timothy J.; Hintz, Christopher J.; Twining, Benjamin S.; Lawrenz, Evelyn; Myrick, Michael L.

    2010-01-01

    Characterization of phytoplankton community composition is critical to understanding the ecology and biogeochemistry of the oceans. One approach to taxonomic characterization takes advantage of differing pigmentation between algal taxa and thus differences in fluorescence excitation spectra. Analyses of bulk water samples, however, may be confounded by interference from chromophoric dissolved organic matter or suspended particulate matter. Here, we describe an instrument that uses a laser trap based on a Nikon TE2000-U microscope to position individual phytoplankton cells for confocal fluorescence excitation spectroscopy, thus avoiding interference from the surrounding medium. Quantitative measurements of optical power give data in the form of photons emitted per photon of exposure for an individual phytoplankton cell. Residence times for individual phytoplankton in the instrument can be as long as several minutes with no substantial change in their fluorescence excitation spectra. The laser trap was found to generate two-photon fluorescence from the organisms so a modification was made to release the trap momentarily during data acquisition. Typical signal levels for an individual cell are in the range of 106 photons∕s of fluorescence using a monochromated 75 W Xe arc lamp excitation source with a 2% transmission neutral density filter. PMID:20113077

  16. Enhanced energy transfer in respiratory-deficient endothelial cells probed by microscopic fluorescence excitation spectroscopy

    NASA Astrophysics Data System (ADS)

    Schneckenburger, Herbert; Gschwend, Michael H.; Bauer, Manfred; Strauss, Wolfgang S. L.; Steiner, Rudolf W.

    1996-12-01

    Mitochondrial malfunction may be concomitant with changes of the redox states of the coenzymes nicotinamide adenine dinucleotide (NAD+/NADH), as well as flavin.mononucleotide or dinucleotide. The intrinsic fluorescence of these coenzymes was therefore proposed to be a measure of malfunction. Since mitochondrial fluorescence is strongly superposed by autofluorescence from various cytoplasmatic fluorophores, cultivated endothelial cells were incubated with the mitochondrial marker rhodamine 123 (R123), and after excitation of flavin molecules, energy transfer to R123 was investigated. Due to spectral overlap of flavin and R123 fluorescence, energy transfer flavin yields R123 could not be detected from their emission spectra. Therefore, the method of microscopic fluorescence excitation spectroscopy was established. When detecting R123 fluorescence, excitation maxima at 370 - 390 nm and 420-460 nm were assigned to flavins, whereas a pronounced excitation band at 465 - 490 nm was attributed to R123. Therefore, excitation at 475 nm reflected the intracellular concentration of R123, whereas excitation at 385 nm reflected flavin excitation with a subsequent energy transfer to R123 molecules. An enhanced energy transfer after inhibition of specific enzyme complexes of the respiratory chain is discussed in the present article.

  17. Fluorescence imaging and spectroscopy of ALA-induced protoporphyrin IX preferentially accumulated in tumor tissue

    NASA Astrophysics Data System (ADS)

    Stepp, Herbert G.; Baumgartner, Reinhold; Beyer, Wolfgang; Knuechel, Ruth; Koerner, T. O.; Kriegmair, M.; Rick, Kai; Steinbach, Pia; Hofstetter, Alfons G.

    1995-12-01

    In a clinical pilot study performed on 104 patients suffering from bladder cancer it could be shown that intravesical instillation of a solution of 5-aminolevulinic acid (5-ALA) induces a tumorselective accumulation of Protoporphyrin IX (PPIX). Malignant lesions could be detected with a sensitivity of 97% and a specificity of 67%. The Kr+-laser as excitation light source could successfully be replaced by a filtered short arc Xe-lamp. Its emission wavelength band (375 nm - 440 nm) leads to an efficiency of 58% for PPIX- excitation compared to the laser. Two-hundred-sixty mW of output power at the distal end of a slightly modified cystoscope could be obtained. This is sufficient for recording fluorescence images with a target integrating color CCD-camera. Red fluorescence and blue remitted light are displayed simultaneously. Standard white light observation is possible with the same instrumentation. Pharmacokinetic measurements were performed on 18 patients after different routes of 5-ALA application (oral, inhalation and intravesical instillation). PPIX-fluorescence measurements were made on the skin and on the blood plasma. Pharmacokinetic of 5-ALA could be performed on blood plasma. Endoscopical florescence spectroscopy showed the high fluorescence contrast between tumor and normal tissue with a mean value of 10.7. Forthcoming clinical multicenter studies require an objective measure of the fluorescence intensity. Monte Carlo computer simulations showed that artifacts due to observation geometry and varying absorption can largely be reduced by ratioing fluorescence (red channel of camera) to remission (blue channel). Real time image ratioing provides false color images with a reliable fluorescence information.

  18. Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy.

    PubMed

    Thompson, N L; Burghardt, T P; Axelrod, D

    1981-03-01

    The theoretical basis of a new technique for measuring equilibrium adsorption/desorption kinetics and surface diffusion of fluorescent-labeled solute molecules at solid surfaces has been developed. The technique combines total internal reflection fluorescence (TIR) with either fluorescence photobleaching recovery (FPR) or fluorescence correlation spectroscopy (FCS). A laser beam totally internally reflects at a solid/liquid interface; the shallow evanescent field in the liquid excites the fluorescence of surface adsorbed molecules. In TIR/FPR, adsorbed molecules are bleaching by a flash of the focused laser beam; subsequent fluorescence recovery is monitored as bleached molecules exchange with unbleached ones from the solution or surrounding nonilluminated regions of the surface. In TIR/FCS, spontaneous fluorescence fluctuations due to individual molecules entering and leaving a well-defined portion of the evanescent field are autocorrelated. Under appropriate experimental conditions, the rate constants and surface diffusion coefficient can be readily obtained from the TIR/FPR and TIR/FCS curves. In general, the shape of the theoretical TIR/FPR and TIR/FCS curves depends in a complex manner upon the bulk and surface diffusion coefficients, the size of the iluminated or observed region, and the adsorption/desorption/kinetic rate constants. The theory can be applied both to specific binding between immobilized receptors and soluble ligands, and to nonspecific adsorption processes. A discussion of experimental considerations and the application of this technique to the adsorption of serum proteins on quartz may be found in the accompanying paper (Burghardt and Axelrod. 1981. Biophys. J. 33:455). PMID:7225515

  19. Characterization of dissolved organic matter in fogwater by excitation-emission matrix fluorescence spectroscopy

    USGS Publications Warehouse

    Birdwell, J.E.; Valsaraj, K.T.

    2010-01-01

    Dissolved organic matter (DOM) present in fogwater samples collected in southeastern Louisiana and central-eastern China has been characterized using excitation-emission matrix fluorescence spectroscopy. The goal of the study was to illustrate the utility of fluorescence for obtaining information on the large fraction of organic carbon in fogwaters (typically >40% by weight) that defies characterization in terms of specific chemical compounds without the difficulty inherent in obtaining sufficient fogwater volume to isolate DOM for assessment using other spectroscopic and chemical analyses. Based on the findings of previous studies using other characterization methods, it was anticipated that the unidentified organic carbon fraction would have characteristic peaks associated with humic substances and fluorescent amino acids. Both humic- and protein-like fluorophores were observed in the fogwater spectra and fluorescence-derived indices for the fogwater had similar values to those of soil and sediment porewater. Greater biological character was observed in samples with higher organic carbon concentrations. Fogwaters are shown to contain a mixture of terrestrially- and microbially-derived fluorescent organic material, which is expected to be derived from an array of different sources, such as suspended soil and dust particles, biogenic emissions and organic substances generated by atmospheric processes. The fluorescence results indicate that much of the unidentified organic carbon present in fogwater can be represented by humic-like and biologically-derived substances similar to those present in other aquatic systems, though it should be noted that fluorescent signatures representative of DOM produced by atmospheric processing of organic aerosols may be contributing to or masked by humic-like fluorophores. ?? 2010.

  20. Fluorescence Imaging In Vivo at Wavelengths beyond 1500 nm.

    PubMed

    Diao, Shuo; Blackburn, Jeffrey L; Hong, Guosong; Antaris, Alexander L; Chang, Junlei; Wu, Justin Z; Zhang, Bo; Cheng, Kai; Kuo, Calvin J; Dai, Hongjie

    2015-12-01

    Compared to imaging in the visible and near-infrared regions below 900 nm, imaging in the second near-infrared window (NIR-II, 1000-1700 nm) is a promising method for deep-tissue high-resolution optical imaging in vivo mainly owing to the reduced scattering of photons traversing through biological tissues. Herein, semiconducting single-walled carbon nanotubes with large diameters were used for in vivo fluorescence imaging in the long-wavelength NIR region (1500-1700 nm, NIR-IIb). With this imaging agent, 3-4 μm wide capillary blood vessels at a depth of about 3 mm could be resolved. Meanwhile, the blood-flow speeds in multiple individual vessels could be mapped simultaneously. Furthermore, NIR-IIb tumor imaging of a live mouse was explored. NIR-IIb imaging can be generalized to a wide range of fluorophores emitting at up to 1700 nm for high-performance in vivo optical imaging. PMID:26460151

  1. Fluorescence and UV/VIS absorption spectroscopy studies on polymer blend films for photovoltaics

    NASA Astrophysics Data System (ADS)

    van Stam, Jan; Lindqvist, Camilla; Hansson, Rickard; Ericsson, Leif; Moons, Ellen

    2015-08-01

    The quinoxaline-based polymer TQ1 (poly[2,3-bis-(3-octyloxyphenyl)quinoxaline-5,8-diyl-alt-thiophene-2,5- diyl]) is a promising candidate as electron donor in organic solar cells. In combination with the electron acceptor [6,6]- phenyl-C71- butyric acid methyl ester (PC70BM), TQ1 has resulted in solar cells with power conversion efficiencies of 7 %. We have studied TQ1 films, with and without PC70BM, spin-casted from different solvents, by fluorescence spectroscopy and UV/VIS absorption spectroscopy. We used chloroform (CF), chlorobenzene (CB), and odichlorobenzene (o-DCB) as solvents for the coating solutions and 1-chloronaphthalene (CN) as solvent additive. CN addition has been shown to enhance photo-conversion efficiency of these solar cells. Phase-separation causes lateral domain formation in the films and the domain size depends on the solvent . These morphological differences coincide with changes in the spectroscopic patterns of the films. From a spectroscopic point of view, TQ1 acts as fluorescent probe and PC70BM as quencher. The degree of fluorescence quenching is coupled to the morphology through the distance between TQ1 and PC70BM. Furthermore, if using a bad solvent for PC70BM, morphological regions rich in the fullerene yield emission characteristic for aggregated PC70BM. Clear differences were found, comparing the TQ1:PC70BM blend films casted from different solvents and at different ratios between the donor and acceptor. The morphology also influences the UV/VIS absorption spectra, yielding further information on the composition. The results show that fluorescence and UV/VIS absorption spectroscopy can be used to detect aggregation in blended films and that these methods extend the morphological information beyond the scale accessible with microscopy.

  2. A novel indocyanine green nanoparticle probe for non invasive fluorescence imaging in vivo

    NASA Astrophysics Data System (ADS)

    Navarro, Fabrice P.; Berger, Michel; Goutayer, Mathieu; Guillermet, Stéphanie; Josserand, Véronique; Rizo, Philippe; Vinet, Françoise; Texier, Isabelle

    2009-02-01

    Fluorescence imaging (FLI) allows the in vivo monitoring of biological events associated with disease and represents a new promising tool for drug discovery. In particular, it speeds up the development and assessment of new therapies in oncology, helps in diagnosis, and improves surgery by fluorescence-guided tumor resection. This technique is highly sensitive, non-ionizing, easy to use and relatively inexpensive. Nevertheless, the main limitation of FLI lies in the optical properties of biological tissues. Mainly because of haemoglobin and water absorption, only near-infrared (NIR) light is adapted to image tissues in depth. Using a contrasting agent absorbing and emitting in the NIR region is therefore necessary to improve the background signal ratio, and thus the image contrast. Among many commercially available NIR optical contrast agents, only indocyanine green (ICG), has been approved by the United State Food and Drug Administration (FDA) for various medical applications. However, its instability (photo-degradation, thermal-degradation and low aqueous solubility) limits its applications as a fluorescent probe for imaging purposes. In order to improve the effectiveness of ICG, we engineered ICG-doped lipid nanoparticles (LNP). In this communication, we will report the design of these novel fluorescent nanoparticle probes. These low cost nanocarriers have numerous advantages, including their high chemical stability and biocompatibility. The characterization of the optical properties of the nanoparticles entrapping ICG will also be discussed. Finally, the biodistribution in mice of ICG when delivered through nanoparticles in comparison to free ICG in solution is presented. It demonstrates the efficient accumulation of ICG-doped nanoparticles in the tumor site.

