Science.gov

Sample records for vivo fluorescence spectroscopy

  1. Toward quantitative "in vivo biochemistry" with fluorescence fluctuation spectroscopy.

    PubMed

    Slaughter, Brian D; Li, Rong

    2010-12-01

    Quantitative description of protein dynamics and interactions in vivo with temporal and spatial resolution is a key step in dissecting molecular mechanisms in cell biology. Fluorescence fluctuation spectroscopy (FFS) has recently emerged as a powerful in vivo tool for assessing molecular concentration and movement and formation of hetero- and homo-oligomeric complexes. This article discusses point FFS-based analysis methods that have proven useful to cell biologists, focusing on the kinds of information they provide, their pros and cons, and the basic instrumentation required. Along the way, we describe briefly a few recent examples where these analyses have helped address important biological questions. PMID:21160072

  2. 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.

  3. Intrinsic photosensitizer fluorescence measured using multi-diameter single-fiber spectroscopy in vivo

    NASA Astrophysics Data System (ADS)

    van Leeuwen-van Zaane, Floor; Gamm, Ute A.; van Driel, Pieter B. A. A.; Snoeks, Thomas J.; de Bruijn, Henriette S.; van der Ploeg-van den Heuvel, Angelique; Sterenborg, Henricus J. C. M.; Löwik, Clemens W.; Amelink, Arjen; Robinson, Dominic J.

    2014-01-01

    Quantification of fluorescence in vivo is complicated by the influence of tissue optical properties on the collected fluorescence signal. When tissue optical properties in the measurement volume are quantified, one can obtain the intrinsic fluorescence, which equals the product of fluorophore absorption coefficient and quantum yield. We applied this method to in vivo single-fiber fluorescence spectroscopy measurements on mouse tongue, skin, liver, and oral squamous cell carcinoma, where we detected intrinsic fluorescence spectra of the photosensitizers chlorin e6 and Bremachlorin at t=[3,4.5,6,24,48] h incubation time. We observed a tissue-dependent maximum of 35% variation in the total correction factor over the visible wavelength range. Significant differences in spectral shape over time between sensitizers were observed. Although the wavelength position of the fluorescence intensity maximum for ce6 shifted to the red, Bremachlorin showed a blue shift. Furthermore, the Bremachlorin peak appeared to be broader than the ce6 fluorescence peak. Intrinsic fluorescence intensity, which can be related to photosensitizer concentration, was decreasing for all time points but showed significantly more Bremachlorin present compared to ce6 at long incubation times. Results from this study can be used to define an optimal treatment protocol for Bremachlorin-based photodynamic therapy.

  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. Stationary spectroscopy of biotissues in vivo: Fluorescent studies of some pathological states

    NASA Astrophysics Data System (ADS)

    Giraev, K. M.; Ashurbekov, N. A.; Medzhidov, R. T.

    2003-11-01

    The stationary spectra of autofluorescence, along with the reflection coefficient at the wavelength of excitation, are measured in vivo for some stomach tissues in the case of different pathological states (dysplasia, superficial gastritis, and cancer) using a nitrogen laser as the source of excitation (λrad=337.1 nm). The fluorescence spectra obtained are decomposed into Gaussian-Lorentzian components. It is found that, in development of dysplasia and tumor processes, at least seven groups of fluorophores can be distinguished that form the entire emission spectrum. The ratio between the fluorescence intensities of flavins and NAD(P)H is determined and the degree of respiratory activity of cells estimated for the states considered. The quantum yields of fluorescence of the biotissues under investigation are estimated.

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

    PubMed Central

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

    2012-01-01

    Abstract. 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. PMID:23235925

  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. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Prostate cancer detection using combined auto-fluorescence and light reflectance spectroscopy: ex vivo study of human prostates

    PubMed Central

    Sharma, Vikrant; Olweny, Ephrem O.; Kapur, Payal; Cadeddu, Jeffrey A.; Roehrborn, Claus G.; Liu, Hanli

    2014-01-01

    This study was conducted to evaluate the capability of detecting prostate cancer (PCa) using auto-fluorescence lifetime spectroscopy (AFLS) and light reflectance spectroscopy (LRS). AFLS used excitation at 447 nm with four emission wavelengths (532, 562, 632, and 684 nm), where their lifetimes and weights were analyzed using a double exponent model. LRS was measured between 500 and 840 nm and analyzed by a quantitative model to determine hemoglobin concentrations and light scattering. Both AFLS and LRS were taken on n = 724 distinct locations from both prostate capsular (nc = 185) and parenchymal (np = 539) tissues, including PCa tissue, benign peripheral zone tissue and benign prostatic hyperplasia (BPH), of fresh ex vivo radical prostatectomy specimens from 37 patients with high volume, intermediate-to-high-grade PCa (Gleason score, GS ≥7). AFLS and LRS parameters from parenchymal tissues were analyzed for statistical testing and classification. A feature selection algorithm based on multinomial logistic regression was implemented to identify critical parameters in order to classify high-grade PCa tissue. The regression model was in turn used to classify PCa tissue at the individual aggressive level of GS = 7,8,9. Receiver operating characteristic curves were generated and used to determine classification accuracy for each tissue type. We show that our dual-modal technique resulted in accuracies of 87.9%, 90.1%, and 85.1% for PCa classification at GS = 7, 8, 9 within parenchymal tissues, and up to 91.1%, 91.9%, and 94.3% if capsular tissues were included for detection. Possible biochemical and physiological mechanisms causing signal differences in AFLS and LRS between PCa and benign tissues were also discussed. PMID:24877012

  15. 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.

  16. Combined fluorescence and reflectance spectroscopy for in vivo quantification of cancer biomarkers in low- and high-grade glioma surgery

    NASA Astrophysics Data System (ADS)

    Valdés, Pablo A.; Kim, Anthony; Leblond, Frederic; Conde, Olga M.; Harris, Brent T.; Paulsen, Keith D.; Wilson, Brian C.; Roberts, David W.

    2011-11-01

    Biomarkers are indicators of biological processes and hold promise for the diagnosis and treatment of disease. Gliomas represent a heterogeneous group of brain tumors with marked intra- and inter-tumor variability. The extent of surgical resection is a significant factor influencing post-surgical recurrence and prognosis. Here, we used fluorescence and reflectance spectral signatures for in vivo quantification of multiple biomarkers during glioma surgery, with fluorescence contrast provided by exogenously-induced protoporphyrin IX (PpIX) following administration of 5-aminolevulinic acid. We performed light-transport modeling to quantify multiple biomarkers indicative of tumor biological processes, including the local concentration of PpIX and associated photoproducts, total hemoglobin concentration, oxygen saturation, and optical scattering parameters. We developed a diagnostic algorithm for intra-operative tissue delineation that accounts for the combined tumor-specific predictive capabilities of these quantitative biomarkers. Tumor tissue delineation achieved accuracies of up to 94% (specificity = 94%, sensitivity = 94%) across a range of glioma histologies beyond current state-of-the-art optical approaches, including state-of-the-art fluorescence image guidance. This multiple biomarker strategy opens the door to optical methods for surgical guidance that use quantification of well-established neoplastic processes. Future work would seek to validate the predictive power of this proof-of-concept study in a separate larger cohort of patients.

  17. In vivo validation of a bimodal technique combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy for diagnosis of oral carcinoma

    NASA Astrophysics Data System (ADS)

    Sun, Yang; Xie, Hongtao; Liu, Jing; Lam, Matthew; Chaudhari, Abhijit J.; Zhou, Feifei; Bec, Julien; Yankelevich, Diego R.; Dobbie, Allison; Tinling, Steven L.; Gandour-Edwards, Regina F.; Monsky, Wayne L.; Gregory Farwell, D.; Marcu, Laura

    2012-11-01

    Tissue diagnostic features generated by a bimodal technique integrating scanning time-resolved fluorescence spectroscopy (TRFS) and ultrasonic backscatter microscopy (UBM) are investigated in an in vivo hamster oral carcinoma model. Tissue fluorescence is excited by a pulsed nitrogen laser and spectrally and temporally resolved using a set of filters/dichroic mirrors and a fast digitizer, respectively. A 41-MHz focused transducer (37-μm axial, 65-μm lateral resolution) is used for UBM scanning. Representative lesions of the different stages of carcinogenesis show that fluorescence characteristics complement ultrasonic features, and both correlate with histological findings. These results demonstrate that TRFS-UBM provide a wealth of co-registered, complementary data concerning tissue composition and structure as it relates to disease status. The direct co-registration of the TRFS data (sensitive to surface molecular changes) with the UBM data (sensitive to cross-sectional structural changes and depth of tumor invasion) is expected to play an important role in pre-operative diagnosis and intra-operative determination of tumor margins.

  18. In vivo validation of a bimodal technique combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy for diagnosis of oral carcinoma.

    PubMed

    Sun, Yang; Xie, Hongtao; Liu, Jing; Lam, Matthew; Chaudhari, Abhijit J; Zhou, Feifei; Bec, Julien; Yankelevich, Diego R; Dobbie, Allison; Tinling, Steven L; Gandour-Edwards, Regina F; Monsky, Wayne L; Farwell, D Gregory; Marcu, Laura

    2012-11-01

    Tissue diagnostic features generated by a bimodal technique integrating scanning time-resolved fluorescence spectroscopy (TRFS) and ultrasonic backscatter microscopy (UBM) are investigated in an in vivo hamster oral carcinoma model. Tissue fluorescence is excited by a pulsed nitrogen laser and spectrally and temporally resolved using a set of filters/dichroic mirrors and a fast digitizer, respectively. A 41-MHz focused transducer (37-μm axial, 65-μm lateral resolution) is used for UBM scanning. Representative lesions of the different stages of carcinogenesis show that fluorescence characteristics complement ultrasonic features, and both correlate with histological findings. These results demonstrate that TRFS-UBM provide a wealth of co-registered, complementary data concerning tissue composition and structure as it relates to disease status. The direct co-registration of the TRFS data (sensitive to surface molecular changes) with the UBM data (sensitive to cross-sectional structural changes and depth of tumor invasion) is expected to play an important role in pre-operative diagnosis and intra-operative determination of tumor margins. PMID:23117798

  19. Fluorescence lifetime spectroscopy of glioblastoma multiforme.

    PubMed

    Marcu, Laura; Jo, Javier A; Butte, Pramod V; Yong, William H; Pikul, Brian K; Black, Keith L; Thompson, Reid C

    2004-01-01

    Fluorescence spectroscopy of the endogenous emission of brain tumors has been researched as a potentially important method for the intraoperative localization of brain tumor margins. We investigated the use of time-resolved, laser-induced fluorescence spectroscopy for demarcation of primary brain tumors by studying the time-resolved spectra of gliomas. The fluorescence of human brain samples (glioblastoma multiforme, cortex and white matter: six patients, 23 sites) was induced ex vivo with a pulsed nitrogen laser (337 nm, 3 ns). The time-resolved spectra were detected in a 360-550 nm wavelength range using a fast digitizer and gated detection. Parameters derived from both the spectral- (intensities from narrow spectral bands) and the time domain (average lifetime) measured at 390 and 460 nm were used for tissue characterization. We determined that high-grade gliomas are characterized by fluorescence lifetimes that varied with the emission wavelength (>3 ns at 390 nm, <1 ns at 460 nm) and their emission is overall longer than that of normal brain tissue. Our study demonstrates that the use of fluorescence lifetime not only improves the specificity of fluorescence measurements but also allows a more robust evaluation of data collected from brain tissue. Combined information from both the spectral- and the time domain can enhance the ability of fluorescence-based techniques to diagnose and detect brain tumor margins intraoperatively. PMID:15339216

  20. 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. PMID:25098859

  1. 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.

  2. In vivo native fluorescence spectroscopy and nicotinamide adinine dinucleotide/flavin adenine dinucleotide reduction and oxidation states of oral submucous fibrosis for chemopreventive drug monitoring

    NASA Astrophysics Data System (ADS)

    Sivabalan, Shanmugam; Vedeswari, C. Ponranjini; Jayachandran, Sadaksharam; Koteeswaran, Dornadula; Pravda, Chidambaranathan; Aruna, Prakasa Rao; Ganesan, Singaravelu

    2010-01-01

    Native fluorescence spectroscopy has shown potential to characterize and diagnose oral malignancy. We aim at extending the native fluorescence spectroscopy technique to characterize normal and oral submucous fibrosis (OSF) patients under pre- and post-treated conditions, and verify whether this method could also be considered in the monitoring of therapeutic prognosis noninvasively. In this study, 28 normal subjects and 28 clinically proven cases of OSF in the age group of 20 to 40 years are diagnosed using native fluorescence spectroscopy. The OSF patients are given dexamethasone sodium phosphate and hyaluronidase twice a week for 6 weeks, and the therapeutic response is monitored using fluorescence spectroscopy. The fluorescence emission spectra of normal and OSF cases of both pre- and post-treated conditions are recorded in the wavelength region of 350 to 600 nm at an excitation wavelength of 330 nm. The statistical significance is verified using discriminant analysis. The oxidation-reduction ratio of the tissue is also calculated using the fluorescence emission intensities of flavin adenine dinucleotide and nicotinamide adinine dinucleotide at 530 and 440 nm, respectively, and they are compared with conventional physical clinical examinations. This study suggests that native fluorescence spectroscopy could also be extended to OSF diagnosis and therapeutic prognosis.

  3. High-Pressure Fluorescence Spectroscopy.

    PubMed

    Maeno, Akihiro; Akasaka, Kazuyuki

    2015-01-01

    The combination of fluorescence and pressure perturbation is a widely used technique to study the effect of pressure on a protein system to obtain thermodynamic, structural and kinetic information on proteins. However, we often encounter the situation where the available pressure range up to 400 MPa of most commercial high-pressure fluorescence spectrometers is insufficient for studying highly pressure-stable proteins like inhibitors and allergenic proteins. To overcome the difficulty, we have recently developed a new high-pressure fluorescence system that allows fluorescence measurements up to 700 MPa. Here we describe the basic design of the apparatus and its application to study structural and thermodynamic properties of a couple of highly stable allergenic proteins, hen lysozyme and ovomucoid, using Tryptophan and Tyrosine/Tyrosinate fluorescence, respectively. Finally, we discuss the utility and the limitation of Trp and Tyr fluorescence. We discuss pitfalls of fluorescence technique and importance of simultaneous use of other high-pressure spectroscopy, particularly high-pressure NMR spectroscopy. PMID:26174405

  4. 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.

  5. 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.

  6. Supercritical Angle Fluorescence Correlation Spectroscopy

    PubMed Central

    Ries, Jonas; Ruckstuhl, Thomas; Verdes, Dorinel; Schwille, Petra

    2008-01-01

    We explore the potential of a supercritical angle (SA) objective for fluorescence correlation spectroscopy (FCS). This novel microscope objective combines tight focusing by an aspheric lens with strong axial confinement of supercritical angle fluorescence collection by a parabolic mirror lens, resulting in a small detection volume. The tiny axial extent of the detection volume features an excellent surface sensitivity, as is demonstrated by diffusion measurements in model membranes with an excess of free dye in solution. All SA-FCS measurements are directly compared to standard confocal FCS, demonstrating a clear advantage of SA-FCS, especially for diffusion measurements in membranes. We present an extensive theoretical framework that allows for accurate and quantitative evaluation of the SA-FCS correlation curves. PMID:17827221

  7. 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

  8. In vivo fluorescence lifetime optical projection tomography

    PubMed Central

    McGinty, James; Taylor, Harriet B.; Chen, Lingling; Bugeon, Laurence; Lamb, Jonathan R.; Dallman, Margaret J.; French, Paul M. W.

    2011-01-01

    We demonstrate the application of fluorescence lifetime optical projection tomography (FLIM-OPT) to in vivo imaging of lysC:GFP transgenic zebrafish embryos (Danio rerio). This method has been applied to unambiguously distinguish between the fluorescent protein (GFP) signal in myeloid cells from background autofluorescence based on the fluorescence lifetime. The combination of FLIM, an inherently ratiometric method, in conjunction with OPT results in a quantitative 3-D tomographic technique that could be used as a robust method for in vivo biological and pharmaceutical research, for example as a readout of Förster resonance energy transfer based interactions. PMID:21559145

  9. 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.

  10. 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.

  11. 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.