  3. A simple preparation of Ag@graphene nanocomposites for surface-enhanced Raman spectroscopy of fluorescent anticancer drug

    NASA Astrophysics Data System (ADS)

    Meng, Ying; Yan, Xueying; Wang, Yi

    2016-05-01

    A simple method was developed to synthesize Ag@graphene nanocomposites with rough Ag nanoparticles (AgNPs) conjugated with graphene nanosheets, and the nanocomposites could be used as substrates for effective surface-enhanced Raman spectroscopy (SERS) of fluorescent anticancer drug (Dox) since they could not only enhance the Raman signals but also suppress the fluorescent signals.

  4. Fluorescence spectroscopy for the detection of potentially malignant disorders of the oral cavity: analysis of 30 cases

    NASA Astrophysics Data System (ADS)

    Francisco, A. L. N.; Correr, W. R.; Azevedo, L. H.; Galletta, V. K.; Pinto, C. A. L.; Kowalski, L. P.; Kurachi, C.

    2014-01-01

    Oral cancer is a major health problem worldwide and although early diagnosis of potentially malignant and malignant diseases is associated with better treatment results, a large number of cancers are initially misdiagnosed, with unfortunate consequences for long-term survival. Fluorescence spectroscopy is a noninvasive modality of diagnostic approach using induced fluorescence emission in tumors that can improve diagnostic accuracy. The objective of this study was to determine the ability to discriminate between normal oral mucosa and potentially malignant disorders by fluorescence spectroscopy. Fluorescence investigation under 408 and 532 nm excitation wavelengths was performed on 60 subjects, 30 with potentially malignant disorders and 30 volunteers with normal mucosa. Data was analyzed to correlate fluorescence patterns with clinical and histopathological diagnostics. Fluorescence spectroscopy used as a point measurement technique resulted in a great variety of spectral information. In a qualitative analysis of the fluorescence spectral characteristics of each type of injury evaluated, it was possible to discriminate between normal and abnormal oral mucosa. The results show the potential use of fluorescence spectroscopy for an improved discrimination of oral disorders.

  5. A reusable sensor for the label-free detection of specific oligonucleotides by surface plasmon fluorescence spectroscopy.

    PubMed

    Nöll, Gilbert; Su, Qiang; Heidel, Björn; Yu, Yaming

    2014-01-01

    The development of a reusable molecular beacon (MB)-based sensor for the label-free detection of specific oligonucleotides using surface plasmon fluorescence spectroscopy (SPFS) as the readout method is described. The MBs are chemisorbed at planar gold surfaces serving as fluorescence quenching units. Target oligonucleotides of 24 bases can be detected within a few minutes at high single-mismatch discrimination rates.

  6. Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways

    NASA Astrophysics Data System (ADS)

    Shirshin, Evgeny; Cherkasova, Olga; Tikhonova, Tatiana; Berlovskaya, Elena; Priezzhev, Alexander; Fadeev, Victor

    2015-05-01

    We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation.

  7. Integrated fingerprint and high wavenumber confocal Raman spectroscopy for in vivo diagnosis of cervical precancer

    NASA Astrophysics Data System (ADS)

    Duraipandian, Shiyamala; Zheng, Wei; Ng, Joseph; Low, Jeffrey J. H.; Ilancheran, A.; Huang, Zhiwei

    2013-03-01

    Raman spectroscopy is a vibrational spectroscopic technique capable of optically probing the compositional, conformational, and structural changes in the tissue associated with disease progression. The main goal of this work is to develop an integrated fingerprint (FP) and high wavenumber (HW) in vivo confocal Raman spectroscopy for simultaneous FP/HW tissue Raman spectral measurements. This work further explores the potential of integrated FP/HW Raman spectroscopy developed as a diagnostic tool for in vivo detection of cervical precancer. A total of 473 in vivo integrated FP/HW Raman spectra (340 normal and 133 precancer) were acquired from 35 patients within 1 s during clinical colposcopy. The major tissue Raman peaks are noticed around 854, 937, 1001, 1095, 1253, 1313, 1445, 1654, 2946 and 3400 cm-1, related to the molecular changes (e.g., proteins, lipids, glycogen, nucleic acids, water, etc.) that accompany the dysplastic transformation of tissue. The FP (800 - 1800 cm-1), HW (2800 - 3800 cm-1) and the integrated FP/HW Raman spectra were analyzed using partial least squares-discriminant analysis (PLS-DA) together with the leave-one patient-out, cross-validation. The developed PLS-DA classification models and receiver operating characteristics (ROC) curves for the FP, HW and integrated FP/HW spectroscopy further discloses that the performance of integrated FP/HW Raman spectroscopy is superior to that of all others in discriminating the dysplastic cervix. The results of this work indicate that the co-contributions of underlying rich biochemical information revealed by the complementary spectral modalities (FP and HW Raman) can improve the in vivo early diagnosis of cervical precancer at clinical colposcopy

  8. Fluorescence spectroscopy of soil pellets : The use of CP/PARAFAC.

    NASA Astrophysics Data System (ADS)

    Mounier, Stéphane; Nicolodeli, Gustavo; Redon, Roland; Hacherouf, Kalhed; Milori, Debora M. B. P.

    2014-05-01

    Fluorescence spectroscopy is one of the most sensitive techniques available for analytical purposes. It is relatively easy to implement, phenomenologically straightforward and well investigated. Largely non-invasive and fast, so that it can be useful for environmental applications. Fluorescence phenomenon is highly probable in molecular systems containing atoms with lone pairs of electrons such as C=O, aromatic, phenolic, quinone and more rigid unsaturated conjugated systems. These functional groups are present in humic substances (HS) from soils (Senesi, 1990; N. Senesi et al., 1991) and represent the main fluorophors of Soil Organic Matter (SOM). The extension of the conjugated electronic system, the level of heteroatom substitution and type and number of substituting groups under the aromatic rings strongly affect the intensity and wavelength of molecular fluorescence. However, to analyse the SOM it is generally done a chemical extraction that allows measuring the fluorescence response of the liquid extract. To avoid this fractionation of the SOM, Milori et al. (2006) proposed the application of laser induced fluorescence spectroscopy (LIFS) in whole soil. This work intends to assess the technical feasibility of 3D fluorescence spectroscopy using lamp for excitation to analyse solids opaque samples prepared with different substances. Seventy four (74) solid samples were prepared from different mixtures of boric acid (BA), humic substance acid and tryptophan (TRP) powder. The compounds were mixture and a pellet was done by using pressure (8 ton). The pellets were measured using a spectrofluorimeter HITACHI F4500, and a 3D fluorescence tensor was done from emission spectra (200-600 nm) with excitation range from 200 to 500 nm. The acquisition parameters were: step at 5 nm, scan speed at 2400 nm.min-1, response time at 0.1 s, excitation and emission slits at 5 nm and photomultiplier voltage at 700 V. Furthermore, measures of Laser-induced Fluorescence were

  9. Spectrally programmable light engine for in vitro or in vivo molecular imaging and spectroscopy

    NASA Astrophysics Data System (ADS)

    MacKinnon, Nicholas; Stange, Ulrich; Lane, Pierre; Macaulay, Calum; Quatrevalet, Mathieu

    2005-04-01

    A spectrally and temporally programmable light engine can create any spectral profile for hyperspectral, fluorescence, or principal-component imaging or with medical photonics devices employing spectroscopy, microscopy, and endoscopy. Multispectral imaging feasibility was demonstrated by capturing nine images at wavelengths from 450 to 650 mm (25-nm FWHM) with a CCD-camera-equipped bronchoscope coupled to the light engine. Selected wavelength regions were combined to produce a color endoscopy image.

  10. Objective Assessment of Endogenous Collagen In Vivo during Tissue Repair by Laser Induced Fluorescence

    PubMed Central

    Prabhu, Vijendra; Rao, Satish B. S.; Fernandes, Edward Mark; Rao, Anuradha C. K.; Prasad, Keerthana; Mahato, Krishna K.

    2014-01-01

    Collagen, a triple helical protein with the primary role of mechanical function, provides tensile strength to the skin, and plays a pivotal task in tissue repair. During tissue regeneration, collagen level increases gradually and therefore, monitoring of such changes in vivo by laser induced fluorescence was the main objective behind the present study. In order to accomplish this, 15 mm diameter excisional wounds were created on six to eight week old Swiss albino mice. The collagen deposition accelerated upon irradiation of single exposure of 2 J/cm2 He-Ne laser dose immediately after wounding was recorded by laser induced autofluorescence in vivo along with un-illuminated and un-wounded controls. Autofluorescence spectra were recorded for each animal of the experimental groups on 0, 5, 10, 30, 45 and 60 days post-wounding, by exciting the granulation tissue/skin with 325 nm He-Cd laser. The variations in the average collagen intensities from the granulation tissue/skin of mice were inspected as a function of age and gender. Further, the spectral findings of the collagen synthesis in wound granulation tissue/un-wounded skin tissues were validated by Picro-Sirius red- polarized light microscopy in a blinded manner through image analysis of the respective collagen birefringence. The in vivo autofluorescence studies have shown a significant increase in collagen synthesis in laser treated animals as compared to the un-illuminated controls. Image analysis of the collagen birefringence further authenticated the ability of autofluorescence in the objective monitoring of collagen in vivo. Our results clearly demonstrate the potential of laser induced autofluorescence in the monitoring of collegen synthesis during tissue regeneration, which may have clinical implications. PMID:24874229

  11. Objective assessment of endogenous collagen in vivo during tissue repair by laser induced fluorescence.

    PubMed

    Prabhu, Vijendra; Rao, Satish B S; Fernandes, Edward Mark; Rao, Anuradha C K; Prasad, Keerthana; Mahato, Krishna K

    2014-01-01

    Collagen, a triple helical protein with the primary role of mechanical function, provides tensile strength to the skin, and plays a pivotal task in tissue repair. During tissue regeneration, collagen level increases gradually and therefore, monitoring of such changes in vivo by laser induced fluorescence was the main objective behind the present study. In order to accomplish this, 15 mm diameter excisional wounds were created on six to eight week old Swiss albino mice. The collagen deposition accelerated upon irradiation of single exposure of 2 J/cm2 He-Ne laser dose immediately after wounding was recorded by laser induced autofluorescence in vivo along with un-illuminated and un-wounded controls. Autofluorescence spectra were recorded for each animal of the experimental groups on 0, 5, 10, 30, 45 and 60 days post-wounding, by exciting the granulation tissue/skin with 325 nm He-Cd laser. The variations in the average collagen intensities from the granulation tissue/skin of mice were inspected as a function of age and gender. Further, the spectral findings of the collagen synthesis in wound granulation tissue/un-wounded skin tissues were validated by Picro-Sirius red- polarized light microscopy in a blinded manner through image analysis of the respective collagen birefringence. The in vivo autofluorescence studies have shown a significant increase in collagen synthesis in laser treated animals as compared to the un-illuminated controls. Image analysis of the collagen birefringence further authenticated the ability of autofluorescence in the objective monitoring of collagen in vivo. Our results clearly demonstrate the potential of laser induced autofluorescence in the monitoring of collegen synthesis during tissue regeneration, which may have clinical implications. PMID:24874229

  12. Tissue classification and diagnostics using a fiber probe for combined Raman and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Cicchi, Riccardo; Anand, Suresh; Crisci, Alfonso; Giordano, Flavio; Rossari, Susanna; De Giorgi, Vincenzo; Maio, Vincenza; Massi, Daniela; Nesi, Gabriella; Buccoliero, Anna Maria; Guerrini, Renzo; Pimpinelli, Nicola; Pavone, Francesco S.