  12. Chromophore detection by fluorescence spectroscopy in tissue-like phantoms

    NASA Astrophysics Data System (ADS)

    Cerussi, Albert E.; Fantini, Sergio; Maier, John S.; Mantulin, William W.; Gratton, Enrico

    1997-08-01

    In the near-infrared spectral region (700 - 900 nm) light penetrates a few centimeters into tissues and hemoglobin dominates the absorption. Consequently, in vivo near-infrared tissue absorption spectroscopy becomes difficult for endogenous compounds of biological interest other than hemoglobin. Exogenous chromophore detection by fluorescence spectroscopy has the potential to provide enhanced sensitivity and specificity for in vivo optical tissue spectroscopy, facilitating the study of many important metabolites in tissues other than hemoglobin. We have performed measurements of the dc fluorescence intensity generated by a fluorophore (rhodamine B) homogeneously dissolved inside a highly scattering tissue-simulating phantom (aqueous suspension of titanium-dioxide particles). The phantom was prepared with optical coefficients (absorption and reduced scattering) similar to those of tissue in the near-infrared; these coefficients were measured with a frequency-domain spectrometer. Measurable dc fluorescence intensity signals from 1 nM rhodamine concentrations inside the phantom are reported. Furthermore, we were able to resolve changes in rhodamine concentration on the order of 1% using the dc fluorescence intensity. This dc fluorescence sensitivity is characterized experimentally at two concentrations (55 and 360 nM) and over a range of source-detector separations. Other aspects of the sensitivity are discussed over a large range of concentrations using a fluorescence photon migration model.

  13. 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.

  14. Fluorescence spectroscopy of rhodopsins: insights and approaches.

    PubMed

    Alexiev, Ulrike; Farrens, David L

    2014-05-01

    Fluorescence spectroscopy has become an established tool at the interface of biology, chemistry and physics because of its exquisite sensitivity and recent technical advancements. However, rhodopsin proteins present the fluorescence spectroscopist with a unique set of challenges and opportunities due to the presence of the light-sensitive retinal chromophore. This review briefly summarizes some approaches that have successfully met these challenges and the novel insights they have yielded about rhodopsin structure and function. We start with a brief overview of fluorescence fundamentals and experimental methodologies, followed by more specific discussions of technical challenges rhodopsin proteins present to fluorescence studies. Finally, we end by discussing some of the unique insights that have been gained specifically about visual rhodopsin and its interactions with affiliate proteins through the use of fluorescence spectroscopy. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks. PMID:24183695

  15. Fluorescence spectroscopy of rhodopsins: Insights and approaches

    PubMed Central

    Alexiev, Ulrike; Farrens, David L.

    2014-01-01

    Fluorescence spectroscopy has become an established tool at the interface of biology, chemistry and physics because of its exquisite sensitivity and recent technical advancements. However, rhodopsin proteins present the fluorescence spectroscopist with a unique set of challenges and opportunities due to the presence of the light-sensitive retinal chromophore. This review briefly summarizes some approaches that have successfully met these challenges and the novel insights they have yielded about rhodopsin structure and function. We start with a brief overview of fluorescence fundamentals and experimental methodologies, followed by more specific discussions of technical challenges rhodopsin proteins present to fluorescence studies. Finally, we end by discussing some of the unique insights that have been gained specifically about visual rhodopsin and its interactions with affiliate proteins through the use of fluorescence spectroscopy. PMID:24183695

  16. Real-Time Fluorescence Tracking of Protoporphyrin Incorporated Thermosensitive Hydrogel and Its Drug Release in Vivo.

    PubMed

    Dong, Xia; Wei, Chang; Liu, Tianjun; Lv, Feng; Qian, Zhiyong

    2016-03-01

    Fluorescence imaging in vivo will pave an important way for the evaluation of biomaterials. The major advantage of fluorescence imaging compared to other imaging modalities is the possibility of tracking two or more fluorescence probes simultaneously with multispectral fluorescence imaging. It is essential to elucidate the location, erosion, drug release and resection of implanted biomaterials in vivo. Herein, a thermosensitive hydrogel with a protoporphyrin core based on a PEG and PCL copolymer (PCL-PEG-PPOR-PEG-PCL) was synthesized by ring-opening polymerization using protoporphyrin as a fluorescence tag. The optical properties of the hydrogel were investigated by UV-vis and fluorescence spectroscopy in vitro and by fluorescence imaging system in vivo. The hydrogel erosion and drug delivery in vivo were monitored and tracked by multispectral fluorescence imaging system in nude mice. The results show that the thermosensitive hydrogel exhibits fluorescence and injectability in vivo with good biocompatibility. Through the modality of fluorescence imaging, the status of the hydrogel is reflected in situ in vivo including its location and erosion. Multispectral analysis separates the autofluorescence signals from the specific label and provides the ability to locate the drug and carrier. The protoporphyrin incorporated thermosensitive hydrogel can be a potential visiable biomedical implant for tissue repair or drug delivery. PMID:26848506

  17. Online fluorescence suppression in modulated Raman spectroscopy.

    PubMed

    De Luca, Anna Chiara; Mazilu, Michael; Riches, Andrew; Herrington, C Simon; Dholakia, Kishan

    2010-01-15

    Label-free chemical characterization of single cells is an important aim for biomedical research. Standard Raman spectroscopy provides intrinsic biochemical markers for noninvasive analysis of biological samples but is often hindered by the presence of fluorescence background. In this paper, we present an innovative modulated Raman spectroscopy technique to filter out the Raman spectra from the fluorescence background. The method is based on the principle that the fluorescence background does not change whereas the Raman scattering is shifted by the periodical modulation of the laser wavelength. Exploiting this physical property and importantly the multichannel lock-in detection of the Raman signal, the modulation technique fulfills the requirements of an effective fluorescence subtraction method. Indeed, once the synchronization and calibration procedure is performed, minimal user intervention is required, making the method online and less time-consuming than the other fluorescent suppression methods. We analyze the modulated Raman signal and shifted excitation Raman difference spectroscopy (SERDS) signal of 2 mum-sized polystyrene beads suspended in a solution of fluorescent dye as a function of modulation rate. We show that the signal-to-noise ratio of the modulated Raman spectra at the highest modulation rate is 3 times higher than the SERDS one. To finally evaluate the real benefits of the modulated Raman spectroscopy, we apply our technique to Chinese hamster ovary cells (CHO). Specifically, by analyzing separate spectra from the membrane, cytoplasm, and nucleus of CHO cells, we demonstrate the ability of this method to obtain localized sensitive chemical information from cells, away from the interfering fluorescence background. In particular, statistical analysis of the Raman data and classification using PCA (principal component analysis) indicate that our method allows us to distinguish between different cell locations with higher sensitivity and

  18. Multipoint fluorescence correlation spectroscopy with total internal reflection fluorescence microscope.

    PubMed

    Ohsugi, Yu; Kinjo, Masataka

    2009-01-01

    We report simultaneous determination of diffusion coefficients at different points of a cell membrane using a multipoint fluorescence correlation spectroscopy (FCS) system. A system carrying seven detection areas in the evanescent field is achieved by using seven optical fibers on the image plane in the detection port of an objective-type total internal reflection FCS (TIR-FCS) system. Fluctuation of fluorescence intensity is monitored and evaluated using seven photomultiplier tubes (PMTs) and a newly constructed multichannel correlator. We demonstrate simultaneous-multipoint FCS, with a 3-mus time resolution, to investigate heterogeneous structures such as cell membranes and membrane-binding molecular dynamics near glass surfaces in live cells. PMID:19256718

  19. 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.

  20. 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.

  1. 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

  2. Fluorescence Correlation Spectroscopy: The Case of Subdiffusion

    PubMed Central

    Lubelski, Ariel; Klafter, Joseph

    2009-01-01

    The theory of fluorescence correlation spectroscopy is revisited here for the case of subdiffusing molecules. Subdiffusion is assumed to stem from a continuous-time random walk process with a fat-tailed distribution of waiting times and can therefore be formulated in terms of a fractional diffusion equation (FDE). The FDE plays the central role in developing the fluorescence correlation spectroscopy expressions, analogous to the role played by the simple diffusion equation for regular systems. Due to the nonstationary nature of the continuous-time random walk/FDE, some interesting properties emerge that are amenable to experimental verification and may help in discriminating among subdiffusion mechanisms. In particular, the current approach predicts 1), a strong dependence of correlation functions on the initial time (aging); 2), sensitivity of correlation functions to the averaging procedure, ensemble versus time averaging (ergodicity breaking); and 3), that the basic mean-squared displacement observable depends on how the mean is taken. PMID:19289033

  3. 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.

  4. Multispectral scanning time-resolved fluorescence spectroscopy (TRFS) technique for intravascular diagnosis

    PubMed Central

    Xie, Hongtao; Bec, Julien; Liu, Jing; Sun, Yang; Lam, Matthew; Yankelevich, Diego R.; Marcu, Laura

    2012-01-01

    This study describes a scanning time-resolved fluorescence spectroscopy (TRFS) system designed to continuously acquire fluorescence emission and to reconstruct fluorescence lifetime images (FLIM) from a luminal surface by using a catheter-based optical probe with rotary joint and pull-back device. The ability of the system to temporally and spectrally resolve the fluorescence emission from tissue was validated using standard dyes and tissue phantoms (e.g., ex vivo pig aorta phantom). Current results demonstrate that this system is capable to reliably resolve the fluorescence emission of multiple fluorophores located in the lumen; and suggest its potential for intravascular detection of distinct biochemical features of atherosclerotic plaques. PMID:22808425

  5. In vivo multiphoton fluorescence microscopy of epithelial precancer

    NASA Astrophysics Data System (ADS)

    Zheng, Wei; Li, Dong; Zeng, Yan; Qu, Jianan Y.

    2011-03-01

    Most human cancers arise from epithelium, the superficial layer covering the exterior of body or lining the internal body cavities. Endogenous fluorophores such as aromatic amino acids, reduced nicotinamide adenine dinucleotide (NADH), flavoprotein (FAD), keratin, collagen, and elastin can provide abundant information to reveal the changes in biochemistry, metabolism, and morphology of living tissues. Thus, autofluorescence spectroscopy and microscopy have been recognized as potential tools for discrimination of cancer from normal tissues. However, current fluorescence diagnostic studies mostly rely on spectral analysis or morphological differentiation. It is challenged since the emission spectra of endogenous fluorophores are broad and usually overlapping with each other and the fluorescence intensity could be affected by many factors. In this study, we instrumented a nonlinear optical microscopy system to characterize the morphologic and biochemical features in the epithelial precancer in vivo. The 7,12-dimethylbenz(a)anthracenetreated hamster cheek pouch were used as a living animal carcinogenesis model. And the autofluorescence signals of NADH, collagen and elastin were recorded by a time- and spectral- resolved detection system. The results show that there are obvious differences in the morphology of three-dimensional autofluorescence images between normal and precancerous epithelial tissues. The fluorescence lifetime of NADH and the SHG signal from collagen could provide additional approaches to identify cancer from normal tissue.

  6. 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

  7. Position-Sensitive Scanning Fluorescence Correlation Spectroscopy

    PubMed Central

    Skinner, Joseph P.; Chen, Yan; Müller, Joachim D.

    2005-01-01

    Fluorescence correlation spectroscopy (FCS) uses a stationary laser beam to illuminate a small sample volume and analyze the temporal behavior of the fluorescence fluctuations within the stationary observation volume. In contrast, scanning FCS (SFCS) collects the fluorescence signal from a moving observation volume by scanning the laser beam. The fluctuations now contain both temporal and spatial information about the sample. To access the spatial information we synchronize scanning and data acquisition. Synchronization allows us to evaluate correlations for every position along the scanned trajectory. We use a circular scan trajectory in this study. Because the scan radius is constant, the phase angle is sufficient to characterize the position of the beam. We introduce position-sensitive SFCS (PSFCS), where correlations are calculated as a function of lag time and phase. We present the theory of PSFCS and derive expressions for diffusion, diffusion in the presence of flow, and for immobilization. To test PSFCS we compare experimental data with theory. We determine the direction and speed of a flowing dye solution and the position of an immobilized particle. To demonstrate the feasibility of the technique for applications in living cells we present data of enhanced green fluorescent protein measured in the nucleus of COS cells. PMID:15894645

  8. Two-Photon Fluorescence Correlation Spectroscopy

    NASA Technical Reports Server (NTRS)

    Zimmerli, Gregory A.; Fischer, David G.

    2002-01-01

    We will describe a two-photon microscope currently under development at the NASA Glenn Research Center. It is composed of a Coherent Mira 900 tunable, pulsed Titanium:Sapphire laser system, an Olympus Fluoview 300 confocal scanning head, and a Leica DM IRE inverted microscope. It will be used in conjunction with a technique known as fluorescence correlation spectroscopy (FCS) to study intracellular protein dynamics. We will briefly explain the advantages of the two-photon system over a conventional confocal microscope, and provide some preliminary experimental results.

  9. In Vivo Fluorescence Reflectance Imaging with Subcutaneous Mouse Tumor Models.

    PubMed

    Cao, Jie; Zhou, Mingzhou

    2016-01-01

    Optical imaging is undoubtedly one of the most versatile and widely used imaging techniques in both research and clinical practice. Among optical imaging technologies, fluorescence imaging is the most popularly used and has become an essential tool in biomedical science. A key component of fluorescence imaging is fluorescence-producing reporters, including fluorescent dyes and conjugates, as well as fluorescent proteins. For in vivo imaging applications, fluorophores with long emission at the near-infrared (NIR) region are generally preferred to overcome the photon attenuation in living tissue. Here, we describe the in vivo fluorescence imaging of an integrin αυβ3 targeted NIR fluorescent probe (cRGD-ICG-Der-02) using subcutaneous mouse tumor models. PMID:27283414

  10. 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.

  11. 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.

  12. Fluorescence Spectroscopy Investigations of Cutaneous Tissues

    NASA Astrophysics Data System (ADS)

    Borisova, E.; Bliznakova, I.; Momchilov, N.; Troyanova, P.; Avramov, L.

    2007-04-01

    Fluorescence Spectroscopy of the human skin is very prominent for early diagnosis and differentiation of cutaneous diseases. Selection of proper excitation sources and sensitive detectors gives wide range of possibilities related to real-time determination of existing pathological conditions. A problem with using laser as an excitation source is the high expenses associated with the operation of these types of installations. This is why we test capability of a cheaper excitation sources - ultraviolet and blue light-emitting diodes. Initially, we investigate the spectral response of normal skin from different anatomic areas, as well as from different phototypes volunteers. Our first results obtained demonstrated promising possibility to implement an inexpensive system for detection of cutaneous lesions with wide clinical applications. The results achieved will be introduced in development of diagnostic algorithms for improvement of diagnostic sensitivity of benign and malignant tumor lesions determination.

  13. 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.

  14. In vivo optical spectroscopy for improved detection of pancreatic adenocarcinoma: a feasibility study

    PubMed Central

    Lloyd, William R.; Wilson, Robert H.; Lee, Seung Yup; Chandra, Malavika; McKenna, Barbara; Simeone, Diane; Scheiman, James; Mycek, Mary-Ann

    2013-01-01

    Pancreatic adenocarcinoma has a five-year survival rate of less than 6%. This low survival rate is attributed to the lack of accurate detection methods, which limits diagnosis to late-stage disease. Here, an in vivo pilot study assesses the feasibility of optical spectroscopy to improve clinical detection of pancreatic adenocarcinoma. During surgery on 6 patients, we collected spectrally-resolved reflectance and fluorescence in vivo. Site-matched in vivo and ex vivo data agreed qualitatively and quantitatively. Quantified differences between adenocarcinoma and normal tissues in vivo were consistent with previous results from a large ex vivo data set. Thus, optical spectroscopy is a promising method for the improved diagnosis of pancreatic cancer in vivo. PMID:24466472

  15. 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.

  16. 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.

  17. Towards a disposable in vivo miniature implantable fluorescence detector

    NASA Astrophysics Data System (ADS)

    Bellis, Stephen; Jackson, J. Carlton; Mathewson, Alan

    2006-02-01

    In the field of fluorescent microscopy, neuronal activity, diabetes and drug treatment are a few of the wide ranging biomedical applications that can be monitored with the use of dye markers. Historically, in-vivo fluorescent detectors consist of implantable probes coupled by optical fibre to sophisticated bench-top instrumentation. These systems typically use laser light to excite the fluorescent marker dies and using sensors, such as the photo-multiplier tube (PMT) or charge coupled devices (CCD), detect the fluorescent light that is filtered from the total excitation. Such systems are large and expensive. In this paper we highlight the first steps toward a fully implantable in-vivo fluorescence detection system. The aim is to make the detector system small, low cost and disposable. The current prototype is a hybrid platform consisting of a vertical cavity surface emitting laser (VCSEL) to provide the excitation and a filtered solid state Geiger mode avalanche photo-diode (APD) to detect the emitted fluorescence. Fluorescence detection requires measurement of extremely low levels of light so the proposed APD detectors combine the ability to count individual photons with the added advantage of being small in size. At present the exciter and sensor are mounted on a hybrid PCB inside a 3mm diameter glass tube.This is wired to external electronics, which provide quenching, photon counting and a PC interface. In this configuration, the set-up can be used for in-vitro experimentation and in-vivo analysis conducted on animals such as mice.

  18. 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

  19. Fluorescence Spectroscopy of Neoplastic and Non-Neoplastic Tissues

    PubMed Central

    Ramanujam, Nirmala

    2000-01-01

    Abstract Fast and non-invasive, diagnostic techniques based on fluorescence spectroscopy have the potential to link the biochemical and morphologic properties of tissues to individual patient care. One of the most widely explored applications of fluorescence spectroscopy is the detection of endoscopically invisible, early neoplastic growth in epithelial tissue sites. Currently, there are no effective diagnostic techniques for these early tissue transformations. If fluorescence spectroscopy can be applied successfully as a diagnostic technique in this clinical context, it may increase the potential for curative treatment, and thus, reduce complications and health care costs. Steady-state, fluorescence measurements from small tissue regions as well as relatively large tissue fields have been performed. To a much lesser extent, time-resolved, fluorescence measurements have also been explored for tissue characterization. Furthermore, sources of both intrinsic (endogenous fluorophores) and extrinsic fluorescence (exogenous fluorophores) have been considered. The goal of the current report is to provide a comprehensive review on steady-state and time-resolved, fluorescence measurements of neoplastic and non-neoplastic, biologic systems of varying degrees of complexity. First, the principles and methodology of fluorescence spectroscopy are discussed. Next, the endogenous fluorescence properties of cells, frozen tissue sections and excised and intact bulk tissues are presented; fluorescence measurements from both animal and human tissue models are discussed. This is concluded with future perspectives. PMID:10933071

  20. Handheld multispectral fluorescence lifetime imaging system for in vivo applications.

    PubMed

    Cheng, Shuna; Cuenca, Rodrigo M; Liu, Boang; Malik, Bilal H; Jabbour, Joey M; Maitland, Kristen C; Wright, John; Cheng, Yi-Shing Lisa; Jo, Javier A

    2014-03-01

    There is an increasing interest in the application of fluorescence lifetime imaging (FLIM) for medical diagnosis. Central to the clinical translation of FLIM technology is the development of compact and high-speed clinically compatible systems. We present a handheld probe design consisting of a small maneuverable box fitted with a rigid endoscope, capable of continuous lifetime imaging at multiple emission bands simultaneously. The system was characterized using standard fluorescent dyes. The performance was then further demonstrated by imaging a hamster cheek pouch in vivo, and oral mucosa tissue both ex vivo and in vivo, all using safe and permissible exposure levels. Such a design can greatly facilitate the evaluation of FLIM for oral cancer imaging in vivo. PMID:24688824

  1. 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.

  2. 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.

  3. 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.

  4. Combined Raman spectroscopy and autofluoresence imaging method for in vivo skin tumor diagnosis

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

    The fluorescence and Raman spectroscopy (RS) combined method of in vivo detection of malignant human skin cancer was demonstrated. The fluorescence analysis was used for detection of abnormalities during fast scanning of large tissue areas. In suspected cases of malignancy the Raman spectrum analysis of biological tissue was performed to determine the type of neoplasm. A special RS phase method was proposed for in vivo identification of skin tumor. Quadratic Discriminant Analysis was used for tumor type classification on phase planes. It was shown that the application of phase method provides a diagnosis of malignant melanoma with a sensitivity of 89% and a specificity of 87%.

  5. Multiphoton excitation fluorescence correlation spectroscopy of fluorescent DNA base analogs

    NASA Astrophysics Data System (ADS)

    Katilius, Evaldas; Woodbury, Neal W.

    2004-06-01

    Two- and three-photon excitation was used to investigate the properties of two fluorescent DNA base analogs: 2-aminopurine and 6-methylisoxanthopterin. 2-aminopurine is a widely used fluorescent analog of the DNA base adenine. Three-photon excitation of 2-aminopurine is achievable by using intense femtosecond laser pulses in 850-950 nm spectral region. Interestingly, the three-photon excitation spectrum is blue-shifted relative to the three-times-wavelength single-photon excitation spectrum. The maximum of the absorbance band in the UV is at 305 nm, while the three-photon excitation spectrum has a maximum at around 880 nm. Fluorescence correlation measurements were attempted to evaluate the feasibility of using three-photon excitation of 2-aminopurine for DNA-protein interaction studies. However, due to relatively small three-photon absorption cross-section, a good signal-to-noise fluorescence correlation curves take very long time to obtain. Fluorescence properties of 6-methylisoxanthopterin, the fluorescent analog of guanine, were investigated using two-photon excitation. This molecule has the lowest energy absorption band centered around 350 nm, thus, two-photon excitation is attainable using 700 to 760 nm output of Ti-sapphire laser. The excitation spectrum of this molecule in the infrared well matches the doubled-wavelength single-photon excitation spectrum in the UV. The high fluorescence quantum yield of 6-methylisoxanthopterin allows efficient fluorescence correlation measurements and makes this molecule a very good candidate for using in in vitro DNA-protein interaction studies.

  6. 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.

  7. 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.

  8. Quantum dots fluorescence quantum yield measured by Thermal Lens Spectroscopy.

    PubMed

    Estupiñán-López, Carlos; Dominguez, Christian Tolentino; Cabral Filho, Paulo E; Fontes, Adriana; de Araujo, Renato E

    2014-01-01

    An essential parameter to evaluate the light emission properties of fluorophores is the fluorescence quantum yield, which quantify the conversion efficiency of absorbed photons to emitted photons. We detail here an alternative nonfluorescent method to determine the absolute fluorescence quantum yield of quantum dots (QDs). The method is based in the so-called Thermal Lens Spectroscopy (TLS) technique, which consists on the evaluation of refractive index gradient thermally induced in the fluorescent material by the absorption of light. Aqueous dispersion carboxyl-coated cadmium telluride (CdTe) QDs samples were used to demonstrate the Thermal Lens Spectroscopy technical procedure. PMID:25103802

  9. 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

  10. Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy.

    PubMed

    Boens, Noël; Qin, Wenwu; Basarić, Nikola; Hofkens, Johan; Ameloot, Marcel; Pouget, Jacques; Lefèvre, Jean-Pierre; Valeur, Bernard; Gratton, Enrico; vandeVen, Martin; Silva, Norberto D; Engelborghs, Yves; Willaert, Katrien; Sillen, Alain; Rumbles, Garry; Phillips, David; Visser, Antonie J W G; van Hoek, Arie; Lakowicz, Joseph R; Malak, Henryk; Gryczynski, Ignacy; Szabo, Arthur G; Krajcarski, Don T; Tamai, Naoto; Miura, Atsushi

    2007-03-01

    A series of fluorophores with single-exponential fluorescence decays in liquid solution at 20 degrees C were measured independently by nine laboratories using single-photon timing and multifrequency phase and modulation fluorometry instruments with lasers as excitation source. The dyes that can serve as fluorescence lifetime standards for time-domain and frequency-domain measurements are all commercially available, are photostable under the conditions of the measurements, and are soluble in solvents of spectroscopic quality (methanol, cyclohexane, water). These lifetime standards are anthracene, 9-cyanoanthracene, 9,10-diphenylanthracene, N-methylcarbazole, coumarin 153, erythrosin B, N-acetyl-l-tryptophanamide, 1,4-bis(5-phenyloxazol-2-yl)benzene, 2,5-diphenyloxazole, rhodamine B, rubrene, N-(3-sulfopropyl)acridinium, and 1,4-diphenylbenzene. At 20 degrees C, the fluorescence lifetimes vary from 89 ps to 31.2 ns, depending on fluorescent dye and solvent, which is a useful range for modern pico- and nanosecond time-domain or mega- to gigahertz frequency-domain instrumentation. The decay times are independent of the excitation and emission wavelengths. Dependent on the structure of the dye and the solvent, the excitation wavelengths used range from 284 to 575 nm, the emission from 330 to 630 nm. These lifetime standards may be used to either calibrate or test the resolution of time- and frequency-domain instrumentation or as reference compounds to eliminate the color effect in photomultiplier tubes. Statistical analyses by means of two-sample charts indicate that there is no laboratory bias in the lifetime determinations. Moreover, statistical tests show that there is an excellent correlation between the lifetimes estimated by the time-domain and frequency-domain fluorometries. Comprehensive tables compiling the results for 20 (fluorescence lifetime standard/solvent) combinations are given. PMID:17269654

  11. 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

  12. 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.

  13. Photouncaging nanoparticles for MRI and fluorescence imaging in vitro and in vivo.

    PubMed

    Shibu, Edakkattuparambil S; Ono, Kenji; Sugino, Sakiko; Nishioka, Ayami; Yasuda, Akikazu; Shigeri, Yasushi; Wakida, Shin-Ichi; Sawada, Makoto; Biju, Vasudevanpillai

    2013-11-26

    Multimodal and multifunctional nanomaterials are promising candidates for bioimaging and therapeutic applications in the nanomedicine settings. Here we report the preparation of photouncaging nanoparticles with fluorescence and magnetic modalities and evaluation of their potentials for in vitro and in vivo bioimaging. Photoactivation of such bimodal nanoparticles prepared using photouncaging ligands, CdSe/ZnS quantum dots, and super paramagnetic iron oxide nanoparticles results in the systematic uncaging of the particles, which is correlated with continuous changes in the absorption, mass and NMR spectra of the ligands. Fluorescence and magnetic components of the bimodal nanoparticles are characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and elemental analyses using energy dispersive X-ray (EDX) spectroscopy and X-ray photoelectron spectroscopy (XPS). Bioconjugation of the nanoparticles with peptide hormones renders them with biocompatibility and efficient intracellular transport as seen in the fluorescence and MRI images of mouse melanoma cells (B16) or human lung epithelial adenocarcinoma cells (H1650). Biocompatibility of the nanoparticles is evaluated using MTT cytotoxicity assays, which show cell viability over 90%. Further, we combine MRI and NIR fluorescence imaging in C57BL/6 (B6) mice subcutaneously or intravenously injected with the photouncaging nanoparticles and follow the in vivo fate of the nanoparticles. Interestingly, the intravenously injected nanoparticles initially accumulate in the liver within 30 min post injection and subsequently clear by the renal excretion within 48 h as seen in the time-dependent MRI and fluorescence images of the liver, urinary bladder, and urine samples. Photouncaging ligands such as the ones reported in this article are promising candidates for not only the site-specific delivery of nanomaterials-based contrast agents and drugs but also the systematic uncaging and renal

  14. Widefield multiphoton excited fluorescence microscopy for animal study in vivo

    NASA Astrophysics Data System (ADS)

    Cheng, L.-C.; Chang, C.-Y.; Lin, C.-H.; Su, Y.-D.; Huang, T.-Y.; Chen, S.-J.

    2010-08-01

    Unlike conventional multiphoton excited microscopy according to pixel-by-pixel point scanning, a widefield multiphoton excited microscopy based on spatiotemporal focusing has been developed to construct three-dimensional (3D) multiphoton fluorescence images only with the need of an axial scanning. By implementing a 4.0 W 10 kHz femtosecond laser amplifier with an instant strong peak power and a fast TE-cooled EMCCD camera with an ultra-sensitive fluorescence detection, the multiphoton excited fluorescence images with the excitation area over 100 μm x 100 μm can be achieved at a frame rate up to 80 Hz. A mechanical shutter is utilized to control the exposure time of 1 ms, i.e. average ten laser pulses reach the fluorescent specimen, and hence an uniform enough multiphoton excited fluorescence image can be attained with less photobleaching. The Brownian motion of microbeads and 3D neuron cells of a rat cerebellum have been observed with a lateral spatial resolution of 0.24 μm and an axial resolution of 2.5 μm. Therefore, the developed widefield multiphoton microscopy can provide fast and high-resolution multiphoton excited fluorescence images for animal study in vivo.

  15. 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.

  16. Fluorescence suppression using wavelength modulated Raman spectroscopy in fiber-probe-based tissue analysis

    NASA Astrophysics Data System (ADS)

    Praveen, Bavishna B.; Ashok, Praveen C.; Mazilu, Michael; Riches, Andrew; Herrington, Simon; Dholakia, Kishan

    2012-07-01

    In the field of biomedical optics, Raman spectroscopy is a powerful tool for probing the chemical composition of biological samples. In particular, fiber Raman probes play a crucial role for in vivo and ex vivo tissue analysis. However, the high-fluorescence background typically contributed by the auto fluorescence from both a tissue sample and the fiber-probe interferes strongly with the relatively weak Raman signal. Here we demonstrate the implementation of wavelength-modulated Raman spectroscopy (WMRS) to suppress the fluorescence background while analyzing tissues using fiber Raman probes. We have observed a significant signal-to-noise ratio enhancement in the Raman bands of bone tissue, which have a relatively high fluorescence background. Implementation of WMRS in fiber-probe-based bone tissue study yielded usable Raman spectra in a relatively short acquisition time (~30 s), notably without any special sample preparation stage. Finally, we have validated its capability to suppress fluorescence on other tissue samples such as adipose tissue derived from four different species.

  17. Fluorescent-Spectroscopic Research of in Vivo Tissues Pathological Conditions

    NASA Astrophysics Data System (ADS)

    Giraev, K. M.; Ashurbekov, N. A.; Medzhidov, R. T.

    The steady-state spectra of autofluorescence and the reflection coefficient on the excitation wavelength of some stomach tissues in vivo with various pathological conditions (surface gastritis, displasia, cancer) are measured under excitation by the nitrogen laser irradiation (λex=337.1 nm). The contour expansion of obtained fluorescence spectra into contributions of components is conducted by the Gaussian-Lorentzian curves method. It is shown that at least 7 groups of fluorophores forming a total luminescence spectrum can be distinguished during the development of displasia and tumor processes. The correlation of intensities of flavins and NAD(P)·H fluorescence is determined and the degree of respiratory activity of cells for the functional condition considered is estimated. The evaluations of the fluorescence quantum yield of the tissue's researched are given.

  18. 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

  19. Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs.

    PubMed

    Visser, A J W G; Laptenok, S P; Visser, N V; van Hoek, A; Birch, D J S; Brochon, J-C; Borst, J W

    2010-01-01

    Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET-FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive. PMID:19693494

  20. 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.

  1. [Study on interaction of caffeine with myoglobin by fluorescence spectroscopy].

    PubMed

    Huang, He-Yong; Gu, Xiao-Tian; Ding, Yan; Zhou, Jia-Hong; Feng, Yu-Ying

    2009-10-01

    The interaction of caffein and myoglobin was investigated by fluorescence spectroscopy and synchronous fluorescence spectroscopy. The intrinsic fluorescence of myoglobin was significantly quenched by caffein under the physiological condition (pH 7.4). The results indicated that caffeine was capable of binding with myoglobin to form a 1:1 complex and the quenching mechanism of myoglobin affected by caffeine was shown to be a static quenching procedure by calculating quenching constant, binding sites and binding constant. According to the thermodynamic parameters, the main binding force of the interaction is electrostatic force and hydrophobic force. The change in the micro-circumstance of aminos of myoglobin was analyzed by synchronous fluorescence spectrometry. The result indicated that caffeine can change the conformation of the protein, leading to the change in the micro-environment of tryptophane and tyrosine residues from hydrophobic environment to hydrophilic environment to different extent. PMID:20038063

  2. Fluorescence correlation spectroscopy: inception, biophysical experimentations, and prospectus.

    PubMed

    Webb, W W

    2001-08-20

    Fluorescence correlation spectroscopy examines the chemical and the photophysical dynamics of dilute molecular solutions by measurement of the dynamic optical fluctuations of the fluorescence of a few molecules, even averaging less than one molecule at a time, in open focal volumes that are usually less than a femtoliter (<10(-18) m(3)). It applies the same principles of statistical thermodynamics as does quasi-elastic light scattering. Molecular interactions, conformational changes, chemical reactions, and photophysical dynamics that are not ordinarily detectable by quasi-elastic light scattering can be analyzed by fluorescence correlation spectroscopy in cases in which molecular fluorescence changes in the dynamic range 10(-7)-10(2) s. PMID:18360431

  3. Double-excitation fluorescence spectral imaging: eliminating tissue auto-fluorescence from in vivo PPIX measurements

    NASA Astrophysics Data System (ADS)

    Torosean, Sason; Flynn, Brendan; Samkoe, Kimberley S.; Davis, Scott C.; Gunn, Jason; Axelsson, Johan; Pogue, Brian W.