    2015-07-01

    Two different optical fiber probes for combined Raman and fluorescence spectroscopic measurements were designed, developed and used for tissue diagnostics. Two visible laser diodes were used for fluorescence spectroscopy, whereas a laser diode emitting in the NIR was used for Raman spectroscopy. The two probes were based on fiber bundles with a central multimode optical fiber, used for delivering light to the tissue, and 24 surrounding optical fibers for signal collection. Both fluorescence and Raman spectra were acquired using the same detection unit, based on a cooled CCD camera, connected to a spectrograph. The two probes were successfully employed for diagnostic purposes on various tissues in a good agreement with common routine histology. This study included skin, brain and bladder tissues and in particular the classification of: malignant melanoma against melanocytic lesions and healthy skin; urothelial carcinoma against healthy bladder mucosa; brain tumor against dysplastic brain tissue. The diagnostic capabilities were determined using a cross-validation method with a leave-one-out approach, finding very high sensitivity and specificity for all the examined tissues. The obtained results demonstrated that the multimodal approach is crucial for improving diagnostic capabilities. The system presented here can improve diagnostic capabilities on a broad range of tissues and has the potential of being used for endoscopic inspections in the near future.

  13. The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy

    PubMed Central

    Kim, Jiho; Doose, Sören; Neuweiler, Hannes; Sauer, Markus

    2006-01-01

    Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC–dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC–dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC–dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides. PMID:16687657

  14. Fluorescence light suppression in Raman spectroscopy using ultrafast time-gated CCD camera

    NASA Astrophysics Data System (ADS)

    Martyshkin, Dmitri V.; Ahuja, Ramesh C.; Kudriavtsev, Anatoliy; Mirov, Sergey B.

    2004-06-01

    A high level of fluorescence background signal rejection was achieved for solid and powder samples by using a combination of simple low-resolution spectrograph and ultrafast intensified/gated CCD camera. The unique timing characteristics of CCD camera match exceptionally well characteristics of Ti:sapphire oscillator allowing fast gated light detection at a repetition rate of up to 110 MHz, making this approach superior in terms of duty cycle in comparison with other time-resolved Raman techniques. The achieved temporal resolution was about 150 ps under 785 nm Ti:sapphire laser excitation. At an average excitation power up to 300 mW there was no noticeable sample damage observed. The strong Hexobenzocoronane (HBC) fluorescence with a lifetime about 2.1 ns was efficiently rejected and Raman spectrum revealed. The combination of spectrometer and ultrafast gated CCD camera allows simultaneous study of spectral and temporal characteristics of emitted light for the fluorophores with a fluorescence lifetime in nanosecond range. It is particularly important in biomedical spectroscopy, since the majority of endogenous fluorophores has a relatively short lifetime of about 1-5 ns. This capability opens an exciting possibility to build a universal instrument for solving multitask problems in applied laser spectroscopy.

  15. [Commercial orange juice beverages detection by fluorescence spectroscopy combined with PCA-ED and PLSR methods].

    PubMed

    Hu, Yang-jun; Zhu, Chun; Chen, Guo-qing; Zhang, Yong; Kong, Fan-biao; Li, Run; Zhu, Zhuo-wei; Wang, Xu; Gao, Shu-mei

    2014-08-01

    In order to classify the orange juiice beverages effectively, the fluorescence character differences of two kinds of orange juice beverages including 100% orange juice and orange drink were analyzed and compared, principal component analysis combined with Euclidean distance was adopted to classify two kinds of orange juice beverages, and ideal classification results were obtained. Meanwhile, the orange juice content estimation model was established by using fluorescence spectroscopy combined with partial least squares regression method, and the correlation coefficient R, root mean square error of calibration RMSEC and root mean square error of prediction RMSEP were 0.997, 0.87% and 2.05%, respectively. The experimental results indicate that the calibration model offers comparatively accurate content estimation, which reflect the actual orange juice content in the commercial orange juice beverages. The exploration to classify orange juice beverages was carried out from two aspects of qualitative and quantitative analysis by employing fluorescence spectroscopy combined with chemometrics method, which can provide a new idea for the classification and adulteration detection of commercial orange juice beverages, and also can give certain reference basis for the quality control of orange juice raw material.

  16. Fluorescence excitation-emission matrix spectroscopy as a tool for determining quality of sparkling wines.

    PubMed

    Elcoroaristizabal, Saioa; Callejón, Raquel M; Amigo, Jose M; Ocaña-González, Juan A; Morales, M Lourdes; Ubeda, Cristina

    2016-09-01

    Browning in sparkling wines was assessed by the use of excitation-emission fluorescence spectroscopy combined with PARAllel FACtor analysis (PARAFAC). Four different cava sparkling wines were monitored during an accelerated browning process and subsequently storage. Fluorescence changes observed during the accelerated browning process were monitored and compared with other conventional parameters: absorbance at 420nm (A420) and the content of 5-hydroxymethyl-2-furfural (5-HMF). A high similarity of the spectral profiles for all sparkling wines analyzed was observed, being explained by a four component PARAFAC model. A high correlation between the third PARAFAC factor (465/530nm) and the commonly used non-enzymatic browning indicators was observed. The fourth PARAFAC factor (280/380nm) gives us also information about the browning process following a first order kinetic reaction. Hence, excitation-emission fluorescence spectroscopy, together with PARAFAC, provides a faster alternative for browning monitoring to conventional methods, as well as useful key indicators for quality control. PMID:27041327

  17. Study of the interaction between N-confused porphyrin and bovine serum albumin by fluorescence spectroscopy.

    PubMed

    Yu, Xianyong; Liu, Ronghua; Yi, Rongqiong; Yang, Fengxian; Huang, Haowen; Chen, Jian; Ji, Danhong; Yang, Ying; Li, Xiaofang; Yi, Pinggui

    2011-04-01

    The fluorescence and ultraviolet spectroscopy were explored to study the interaction between N-confused porphyrins (NCP) and bovine serum albumin (BSA) under imitated physiological condition. The experimental results indicated that the fluorescence quenching mechanism between BSA and NCP was static quenching procedure at low NCP concentration at 293 and 305 K or a combined quenching (static and dynamic) procedure at higher NCP concentration at 305 K. The binding constants, binding sites and the corresponding thermodynamic parameters ΔH, ΔS, and ΔG were calculated at different temperatures. The comparison of binding potency of the three NCP to BSA showed that the substituting groups in benzene ring could enhance the binding affinity. From the thermodynamic parameters, we concluded that the action force was mainly hydrophobic interaction. The binding distances between NCP and BSA were calculated using Förster non-radiation energy transfer theory. In addition, the effect of NCP on the conformation of BSA was analyzed using synchronous fluorescence spectroscopy.

  18. Investigation of the Dimerization of Proteins from the Epidermal Growth Factor Receptor Family by Single Wavelength Fluorescence Cross-Correlation Spectroscopy

    PubMed Central

    Liu, Ping; Sudhaharan, Thankiah; Koh, Rosita M. L.; Hwang, Ling C.; Ahmed, Sohail; Maruyama, Ichiro N.; Wohland, Thorsten

    2007-01-01

    Single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS), introduced to study biomolecular interactions, has recently been reported to monitor enzyme activity by using a newly developed fluorescent protein variant together with cyan fluorescent protein. Here, for the first time to our knowledge, SW-FCCS is applied to detect interactions between membrane receptors in vivo by using the widely used enhanced green fluorescent protein and monomeric red fluorescent protein. The biological system studied here is the epidermal growth factor/ErbB receptor family, which plays pivotal roles in the development of organisms ranging from worms to humans. It is widely thought that a ligand binds to the monomeric form of the receptor and induces its dimeric form for activation. By using SW-FCCS and Förster resonance energy transfer, we show that the epidermal growth factor receptor and ErbB2 have preformed homo- and heterodimeric structures on the cell surface and quantitation of dimer fractions is performed by SW-FCCS. These receptors are major targets of anti-cancer drug development, and the receptors' homo- and heterodimeric structures are relevant for such developments. PMID:17468161

  19. Study of the potential of stratum corneum lipids and exogenous molecules interaction by fluorescence spectroscopy for the estimation of percutaneous penetration.

    PubMed

    Jungman, Elsa; Laugel, Cécile; Kasselouri, Athéna; Baillet-Guffroy, Arlette

    2012-09-15

    Considering that the skin barrier properties are closely linked to the ceramides composition and conformation within the SC, our work focused on developing a new evaluation criterion in complement of the Log Pow and MW: lipids retentive role within the SC. We developed an in vitro model to study exogenous molecules (Mol) and SC lipids interaction by fluorescence spectroscopy. As ceramides do not fluoresce, fluorescence probes that emit a fluorescence signal in contact with lipidic chains were selected for the study. A protocol was developed based on the exogenous molecule (cosmetic actives) affinity for the SC lipids. A fluorescence criterion (ΔI) was calculated from our results and compared to ex vivo skin penetration measurements realized with a Franz cell device. Our results indicated that polarity seems to be very representative of the ceramide and exogenous molecule interaction for most of the molecules tested. However, the ΔI calculated highlighted the particular interaction of some exogenous molecules with ceramides and their skin distribution. This particular behavior was not initially possible to estimate with the Log Pow and MW. This work aimed to develop a new alternative method to enhance the percutaneous penetration estimation of exogenous molecules for the risk analysis.

  20. Amyloid-β Deposits Target Efficient Near-Infrared Fluorescent Probes: Synthesis, in Vitro Evaluation, and in Vivo Imaging.

    PubMed

    Fu, Hualong; Tu, Peiyu; Zhao, Liu; Dai, Jiapei; Liu, Boli; Cui, Mengchao

    2016-02-01

    The formation of extracellular amyloid-β (Aβ) plaques is a common molecular change that underlies several debilitating human conditions, including Alzheimer's disease (AD); however, the existing near-infrared (NIR) fluorescent probes for the in vivo detection of Aβ plaques are limited by undesirable fluorescent properties and poor brain kinetics. In this work, we designed, synthesized, and evaluated a new family of efficient NIR probes that target Aβ plaques by incorporating hydroxyethyl groups into the ligand structure. Among these probes, DANIR 8c showed excellent fluorescent properties with an emission maximum above 670 nm upon binding to Aβ aggregates and also displayed a high sensitivity (a 629-fold increase in fluorescence intensity) and affinity (Kd = 14.5 nM). Because of the improved hydrophilicity that was induced by hydroxyls, 8c displayed increased initial brain uptake and a fast washout from the brain, as well as an acceptable biostability in the brain. In vivo NIR fluorescent imaging revealed that 8c could efficiently distinguish between AD transgenic model mice and normal controls. Overall, 8c is an efficient and veritable NIR fluorescent probe for the in vivo detection of Aβ plaques in the brain. PMID:26717442

  1. In vivo Raman spectroscopy for breast cancer: diagnosis in animal model

    NASA Astrophysics Data System (ADS)

    Bitar, R.; Martins, M. A.; Ribeiro, D.; Carvalho, C.; Santos, E. A. P.; Ramalho, L. N. Z.; Ramalho, F.; Martinho, H.; Martin, A. A.

    2008-02-01

    Raman spectroscopy has been well established as a powerful method for studying biological tissues and diagnosing diseases. In this study we have developed a breast cancer animal model and collected in vivo Raman spectra of mammary glands of 27 Sprague-Dawley female rats treated with DMBA and 5 non-treated used as control group. A dispersive Raman spectrometer with a @785 nm laser excitation coupled a fiber optic probe and a CCD detector was used to obtain the spectra. The obtained in vivo transcutaneous Raman spectra have shown important differences between normal and abnormal tissues when acquired from one side to the other side of the lesion.