    2012-02-01

    An ultrasound coupled handheld-probe-based optical fluorescence molecular tomography (FMT) system has been in development for the purpose of quantifying the production of Protoporphyrin IX (PPIX) in aminolevulinic acid treated (ALA), Basal Cell Carcinoma (BCC) in vivo. The design couples fiber-based spectral sampling of PPIX fluorescence emission with a high frequency ultrasound imaging system, allowing regionally localized fluorescence intensities to be quantified [1]. The optical data are obtained by sequential excitation of the tissue with a 633nm laser, at four source locations and five parallel detections at each of the five interspersed detection locations. This method of acquisition permits fluorescence detection for both superficial and deep locations in ultrasound field. The optical boundary data, tissue layers segmented from ultrasound image and diffusion theory are used to estimate the fluorescence in tissue layers. To improve the recovery of the fluorescence signal of PPIX, eliminating tissue autofluorescence is of great importance. Here the approach was to utilize measurements which straddled the steep Qband excitation peak of PPIX, via the integration of an additional laser source, exciting at 637 nm; a wavelength with a 2 fold lower PPIX excitation value than 633nm.The auto-fluorescence spectrum acquired from the 637 nm laser is then used to spectrally decouple the fluorescence data and produce an accurate fluorescence emission signal, because the two wavelengths have very similar auto-fluorescence but substantially different PPIX excitation levels. The accuracy of this method, using a single source detector pair setup, is verified through animal tumor model experiments, and the result is compared to different methods of fluorescence signal recovery.

  4. 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.

  5. Validation of temperature-modulated fluorescence tomography in vivo

    NASA Astrophysics Data System (ADS)

    Kwong, Tiffany C.; Nouizi, Farouk; Lin, Yuting; Rajyaguru, Rushi; Nguyen, Trinh; Alptekin, Lara; Sampathkumaran, Uma; Zhu, Yue; Ahmed, Shaaz; Gulsen, Gultekin

    2014-02-01

    To overcome the strong scattering in biological tissue that has long afflicted fluorescence tomography, we have developed a novel technique, "temperature-modulated fluorescence tomography" (TM-FT) to combine the sensitivity of fluorescence imaging with focused ultrasound resolution. TM-FT relies on two key elements: temperature sensitive ICG loaded pluronic nanocapsules we termed ThermoDots and high intensity focused ultrasound (HIFU). TM-FT localizes the position of the fluorescent ThermoDots by irradiating and scanning a HIFU beam across the tissue while conventional fluorescence tomography measurements are acquired. The HIFU beam produces a local hot spot, in which the temperature suddenly increases changing the quantum efficiency of the ThermoDots. The small size of the focal spot (~1 mm) up to a depth of 6 cm, allows imaging the distribution of these temperature sensitive agents with not only high spatial resolution but also high quantitative accuracy in deep tissue using a proper image reconstruction algorithm. Previously we have demonstrated this technique with a phantom study with ThermoDots sensitive in the 20-25°C range. We recently optimized the ThermoDots for physiological temperatures. In this work, we will demonstrate a new HIFU scanning method which is optimized for in vivo studies. The performance of the system is tested using a phantom that resembles a small animal bearing a small tumor targeted by ThermoDots.

  6. 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.

  7. Intramolecular Fluorescence Correlation Spectroscopy in a Feedback Tracking Microscope

    NASA Astrophysics Data System (ADS)

    McHale, Kevin; Mabuchi, Hideo

    2010-07-01

    We derive the statistics of the signals generated by shape fluctuations of large molecules studied by feedback tracking microscopy. We account for the influence of intramolecular dynamics on the response of the tracking system, and derive a general expression for the fluorescence autocorrelation function that applies when those dynamics are linear. We show that tracking provides enhanced sensitivity to translational diffusion, molecular size, heterogeneity and long time-scale decays in comparison to traditional fluorescence correlation spectroscopy. We demonstrate our approach by using a three-dimensional tracking microscope to study genomic $\\lambda$-phage DNA molecules with various fluorescence label configurations.

  8. 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.

  9. 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.

  10. Spectral unmixing of multi-color tissue specific in vivo fluorescence in mice

    NASA Astrophysics Data System (ADS)

    Zacharakis, Giannis; Favicchio, Rosy; Garofalakis, Anikitos; Psycharakis, Stylianos; Mamalaki, Clio; Ripoll, Jorge

    2007-07-01

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

  11. 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.

  12. 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

  13. "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.

  14. Fibre optic fluorescence spectroscopy for monitoring fish freshness

    NASA Astrophysics Data System (ADS)

    Wu, Chi-Wu; Hsiao, Tzu-Chien; Chu, Shou-Chia; Hu, Hung-Hsi; Chen, Jyh-Cheng

    2012-01-01

    In this study, a portable Y-type fibreoptic fluorescence spectroscopy measurement system was used to evaluate the freshness of eight cobias (Rachycentron canadum). The results showed that the ratio of fluorescent intensity, which F480 nm/Fexci+50 nm was belong with the range of collagen type I and type V characteristic spectra, was positive correlated to the frozen time by hours. It was a strong approach to be a potential index for differentiating the fish freshness during delivery process. Besides, the different pattern results of dorsum and abdomen were shown in this study. In further, fibreoptic fluorescence spectroscopy could be a way not only to measure and quantify the freshness of different fish body but also to verify the level of taste.

  15. 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.

  16. 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.

  17. Solid-phase fluorescence spectroscopy to characterize organic wastes.

    PubMed

    Muller, Mathieu; Milori, Débora Marcondes Bastos Pereira; Déléris, Stéphane; Steyer, Jean-Philippe; Dudal, Yves

    2011-01-01

    The production of solid organic waste (SOW) such as sewage sludge (SS) or municipal solid waste (MSW) has been continuously increasing in Europe since the beginning of the 1990'. Today, the European Union encourages the stabilization of these wastes using biologic processes such as anaerobic digestion and/or composting to produce bio-energy and organic fertilizers. However, the design and management of such biologic processes require knowledge about the quantity and quality of the organic matter (OM) contained in the SOW. The current methods to characterize SOW are tedious, time-consuming and often insufficiently informative. In this paper, we assess the potential of solid-phase fluorescence (SPF) spectroscopy to quickly provide a relevant characterization of SOW. First, we tested well known model compounds (tryptophan, bovine serum albumin, lignin and humic acid) and biologic matrix (Escherichia coli) in three dimensional solid-phase fluorescence (3D-SPF) spectroscopy. We recorded fluorescence spectra from proteinaceous samples but we could not record the fluorescence emitted by lignin and humic acid powders. For SOW samples, fluorescence spectra were successfully recorded for MSW and most of its sub-components (foods, cardboard) but impossible for SS, sludge compost (SC) and ligno-cellulosic wastes. Based on visual observations and additional assays, we concluded that the presence of highly light-absorptive chemical structures in such dark-colored samples was responsible for this limitation. For such samples, i.e. lignin, humic acid, SS, SC and ligno-cellulosic wastes, we show that laser induced fluorescence (LIF) spectroscopy enables the acquisition of 2D fluorescence spectra. PMID:21696938

  18. Feasibility of Raman spectroscopy in vitro after 5-ALA-based fluorescence diagnosis in the bladder

    NASA Astrophysics Data System (ADS)

    Grimbergen, M. C. M.; van Swol, C. F. P.; van Moorselaar, R. J. A.; Mahadevan-Jansen, A.,; Stone, N.

    2006-02-01

    Photodynamic diagnosis (PDD) has become popular in bladder cancer detection. Several studies have however shown an increased false positive biopsies rate under PDD guidance compared to conventional cystoscopy. Raman spectroscopy is an optical technique that utilizes molecular specific, inelastic scattering of light photons to interrogate biological tissues, which can successfully differentiate epithelial neoplasia from normal tissue and inflammations in vitro. This investigation was performed to show the feasibility of NIR Raman spectroscopy in vitro on biopsies obtained under guidance of 5-ALA induced PPIX fluorescence imaging. Raman spectra of a PPIX solution was measured to obtain a characteristic signature for the photosensitzer without contributions from tissue constituents. Biopsies were obtained from patients with known bladder cancer instilled with 50ml, 5mg 5-ALA two hours prior to trans-urethral resection of tumor (TURT). Additional biopsies were obtained at a fluorescent and non-fluorescent area, snap-frozen in liquid nitrogen and stored at -80 °C. Each biopsy was thawed before measurements (10sec integration time) with a confocal Raman system (Renishaw Gloucestershire, UK). The 830 nm excitation (300mW) source is focused on the tissue by a 20X ultra-long-working-distance objective. Differences in fluorescence background between the two groups were removed by means of a special developed fluorescence subtraction algorithm. Raman spectra from ALA biopsies showed different fluorescence background which can be effectively removed by a fluorescence subtraction algorithm. This investigation shows that the interaction of the ALA induced PPIX with Raman spectroscopy in bladder samples. Combination of these techniques in-vivo may lead to a viable method of optical biopsies in bladder cancer detection.

  19. Diagnosis of corneal pathology by laser fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Salmin, V. V.; Lazarenko, V. I.; Salmina, A. B.; Hovalyg, M. Sh.; Vladimirova, E. S.

    2012-09-01

    We have studied the difference between the fluorescence spectra of the human cornea in vivo under normal conditions and after contact lenses have been worn for different lengths of time, with excitation by emission from a nitrogen laser (337 nm). The most significant sections of the difference spectrum were identified, corresponding to peaks for endogenous fluorophores (NADH and collagen). A high correlation was found between how long the contact lenses have been worn and the fluorescence intensity ratio for wavelengths 460 nm and 410 nm.

  20. Fluorescence Lifetime Correlation Spectroscopy (FLCS): Concepts, Applications and Outlook

    PubMed Central

    Kapusta, Peter; Macháň, Radek; Benda, Aleš; Hof, Martin

    2012-01-01

    Fluorescence Lifetime Correlation Spectroscopy (FLCS) is a variant of fluorescence correlation spectroscopy (FCS), which uses differences in fluorescence intensity decays to separate contributions of different fluorophore populations to FCS signal. Besides which, FLCS is a powerful tool to improve quality of FCS data by removing noise and distortion caused by scattered excitation light, detector thermal noise and detector after pulsing. We are providing an overview of, to our knowledge, all published applications of FLCS. Although these are not numerous so far, they illustrate possibilities for the technique and the research topics in which FLCS has the potential to become widespread. Furthermore, we are addressing some questions which may be asked by a beginner user of FLCS. The last part of the text reviews other techniques closely related to FLCS. The generalization of the idea of FLCS paves the way for further promising application of the principle of statistical filtering of signals. Specifically, the idea of fluorescence spectral correlation spectroscopy is here outlined. PMID:23202928

  1. In vivo resolution of oligomers with fluorescence photobleaching recovery histograms

    PubMed Central

    Youn, B.S.; Lepock, J.R.; Borrelli, M.J.; Jervis, E.J.

    2006-01-01

    Simple independent enzyme-catalyzed reactions distributed homogeneously throughout an aqueous environment cannot adequately explain the regulation of metabolic and other cellular processes in vivo. Such an unstructured system results in unacceptably slow substrate turnover rates and consumes inordinate amounts of cellular energy. Current approaches to resolving compartmentalization in living cells requires the partitioning of the molecular species in question such that its localization can be resolved with fluorescence microscopy. Standard imaging approaches will not resolve localization of protein activity for proteins that are ubiquitously distributed, but whose function requires a change in state of the protein. The small heat shock protein sHSP27 exists as both dimers and large multimers and is distributed homogeneously throughout the cytoplasm. A fusion of the green fluorescent protein variant S65T and sHSP27 is used to assess the ability of diffusion rate histograms to resolve compartmentalization of the 2 dominant oligomeric species of sHSP27. Diffusion rates were measured by multiphoton fluorescence photobleaching recovery. Under physiologic conditions, diffusion rate histograms resolved at least 2 diffusive transport rates within a living cell potentially corresponding to the large and small oligomers of sHSP27. Given that oligomerization is often a means of regulation, compartmentalization of different oligomer species could provide a means for efficient regulation and localization of sHsp27 activity. PMID:16817323

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

    PubMed Central

    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

  10. 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.

  11. Investigation on the interaction of cefpirome sulfate with lysozyme by fluorescence quenching spectroscopy and synchronous fluorescence spectroscopy.

    PubMed

    Han, Rong; Liu, Baosheng; Li, Gaixia; Zhang, Qiuju

    2016-03-01

    The reaction mechanism of cefpirome sulfate with lysozyme at different temperatures (298, 310 and 318 K) was investigated using fluorescence quenching and synchronous fluorescence spectroscopy under simulated physiological conditions. The results clearly demonstrated that cefpirome sulfate caused strong quenching of the fluorescence of lysozyme by a static quenching mechanism. The binding constants obtained using the above methods were of the same order of magnitude and very similar. Static electric forces played a key role in the interaction between cefpirome sulfate and lysozyme, and the number of binding sites in the interaction was close to 1. The values of Hill's coefficients were > 1, indicating that drugs or proteins showed a very weakly positive cooperativity in the system. In addition, the conclusions obtained from the two methods using the same equation were consistent. The results indicated that synchronous fluorescence spectrometry could be used to study the binding mechanism between drug and protein, and was a useful supplement to the fluorescence quenching method. In addition, the effect of cefpirome sulfate on the secondary structure of lysozyme was analyzed using circular dichroism spectroscopy. PMID:26304690

  12. 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

  13. Simultaneous Surface-Near and Solution Fluorescence Correlation Spectroscopy.

    PubMed

    Winterflood, Christian M; Seeger, Stefan

    2016-05-01

    We report the first simultaneous measurement of surface-confined and solution fluorescence correlation spectroscopy (FCS). We use an optical configuration for tightly focused excitation and separate detection of light emitted below (undercritical angle fluorescence, UAF) and above (supercritical angle fluorescence, SAF) the critical angle of total internal reflection of the coverslip/sample interface. This creates two laterally coincident detection volumes which differ in their axial extent. While detection of far-field UAF emission producesa standard confocal volume, near-field-mediated SAF produces a highly surface-confined detection volume at the coverslip/sample interface which extends only ~200 nm into the sample. A characterization of the two detection volumes by FCS of free diffusion is presented and compared with analytical models and simulations. The presented FCS technique allows to determine bulk solution concentrations and surface-near concentrations at the same time. PMID:27001472

  14. Spectroscopy analysis of tissues in vivo

    NASA Astrophysics Data System (ADS)

    Loschenov, Victor B.; Poleshkin, P. V.; Stratonnikov, Alexander A.; Torshina, Nadezgda L.

    1995-01-01

    The spectral analysis of biological tissues in vivo is widely used in various fields particularly in medical diagnostics and therapy control. Great possibilities of spectral tissue analysis exist to be realized in the future. Among them are the complete non-invasive clinical blood analysis with evaluation of, for example, sugar concentration in blood; the evaluation of chemical state and localization on subcell level of various drugs binded with biological structures. These facts were shown to affect drastically the drug therapeutic activity. The main advantage of spectral analysis of tissues in vivo is its noninvasivity. This allows one to get information about tissue condition without affecting the dynamic of various biological processes. Another advantage of optical tissue analysis is the possibility to process data in real time and to control parameters of therapy process according to information acquired. For example the in situ analysis of photosensitizer concentration and its chemical state during photodynamic therapy makes it possible to correct the laser irradiation intensity (the photobleaching of photosensitizer requires the decrease in laser intensity).