  2. High pressure sample cell for total internal reflection fluorescence spectroscopy at pressures up to 2500 bar

    NASA Astrophysics Data System (ADS)

    Koo, Juny; Czeslik, Claus

    2012-08-01

    Total internal reflection fluorescence (TIRF) spectroscopy is a surface sensitive technique that is widely used to characterize the structure and dynamics of molecules at planar liquid-solid interfaces. In particular, biomolecular systems, such as protein adsorbates and lipid membranes can easily be studied by TIRF spectroscopy. Applying pressure to molecular systems offers access to all kinds of volume changes occurring during assembly of molecules, phase transitions, and chemical reactions. So far, most of these volume changes have been characterized in bulk solution, only. Here, we describe the design and performance of a high pressure sample cell that allows for TIRF spectroscopy under high pressures up to 2500 bar (2.5 × 108 Pa), in order to expand the understanding of volume effects from the bulk phase to liquid-solid interfaces. The new sample cell is based on a cylindrical body made of Nimonic 90 alloy and incorporates a pressure transmitting sample cuvette. This cuvette is composed of a fused silica prism and a flexible rubber gasket. It contains the sample solution and ensures a complete separation of the sample from the liquid pressure medium. The sample solution is in contact with the inner wall of the prism forming the interface under study, where fluorescent molecules are immobilized. In this way, the new high pressure TIRF sample cell is very useful for studying any biomolecular layer that can be deposited at a planar water-silica interface. As examples, high pressure TIRF data of adsorbed lysozyme and two phospholipid membranes are presented.

  3. In Vivo X-Ray Fluorescence Microtomographic Imaging of Elements in Single-Celled Fern Spores

    SciTech Connect

    Hirai, Yasuharu; Yoneyama, Akio; Hisada, Akiko; Uchida, Kenko

    2007-01-19

    We have observed in vivo three-dimensional distributions of constituent elements of single-celled spores of the fern Adiantum capillus-veneris using an X-ray fluorescence computed microtomography method. The images of these distributions are generated from a series of slice data, each of which is acquired by a sample translation-rotation method. An incident X-ray microbeam irradiates the sample with a spot size of 1 {mu}m. The high Ca concentration in the testa and the localized and overlapping Fe and Zn concentrations inside the spore are shown in three-dimensional images. The K concentration is high throughout the cell, and there are localized regions of higher density. The atomic number densities of these elements in the testa and inside the cell in a tomographic slice are estimated with a resolution of about 1 {mu}m.

  4. In vivo nanoparticle-mediated radiopharmaceutical-excited fluorescence molecular imaging

    PubMed Central

    Hu, Zhenhua; Qu, Yawei; Wang, Kun; Zhang, Xiaojun; Zha, Jiali; Song, Tianming; Bao, Chengpeng; Liu, Haixiao; Wang, Zhongliang; Wang, Jing; Liu, Zhongyu; Liu, Haifeng; Tian, Jie

    2015-01-01

    Cerenkov luminescence imaging utilizes visible photons emitted from radiopharmaceuticals to achieve in vivo optical molecular-derived signals. Since Cerenkov radiation is weak, non-optimum for tissue penetration and continuous regardless of biological interactions, it is challenging to detect this signal with a diagnostic dose. Therefore, it is challenging to achieve useful activated optical imaging for the acquisition of direct molecular information. Here we introduce a novel imaging strategy, which converts γ and Cerenkov radiation from radioisotopes into fluorescence through europium oxide nanoparticles. After a series of imaging studies, we demonstrate that this approach provides strong optical signals with high signal-to-background ratios, an ideal tissue penetration spectrum and activatable imaging ability. In comparison with present imaging techniques, it detects tumour lesions with low radioactive tracer uptake or small tumour lesions more effectively. We believe it will facilitate the development of nuclear and optical molecular imaging for new, highly sensitive imaging applications. PMID:26123615

  5. Metal-Enhanced Fluorescence Lifetime Imaging and Spectroscopy on a Modified SERS Substrate

    PubMed Central

    Ray, Krishanu; Lakowicz, Joseph R.

    2013-01-01

    In this paper, we developed a metal-enhanced fluorescence (MEF) substrate by modification of the commercially available surface enhanced Raman spectroscopy (SERS) substrate that may meet the reproducibility and sensitivity challenge of MEF. In spite of many studies and interest on MEF from a number of research groups, application to real-world situations and its commercial use remain challenging mainly due to the difficulties in fabricating reproducible MEF substrates. Specifically, one of the challenges is achieving a standardized MEF substrate for reproducible fluorescence intensity enhancement and/or changes in lifetime. The gold standard klarite substrates for SERS were coated with a thin layer of silver nanoparticles for MEF studies. To test the newly developed MEF substrates, a monolayer of streptavidin conjugated Alexa-647 was assembled on biotinylated-glass or MEF substrates. We observed over 50-fold increase in the fluorescence intensity from a monolayer of streptavidin conjugated Alexa-647 on the biotinylated MEF substrate compared to the same on glass substrate. A significant reduction in the lifetime and increased photostability of Alexa-647 on MEF substrate was observed. Fluorescence lifetime imaging was performed on the monolayer of dye assembled on the modified SERS substrates. We expect this study will serve as a platform to encourage the future use of a standardized MEF substrate for a plethora of sensing applications. PMID:24416457

  6. Substrate-Supported Phospholipid Membranes Studied by Surface Plasmon Resonance and Surface Plasmon Fluorescence Spectroscopy

    PubMed Central

    Tawa, Keiko; Morigaki, Kenichi

    2005-01-01

    Substrate-supported planar lipid bilayer membranes are attractive model cellular membranes for biotechnological applications such as biochips and sensors. However, reliable fabrication of the lipid membranes on solid surfaces still poses significant technological challenges. In this study, simultaneous surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) measurements were applied to the monitoring of adsorption and subsequent reorganization of phospholipid vesicles on solid substrates. The fluorescence intensity of SPFS depends very sensitively on the distance between the gold substrate and the fluorophore because of the excitation energy transfer to gold. By utilizing this distance dependency, we could obtain information about the topography of the adsorbed membranes: Adsorbed vesicles could be clearly distinguished from planar bilayers due to the high fluorescence intensity. SPSF can also incorporate various analytical techniques to evaluate the physicochemical properties of the adsorbed membranes. As an example, we demonstrated that the lateral mobility of lipid molecules could be estimated by observing the recovery of fluorescence after photobleaching. Combined with the film thickness information obtained by SPR, SPR-SPFS proved to be a highly informative technique to monitor the lipid membrane assembly processes on solid substrates. PMID:16040759

  7. Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

    PubMed

    Laňková, Martina; Humpolíčková, Jana; Vosolsobě, Stanislav; Cit, Zdeněk; Lacek, Jozef; Čovan, Martin; Čovanová, Milada; Hof, Martin; Petrášek, Jan

    2016-04-01

    A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants. PMID:27041337

  8. Fluorescence spectroscopy of collagen crosslinking: non-invasive and in situ evaluation of corneal stiffness

    NASA Astrophysics Data System (ADS)

    Franco, Walfre; Ortega-Martinez, Antonio; Zhu, Hong; Wang, Ruisheng; Kochevar, Irene E.

    2015-03-01

    Collagen is a long fibrous structural protein that imparts mechanical support, strength and elasticity to many tissues. The state of the tissue mechanical environment is related to tissue physiology, disease and function. In the cornea, the collagen network is responsible for its shape and clarity; disruption of this network results in degradation of visual acuity, for example in the keratoconus eye disease. The objective of the present study is to investigate the feasibility of using the endogenous fluorescence of collagen crosslinks to evaluate variations in the mechanical state of tissue, in particular, the stiffness of cornea in response to different degrees of photo-crosslinking or RGX treatment—a novel keratoconus treatment. After removing the epithelium, rabbit corneas were stained with Rose Bengal and then irradiated with a 532 nm solid-state laser. Analysis of the excitation spectra obtained by fluorescence spectroscopy shows a correlation between the fluorescence intensity at 370/460 nm excitation/emission wavelengths and the mechanical properties. In principle, it may be feasible to use the endogenous fluorescence of collagen crosslinks to evaluate the mechanical stiffness of cornea non-invasively and in situ.

  9. Applications of time-resolved laser fluorescence spectroscopy to the environmental biogeochemistry of actinides.

    PubMed

    Collins, Richard N; Saito, Takumi; Aoyagi, Noboru; Payne, Timothy E; Kimura, Takaumi; Waite, T David

    2011-01-01

    Time-resolved laser fluorescence spectroscopy (TRLFS) is a useful means of identifying certain actinide species resulting from various biogeochemical processes. In general, TRLFS differentiates chemical species of a fluorescent metal ion through analysis of different excitation and emission spectra and decay lifetimes. Although this spectroscopic technique has largely been applied to the analysis of actinide and lanthanide ions having fluorescence decay lifetimes on the order of microseconds, such as UO , Cm, and Eu, continuing development of ultra-fast and cryogenic TRLFS systems offers the possibility to obtain speciation information on metal ions having room-temperature fluorescence decay lifetimes on the order of nanoseconds to picoseconds. The main advantage of TRLFS over other advanced spectroscopic techniques is the ability to determine in situ metal speciation at environmentally relevant micromolar to picomolar concentrations. In the context of environmental biogeochemistry, TRLFS has principally been applied to studies of (i) metal speciation in aqueous and solid phases and (ii) the coordination environment of metal ions sorbed to mineral and bacterial surfaces. In this review, the principles of TRLFS are described, and the literature reporting the application of this methodology to the speciation of actinides in systems of biogeochemical interest is assessed. Significant developments in TRLFS methodology and advanced data analysis are highlighted, and we outline how these developments have the potential to further our mechanistic understanding of actinide biogeochemistry.

  10. Multicolor whole-cell bacterial sensing using a synchronous fluorescence spectroscopy-based approach.

    PubMed

    Parrello, Damien; Mustin, Christian; Brie, David; Miron, Sebastian; Billard, Patrick

    2015-01-01

    The wide collection of currently available fluorescent proteins (FPs) offers new possibilities for multicolor reporter gene-based studies of bacterial functions. However, the simultaneous use of multiple FPs is often limited by the bleed-through of their emission spectra. Here we introduce an original approach for detection and separation of multiple overlapping fluorescent signals from mixtures of bioreporters strains. The proposed method relies on the coupling of synchronous fluorescent spectroscopy (SFS) with blind spectral decomposition achieved by the Canonical Polyadic (CP) decomposition (also known as Candecomp/Parafac) of three-dimensional data arrays. Due to the substantial narrowing of FP emission spectra and sensitive detection of multiple FPs in a one-step scan, SFS reduced spectral overlap and improved the selectivity of the CP unmixing procedure. When tested on mixtures of labeled E. coli strains, the SFS/CP approach could easily extract the contribution of at least four overlapping FPs. Furthermore, it allowed to simultaneously monitor the expression of three iron responsive genes and pyoverdine production in P. aeruginosa. Implemented in a convenient microplate format, this multiplex fluorescent reporter method provides a useful tool to study complex processes with different variables in bacterial systems. PMID:25822488

  11. Monitoring changes in sponge cakes during aging by front face fluorescence spectroscopy and instrumental techniques.

    PubMed

    Botosoa, Eliot Patrick; Chénè, Christine; Karoui, Romdhane

    2013-03-20

    In the present study, sponge cakes, produced at the pilot scale, were monitored during aging (i.e., 1, 3, 6, 9, 16, and 20 days) by three different analytical techniques. For the texture analyzer, the hardness and elasticity of crumb cakes were found to significantly increase and decrease, respectively, throughout aging. Color parameters (L*, a*, and b*) showed only slight change throughout aging, and a high correlation (R(2) = 0.88) was observed between the whiteness and the yellowness. Tryptophan fluorescence spectra (excitation, 290 nm; emission, 305-490 nm) recorded on cakes exhibited three maxima located at 382, 435, and 467 nm that were attributed to maximum emission of tryptophan (382 nm) and fluorescent Maillard reaction products (435 and 467 nm). The principal component analysis (PCA) applied to the tryptophan spectra allowed a clear discrimination of cakes aged for 1, 3, and 6 days from those aged for 9, 16, and 20 days. Finally, canonical correlation analysis (CCA) performed on the textural and tryptophan fluorescence spectral data sets showed that the two groups of variables were highly correlated because the squared canonical coefficients for canonical variates were 0.99, indicating that cake texture determined at the macroscopic level by texture analyzer is a reflection of its structure at the molecular level determined by fluorescence spectroscopy.