  15. 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.

  16. 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.

  17. 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

  18. In vivo monitoring of photosensitizer fluorescence during photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Stringer, Mark R.; Robinson, Dominic J.; Hudson, Emma J.; Smith, Michael A.

    1995-03-01

    A method is presented of monitoring the low level fluorescence emitted by the photosensitizing agent protoporphyrin IX during superficial photodynamic therapy of skin carcinomas, using 630 nm illumination. A fiber optic probe samples the light field which is filtered and recorded by an optical spectrum analyzer. The technique is minimally invasive and can proceed concurrently with light dosimetry measurements. This paper presents in vitro data that define the sensitivity and selectivity of the technique, along with preliminary in vivo measurements. These indicate that it is the rate of phototransformation of the photosensitizer, rather than the total light dose, that determines the optimum treatment duration. Clinically effective treatment therefore depends upon achieving a threshold concentration of drug throughout the volume of the lesion. In this way the effect of phototransformation does not inactivate the drug before complete tumor necrosis occurs.

  19. 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.

  20. Lifetime fluorescence spectroscopy for in situ investigation of osteogenic differentiation

    NASA Astrophysics Data System (ADS)

    Marcu, Laura; Elbarbary, Amir; Zuk, Patricia; De Ugarte, Daniel A.; Benhaim, Prosper; Kurt, Hamza; Hedrick, Marc H.; Ashjian, Peter

    2003-07-01

    Time-Resolved Laser-Induced Fluorescence Spectroscopy (TR-LIFS) represents a potential tool for the in-situ characterization of bioengineered tissues. In this study, we evaluate the application of TR-LIFS to non-intrusive monitoring of matrix composition during osteogenetic differentiation. Human adipose-derived stem cells, harvested from 3 patients, were induced in osteogenic media for 3, 5, and 7 weeks. Samples were subsequently collected and probed for time-resolved fluorescence emission with a pulsed nitrogen laser. Fluorescence parameters, derived from both spectral- and time-domain, were used for sample characterization. The samples were further analyzed using Western blot analysis and computer-based densitometry. A significant change in the fluorescence parameters was detected for samples beyond 3 weeks of osteogenic differentiation. The spectroscopic observations: 1) show increase of collagen I when contrasted against the time-resolved fluorescence spectra of commercially available collagens; and 2) are in agreement with Western blot analysis that demonstrated significant increase in collagen I content between 3- vs. 5-weeks and 3- vs. 7-weeks and no changes for collagens III, IV, and V. Our results suggest that TR-LIFS can be used as a non-invasive means for the detection of specific collagens in maturing connective tissues.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. Coded spectroscopy for ethanol detection in diffuse, fluorescent media

    NASA Astrophysics Data System (ADS)

    McCain, Scott Thomas

    Optical sensing in the visible and near-infrared regions of the electromagnetic spectrum has many useful applications. One particularly interesting one is the non-invasive analysis of tissue since a high penetration depth is possible. With the use of Raman spectroscopy, a high degree of chemical specificity is available with laser powers that are harmless to living tissue. Such systems, however, are plagued by the low efficiency of the Raman scattering process by molecules and the intense background fluorescence from some biological materials. To address these drawbacks, we have investigated the use of coded spectroscopy to make Raman spectroscopy more feasible in routine use. By coding the input aperture of a dispersive spectrometer, throughput gains of 10-100 are possible over a traditional slit spectrometer. The theory, design, and performance characteristics of this static aperture coding will be discussed in this thesis. In addition, by coding the excitation light sources one can filter out the shifting Raman signals from the stationary fluorescent background. The theory and implementation of an expectation maximization algorithm for Raman signal reconstruction will be analyzed. In addition, the design of a multi-excitation, coded-aperture Raman spectrometer will be described, which uses both of the coding mechanisms described.

  6. 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.

  7. 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.

  8. New approach on fluorescence spectroscopy for caries detection

    NASA Astrophysics Data System (ADS)

    Hibst, Raimund; Paulus, Robert

    1999-05-01

    Today the diagnosis of caries is based mainly on examinations by visual inspection, dental probe or by x- rays. All methods are very limited when either initial or undermining caries have to be found. For initial caries promising results have been demonstrated by fluorescence spectroscopy with excitation wavelengths in the (ultra-)violet to green spectral region, especially 406 nm or 488 nm. In our investigations, we extended the considered excitation wavelength range into red. As expected, total fluorescence yield is decreasing with increasing wavelength, but this decrease is much more pronounced for sound compared to carious enamel or dentin. For 640 nm or 655 nm excitation for example, integral (λ>680nm) fluorescence intensity of cares can exceed that of healthy tissue by about one order of magnitude. This allows to detect caries by fluorescence intensity rather than by spectral analysis. On the basis of these results we have built up a system using a diode laser as light source, and a photo diode combined with a long pass filter as detector. It provides quantitatively reproducible measurements and detection even through sound enamel of 1 mm thickness. Clinical applications include detection of undermining caries and monitoring of the decay process.

  9. Investigation of asphaltene association by front-face fluorescence spectroscopy.

    PubMed

    Albuquerque, Flávio Cortiñas; Nicodem, David E; Rajagopal, Krishnaswamy

    2003-07-01

    The tendency of asphaltenes to aggregate and form clusters in solvents was studied by fluorescence spectroscopy. This was done by evaluating the relative fluorescence quantum yield of asphaltenes diluted at several concentrations in toluene and by studying the changes in the fluorescence spectra of asphaltene solutions as the composition of the solvent, toluene and cyclohexane, is changed. The asphaltene fraction (heptane insoluble) was collected from a Brazilian heavy crude oil, and solutions of this material varying from 0.016 g/L up to 10 g/L were prepared in toluene. Front-face emission spectra were obtained in two wavelength ranges, from 310 to 710 nm, excited at 300 nm (short range), and from 410 to 710 nm, excited at 400 nm (long range). Severe quenching was observed at concentrations above about 0.1 g/L. Stern-Volmer plots (reciprocal of quantum yield against concentration) exhibited nonlinear, downward-curved behavior, indicating that a more complex suppression mechanism, probably influenced by the association of the asphaltene molecules, is taking place. The same asphaltenes were dissolved (0.1 g/L) in binary mixtures of toluene and cyclohexane, and emission spectra in both the short range and long range were obtained. Fluorescence was progressively quenched at longer wavelengths of the spectra as the proportion of cyclohexane in the solvent grew. Cyclohexane, a poor asphaltene solvent, is probably inducing static quenching through association of asphaltenes. PMID:14658659

  10. Single-molecule fluorescence spectroscopy in (bio)catalysis

    PubMed Central

    Roeffaers, Maarten B. J.; De Cremer, Gert; Uji-i, Hiroshi; Muls, Benîot; Sels, Bert F.; Jacobs, Pierre A.; De Schryver, Frans C.; De Vos, Dirk E.; Hofkens, Johan

    2007-01-01

    The ever-improving time and space resolution and molecular detection sensitivity of fluorescence microscopy offer unique opportunities to deepen our insights into the function of chemical and biological catalysts. Because single-molecule microscopy allows for counting the turnover events one by one, one can map the distribution of the catalytic activities of different sites in solid heterogeneous catalysts, or one can study time-dependent activity fluctuations of individual sites in enzymes or chemical catalysts. By experimentally monitoring individuals rather than populations, the origin of complex behavior, e.g., in kinetics or in deactivation processes, can be successfully elucidated. Recent progress of temporal and spatial resolution in single-molecule fluorescence microscopy is discussed in light of its impact on catalytic assays. Key concepts are illustrated regarding the use of fluorescent reporters in catalytic reactions. Future challenges comprising the integration of other techniques, such as diffraction, scanning probe, or vibrational methods in single-molecule fluorescence spectroscopy are suggested. PMID:17664433

  11. Cytoskeleton dynamics studied by dispersion-relation fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Wang, Ru; Lei, Lei; Wang, Yingxiao; Levine, Alex; Popescu, Gabriel

    2013-03-01

    Fluorescence is the most widely used microscopy technique for studying the dynamics and function in both medical and biological sciences due to its sensitivity and specificity. Inspired by the spirit of spatial fluorescence correlation spectroscopy, we propose a new method to study the transport dynamics over a broad range of spatial and temporal scales. The molecules of interest are labeled with a fluorophore whose motion gives rise to spontaneous fluorescence intensity fluctuations that can be further analyzed to quantify the governing molecular mass transport dynamics. We analyze these data by the dispersion relation in the form of a power law, Γ(q) ~qα , which describe the relaxation rate of fluorescence intensity fluctuations, Γ, vs. the wavenumber, q. We used this approach to study the interplay of various cytoskeletal components in intracellular transport under the influence of protein-motor inhibitors. We found that after actin is depolymerized, the transport becomes completely random for a few minutes and then it starts to organize deterministically again. We conclude that the disrupted cytoskeletal components first diffuse in the cytoplasm, but then become attached to microtubules and get transported deterministically.

  12. 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

  13. An analog filter approach to frequency domain fluorescence spectroscopy

    DOE PAGESBeta

    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

  14. 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.

  15. 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.

  16. Transient Fluorescence Spectroscopy and laser induced fluorescence lifetimes of terbium doped dipicolinic acid

    NASA Astrophysics Data System (ADS)

    Makoui, Anali

    We have investigated the use of deep UV laser induced fluorescence for the sensitive detection and spectroscopic lifetime studies of terbium doped dipicolinic acid (DPA-Tb) and used this to study the optical characteristics of DPA which is a chemical surrounding most bacterial spores. Background absorption spectra, fluorescence spectra, and Excitation Emission Matrix (EEM) spectra were made of the DPA-Tb complex, using both fixed 266 nm wavelength and tunable (220 nm--280 nm) UV laser excitations. Of importance, the fluorescence lifetimes of the four main fluorescence peaks (488 nm, 543 nm, 581 nm, and 618 nm) of the DPA-Tb complex have been measured for the first time to our knowledge. The lifetimes of all the fluorescing lines have been measured as a function of DPA-Tb concentration, solvent pH, and solvent composition, including that for the weakest fluorescing line of DPA-Tb at 618 nm. In addition, a new spectroscopic lifetime measurement technique, which we call "Transient Fluorescence Spectroscopy", was developed. In this technique, a weak, quasi-CW, amplitude modulated UV laser (8.5 kHz) was used to measure the lifetimes of the fluorescence lines, and yields insight into energy transfer and excitation lifetimes within the system. This technique is especially useful when a high power laser is not either available or not suitable. In the latter case, this would be when a high power pulsed deep-UV laser could produce bleaching or destruction of the biological specimen. In addition, this technique simulated the excitation and fluorescence emission of the DPA-Tb using a 4-level energy model, and solved the dynamic transient rate equations to predict the temporal behavior of the DPA-Tb emitted fluorescence. Excellent agreement between the experiments and the simulation were found. This technique has the potential to provide a more accurate value for the fluorescence lifetime values. In addition, with the use of asymmetric excitation waveforms, the dynamic

  17. Stochastic fractal behavior in concentration fluctuation and fluorescence correlation spectroscopy

    PubMed Central

    Raymond, Gary M.; Bassingthwaighte, James B.

    2010-01-01

    Fluctuations in the concentration of Brownian particles in one and two dimensions, or any reasonable measurement of the concentration such as in fluorescence correlation spectroscopy, is shown to be a stochastic fractal with a long tail. Being singular at ω = 0, the power spectrum of the fluctuation S(ω) ~ ω −1/2 for diffusion in one dimension, ~ log ω in two dimensions, but non-singular in three dimensions. This discovery provides one simple physical mechanism for possible long-memory fractal behavior, and its implications to various biological processes are discussed. PMID:10457592

  18. 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.

  19. 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.

  20. 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

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

    PubMed

    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

  2. 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.

  3. 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

  4. A comparative evaluation of Raman and fluorescence spectroscopy for optical diagnosis of oral neoplasia

    NASA Astrophysics Data System (ADS)

    Majumder, S. K.; Krishna, H.; Sidramesh, M.; Chaturvedi, P.; Gupta, P. K.

    2011-08-01

    We report the results of a comparative evaluation of in vivo fluorescence and Raman spectroscopy for diagnosis of oral neoplasia. The study carried out at Tata Memorial Hospital, Mumbai, involved 26 healthy volunteers and 138 patients being screened for neoplasm of oral cavity. Spectral measurements were taken from multiple sites of abnormal as well as apparently uninvolved contra-lateral regions of the oral cavity in each patient. The different tissue sites investigated belonged to one of the four histopathology categories: 1) squamous cell carcinoma (SCC), 2) oral sub-mucous fibrosis (OSMF), 3) leukoplakia (LP) and 4) normal squamous tissue. A probability based multivariate statistical algorithm utilizing nonlinear Maximum Representation and Discrimination Feature for feature extraction and Sparse Multinomial Logistic Regression for classification was developed for direct multi-class classification in a leave-one-patient-out cross validation mode. The results reveal that the performance of Raman spectroscopy is considerably superior to that of fluorescence in stratifying the oral tissues into respective histopathologic categories. The best classification accuracy was observed to be 90%, 93%, 94%, and 89% for SCC, SMF, leukoplakia, and normal oral tissues, respectively, on the basis of leave-one-patient-out cross-validation, with an overall accuracy of 91%. However, when a binary classification was employed to distinguish spectra from all the SCC, SMF and leukoplakik tissue sites together from normal, fluorescence and Raman spectroscopy were seen to have almost comparable performances with Raman yielding marginally better classification accuracy of 98.5% as compared to 94% of fluorescence.

  5. A comparative evaluation of Raman and fluorescence spectroscopy for optical diagnosis of oral neoplasia

    NASA Astrophysics Data System (ADS)

    Majumder, S. K.; Krishna, H.; Sidramesh, M.; Chaturvedi, P.; Gupta, P. K.

    2010-12-01

    We report the results of a comparative evaluation of in vivo fluorescence and Raman spectroscopy for diagnosis of oral neoplasia. The study carried out at Tata Memorial Hospital, Mumbai, involved 26 healthy volunteers and 138 patients being screened for neoplasm of oral cavity. Spectral measurements were taken from multiple sites of abnormal as well as apparently uninvolved contra-lateral regions of the oral cavity in each patient. The different tissue sites investigated belonged to one of the four histopathology categories: 1) squamous cell carcinoma (SCC), 2) oral sub-mucous fibrosis (OSMF), 3) leukoplakia (LP) and 4) normal squamous tissue. A probability based multivariate statistical algorithm utilizing nonlinear Maximum Representation and Discrimination Feature for feature extraction and Sparse Multinomial Logistic Regression for classification was developed for direct multi-class classification in a leave-one-patient-out cross validation mode. The results reveal that the performance of Raman spectroscopy is considerably superior to that of fluorescence in stratifying the oral tissues into respective histopathologic categories. The best classification accuracy was observed to be 90%, 93%, 94%, and 89% for SCC, SMF, leukoplakia, and normal oral tissues, respectively, on the basis of leave-one-patient-out cross-validation, with an overall accuracy of 91%. However, when a binary classification was employed to distinguish spectra from all the SCC, SMF and leukoplakik tissue sites together from normal, fluorescence and Raman spectroscopy were seen to have almost comparable performances with Raman yielding marginally better classification accuracy of 98.5% as compared to 94% of fluorescence.

  6. 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.

  7. 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

  8. 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.

  9. 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. PMID:26593555

  10. 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.

  11. 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.

  12. Preparation, fluorescence spectroscopy, and AFM analysis of erbium oxide nanocolloid

    NASA Astrophysics Data System (ADS)

    Patel, Darayas; Vance, Calvin; King, Newton; Jessup, Malcolm; Sarkisov, Sergey

    2009-02-01

    Nanocolloids of compounds containing fluorescent rare earth ions have recently attracted significant attention as agents for biolabeling, bioimaging, bio- and chemical sensing, and other applications. Erbium oxide nanocolloids have been prepared for the first time in water and gammabutyrolactone. Optical dynamic scatterometry and atomic force microscopy determined an average size (average mean height) of erbium oxide nanoparticles to be 10-11 nm. Prominent optical absorption peaks of the nanocolloids at 442.5 nm, 450.0 nm, 487.2 nm (strong), 492.0 nm, 523.0 nm (strong), 541.6 nm, 548.6 nm, 652.6 nm, and 665.7 nm (strong) can be attributed to erbium ions hosted within nanoparticles. Laser fluorescence spectroscopy of the nanocolloids was conducted using excitations with the lines of argon-ion laser (514 nm, 488 nm, 476 nm, and 458 nm) and 980-nm semiconductor laser. Strong green emission at 571 nm is more likely from transition between 4S3/2 and 4I15/2 levels and relatively weak red emissions from transition between 4I9/2 and 4I15/2 level of erbium was observed at excitation with visible laser radiation 488 nm and 476 nm. The reported nanocolloids thus showed to be good candidates for fluorescent biosensing applications and also as a new lasing filling medium in fiber lasers.