  12. Multicolor Whole-Cell Bacterial Sensing Using a Synchronous Fluorescence Spectroscopy-Based Approach

    PubMed Central

    Parrello, Damien; Mustin, Christian; Brie, David; Miron, Sebastian; Billard, Patrick

    2015-01-01

    The wide collection of currently available fluorescent proteins (FPs) offers new possibilities for multicolor reporter gene-based studies of bacterial functions. However, the simultaneous use of multiple FPs is often limited by the bleed-through of their emission spectra. Here we introduce an original approach for detection and separation of multiple overlapping fluorescent signals from mixtures of bioreporters strains. The proposed method relies on the coupling of synchronous fluorescent spectroscopy (SFS) with blind spectral decomposition achieved by the Canonical Polyadic (CP) decomposition (also known as Candecomp/Parafac) of three-dimensional data arrays. Due to the substantial narrowing of FP emission spectra and sensitive detection of multiple FPs in a one-step scan, SFS reduced spectral overlap and improved the selectivity of the CP unmixing procedure. When tested on mixtures of labeled E. coli strains, the SFS/CP approach could easily extract the contribution of at least four overlapping FPs. Furthermore, it allowed to simultaneously monitor the expression of three iron responsive genes and pyoverdine production in P. aeruginosa. Implemented in a convenient microplate format, this multiplex fluorescent reporter method provides a useful tool to study complex processes with different variables in bacterial systems. PMID:25822488

  13. Preliminary studies of fluorescence image-guided photothermal therapy of human oesophageal adenocarcinoma in vivo using multifunctional gold nanorods

    NASA Astrophysics Data System (ADS)

    Nabavi, Elham; Singh, Mohan; Zhou, Yu; Gallina, Maria Elena; Zhao, Hailin; Ma, Daqing; Cass, Anthony; Hanna, George; Elson, Daniel S.

    2016-03-01

    We present a preliminary in vivo study of fluorescence imaging and photothermal therapy (PTT) of human oesophageal adenocarcinoma using multi-functionalised gold nanorods (GNRs). After establishing tumour xenograft in mouse functionalised GNRs were administrated intravenously (IV). Fluorescence imaging was performed to detect the tumour area. The intensity of the fluorescence signal varied significantly across the tumour site and surrounding tissues. PTT was then performed using a 808 nm continuous wave diode laser to irradiate the tumour for 3 minutes, inducing a temperature rise of ~44°C, which photothermally ablated the tumour.

  14. Using Fluorescence Recovery After Photobleaching (FRAP) to Study Dynamics of the Structural Maintenance of Chromosome (SMC) Complex In Vivo.

    PubMed

    Badrinarayanan, Anjana; Leake, Mark C

    2016-01-01

    The SMC complex, MukBEF, is important for chromosome organization and segregation in Escherichia coli. Fluorescently tagged MukBEF forms distinct spots (or "foci") in the cell, where it is thought to carry out most of its chromosome associated activities. This chapter outlines the technique of Fluorescence Recovery After Photobleaching (FRAP) as a method to study the properties of YFP-tagged MukB in fluorescent foci. This method can provide important insight into the dynamics of MukB on DNA and be used to study its biochemical properties in vivo. PMID:27283300

  15. Detection of non-melanoma skin cancer by in vivo fluorescence imaging with fluorocoxib A.

    PubMed

    Ra, Hyejun; González-González, Emilio; Uddin, Md Jashim; King, Bonnie L; Lee, Alex; Ali-Khan, Irfan; Marnett, Lawrence J; Tang, Jean Y; Contag, Christopher H

    2015-02-01

    Non-melanoma skin cancer (NMSC) is the most common form of cancer in the US and its incidence is increasing. The current standard of care is visual inspection by physicians and/or dermatologists, followed by skin biopsy and pathologic confirmation. We have investigated the use of in vivo fluorescence imaging using fluorocoxib A as a molecular probe for early detection and assessment of skin tumors in mouse models of NMSC. Fluorocoxib A targets the cyclooxygenase-2 (COX-2) enzyme that is preferentially expressed by inflamed and tumor tissue, and therefore has potential to be an effective broadly active molecular biomarker for cancer detection. We tested the sensitivity of fluorocoxib A in a BCC allograft SCID hairless mouse model using a wide-field fluorescence imaging system. Subcutaneous allografts comprised of 1000 BCC cells were detectable above background. These BCC allograft mice were imaged over time and a linear correlation (R(2) = 0.8) between tumor volume and fluorocoxib A signal levels was observed. We also tested fluorocoxib A in a genetically engineered spontaneous BCC mouse model (Ptch1(+/-) K14-Cre-ER2 p53(fl/fl)), where sequential imaging of the same animals over time demonstrated that early, microscopic lesions (100 μm size) developed into visible macroscopic tumor masses over 11 to 17 days. Overall, for macroscopic tumors, the sensitivity was 88% and the specificity was 100%. For microscopic tumors, the sensitivity was 85% and specificity was 56%. These results demonstrate the potential of fluorocoxib A as an in vivo imaging agent for early detection, margin delineation and guided biopsies of NMSCs. PMID:25748239

  16. Fluorescence and photodynamic effects of bacteriochlorin a observed in vivo in 'sandwich' observation chambers.

    PubMed

    van Leengoed, H L; Schuitmaker, J J; van der Veen, N; Dubbelman, T M; Star, W M

    1993-05-01

    Bacteriochlorin a (BCA), a derivative of bacteriochlorphyll a, is an effective photosensitiser in vitro and in vivo. BCA has a major absorption peak at 760 nm where tissue penetration is optimal. This property, together with rapid tissue clearance promises minor skin photosensitivity. The tissue localising and photodynamic properties of BCA were studied using isogeneic RMA mammary tumours, transplanted into subcutaneous tissue in transparent 'sandwich' observation chambers on the back of WAG/Rij rats. The fluorescence kinetics following an i.v. administration of 20 mg kg-1 BCA was assessed in blood vessels, tumour and normal tissue. Subsequently, the development of vascular- and tissue damage after a therapeutic light dose (760 nm, 600 J cm-2) was observed. Fifteen minutes post injection (p.i.), the fluorescence of BCA in the tumour reached a plateau value of 2.5 times the fluorescence in the normal tissue. From 1 h post injection the tumour fluorescence diminished gradually; after 24 h, the tumour fluorescence signal did not exceed that of the normal tissue. Following photodynamic therapy (PDT), 24 h p.i., complete vascular stasis was observed 2 h post treatment in the tumour only, with subsequent recovery. The presence of viable tumour cells following PDT was assessed by histology and re-transplantation of treated tumour tissue from the chamber into the flank immediately or 7 days after treatment. In both cases tumour regrowth was observed. BCA-PDT (20 mg kg-1, 760 nm, 100 J cm-2) 1 h after BCA administration, an interval which gives the optimal differential between tumour and normal tissue, was sufficient to prevent tumour regrowth. However, this only occurred when re-transplantation was performed 7 days after PDT. During PDT, 1 h p.i., vascular damage in tumour and normal tissue was considerable. Complete vascular shut-down was observed in the tumour 2 h after therapy and in the surrounding tissues at 24 h. Circulation damage was associated with vascular spasm

  17. Structure and dynamics of fluorescently labeled complex fluids by fourier imaging correlation spectroscopy

    PubMed

    Grassman; Knowles; Marcus

    2000-12-01

    We present a method of Fourier imaging correlation spectroscopy (FICS) that performs phase-sensitive measurements of modulated optical signals from fluorescently labeled complex fluids. FICS experiments probe the time-dependent trajectory of a spatial Fourier component of the fluid particle density at a specified wave number k, and provide a direct route to the intermediate scattering function. The FICS approach overcomes signal sensitivity problems associated with dynamic light scattering, while offering a means to acquire time-dependent information about spatial distributions of fluorescent particles, superior in efficiency to direct imaging methods. We describe the instrumental setup necessary to perform FICS experiments, and outline the theory that establishes the connection between FICS observables and statistical mechanical quantities describing liquid state dynamics. Test measurements on monolayer suspensions of rhodamine labeled polystyrene spheres are detailed.

  18. Synthesis of Ag clusters in microemulsions: A time-resolved UV vis and fluorescence spectroscopy study

    NASA Astrophysics Data System (ADS)

    Ledo, Ana; Martínez, F.; López-Quintela, M. A.; Rivas, J.

    2007-09-01

    The combined use of the microemulsion technique and the kinetic control allows the preparation of small silver clusters. By using UV-vis and fluorescence spectroscopy the main stages by which the clusters grow, before the formation of nanoparticles, were elucidated. Transmission electron microscopy (TEM) and scanning tunnelling microscopy (STM) were used to further characterize the samples. Two main stages were clearly identified, which are associated with: (1) the formation of Ag n clusters with n<10, which self-aggregate into one atom high 2D nanodiscs of 3.2 nm size and (2) Ag n clusters, which self-aggregate into 3D nanostructures of 1.5 nm in size. The fluorescence properties observed with both stages show that the formed clusters are small enough to display a molecule-like behaviour.

  19. Comparing Raman and fluorescence lifetime spectroscopy from human atherosclerotic lesions using a bimodal probe.

    PubMed

    Dochow, Sebastian; Fatakdawala, Hussain; Phipps, Jennifer E; Ma, Dinglong; Bocklitz, Thomas; Schmitt, Michael; Bishop, John W; Margulies, Kenneth B; Marcu, Laura; Popp, Jürgen

    2016-09-01

    Fluorescence lifetime imaging (FLIm) and Raman spectroscopy are two promising methods to support morphological intravascular imaging techniques with chemical contrast. Both approaches are complementary and may also be used in combination with OCT/IVUS to add chemical specificity to these morphologic intravascular imaging modalities. In this contribution, both modalities were simultaneously acquired from two human coronary specimens using a bimodal probe. A previously trained SVM model was used to interpret the fluorescence lifetime data; integrated band intensities displayed in RGB false color images were used to interpret the Raman data. Both modalities demonstrate unique strengths and weaknesses and these will be discussed in comparison to histologic analyses from the two coronary arteries imaged. PMID:27003796

  20. Homodimerization of glucocorticoid receptor from single cells investigated using fluorescence correlation spectroscopy and microwells.

    PubMed

    Oasa, Sho; Sasaki, Akira; Yamamoto, Johtaro; Mikuni, Shintaro; Kinjo, Masataka

    2015-08-01

    Glucocorticoid receptor α (GR) binds to the promoter regions of target genes as a homodimer and activates its transcriptional process. Though the homodimerization is thought to be the initial and essential process, the dissociation constant for homodimerization of GR remains controversial. To quantify homodimerization of (enhanced green fluorescence protein) EGFP-(glucocorticoid receptor) GR, the particle brightness in lysates from single cell was estimated for the fraction of homodimeric EGFP-GR using fluorescence correlation spectroscopy and microwells. Fitting the data with a bimolecular reaction model, the dissociation constant was determined. Moreover slow-diffusion complex was observed. These results suggest that EGFP-GR forms not only a monomer-dimer equivalent state but also a large-molecular-weight complex. PMID:26183204

  1. What information is contained in the fluorescence correlation spectroscopy curves, and where

    NASA Astrophysics Data System (ADS)

    Khadem, S. M. J.; Hille, C.; Löhmannsröben, H.-G.; Sokolov, I. M.

    2016-08-01

    We discuss the application of fluorescence correlation spectroscopy (FCS) for characterization of anomalous diffusion of tracer particles in crowded environments. While the fact of anomaly may be detected by the standard fitting procedure, the value of the exponent α of anomalous diffusion may be not reproduced correctly for non-Gaussian anomalous diffusion processes. The important information is however contained in the asymptotic behavior of the fluorescence autocorrelation function at long and at short times. Thus, analysis of the short-time behavior gives reliable values of α and of lower moments of the distribution of particles' displacement, which allows us to confirm or reject its Gaussian nature. The method proposed was tested on the FCS data obtained in artificial crowded fluids and in living cells.