  13. 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

  14. Single-Molecule Fluorescence Spectroscopy using Phospholipid Bilayer Nanodiscs

    PubMed Central

    Nath, Abhinav; Trexler, Adam J.; Koo, Peter; Miranker, Andrew D.; Atkins, William M.; Rhoades, Elizabeth

    2012-01-01

    Nanodiscs are a new class of model membranes that are being used to solubilize and study a range of integral membrane proteins and membrane-associated proteins. Unlike other model membranes, the Nanodisc bilayer is bounded by a scaffold protein coat that confers enhanced stability and a narrow particle size distribution. The bilayer diameter can be precisely controlled by changing the diameter of the protein coat. All these properties make Nanodiscs excellent model membranes for single molecule fluorescence applications. In this chapter, we describe our work using Nanodiscs to apply total internal reflection fluorescence microscopy (TIRFM), fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) to study the integral membrane protein cytochrome P450 3A4 and the membrane-binding proteins islet amyloid popypeptide (IAPP) and α-synuclein, respectively. The monodisperse size distribution of Nanodiscs enhances control over the oligomeric state of the membrane protein of interest, and also facilitates accurate solution-based measurements. Nanodiscs also comprise an excellent system to stably immobilize integral membrane proteins in a bilayer without covalent modification, enabling a range of surface-based experiments where accurate localization of the protein of interest is required. PMID:20580961

  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. 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

  17. Studies in atomic-fluorescence spectroscopy-V The fluorescence characteristics and determination of antimony.

    PubMed

    Dagnall, R M; Thompson, K C; West, T S

    1967-10-01

    Atomic-fluorescence of antimony may be generated in an air-propane flame by nebulizing aqueous solutions of antimony salts whilst irradiating the flame by means of a microwave-excited electrode-less discharge tube operating at 30 W. The strongest fluorescence is exhibited by the (4)S(11 2 ) --> (4)P(1 3 ) 2311 A resonance line and weaker signals are observed at the 2068 and 2176 A resonance lines and at four intercombination lines, at 2598, 2671, 2770 and 2878 A. A process of thermally assisted direct-line fluorescence is postulated to account for the otherwise inexplicable intensity of the 2598 A line emission. Atomic-fluorescence spectroscopy at 2176 A permits the determination of antimony in the range 0.1-120 ppm with a detection limit of 0.05 ppm. With the same equipment and source, the range of measurement for atomic-absorption was 6-120 ppm and the detection limit was 1 ppm. No interferences were observed from 100-fold molar amounts of Cd, Co, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, NH(4), Pb and Zn or from arsenate, chloride, nitrate, phosphate and sulphate. PMID:18960212

  18. Two-dimensional fluorescence lifetime correlation spectroscopy. 2. Application.

    PubMed

    Ishii, Kunihiko; Tahara, Tahei

    2013-10-01

    In the preceding article, we introduced the theoretical framework of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS). In this article, we report the experimental implementation of 2D FLCS. In this method, two-dimensional emission-delay correlation maps are constructed from the photon data obtained with the time-correlated single photon counting (TCSPC), and then they are converted to 2D lifetime correlation maps by the inverse Laplace transform. We develop a numerical method to realize reliable transformation, employing the maximum entropy method (MEM). We apply the developed actual 2D FLCS to two real systems, a dye mixture and a DNA hairpin. For the dye mixture, we show that 2D FLCS is experimentally feasible and that it can identify different species in an inhomogeneous sample without any prior knowledge. The application to the DNA hairpin demonstrates that 2D FLCS can disclose microsecond spontaneous dynamics of biological molecules in a visually comprehensible manner, through identifying species as unique lifetime distributions. A FRET pair is attached to the both ends of the DNA hairpin, and the different structures of the DNA hairpin are distinguished as different fluorescence lifetimes in 2D FLCS. By constructing the 2D correlation maps of the fluorescence lifetime of the FRET donor, the equilibrium dynamics between the open and the closed forms of the DNA hairpin is clearly observed as the appearance of the cross peaks between the corresponding fluorescence lifetimes. This equilibrium dynamics of the DNA hairpin is clearly separated from the acceptor-missing DNA that appears as an isolated diagonal peak in the 2D maps. The present study clearly shows that newly developed 2D FLCS can disclose spontaneous structural dynamics of biological molecules with microsecond time resolution. PMID:23977902

  19. 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.

  20. Fluorescence spectroscopy: considerations for highly absorbing dissolved organic matter samples

    NASA Astrophysics Data System (ADS)

    Simone, B. E.; Miller, M.; McKnight, D. M.

    2009-12-01

    Fluorescence spectroscopy is a robust method for characterizing organic matter (OM). However, proper collection and correction of spectra are necessary to provide useful data. One important correction is the inner-filter correction, which primarily accounts for the inner-filter effect by adjusting for the wavelength dependent attenuation of emitted light by the solution prior to detection by the fluorometer. The most commonly used correction is based on an assumption that light is emitted at the center of the pathlength. Thus, the inner-filter effect is more pronounced in highly absorbing samples, and has the potential to skew the fluorescence spectra. For this study, the terrestrially derived Suwannee River fulvic acid (SRFA) and microbially derived Pony Lake fulvic acid (PLFA), from the International Humic Substances Society (IHSS), were diluted to incremental absorbances at a wavelength of 254 nm from 0.05 to 1.0 at pH 4 and 7. Three dimensional fluorescence spectra were measured and modeled with the Cory and McKnight (2005) parallel factor analysis (PARAFAC) model which resolves the fluorescence spectra into 13 components, including quinone-like and protein-like components. In the absence of inner-filter effects, plots of absorbance vs. loadings should be linear. Using the data from absorbance of 0.05 to 0.3, where the inner-filter affect is least pronounced, a linear regression was created and used as a baseline to predict component loadings at higher absorbance values in the absence of inner-filter effects. Results indicate that at absorbance values greater than 0.3, the commonly-used inner-filter correction is not able to remove the inner-filter effect. Therefore, in order to obtain reliable component loadings and correctly interpret the spectra, samples should be diluted to absorbance values less than 0.3 at 254 nm prior to collection of three dimensional fluorescence scans. The recommendation of a maximum absorbance of 0.3 agrees with the results of a

  1. In vivo and ex vivo magnetic resonance spectroscopy in the characterization of hemangioblastoma cyst fluid.

    PubMed

    Crisi, Girolamo; Filice, Silvano; Pertinhez, Thelma A; Ventura, Elisa; Servadei, Franco

    2014-01-01

    Peritumoral cyst formation is commonly associated with hemangioblastomas of the central nervous system. Results of a proteomic profiling of hemangioblastoma cyst fluid suggested that cyst formation, whether intratumoral or peritumoral, is a consequence of vascular leakage because protein profiles of cyst fluid and blood serum were similar. To the best of our knowledge, this is the first report of in vivo and ex vivo magnetic resonance spectroscopy analyses of hemangioblastoma cyst fluid that investigates on the mechanism leading to peritumoral cyst formation. PMID:24424553

  2. Intraoperative delineation of primary brain tumors using time-resolved fluorescence spectroscopy.

    PubMed

    Butte, Pramod V; Fang, Qiyin; Jo, Javier A; Yong, William H; Pikul, Brian K; Black, Keith L; Marcu, Laura

    2010-01-01

    The goal of this study is to determine the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for delineation of brain tumor from surrounding normal tissue in order to assist the neurosurgeon in near-complete tumor excision. A time-domain TR-LIFS prototype apparatus (gated photomultiplier detection, fast digitizer) was used for recording tissue autofluorescence in normal cortex (NC), normal white matter (NWM), and various grades of gliomas intraoperatively. Tissue fluorescence was induced with a pulsed nitrogen laser (337 nm, 700 ps), and the intensity decay profiles were recorded in the 360- to 550-nm spectral range (10-nm interval). Histopathological analysis (hematoxylin & eosin) of the biopsy samples taken from the site of TR-LIFS measurements was used for validation of spectroscopic results. Preliminary results on 17 patients demonstrate that normal cortex (N=16) and normal white matter (N=3) show two peaks of fluorescence emission at 390 nm (lifetime=1.8+/-0.3 ns) and 460 nm (lifetime=0.8+/-0.1 ns). The 390-nm emission peak is absent in low-grade glioma (N=5; lifetime=1.1 ns) and reduced in high-grade glioma (N=9; lifetime=1.7+/-0.4 ns). The emission characteristics at 460 nm in all tissues correlated with the nicotinamide adenine dinucleotide fluorescence (peak: 440 to 460 nm; lifetime: 0.8 to 1.0 ns). These findings demonstrate the potential of using TR-LIFS as a tool for enhanced delineation of brain tumors during surgery. In addition, this study evaluates similarities and differences between TR-LIFS signatures of brain tumors obtained in vivo and those previously reported in ex vivo brain tumor specimens. PMID:20459282

  3. Intraoperative delineation of primary brain tumors using time-resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Butte, Pramod V.; Fang, Qiyin; Jo, Javier A.; Yong, William H.; Pikul, Brian K.; Black, Keith L.; Marcu, Laura

    2010-03-01

    The goal of this study is to determine the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for delineation of brain tumor from surrounding normal tissue in order to assist the neurosurgeon in near-complete tumor excision. A time-domain TR-LIFS prototype apparatus (gated photomultiplier detection, fast digitizer) was used for recording tissue autofluorescence in normal cortex (NC), normal white matter (NWM), and various grades of gliomas intraoperatively. Tissue fluorescence was induced with a pulsed nitrogen laser (337 nm, 700 ps), and the intensity decay profiles were recorded in the 360- to 550-nm spectral range (10-nm interval). Histopathological analysis (hematoxylin & eosin) of the biopsy samples taken from the site of TR-LIFS measurements was used for validation of spectroscopic results. Preliminary results on 17 patients demonstrate that normal cortex (N=16) and normal white matter (N=3) show two peaks of fluorescence emission at 390 nm (lifetime=1.8+/-0.3 ns) and 460 nm (lifetime=0.8+/-0.1 ns). The 390-nm emission peak is absent in low-grade glioma (N=5; lifetime=1.1 ns) and reduced in high-grade glioma (N=9; lifetime=1.7+/-0.4 ns). The emission characteristics at 460 nm in all tissues correlated with the nicotinamide adenine dinucleotide fluorescence (peak: 440 to 460 nm lifetime: 0.8 to 1.0 ns). These findings demonstrate the potential of using TR-LIFS as a tool for enhanced delineation of brain tumors during surgery. In addition, this study evaluates similarities and differences between TR-LIFS signatures of brain tumors obtained in vivo and those previously reported in ex vivo brain tumor specimens.

  4. 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.

  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. PMID:27037785

  6. 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.

  7. In Vivo Fluorescence Imaging in the Second Near-Infrared Window Using Carbon Nanotubes.

    PubMed

    Hong, Guosong; Dai, Hongjie

    2016-01-01

    In vivo fluorescence imaging in the second near-infrared window (NIR-II window, 1000-1700 nm) is a powerful imaging technique that emerged in recent years. This imaging tool allows for noninvasive, deep-tissue visualization and interrogation of anatomical features and functions with improved imaging resolution and contrast at greater tissue penetration depths than traditional fluorescence imaging. Here, we present the detailed protocol for conducting NIR-II fluorescence imaging in live animals, including the procedures for preparation of biocompatible and NIR-II fluorescent carbon nanotube solution, live animal administration and NIR-II fluorescence image acquisition. PMID:27283426

  8. 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.

  9. Fluorescence and UV-vis Spectroscopy of Synovial Fluids

    NASA Astrophysics Data System (ADS)

    Pinti, Marie J.; Stojilovic, Nenad; Kovacik, Mark W.

    2009-10-01

    Total joint arthroplasty involves replacing the worn cartilaginous surfaces of the joint with man-made materials that are designed to be biocompatible and to withstand mechanical stresses. Commonly these bearing materials consist of metallic alloys (TiAlV or CoCrMo) and UHMWPE. Following joint arthroplasty, the normal generation of micro-metallic wear debris particles that dislodge from the prosthesis has been shown to cause inflammatory aseptic osteolysis (bone loss) that ultimately results in the failure of the implant. Here we report our results on the novel use of Fluorescence and UV-vis spectroscopy to investigate the metallic content of synovial fluid specimens taken from postoperative total knee arthroplasties. Preliminary finding showed presence of alumina and chromium is some specimens. The ability to detect and monitor the wear rate of these implants could have far reaching implications in the prevention of metallic wear-debris induced osteolysis and impending implant failure.

  10. 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

  11. 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 +}.

  12. 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.

  13. Trimodal detection of early childhood caries using laser light scanning and fluorescence spectroscopy: clinical prototype

    PubMed Central

    Kim, Amy S.; Ridge, Jeremy S.; Nelson, Leonard Y.; Berg, Joel H.; Seibel, Eric J.

    2013-01-01

    Abstract. There is currently a need for a safe and effective way to detect and diagnose early stages of childhood caries. A multimodal optical clinical prototype for diagnosing caries demineralization in vivo has been developed. The device can be used to quickly image and screen for any signs of demineralized enamel by obtaining high-resolution and high-contrast surface images using a 405-nm laser as the illumination source, as well as obtaining autofluorescence and bacterial fluorescence images. When a suspicious region of demineralization is located, the device also performs dual laser fluorescence spectroscopy using 405- and 532-nm laser excitation. An autofluorescence ratio of the two excitation lasers is computed and used to quantitatively diagnose enamel health. The device was tested on five patients in vivo as well as on 28 extracted teeth with clinically diagnosed carious lesions. The device was able to provide detailed images that highlighted the lesions identified by the clinicians. The autofluorescence spectroscopic ratios obtained from the extracted teeth successfully quantitatively discriminated between sound and demineralized enamel. PMID:23986369

  14. Continuous-wave laser fluorescence spectroscopy of impurities in tokamaks

    SciTech Connect

    Young, C.E.; Pellin, M.J.; Gruen, D.M.; Norem, J.H.

    1982-07-01

    Laser-induced fluorescence spectroscopy has been applied as an in-situ diagnostic for impurity atoms in the edge region of the plasma in the Argonne Plasma Engineering Experiment (APEX) tokamak. Zirconium atoms introduced from a moveable probe were excited by a cw single-mode ring dye laser and monitored on lines of the a/sup 3/F-z/sup 3/F/sup 0/ manifold. The fluorescence signal from a 0.03 cm/sup 3/ volume was recorded at 1-ms intervals with a computer-controlled 4-channel 100-MHz scaler system. Acousto-optic modulation of the laser beam at 100 kHz allowed subtraction of plasma background light. Absolute calibration by Rayleigh scattering gave a detectability limit approx.10/sup 10/ Zr atoms/cm/sup 3/ in this apparatus. The detectability limit was determined by a detailed consideration of power and transit time broadening. The effects of several experimental parameters were examined and suggestions for increasing detection sensitivity are presented. Doppler-shift experiments indicated a thermal-velocity distribution for the detected Zr atoms. Intrinsic-velocity resolution of the experiments, calculated from effective excitation linewidths, was approx.25 m/s.

  15. Continuous-wave laser fluorescence spectroscopy of impurities in tokamaks

    NASA Astrophysics Data System (ADS)

    Young, C. E.; Pellin, M. J.; Gruen, D. M.; Norem, J. H.