  2. Application of X-ray Fluorescence Spectroscopy in Analysis of Oil Paint Pigments

    NASA Astrophysics Data System (ADS)

    Major, Cassandra; Formica, Sarah

    2011-10-01

    X-ray Fluorescence (XRF) spectroscopy is a rapid, noninvasive technique for both detecting and identifying chemical elements within a given sample. At North Georgia College and State University, a sealed tube x-ray source and slightly focusing polycapillary optic are used in nondestructive XRF analysis of oil paint pigments. Oil paints contain both organic and inorganic matter, and the inorganic ingredients such as titanium, vanadium, iron, zinc, and other elements are easily detected by XRF, which can be used to uniquely differentiate between various paint pigments. To calibrate the XRF system for paint color identification, six different colors of oil paint were fluoresced and identified based off of their characteristic spectra. By scanning the paint sample in two dimensions, the characteristic XRF spectra obtained were compiled to produce an XRF replica of the painting.

  3. [Measurement and analysis of lead in soil using X-ray fluorescence spectroscopy].

    PubMed

    Zhang, Rong; Zhang, Yu-Jun; Zhang, Wei; Chen, Dong; Yu, Xiao-Ya; Gao, Yan-Wei

    2013-02-01

    The present paper analyzed the characteristics of X-ray fluorescence spectroscopy (XRF) of metal element lead in soil using the NITON XLt793 portable X-ray fluorescence spectra of heavy metal analyzer under laboratory conditions. The characteristic spectral lines of L(alpha) (energy: 10. 55 keV) and L(beta) (energy: 12. 61 keV) with different matrix elements were selected respectively for lead in the experiment. By measuring the intensities of the characteristic spectral line with different Pb concentration, the results demonstrate that the relation between concentration [mass fraction 10 x 10(-6) - 1 800 x 10(-6)] of Pb element and the intensity of the characteristic spectrum is well linear. The calibration curve of Pb was plotted based on the different concentration measurement results, and the limit of detection of 7.89 x 10(-6) was obtained for Pb in soil.

  4. Fluorescence Spectroscopy of the Retina for the Screening of Bovine Spongiform Encephalopathy.

    PubMed

    Bhattacharjee, Ujjal; Graham, Catherine; Czub, Stefanie; Dudas, Sandor; Rasmussen, Mark A; Casey, Thomas A; Petrich, Jacob W

    2016-01-13

    Transmissible spongiform encephalopathies (TSE) are progressive, neurodegenerative disorders, of which bovine spongiform encephalopathy (BSE) is of special concern because it is infectious and debilitating to humans. The possibility of using fluorescence spectroscopy to screen for BSE in cattle was explored. Fluorescence spectra from the retinas of experimentally infected BSE-positive cattle with clinical disease were compared with those from both sham-inoculated and non-inoculated BSE-negative cattle. The distinct intensity difference of about 4-10-fold between the spectra of the BSE-positive and the BSE-negative (sham-inoculated and non-inoculated) eyes suggests the basis for a means of developing a rapid, noninvasive examination of BSE in particular and TSEs in general.

  5. Fluorescence Spectroscopy of the Retina for the Screening of Bovine Spongiform Encephalopathy.

    PubMed

    Bhattacharjee, Ujjal; Graham, Catherine; Czub, Stefanie; Dudas, Sandor; Rasmussen, Mark A; Casey, Thomas A; Petrich, Jacob W

    2016-01-13

    Transmissible spongiform encephalopathies (TSE) are progressive, neurodegenerative disorders, of which bovine spongiform encephalopathy (BSE) is of special concern because it is infectious and debilitating to humans. The possibility of using fluorescence spectroscopy to screen for BSE in cattle was explored. Fluorescence spectra from the retinas of experimentally infected BSE-positive cattle with clinical disease were compared with those from both sham-inoculated and non-inoculated BSE-negative cattle. The distinct intensity difference of about 4-10-fold between the spectra of the BSE-positive and the BSE-negative (sham-inoculated and non-inoculated) eyes suggests the basis for a means of developing a rapid, noninvasive examination of BSE in particular and TSEs in general. PMID:26623498

  6. X-ray fluorescence/Auger-electron coincidence spectroscopy of vacancy cascades in atomic argon

    SciTech Connect

    Arp, U.; LeBrun, T.; Southworth, S.H.; Jung, M.; MacDonald, M.A.

    1996-12-01

    Argon L{sub 2.3}-M{sub 2.3}M{sub 2.3} Auger-electron spectra were measured in coincidence with K{alpha} fluorescent x-rays in studies of Ar K-shell vacancy decays at several photon energies above the K-threshold and on the 1s-4p resonance in atomic argon. The complex spectra recorded by conventional electron spectroscopy are greatly simplified when recorded in coincidence with fluorescent x-rays, allowing a more detailed analysis of the vacancy cascade process. The resulting coincidence spectra are compared with Hartree-Fock calculations which include shake-up transitions in the resonant case. Small energy shifts of the coincidence electron spectra are attributed to post-collision interaction with 1s photoelectrons.

  7. What information is contained in the fluorescence correlation spectroscopy curves, and where.

    PubMed

    Khadem, S M J; Hille, C; Löhmannsröben, H-G; Sokolov, I M

    2016-08-01

    We discuss the application of fluorescence correlation spectroscopy (FCS) for characterization of anomalous diffusion of tracer particles in crowded environments. While the fact of anomaly may be detected by the standard fitting procedure, the value of the exponent α of anomalous diffusion may be not reproduced correctly for non-Gaussian anomalous diffusion processes. The important information is however contained in the asymptotic behavior of the fluorescence autocorrelation function at long and at short times. Thus, analysis of the short-time behavior gives reliable values of α and of lower moments of the distribution of particles' displacement, which allows us to confirm or reject its Gaussian nature. The method proposed was tested on the FCS data obtained in artificial crowded fluids and in living cells. PMID:27627335

  8. Fluorescence spectroscopy to study dissolved organic matter interactions with agrochemicals applied in Swiss vineyards.

    PubMed

    Daouk, Silwan; Frege, Carla; Blanc, Nicolas; Mounier, Stéphane; Redon, Roland; Merdy, Patricia; Lucas, Yves; Pfeifer, Hans-Rudolf

    2015-06-01

    UV/Vis fluorescence spectroscopy was used to study the possible interactions of dissolved organic matter (DOM) with the herbicide glyphosate and copper-based fungicide used in vineyards. The study focused on the role of DOM in the transport of these micropollutants from parcels to surface waters (river, lake). Soil solution and river water samples were collected in the Lavaux vineyard area, western Switzerland. Their fluorescence excitation emission matrices (EEM) were decomposed using parallel factor (PARAFAC) analysis, and compared to their content in glyphosate and copper. PARAFAC analysis of EEM of both types of samples showed the contribution of protein-like and humic-like fluorophores. In soil water samples, complexes between fulvic-like and humic-like fluorophores of DOM, copper, and glyphosate were likely formed. In surface water, DOM-copper and glyphosate-copper interactions were observed, but not between glyphosate and DOM.

  9. Measurement of the temperature-dependent diffusion properties of nanoparticles by using fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Jung, Chanbae; Lee, Jaeran; Kang, Manil; Kim, Sok Won

    2014-10-01

    Changes in the diffusion properties of three kinds of fluorescent particles, Alexa Fluor 647, Q-dots (quantum dots), and beads, with temperature were investigated with a home-built fluorescence correlation spectroscopy (FCS) system based on a confocal microscope. In all samples, as the temperature was increased, the diffusion times were reduced, indicating an increase in the diffusion coefficient. In particular, of all the particles, Alexa Fluor 647 having the smallest size of ˜1 nm, showed a hydrodynamic radius that increased with increasing temperature of the solvent. However, for the Q-dots and beads with larger sizes, the hydrodynamic radius of the particles was inversely proportional to the temperature. These results show that diffusion coefficient obtained by changing the temperature has an influence on the hydrodynamic radius of the particles.

  10. Fluorescence spectroscopy as a tool for quality assessment of humic substances

    NASA Astrophysics Data System (ADS)

    Boguta, Patrycja

    2016-04-01

    *The studies were partly carried out within the framework of a research project. The project was financed from funds of National Science Center on the base of decision number DEC-2013/11/D/NZ9/02545. Fluorescence spectroscopy belongs to modern, non-destructive, rapid and relatively cheap methods, as well as for many years it was successfully used in studies of organic compounds in the fields of medicine, biology and chemistry. On the other hand, soil organic matter is a group of compounds with a complex spatial structure showing a large number of groups with different kinds of fluorophores. This could suggest the possibility of application of fluorescence spectroscopy in assessing the quality of humic substances as well as in monitoring of their chemical transformations. The aim of study was chemical description of humic and fulvic acids based on fluorescence spectra, as well as an attempt of evaluation of changes occurring under the influence of different pH and during interactions with various concentrations of metal. The humic and fulvic acids were isolated from chemically different soils. The measurements were carried out on Hitachi fluorescence spectrometer in solutions with a concentration of humic acids 40mg dm-3, at pH from 3 to 7, and for the evaluation of the metal impact: with increasing Zn concentrations (0-50mg dm-3). The fluorescence spectra were recorded in the form of synchronous and emission-excitation matrices (EEM). Studies have shown the presence of different groups of fluorophores. Synchronous spectra were characterized by a well-separated bands showing fluorescence in the area of low, medium and high wavelengths, suggesting the presence of structures, both weakly and strongly humified. EEM spectra revealed map of fluorophores within wide ranges of emission and excitation. Fluorophores differed in both position and intensity. The highest intensity was observed for compounds with the lowest humification degree which might be due to high amount

  11. Identification of a Novel Indoline Derivative for in Vivo Fluorescent Imaging of Blood-Brain Barrier Disruption in Animal Models

    PubMed Central

    2013-01-01

    Disruption of the blood-brain barrier (BBB) can occur in various pathophysiological conditions. Administration of extraneous tracers that can pass the disrupted, but not the intact, BBB and detection of the extravasation have been widely used to assess BBB disruption in animal models. Although several fluorescent tracers have been successfully used, the administration of these tracers basically requires intravascular injection, which can be laborious when using small animals such as zebrafish. To identify fluorescent tracers that could be easily administered into various animal models and visualize the BBB disruption in vivo, we prepared nine structurally related indoline derivatives (IDs) as a minimum set of diverse fluorescent compounds. We found that one ID, ZMB741, had the highest affinity for serum albumin and emitted the strongest fluorescence in the presence of serum albumin of the nine IDs tested. The affinity to serum albumin and the fluorescence intensity was superior to those of Evans blue and indocyanine green that have been conventionally used to assess the BBB disruption. We showed that ZMB741 could be administered into zebrafish by static immersion or mice by intraperitoneal injection and visualizes the active disruption of their BBB. These results suggest that ZMB741 can be a convenient and versatile tool for in vivo fluorescent imaging of BBB disruption in various animal models. The strategy used in this study can also be applied to diversity-oriented libraries to identify novel fluorescent tracers that may be superior to ZMB741. PMID:23668665

  12. Intermolecular disulfide bond formation promotes immunoglobulin aggregation: investigation by fluorescence correlation spectroscopy.

    PubMed

    Nag, Moupriya; Bera, Kallol; Basak, Soumen

    2015-01-01

    Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (τ(D)) of ∼200 µsec for Rhodamine-labeled IgG, corresponding to a hydrodynamic radius (R(H)) of 56 Å for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of τ(D) under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation-dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation. PMID:25371040

  13. In vivo simulation environment for fluorescence molecular tomography using Monte Carlo method

    NASA Astrophysics Data System (ADS)

    Zhang, Yizhai; Xu, Qiong; Li, Jin; Tang, Shaojie; Zhang, Xin

    2008-12-01

    Optical sensing of specific molecular target using near-infrared light has been recognized to be the crucial technology, have changing human's future. The imaging of Fluorescence Molecular Tomography is the most novel technology in optical sensing. It uses near-infrared light(600-900nm) as instrument and utilize fluorochrome as probe to take noncontact three-dimensional imaging for live molecular targets and to exhibit molecular process in vivo. In order to solve the problem of forward simulation in FMT, this paper mainly introduces a new simulation modeling. The modeling utilizes Monte Carlo method and is implemented in C++ programming language. Ultimately its accuracy has been testified by comparing with analytic solutions and MOSE from University of Iowa and Chinese Academy of Science. The main characters of the modeling are that it can simulate both of bioluminescent imaging and FMT and take analytic calculation and support more than one source and CCD detector simultaneously. It can generate sufficient and proper data and pre-preparation for the study of fluorescence molecular tomography.