    1982-07-01

    Laser-induced fluorescence spectroscopy has been applied as an in-situ diagnostic for impurity atoms in the edge region of the plasma in the Argonne Plasma Engineering Experiment (APEX) tokamak. Zirconium atoms introduced from a moveable probe were excited by a cw single-mode ring dye laser and monitored on lines of the a3F-z3F0 manifold. The fluorescence signal from a 0.03 cm3 volume was recorded at 1-ms intervals with a computer-controlled 4-channel 100-MHz scaler system. Acousto-optic modulation of the laser beam at 100 kHz allowed subtraction of plasma background light. Absolute calibration by Rayleigh scattering gave a detectability limit ˜1010 Zr atoms/cm3 in this apparatus. The detectability limit was determined by a detailed consideration of power and transit time broadening. The effects of several experimental parameters were examined and suggestions for increasing detection sensitivity are presented. Doppler-shift experiments indicated a thermal-velocity distribution for the detected Zr atoms. Intrinsic-velocity resolution of the experiments, calculated from effective excitation linewidths, was ˜25 m/s.

  16. 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.

  17. 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. PMID:25404711

  18. Diagnosis of meningioma by time-resolved fluorescence spectroscopy.

    PubMed

    Butte, Pramod V; Pikul, Brian K; Hever, Aviv; Yong, William H; Black, Keith L; Marcu, Laura

    2005-01-01

    We investigate the use of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for the intraoperative rapid evaluation of tumor specimens and delineation of tumor from surrounding normal tissue. Tissue autofluorescence is induced with a pulsed nitrogen laser (337 nm, 1.2 ns) and the intensity decay profiles are recorded in the 370 to 500 nm spectral range with a fast digitizer (0.2 ns resolution). Experiments are conducted on excised specimens (meningioma, dura mater, cerebral cortex) from 26 patients (97 sites). Spectral intensities and time-dependent parameters derived from the time-resolved spectra of each site are used for tissue characterization. A linear discriminant analysis algorithm is used for tissue classification. Our results reveal that meningioma is characterized by unique fluorescence characteristics that enable discrimination of tumor from normal tissue with high sensitivity (>89%) and specificity (100%). The accuracy of classification is found to increase (92.8% cases in the training set and 91.8% in the cross-validated set correctly classified) when parameters from both the spectral and the time domain are used for discrimination. Our findings establish the feasibility of using TR-LIFS as a tool for the identification of meningiomas and enables further development of real-time diagnostic tools for analyzing surgical tissue specimens of meningioma or other brain tumors. PMID:16409091

  19. 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. PMID:17704168

  20. 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.

  1. Fluorescence correlation spectroscopy in biology, chemistry, and medicine.

    PubMed

    Perevoshchikova, I V; Kotova, E A; Antonenko, Y N

    2011-05-01

    This review describes the method of fluorescence correlation spectroscopy (FCS) and its applications. FCS is used for investigating processes associated with changes in the mobility of molecules and complexes and allows researchers to study aggregation of particles, binding of fluorescent molecules with supramolecular complexes, lipid vesicles, etc. The size of objects under study varies from a few angstroms for dye molecules to hundreds of nanometers for nanoparticles. The described applications of FCS comprise various fields from simple chemical systems of solution/micelle to sophisticated regulations on the level of living cells. Both the methodical bases and the theoretical principles of FCS are simple and available. The present review is concentrated preferentially on FCS applications for studies on artificial and natural membranes. At present, in contrast to the related approach of dynamic light scattering, FCS is poorly known in Russia, although it is widely employed in laboratories of other countries. The goal of this review is to promote the development of FCS in Russia so that this technique could occupy the position it deserves in modern Russian science. PMID:21639831

  2. Fluorescence correlation spectroscopy: Statistical analysis and biological applications

    NASA Astrophysics Data System (ADS)

    Saffarian, Saveez

    2002-01-01

    The experimental design and realization of an apparatus which can be used both for single molecule fluorescence detection and also fluorescence correlation and cross correlation spectroscopy is presented. A thorough statistical analysis of the fluorescence correlation functions including the analysis of bias and errors based on analytical derivations has been carried out. Using the methods developed here, the mechanism of binding and cleavage site recognition of matrix metalloproteinases (MMP) for their substrates has been studied. We demonstrate that two of the MMP family members, Collagenase (MMP-1) and Gelatinase A (MMP-2) exhibit diffusion along their substrates, the importance of this diffusion process and its biological implications are discussed. We show through truncation mutants that the hemopexin domain of the MMP-2 plays and important role in the substrate diffusion of this enzyme. Single molecule diffusion of the collagenase MMP-1 has been observed on collagen fibrils and shown to be biased. The discovered biased diffusion would make the MMP-1 molecule an active motor, thus making it the first active motor that is not coupled to ATP hydrolysis. The possible sources of energy for this enzyme and their implications are discussed. We propose that a possible source of energy for the enzyme can be in the rearrangement of the structure of collagen fibrils. In a separate application, using the methods developed here, we have observed an intermediate in the intestinal fatty acid binding protein folding process through the changes in its hydrodynamic radius also the fluctuations in the structure of the IFABP in solution were measured using FCS.

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

    PubMed Central

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

    2013-01-01

    Fluorescence Correlation Spectroscopy (FCS) is widely used to quantitate 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. PMID:23525193

  4. Dynamic nuclear protein interactions investigated using fluorescence lifetime and fluorescence fluctuation spectroscopy

    NASA Astrophysics Data System (ADS)

    Siegel, Amanda P.; Hays, Nicole M.; Day, Richard N.

    2012-03-01

    The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for Förster resonance energy transfer (FRET) microscopy and fluorescence fluctuation spectroscopy (FFS) provide important tools for monitoring dynamic protein interactions inside living cells. Fluorescence lifetime imaging microscopy (FLIM) quantitatively maps changes in the spatial distribution of donor FP lifetimes that result from FRET with acceptor FPs. FFS probes dynamic protein associations through its capacity to monitor localized protein diffusion. Here, we use FRET-FLIM combined with FFS in living cells to investigate changes in protein mobility due to protein-protein interactions involving transcription factors and chromatin modifying proteins that function in anterior pituitary gene regulation. The heterochromatin protein 1 alpha (HP1α) plays a key role in the establishment and maintenance of heterochromatin through its interactions with histone methyltransferases. Recent studies, however, also highlight the importance of HP1α as a positive regulator of active transcription in euchromatin. Intriguingly, we observed that the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) interacts with HP1α in regions of pericentromeric heterochromatin in mouse pituitary cells. These observations prompted us to investigate the relationship between HP1α dynamic interactions in pituitary specific gene regulation.

  5. Terahertz spectroscopy for the assessment of burn injuries in vivo.

    PubMed

    Arbab, M Hassan; Winebrenner, Dale P; Dickey, Trevor C; Chen, Antao; Klein, Matthew B; Mourad, Pierre D

    2013-07-01

    A diagnosis criterion is proposed for noninvasive grading of burn injuries using terahertz radiation. Experimental results are presented from in vivo terahertz time-domain spectroscopy of second- and third-degree wounds, which are obtained in a 72-hour animal study. During this period, the change in the spectroscopic response of the burned tissue is studied. It is shown that terahertz waves are sensitive not only to the postburn formation of interstitial edema, but also to the density of skin structures derived from image processing analysis of histological sections. Based on these preliminary results, it is suggested that the combination of these two effects, as probed by terahertz spectroscopy of the tissue, may ultimately be used to differentiate partial-thickness burns that will naturally heal from those that will require surgical intervention. PMID:23860943

  6. 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)

  7. Pancreatic tumor detection using hypericin-based fluorescence spectroscopy and cytology

    NASA Astrophysics Data System (ADS)

    Lavu, Harish; Geary, Kevin; Fetterman, Harold R.; Saxton, Romaine E.

    2005-04-01

    Hypericin is a novel, highly fluorescent photosensitizer that exhibits selective tumor cell uptake properties and is particularly resistant to photobleaching. In this study, we have characterized hypericin uptake in human pancreatic tumor cells with relation to incubation time, cell number, and drug concentration. Ex vivo hypericin based fluorescence spectroscopy was performed to detect the presence of MIA PaCa-2 pancreatic tumor cells in the peritoneal cavity of BALB/c nude mice, as well as to quantify gross tumor burden. Hypericin based cytology of peritoneal lavage samples, using both one and two photon laser confocal microscopy, demonstrated more than a two-fold increase in fluorescence emission of pancreatic tumor cells as compared to control samples. In vitro treatment of pancreatic cancer cells with hypericin based photodynamic therapy showed tumor cell cytotoxicity in a drug dose, incident laser power, and time dependent manner. For these experiments, a continuous wavelength solid-state laser source (532 nm) was operated at power levels in the range of 100-400 mW. Potential applications of hypericin in tumor diagnosis, staging, and therapy will be presented.

  8. 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.

  9. 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.

  10. Fluorescence Spectroscopy of Human Nonmalignant and Malignant Cells and Tissues.

    NASA Astrophysics Data System (ADS)

    Glassman, Wenling Sha

    This thesis explores steady state and time resolved fluorescence spectroscopy from human malignant and non -malignant cells and tissues. The focus of these studies are the analysis of the excitation spectra, emission spectra, and decay time based on the contribution from several key intrinsic fluorophors: NAD(P)H, flavins, tryptophan, elastin and collagen that exist in different amounts in the human tissues and cells. The comparison between the spectra from malignant and non-malignant cells and tissues gives information on the changes that occur from non-malignancy to malignancy in the cells and tissues. The spectra of tissues and cells are also compared to help in understanding what fluorophors are responsible for fluorescence spectral differences between the malignant and non-malignant tissues and cells. The results in this thesis show that the spectral differences between the normal and cancerous tissues and cells exist in various wavelength ranges. The experimental data from GYN tissues have shown with over 95% of the sensitivity and specificity to separate malignant from non-malignant tissues using 300nm excitation. The 340nm band, which is mostly in response to intrinsic fluorophor (amino acid tryptophan), from malignant tissues were relatively higher then that from the non-malignant tissues. This might have been caused by the higher concentration of free tryptophan in the malignant tumor when compared to that of the normal tissue. This has been found in medical clinical study. The experimental data in this thesis also show that the fluorescence intensities around 450nm-460nm, which are mostly due to the intrinsic fluorophor coenzyme NADH, from both malignant cells in vitro and tissues in vitro are relatively higher than from non-malignant cells in vitro and tissues in vitro. These findings are reinforced by the faster decay time of the NADH fluorescence from normal cells in vitro than from neoplasm cells in vitro. Thus, the NADH in the mitochondria might be

  11. In vivo multiphoton tomography and fluorescence lifetime imaging of human brain tumor tissue.

    PubMed

    Kantelhardt, Sven R; Kalasauskas, Darius; König, Karsten; Kim, Ella; Weinigel, Martin; Uchugonova, Aisada; Giese, Alf

    2016-05-01

    High resolution multiphoton tomography and fluorescence lifetime imaging differentiates glioma from adjacent brain in native tissue samples ex vivo. Presently, multiphoton tomography is applied in clinical dermatology and experimentally. We here present the first application of multiphoton and fluorescence lifetime imaging for in vivo imaging on humans during a neurosurgical procedure. We used a MPTflex™ Multiphoton Laser Tomograph (JenLab, Germany). We examined cultured glioma cells in an orthotopic mouse tumor model and native human tissue samples. Finally the multiphoton tomograph was applied to provide optical biopsies during resection of a clinical case of glioblastoma. All tissues imaged by multiphoton tomography were sampled and processed for conventional histopathology. The multiphoton tomograph allowed fluorescence intensity- and fluorescence lifetime imaging with submicron spatial resolution and 200 picosecond temporal resolution. Morphological fluorescence intensity imaging and fluorescence lifetime imaging of tumor-bearing mouse brains and native human tissue samples clearly differentiated tumor and adjacent brain tissue. Intraoperative imaging was found to be technically feasible. Intraoperative image quality was comparable to ex vivo examinations. To our knowledge we here present the first intraoperative application of high resolution multiphoton tomography and fluorescence lifetime imaging of human brain tumors in situ. It allowed in vivo identification and determination of cell density of tumor tissue on a cellular and subcellular level within seconds. The technology shows the potential of rapid intraoperative identification of native glioma tissue without need for tissue processing or staining. PMID:26830089

  12. 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

  13. 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

  14. 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

  15. 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.)

  16. Two-photon excited fluorescence microscopy application for ex vivo investigation of ocular fundus samples

    NASA Astrophysics Data System (ADS)

    Peters, Sven; Hammer, Martin; Schweitzer, Dietrich

    2011-07-01

    Two-photon excited fluorescence (TPEF) imaging of ocular tissue has recently become a promising tool in ophthalmology for diagnostic and research purposes. The feasibility and the advantages of TPEF imaging, namely deeper tissue penetration and improved high-resolution imaging of microstructures, have been demonstrated lately using human ocular samples. The autofluorescence properties of endogenous fluorophores in ocular fundus tissue are well known from spectrophotometric analysis. But fluorophores, especially when it comes to fluorescence lifetime, typically display a dependence of their fluorescence properties on local environmental parameters. Hence, a more detailed investigation of ocular fundus autofluorescence ideally in vivo is of utmost interest. The aim of this study is to determine space-resolved the stationary and time-resolved fluorescence properties of endogenous fluorophores in ex vivo porcine ocular fundus samples by means of two-photon excited fluorescence spectrum and lifetime imaging microscopy (FSIM/FLIM). By our first results, we characterized the autofluorescence of individual anatomical structures of porcine retina samples excited at 760 nm. The fluorescence properties of almost all investigated retinal layers are relatively homogenous. But as previously unknown, ganglion cell bodies show a significantly shorter fluorescence lifetime compared to the adjacent mueller cells. Since all retinal layers exhibit bi-exponential autofluorescence decays, we were able to achieve a more precise characterization of fluorescence properties of endogenous fluorophores compared to a present in vivo FLIM approach by confocal scanning laser ophthalmoscope (cSLO).

  17. 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.

  18. 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

  19. In vivo near-infrared fluorescence three-dimensional positioning system with binocular stereovision

    NASA Astrophysics Data System (ADS)

    Song, Bofan; Jin, Wei; Wang, Ying; Jin, Qinhan; Mu, Ying

    2014-11-01

    Fluorescence is a powerful tool for in-vivo imaging in living animals. The traditional in-vivo fluorescence imaging equipment is based on single-view two-dimensional imaging systems. However, they cannot meet the needs for accurate positioning during modern scientific research. A near-infrared in-vivo fluorescence imaging system is demonstrated, which has the capability of deep source signal detecting and three-dimensional positioning. A three-dimensional coordinates computing (TDCP) method including a preprocess algorithm is presented based on binocular stereo vision theory, to figure out the solution for diffusive nature of light in tissue and the emission spectra overlap of fluorescent labels. This algorithm is validated to be efficient to extract targets from multispectral images and determine the spot center of biological interests. Further data analysis indicates that this TDCP method could be used in three-dimensional positioning of the fluorescent target in small animals. The study also suggests that the combination of a large power laser and deep cooling charge-coupled device will provide an attractive approach for fluorescent detection from deep sources. This work demonstrates the potential of binocular stereo vision theory for three-dimensional positioning for living animal in-vivo imaging.

  20. 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

  1. Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

    DOE PAGESBeta

    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

  2. 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.

  3. Inference of protein diffusion probed via fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Tsekouras, Konstantinos

    2015-03-01

    Fluctuations are an inherent part of single molecule or few particle biophysical data sets. Traditionally, ``noise'' fluctuations have been viewed as a nuisance, to be eliminated or minimized. Here we look on how statistical inference methods - that take explicit advantage of fluctuations - have allowed us to draw an unexpected picture of single molecule diffusional dynamics. Our focus is on the diffusion of proteins probed using fluorescence correlation spectroscopy (FCS). First, we discuss how - in collaboration with the Bustamante and Marqusee labs at UC Berkeley - we determined using FCS data that individual enzymes are perturbed by self-generated catalytic heat (Riedel et al, Nature, 2014). Using the tools of inference, we found how distributions of enzyme diffusion coefficients shift in the presence of substrate revealing that enzymes performing highly exothermic reactions dissipate heat by transiently accelerating their center of mass following a catalytic reaction. Next, when molecules diffuse in the cell nucleus they often appear to diffuse anomalously. We analyze FCS data - in collaboration with Rich Day at the IU Med School - to propose a simple model for transcription factor binding-unbinding in the nucleus to show that it may give rise to apparent anomalous diffusion. Here inference methods extract entire binding affinity distributions for the diffusing transcription factors, allowing us to precisely characterize their interactions with different components of the nuclear environment. From this analysis, we draw key mechanistic insight that goes beyond what is possible by simply fitting data to ``anomalous diffusion'' models.