  14. A new fluorescent dye for cell tracing and mitochondrial imaging in vitro and in vivo.

    PubMed

    Press, Adrian T; Ungelenk, Luisa; Rinkenauer, Alexandra C; Gröger, Marko; Lehmann, Frank; Mosig, Alexander; Schubert, Ulrich S; Clemens, Mark G; Bauer, Michael

    2016-09-01

    Mitochondria contribute to redox and calcium balance, and apoptosis thus regulating cellular fate. In the present study, mitochondrial staining applying a novel dye, V07-07059, was performed in human embryonic kidney cells, a human vascular endothelial cell line and primary human mononuclear cells. The new fluorescent mega Stokes dye (peak excitation: 488 nm, peak emission: 554 nm) showed superior fluorescent properties and stability. V07-07059 stains mitochondria dependent on their membrane potential and is safe to use in vitro and in vivo. Unlike other dyes applied in this context (e.g. Tetramethylrhodamine methyl ester), V07-07059 only marginally inhibits mitochondrial respiration and function. V07-07059 enables real time imaging of mitochondrial trafficking and remodeling. Prolonged staining with V07-07059 demonstrated the dyes suitability as a novel probe to track cells. In comparison to the widely used standard for cell proliferation and tracking studies 5(6)-diacetate N-succinimidyl ester, V07-07059 proved superior regarding toxicity and photostability. PMID:26563981

  15. Volatile fractions of landfill leachates and their effect on Chlamydomonas reinhardtii: In vivo chlorophyll a fluorescence

    SciTech Connect

    Brack, W.; Rottler, H.; Frank, H.

    1998-10-01

    Volatile organic compounds such as short-chain halogenated hydrocarbons and alkylated benzenes are widely used as solvents or as intermediates in the chemical industry, and some of them are fuel components. Dichloromethane, trichloroethene, 1,1,1-trichloroethane, and tetrachloroethene have been produced in amounts of 500,000 to 1 million t/year, 80 to 100% of which are released to the environment. The production of toluene, a major component of fuels for internal combustion engines, amounts to about 30 million t/year. A method for identification of toxic volatile constituents of landfill leachates is presented that combines bioassay-compatible sample preparation, chemical analysis, and a bioassay based on in vivo chlorophyll a fluorescence of the green alga Chlamydomonas reinhardtii. Two major pathways of toxicity were identified by comparing fluorescence patterns: specific toxicity of hydrogen sulfide, and narcotic action of nonreactive organic compounds. For quantification, the contributions of identified compounds were calculated using toxic units. The ecotoxicologic relevance of volatile fractions from hazardous waste leachates was shown.

  16. Laguerre-based method for analysis of time-resolved fluorescence data: application to in-vivo characterization and diagnosis of atherosclerotic lesions

    NASA Astrophysics Data System (ADS)

    Jo, Javier A.; Fang, Qiyin; Papaioannou, Thanassis; Baker, J. Dennis; Dorafshar, Amir; Reil, Todd; Qiao, Jianhua; Fishbein, Michael C.; Freischlag, Julie A.; Marcu, Laura

    2006-03-01

    We report the application of the Laguerre deconvolution technique (LDT) to the analysis of in-vivo time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data and the diagnosis of atherosclerotic plaques. TR-LIFS measurements were obtained in vivo from normal and atherosclerotic aortas (eight rabbits, 73 areas), and subsequently analyzed using LDT. Spectral and time-resolved features were used to develop four classification algorithms: linear discriminant analysis (LDA), stepwise LDA (SLDA), principal component analysis (PCA), and artificial neural network (ANN). Accurate deconvolution of TR-LIFS in-vivo measurements from normal and atherosclerotic arteries was provided by LDT. The derived Laguerre expansion coefficients reflected changes in the arterial biochemical composition, and provided a means to discriminate lesions rich in macrophages with high sensitivity (>85%) and specificity (>95%). Classification algorithms (SLDA and PCA) using a selected number of features with maximum discriminating power provided the best performance. This study demonstrates the potential of the LDT for in-vivo tissue diagnosis, and specifically for the detection of macrophages infiltration in atherosclerotic lesions, a key marker of plaque vulnerability.

  17. Laser-assisted cryosurgery in ex vivo mice hepatic tissue: viability assays using green fluorescent protein.

    PubMed

    Martínez-Suástegui, L; Duperray, B; Godinez, F; Guillén, G; Slade, A; Aguilar, G

    2011-02-01

    An experimental investigation is carried out to develop a novel approach to cryosurgery, where laser heating counteracts tissue freezing to better confine damage to the targeted cancerous tissue within a lethal low-temperature isothermal boundary-an approach we refer to as laser-assisted cryosurgery (LAC). The advantage of this procedure relative to conventional cryosurgery assisted with urethral warmers or cryoheaters is that laser heating provides volumetric rather than superficial heating, which leads to deeper penetration, more homogeneous tissue protection and better demarcation of the destructive freezing effect to a well-defined targeted volume. Tissue viability assays are performed using green fluorescence protein (GFP) as a viability marker and correlated with temperature history after performing LAC procedures on ex vivo mice hepatic tissue. The limit for cell denaturation at the irradiated surface predicted by GFP analysis is further confirmed using reverse transcription polymerase chain reaction (RT-PCR). In addition, the correlation between GFP fluorescence and cell viability and loss of GFP fluorescence in non-viable cells has been tested and validated by histological analysis using a standard cell viability measuring method (hematoxylin and eosin staining). Analysis of our experimental measurements show that reproducible thermal gradients (of 236 °C/cm) and predictable tissue necrosis can be reliably produced by LAC without exceeding temperature thresholds for cell denaturation (of T (surf) ≈ 48 °C) beyond preset tissue boundaries (with resolution of 0.1 °C/mm). The results have shown the feasibility of controlling temperatures at specified tissue locations to prevent hyperthermal or freezing damage. PMID:20963494

  18. Fluorescence spectroscopy of soil pellets : The use of CP/PARAFAC.

    NASA Astrophysics Data System (ADS)

    Mounier, Stéphane; Nicolodeli, Gustavo; Redon, Roland; Hacherouf, Kalhed; Milori, Debora M. B. P.

    2014-05-01

    Fluorescence spectroscopy is one of the most sensitive techniques available for analytical purposes. It is relatively easy to implement, phenomenologically straightforward and well investigated. Largely non-invasive and fast, so that it can be useful for environmental applications. Fluorescence phenomenon is highly probable in molecular systems containing atoms with lone pairs of electrons such as C=O, aromatic, phenolic, quinone and more rigid unsaturated conjugated systems. These functional groups are present in humic substances (HS) from soils (Senesi, 1990; N. Senesi et al., 1991) and represent the main fluorophors of Soil Organic Matter (SOM). The extension of the conjugated electronic system, the level of heteroatom substitution and type and number of substituting groups under the aromatic rings strongly affect the intensity and wavelength of molecular fluorescence. However, to analyse the SOM it is generally done a chemical extraction that allows measuring the fluorescence response of the liquid extract. To avoid this fractionation of the SOM, Milori et al. (2006) proposed the application of laser induced fluorescence spectroscopy (LIFS) in whole soil. This work intends to assess the technical feasibility of 3D fluorescence spectroscopy using lamp for excitation to analyse solids opaque samples prepared with different substances. Seventy four (74) solid samples were prepared from different mixtures of boric acid (BA), humic substance acid and tryptophan (TRP) powder. The compounds were mixture and a pellet was done by using pressure (8 ton). The pellets were measured using a spectrofluorimeter HITACHI F4500, and a 3D fluorescence tensor was done from emission spectra (200-600 nm) with excitation range from 200 to 500 nm. The acquisition parameters were: step at 5 nm, scan speed at 2400 nm.min-1, response time at 0.1 s, excitation and emission slits at 5 nm and photomultiplier voltage at 700 V. Furthermore, measures of Laser-induced Fluorescence were

  19. Field confinement with aberration correction for solid immersion lens based fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Rao, Ramachandra; Mitic, Jelena; Serov, Alexandre; Leitgeb, Rainer A.; Lasser, Theo

    2007-03-01

    The solid immersion lens (SIL) as a tool for increasing the field confinement as well as providing optimal performance by aberration compensation in a confocal fluorescence correlation spectroscopy (FCS) system is illustrated here. Using Zernike polynomials we show that aberration compensation and the resultant pre-shaping of the incident wavefront enables near diffraction-limited performance. This is explained based on vectorial computations for high apertures in the Debye approximation. The obtained axial resolution parameters are compared with the obtained diffusion times in a SIL-FCS experiment for measurements in solutions done at the single molecule level.

  20. The Effect of the Refractive Index of the Medium in Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Cha, Seoncheol; Kim, Sung Hyun; Kim, Doseok

    2010-03-01

    Fluorescence correlation spectroscopy (FCS) is a useful tool to study diffusional motion in liquids as it measures resident time of a dye molecule in a small excitation volume made by confocal microscopy. Some reports recently predicted that the measurement result of FCS is affected sensitively by the refractive index of liquid medium. To check for this possibility, several liquids having the same viscosity values but different refractive indices were chosen to dissolve dye molecules. The change in the observed diffusion coefficients in solutions having the same viscosity value manifests that care needs to be taken in the common practice of using sucrose to change the viscosity in the FCS experiment.

  1. Study on the interaction of anticancer drug mitoxantrone with DNA by fluorescence and Raman spectroscopies

    NASA Astrophysics Data System (ADS)

    Tang, Lingjuan; Sun, Zhenrong; Guo, Jianyu; Wang, Zugeng

    2006-02-01

    Mitoxantrone, a clinically useful antitumour antibiotic for leukaemia and breast cancer, has received more attentions. In this paper, the interaction between mitoxantrone and calf thymus DNA is investigated by Raman and fluorescence spectroscopies, and the binding site of mitoxantrone to calf thymus DNA is explored. The results showed that mitoxantrone interacts with calf thymus DNA bases by the intercalation of anthracycline into the base pair plane of adenine (A) and thymine (T), and it results in the disruption of the hydrogen bonds between calf thymus DNA bases, and thus the calf thymus DNA double-strand can be disrupted into the B-form DNA double-strand segments.

  2. Discrimination of normal and colorectal cancer using Raman spectroscopy and fluorescence

    NASA Astrophysics Data System (ADS)

    Li, Xiaozhou; Wang, Deli; Wang, Yue

    2007-07-01

    Laser-induced fluorescence spectroscopy (LIF) and Raman spectrum of serum for diagnosis of colon cancer and rectum cancer were investigated in this paper. The aim of this study was that using Raman spectrum and LIF analysis the serum of colon cancer and rectum cancer for found the difference compared to normal, the difference was found. For example: the intensity and red shift both different In this paper we investigated 82 colon cancers, 69 rectum cancers and obtained 80.7%, 82.5% accuracy to rectum cancer and colon cancer separately compared to clinical diagnostic. It is exploring that use Raman spectrum and LIF to detection of cancer.

  3. Ultrasensitive detection of waste products in water using fluorescence emission cavity-enhanced spectroscopy.

    PubMed

    Bixler, Joel N; Cone, Michael T; Hokr, Brett H; Mason, John D; Figueroa, Eleonora; Fry, Edward S; Yakovlev, Vladislav V; Scully, Marlan O

    2014-05-20

    Clean water is paramount to human health. In this article, we present a technique for detection of trace amounts of human or animal waste products in water using fluorescence emission cavity-enhanced spectroscopy. The detection of femtomolar concentrations of urobilin, a metabolic byproduct of heme metabolism that is excreted in both human and animal waste in water, was achieved through the use of an integrating cavity. This technique could allow for real-time assessment of water quality without the need for expensive laboratory equipment.

  4. Recent Developments in Fluorescence Correlation Spectroscopy for Diffusion Measurements in Planar Lipid Membranes

    PubMed Central

    Macháň, Radek; Hof, Martin

    2010-01-01

    Fluorescence correlation spectroscopy (FCS) is a single molecule technique used mainly for determination of mobility and local concentration of molecules. This review describes the specific problems of FCS in planar systems and reviews the state of the art experimental approaches such as 2-focus, Z-scan or scanning FCS, which overcome most of the artefacts and limitations of standard FCS. We focus on diffusion measurements of lipids and proteins in planar lipid membranes and review the contributions of FCS to elucidating membrane dynamics and the factors influencing it, such as membrane composition, ionic strength, presence of membrane proteins or frictional coupling with solid support. PMID:20386647

  5. Ultrasensitive detection of waste products in water using fluorescence emission cavity-enhanced spectroscopy

    PubMed Central

    Bixler, Joel N.; Cone, Michael T.; Hokr, Brett H.; Mason, John D.; Figueroa, Eleonora; Fry, Edward S.; Yakovlev, Vladislav V.; Scully, Marlan O.

    2014-01-01

    Clean water is paramount to human health. In this article, we present a technique for detection of trace amounts of human or animal waste products in water using fluorescence emission cavity-enhanced spectroscopy. The detection of femtomolar concentrations of urobilin, a metabolic byproduct of heme metabolism that is excreted in both human and animal waste in water, was achieved through the use of an integrating cavity. This technique could allow for real-time assessment of water quality without the need for expensive laboratory equipment. PMID:24799690

  6. Detection of orange juice frauds using front-face fluorescence spectroscopy and Independent Components Analysis.

    PubMed

    Ammari, Faten; Redjdal, Lamia; Rutledge, Douglas N

    2015-02-01

    The aim of this study was to find simple objective analytical methods to assess the adulteration of orange juice by grapefruit juice. The adulterations by addition of grapefruit juice were studied by 3D-front-face fluorescence spectroscopy followed by Independent Components Analysis (ICA) and by classical methods such as free radical scavenging activity and total flavonoid content. The results of this study clearly indicate that frauds by adding grapefruit juice to orange juice can be detected at percentages as low as 1%.

  7. Ultrasensitive detection of waste products in water using fluorescence emission cavity-enhanced spectroscopy.

    PubMed

    Bixler, Joel N; Cone, Michael T; Hokr, Brett H; Mason, John D; Figueroa, Eleonora; Fry, Edward S; Yakovlev, Vladislav V; Scully, Marlan O

    2014-05-20

    Clean water is paramount to human health. In this article, we present a technique for detection of trace amounts of human or animal waste products in water using fluorescence emission cavity-enhanced spectroscopy. The detection of femtomolar concentrations of urobilin, a metabolic byproduct of heme metabolism that is excreted in both human and animal waste in water, was achieved through the use of an integrating cavity. This technique could allow for real-time assessment of water quality without the need for expensive laboratory equipment. PMID:24799690

  8. Transient absorption spectroscopy detection of sensitized delayed fluorescence in chiral benzophenone/naphthalene systems

    NASA Astrophysics Data System (ADS)

    Bonancía, Paula; Jiménez, M. Consuelo; Miranda, Miguel A.

    2011-10-01

    Transient absorption spectroscopy has proven to be a powerful tool to investigate the formation and decay of excited singlet states upon triplet-triplet annihilation, following T-T energy transfer from a selectively excited sensitizer. Thus, upon selective excitation of benzophenone (BZP) by laser flash photolysis (LFP) at λ = 355 nm in the presence of naphthalene (NPT), a negative band centered at 340 nm has been detected, with growth and decay in the microsecond timescale. It has been assigned to the P-type NPT delayed-fluorescence. In the case of chiral BZP/NPT systems, stereodifferentiation has been observed in the kinetics of the involved photophysical processes.

  9. Quantification of transport and binding parameters using fluorescence recovery after photobleaching. Potential for in vivo applications.

    PubMed Central

    Kaufman, E N; Jain, R K

    1990-01-01

    Fluorescence Recovery After Photobleaching (FRAP) has been used extensively in the study of transport and binding in biological media in vitro. The present study adapts and further develops FRAP so that it may be utilized for the in vivo quantification of binding parameters. The technique is validated in vitro by measuring mass transport and binding parameters for the Concanavalin A/Mannose binding system (a diffusion-limited system). The pseudo-equilibrium constant (the product of the equilibrium constant and the total concentration of binding sites) for this system was determined to be 26 +/- 15 which compares favorably with literature values ranging between 16 and 32. The applicability of this technique to measure parameters for monoclonal antibody/antigen interactions in a thin tissue preparation such as the rabbit ear chamber tissue preparation is also examined. Unlike other methods for measuring binding parameters, this is the only technique which has the potential to measure parameters relevant to antibody delivery in vivo. The proposed technique is noninvasive and does not require a priori knowledge of, independent measurement of, or variation in the concentration of binding sites or total concentration of binding species. PMID:2248992

  10. In vivo deep tissue fluorescence imaging of the murine small intestine and colon

    NASA Astrophysics Data System (ADS)

    Crosignani, Viera; Dvornikov, Alexander; Aguilar, Jose S.; Stringari, Chiara; Edwards, Roberts; Mantulin, Williams; Gratton, Enrico

    2012-03-01

    Recently we described a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. This technique was applied to in vivo imaging of murine small intestine and colon. Individuals with Inflammatory Bowel Disease (IBD), commonly presenting as Crohn's disease or Ulcerative Colitis, are at increased risk for developing colorectal cancer. We have developed a Giα2 gene knock out mouse IBD model that develops colitis and colon cancer. The challenge is to study the disease in the whole animal, while maintaining high resolution imaging at millimeter depth. In the Giα2-/- mice, we have been successful in imaging Lgr5-GFP positive stem cell reporters that are found in crypts of niche structures, as well as deeper structures, in the small intestine and colon at depths greater than 1mm. In parallel with these in vivo deep tissue imaging experiments, we have also pursued autofluorescence FLIM imaging of the colon and small intestine-at more shallow depths (roughly 160μm)- on commercial two photon microscopes with excellent structural correlation (in overlapping tissue regions) between the different technologies.

  11. In vivo wound healing diagnosis with second harmonic and fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Deka, Gitanjal; Wu, Wei-Wen; Kao, Fu-Jen

    2013-06-01

    Skin wounds heal when a series of cell lineages are triggered, followed by collagen deposition, to reconstruct damaged tissues. This study evaluates the regeneration of collagen and change in cellular metabolic rate in vivo during wound healing in rats, with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy respectively. The metabolic rate of cells is reflected through the lifetime of the autofluorescence from the co-enzyme protein, reduced nicotinamide adenine dinucleotide, due to its change in the relative concentration of bound and free forms. A higher than normal cellular metabolic rate is observed during the first week of healing, which decreases gradually after eight days of wound formation. SHG signal intensity change indicates the net degradation of collagen during the inflammatory phase, and net regeneration begins on day five. Eventually, the quantity of collagen increases gradually to form a scar tissue as the final product. Importantly, this work demonstrates the feasibility of an in vivo imaging approach for a normal wound on rat skin, which has the potential to supplement the noninvasive clinical diagnosis of wounds.

  12. In vivo imaging of small animals with optical tomography and near-infrared fluorescent probes

    NASA Astrophysics Data System (ADS)

    Palmer, Matthew R.; Shibata, Yasushi; Kruskal, Jonathan B.; Lenkinski, Robert E.

    2002-06-01

    A developmental optical tomography has been designed for imaging small animals in vivo using near IR fluorophores. The system employs epi-illumination via a 450 W Xe arc lamp, filtered and collimated to illuminate a 10 cm square movable stage. Emission light is filtered then collected by a high- resolution, high quantum efficiency, cooled CCD camera. Stage movement and image acquisition are under the control of a personal computer running system integration and automation software. During an experiment, the anesthetized animal is secured to the stage and up to 200 projections can be acquired over 180 degrees rotation. Angular sampling of the light distribution at a point on the surface is used to determine relative contributions form ballistic and diffuse photons. We have employed the system to investigate a number of applications of in-vivo fluorescent imaging. In dynamic studies, hepatic function has been visualized in nude mice following intravenous injection of indocyanine green (ICG) and cerebrospinal fluid flow as been measured by injection of ICG-lipoprotein conjugate in the subarachnoid space of the lumbar spine followed by dynamic imaging of the brain. Further applications in physiological imaging, cancer detection, and molecular imaging are under investigation in our laboratory.

  13. Fluorescence Tomography of Rapamycin-Induced Autophagy and Cardioprotection In Vivo

    PubMed Central

    Chen, Howard H.; Mekkaoui, Choukri; Cho, Hoonsung; Ngoy, Soeun; Marinelli, Brett; Waterman, Peter; Nahrendorf, Matthias; Liao, Ronglih; Josephson, Lee; Sosnovik, David E.

    2013-01-01

    Background Autophagy is a biological process during which cells digest organelles in their cytoplasm and recycle the constituents. The impact of autophagy in the heart, however, remains unclear in part due to the inability to noninvasively image this process in living animals. Methods and Results Here, we report the use of fluorescence molecular tomography (FMT) and a cathepsin activatable fluorochrome to image autophagy in the heart in vivo following ischemia-reperfusion and rapamycin therapy. We show that cathepsin-B activity in the lysosome is upregulated by rapamycin and that this allows the expanded lysosomal compartment in autophagy to be imaged in vivo with FMT. We further demonstrate that the delivery of diagnostic nanoparticles to the lysosome by endocytosis is enhanced during autophagy. The upregulation of autophagy by rapamycin was associated with a 23% reduction (p<0.05) of apoptosis in the area-at-risk (AAR), and a 45% reduction in final infarct size (19.6 +/− 5.6% of AAR with rapamycin versus 35.9 +/− 9.1% of AAR without rapamycin, p<0.05). Conclusions The ability to perform noninvasive tomographic imaging of autophagy in the heart has the potential to provide valuable insights into the pathophysiology of autophagy, particularly its role in cardiomyocyte salvage. While additional data are needed, our study supports the investigation of rapamycin therapy in patients with acute coronary syndromes. PMID:23537953

  14. Kinetic study of delta-Ala induced porphyrins in mice using photoacoustic and fluorescence spectroscopies.

    PubMed

    Stolik, Suren; Tomás, Sergio A; Ramón-Gallegos, Eva; Sánchez, Feliciano

    2002-11-01

    The production of delta-aminolevulinic acid (ALA)-induced porphyrins in mice skin and blood was studied by photoacoustic and fluorescence spectroscopies. Mice were intraperitoneally administered with 30 mg/kg of ALA. The abdominal skin was subsequently excised at specific times within an 8-h interval and its absorption spectrum obtained by photoacoustics. The highest porphyrins concentration in skin, determined from the optical absorption of the Soret band at 410 nm, was found to occur nearly 2 h after ALA administration, but a first peak was also observed at approximately 15 min. Our hypothesis that the first peak represents the porphyrins content in blood vessels within the skin, whereas the second peak corresponds to porphyrins production in skin tissue, was confirmed by analysing the evolution of protoporphyrin IX content in plasma extracted intracardiacally. By finally applying phase resolved photoacoustic spectroscopy, we were able to evaluate the mean depth at which porphyrins are generated.

  15. Raman Spectroscopy of Lithium Hydride Corrosion: Selection of an Appropriate Excitation Wavelength to Minimize Fluorescence

    SciTech Connect

    Stowe, A. C.; Smyrl, N. R.

    2011-05-26

    The recent interest in a hydrogen-based fuel economy has renewed research into metal hydride chemistry. Many of these compounds react readily with water to release hydrogen gas and form a caustic. Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFT) has been used to study the hydrolysis reaction. The LiOH stretch appears at 3670 cm{sup -1}. Raman spectroscopy is a complementary technique that employs monochromatic excitation (laser) allowing access to the low energy region of the vibrational spectrum (<600 cm{sup -1}). Weak scattering and fluorescence typically prevent Raman from being used for many compounds. The role of Li{sub 2}O in the moisture reaction has not been fully studied for LiH. Li{sub 2}O can be observed by Raman while being hidden in the Infrared spectrum.

  16. Tissue diagnosis using power-sharing multifocal Raman micro-spectroscopy and auto-fluorescence imaging.

    PubMed

    Sinjab, Faris; Kong, Kenny; Gibson, Graham; Varma, Sandeep; Williams, Hywel; Padge