  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. Laboratory studies of in vivo fluorescence of phytoplankton

    NASA Technical Reports Server (NTRS)

    Brown, C. A., Jr.; Farmer, F. H.; Jarrett, O., Jr.; Staton, W. L.

    1978-01-01

    A lidar system is developed that uses four selected excitation wavelengths to induce chlorophyll 'a' fluorescence which is indicative of both the concentration and diversity of phytoplankton. The operating principles of the system and the results of measurements of phytoplankton fluorescence in a controlled laboratory environment are presented. A comparative study of results from lidar fluorosensor laboratory tank tests using representative species of phytoplankton in single and multispecies cultures from each of four color groups reveals that (1) there is good correlation between the fluorescence of chlorophyll 'a' remotely simulated and detected by the lidar system and in-situ measurements using four similar excitation wavelengths in a flow-through fluorometer; (2) good correlation exists between the total chlorophyll 'a' calculated from lidar-fluorosensor data and measurements obtained by the Strickland-Parsons method; and (3) the lidar fluorosensor can provide an index of population diversity.

  6. 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

  7. 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.

  8. Comparison of in vivo optical systems for bioluminescence and fluorescence imaging.

    PubMed

    Cool, Steven K; Breyne, Koen; Meyer, Evelyne; De Smedt, Stefaan C; Sanders, Niek N

    2013-09-01

    In vivo optical imaging has become a popular tool in animal laboratories. Currently, many in vivo optical imaging systems are available on the market, which often makes it difficult for research groups to decide which system fits their needs best. In this work we compared different commercially available systems, which can measure both bioluminescent and fluorescent light. The systems were tested for their bioluminescent and fluorescent sensitivity both in vitro and in vivo. The IVIS Lumina II was found to be most sensitive for bioluminescence imaging, with the Photon Imager a close second. Contrary, the Kodak system was, in vitro, the most sensitive system for fluorescence imaging. In vivo, the fluorescence sensitivity of the systems was similar. Finally, we examined the added value of spectral unmixing algorithms for in vivo optical imaging and demonstrated that spectral unmixing resulted in at least a doubling of the in vivo sensitivity. Additionally, spectral unmixing also enabled separate imaging of dyes with overlapping spectra which were, without spectral unmixing, not distinguishable. PMID:23579930

  9. 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

  10. Fluorescence fluctuation spectroscopy: ushering in a new age of enlightenment for cellular dynamics

    PubMed Central

    Jameson, David M.; Ross, Justin A.; Albanesi, Joseph P.

    2011-01-01

    Originally developed for applications in physics and physical chemistry, fluorescence fluctuation spectroscopy is becoming widely used in cell biology. This review traces the development of the method and describes some of the more important applications. Specifically, the methods discussed include fluorescence correlation spectroscopy (FCS), scanning FCS, dual color cross-correlation FCS, the photon counting histogram and fluorescence intensity distribution analysis approaches, the raster scanning image correlation spectroscopy method, and the Number and Brightness technique. The physical principles underlying these approaches will be delineated, and each of the methods will be illustrated using examples from the literature. PMID:21547245

  11. In vivo Raman spectroscopy for oral cancers diagnosis

    NASA Astrophysics Data System (ADS)

    Singh, S. P.; Deshmukh, Atul; Chaturvedi, Pankaj; Krishna, C. Murali

    2012-01-01

    Oral squamous cell carcinoma is sixth among the major malignancies worldwide. Tobacco habits are known as major causative factor in tumor carcinogenesis in oral cancer. Optical spectroscopy methods, including Raman, are being actively pursued as alternative/adjunct for cancer diagnosis. Earlier studies have demonstrated the feasibility of classifying normal, premalignant and malignant oral ex-vivo tissues. In the present study we have recorded in vivo spectra from contralateral normal and diseased sites of 50 subjects with pathologically confirmed lesions of buccal mucosa using fiber-optic-probe-coupled HE-785 Raman spectrometer. Spectra were recorded on similar points as per teeth positions with an average acquisition time of 8 seconds. A total of 215 and 225 spectra from normal and tumor sites, respectively, were recorded. Finger print region (1200-1800 cm-1) was utilized for classification using LDA. Standard-model was developed using 125 normal and 139 tumor spectra from 27 subjects. Two separate clusters with an efficiency of ~95% were obtained. Cross-validation with leave-one-out yielded ~90% efficiency. Remaining 90 normal and 86 tumor spectra were used as test data and predication efficiency of model was evaluated. Findings of the study indicate that Raman spectroscopic methods in combination with appropriate multivariate tool can be used for objective, noninvasive and rapid diagnosis.

  12. Photoresponsive fluorescent reduced graphene oxide by spiropyran conjugated hyaluronic acid for in vivo imaging and target delivery.

    PubMed

    Nahain, Abdullah-Al; Lee, Jung-Eun; Jeong, Ji Hoon; Park, Sung Young

    2013-11-11

    This present article demonstrates the strategy to prepare photoresponsive reduced graphene oxide with mussel inspired adhesive material dopamine (DN) and photochromic dye spiropyran (SP) conjugated to the backbone of the targeting ligand hyaluronic acid (HA; HA-SP). Graphene oxide (GO) was reduced by prepared HA-SP accepting the advantages of catechol chemistry under mildly alkaline condition enabling to achieve functionalized graphene (rGO/HA-SP) as fluorescent nanoparticles. Due to containing HA, rGO/HA-SP can bind to the CD44 cell receptors. The prepared rGO/HA-SP is able to retain its photochromic features and can be converted to merocyanine (MC) form upon irradiation with UV light (wavelength: 365 nm) displaying purple color. Photochromic behavior of rGO/HA-SP was monitored by UV-vis and fluorescence spectroscopy. In vitro fluorescence behavior, examined by confocal laser scanning microscope (CLSM), of rGO/HA-SP in cancerous A549 cell lines assured that efficient delivery of rGO/HA-SP was gained due to HA as targeting ligand. In this work, we have shown that in vivo fluorescence image of spiropyran is possible by administrating MC form solution of rGO/HA-SP using Balb/C mice as in vivo modal. Accumulation of rGO/HA-SP in tumor tissue from biodistribution analysis strongly supports the specific delivery of prepared graphene to the target destination. The well tuned drug release manner from the surface of rGO/HA-SP strongly recommends the developed material not only as fluorescent probe for diagnosis but also as a drug carrier in drug delivery system. PMID:24106989

  13. 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.

  14. 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.

  15. Poly(dimethylsiloxane) microlens array integrated with microfluidic channel for fluorescence spectroscopy detection

    NASA Astrophysics Data System (ADS)

    Rujihan, Suparat; Damrongsak, Badin; Kittidachachan, Pattareeya

    2013-06-01

    Fluorescence spectroscopy detection has been commonly used in chemical and biochemical applications as it provides a good reliability and high sensitivity. Commercially available fluorescence spectroscopy system is typically bulky and expensive, hence making it inconvenience for on-site measurement which requires portable systems. However, the drawback of small devices is that it has a low detection volume, resulting in low fluorescence signal. In this paper, we report a microfluidic channel implemented with a microlens array for enhancing the performance of fluorescence spectroscopy detection. The microlens array was used to focus an excitation light onto the microchannel, thus expecting the increase in fluorescence detection signal. Both microchannels and microlens arrays were individually fabricated from poly-dimethylsiloxane (PDMS) using low-cost printed-circuit-board master molds. The fabrication and characterization of PDMS-based microlens arrays are discussed. In short, the microlens in plano-convex shape was designed with diameters of 700, 800 and 900 microns. The fabricated microlens arrays were characterized for radius of curvatures, SAGs and focal lengths. The plano-convex microlens array was then integrated into a microfluidic system in order to investigate the overall performance of fluorescence spectroscopy detection. Experiments were conducted with two fluorescence dyes, i.e. Rhodamine 6G and Coumarin 153. The preliminary results revealed that the PDMS microlens array implemented on the designed system shows potential for improving excitation and emission light intensity and, as a consequence, signal to background ratio of the fluorescence spectroscopy detection.

  16. 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

  17. 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

  18. 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.

  19. A pyrene derivative for Hg(2+) -selective fluorescent sensing and its application in in vivo imaging.

    PubMed

    Wang, Guang Ke; Mi, Qi Li; Zhao, Li Yun; Hu, Jun Jie; Guo, Lin E; Zou, Xiao Ju; Liu, Bo; Xie, Xiao Guang; Zhang, Jun Feng; Zhao, Qi Hua; Zhou, Ying

    2014-03-01

    An Hg(2+) -selective fluorescent sensor (1) bearing pyrene as a fluorophore was synthesized. A sandwich-stacking binding mode was formed during the binding process, which increased the excimer fluorescence 22-fold at 490 nm. Compound 1 was successfully applied in in vivo imaging to trace the enrichment and distribution of mercury in the nervous system, digestive system, and reproductive system of Caenorhabditis elegans, as well as the organs of zebrafish. PMID:24323430

  20. 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.

  1. 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.

  2. 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

  3. Noninvasive imaging in vivo with fluorescent proteins from centimeters to micrometers

    NASA Astrophysics Data System (ADS)

    Yang, Meng; Jiang, Ping; Al-Zaid, Manal; Hoffman, Robert M.

    2008-02-01

    Whole-body imaging with fluorescent proteins has been shown to be a powerful technology with many applications in small animals. Our laboratory pioneered in vivo imaging with fluorescent proteins (1) including noninvasive whole-body imaging (2). Whole-body imaging with fluorescent proteins depends in large part on the brightness of the protein. Brighter, red-shifted proteins can make whole-body imaging more sensitive due to reduced absorption by tissues and less scatter. Non-invasive imaging with fluorescent proteins has been shown to be able to quantitatively track tumor growth and metastasis, gene expression, angiogenesis, and bacterial infection (3) even at subcellular resolution depending on the position of the cells in the animal. Interference by skin autofluorescence is kept to a minimum with the use of proper filters. To noninvasively image cancer cell/stromal cell interaction in the tumor microenvironment and drug response at the cellular level in live animals in real time, we developed a new imageable three-color animal model. The model consists of green fluorescent protein (GFP)-expressing mice transplanted with dual-color cancer cells labeled with GFP in the nucleus and red fluorescent protein (RFP) in the cytoplasm. Various in vivo phenomena of tumor-host interaction and cellular dynamics were imaged, including mitotic and apoptotic tumor cells, stromal cells interacting with the tumor cells, tumor vasculature, and tumor blood flow as well as drug response. This imageable technology should lead to many new insights of in vivo cancer cell biology.

  4. Tomographic Diffuse Fluorescence Flow Cytometry for Enumeration of Rare Circulating Cells in Vitro and in Vivo

    NASA Astrophysics Data System (ADS)

    Zettergren, Eric William

    2011-12-01

    Accurate quantification of circulating cell populations is important in many areas of preclinical and clinical biomedical research including the study of metastasized cancers, T-Lymphotocyes and hematopoietic stem cells. Normally this is done either by extraction and analysis of small blood samples or more recently using microscopy-based in vivo fluorescence flow cytometry. In this thesis, a new technological approach to this problem is described using detection of diffuse fluorescent light from relatively large blood vessels in vivo. The 'tomographic diffuse fluorescence flow cytometer' (TDFFC) uses modulated lasers 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. It is first demonstrated that the TDFFC instrument is capable of detecting fluorescent microspheres and Vybrant-DiD labeled cells with excellent accuracy in an optical flow phantom with similar size, optical properties, linear flow rates and autofluorescence as a mouse limb. Preliminary data demonstrating that the TDFFC is capable of detecting circulating cells in nude mice in vivo is also shown. Finally, a number of methods for performing coarse tomographic localization of fluorescent cells within the cross-section of a mouse limb using TDFFC data sets are described, and the feasibility of this approach is demonstrated using in vitro data sets. In principle, this device would allow interrogation of the whole blood volume of a mouse in minutes, with several orders of magnitude sensitivity improvement compared with current approaches.

  5. 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.

  6. Simulation of autocorrelation function and photon counting distribution in fluorescence fluctuation spectroscopy.

    PubMed

    Shingaryov, Igor P; Skakun, Victor V; Apanasovich, Vladimir V

    2014-01-01

    In modern fluorescence fluctuation spectroscopy, the autocorrelation function and photon counting distribution are two widely used statistical characteristics of the measured fluctuating fluorescence intensity signal. Applying special analysis methods such as fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) to these properties, it is possible to recover values of different parameters of fluorescent molecules such as the concentration, diffusion coefficient, molecular brightness, and kinetic rate constants. The development of new analysis methods is senseless without testing their validity, accuracy, and robustness. The most appropriate check of a method is its application to experimental data. However, sometimes it is more convenient and easier to verify a method on simulated data. Simulation is also useful for better understanding the processes that were modeled during the development of analysis methods. Here, we present two simulation models providing an autocorrelation function and photon counting distribution of a sequence of photon arrival times detected in fluorescence fluctuation spectroscopy. PMID:24108653

  7. Characterization of humic acids by two-dimensional correlation fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Nakashima, K.; Xing, Shaoyong; Gong, Yongkuan; Miyajima, Toru

    2008-07-01

    We have investigated interaction between humic acids and heavy metal ions by fluorescence spectroscopy. The humic acids examined are Aldrich humic acid (AHA) and Dando humic acid (DHA), and heavy metal ions are Cu 2+ and Pb 2+. The binding constants between the humic acids and the heavy metal ions are obtained by a conventional fluorescence quenching technique. The two prominent bands in the fluorescence spectra of the humic acids give different binding constants, implying that the two bands are originated from different fluorescent species in the matrices of the humic acids. This was confirmed by two-dimensional correlation analysis based on the quenching perturbation on the fluorescence spectra. Two prominent cross peaks corresponding to the two fluorescence bands are obtained in the asynchronous maps, indicating that the two fluorescence bands belong to different species. The order of the response of the two fluorescence bands to the quenching perturbation is also elucidated based on Noda's rule.

  8. Protein oligomerization monitored by fluorescence fluctuation spectroscopy: Self-assembly of Rubisco activase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A methodology is presented to characterize complex protein assembly pathways by fluorescence correlation spectroscopy. We have derived the total autocorrelation function describing the behavior of mixtures of labeled and unlabeled protein under equilibrium conditions. Our modeling approach allows us...

  9. Sensitivity of in vivo X-ray fluorescence determination of skeletal lead stores

    SciTech Connect

    Sokas, R.K.; Besarab, A.; McDiarmid, M.A.; Shapiro, I.M.; Bloch, P. )

    1990-09-01

    Eighteen patients with known past occupational lead exposure underwent parenteral diagnostic chelation with ethylenediaminetetraacetic acid and x-ray fluorescent determination of in vivo skeletal lead stores at the distal styloid process of the ulna and at the temporal base bone using a cobalt 57 source and measuring lead Ka x-rays. X-ray fluorescent lead measurements in both locations correlated with results of diagnostic chelation. Using a post-chelation urinary excretion of greater than 600 micrograms lead/24 h as the definition of high-lead stores, sensitivity of x-ray fluorescence at the wrist and temple was 56% and 39%, respectively.

  10. 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.

  11. In vivo imaging of the mammalian nervous system using fluorescent proteins.

    PubMed

    Tucker, K L

    2001-01-01

    The recent development of fluorescent proteins has rapidly and radically altered the way cell biology is performed by allowing simple, non-invasive imaging of cellular processes in real time. The special properties of the nervous system, such as synaptic morphology, axonal/dendritic maturation, and neuronal migration are especially amenable to investigation using fluorescent proteins. This review focuses on the various genetic and viral vectors used to express fluorescent proteins in vivo and in slice culture, and the strengths and limitations associated with them. PMID:11219606

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. Temperature-modulated fluorescence tomography: modulating tissue temperature using HIFU for high-resolution in vivo fluorescence tomography

    NASA Astrophysics Data System (ADS)

    Kwong, Tiffany C.; Nouizi, Farouk; Lin, Yuting; Sampathkumaran, Uma; Ahmed, Shaaz; Gulsen, Gultekin

    2013-03-01

    Low spatial resolution due to strong tissue scattering is one of the main barriers that prevent the wide-spread use of fluorescence tomography. To overcome this limitation, we previously demonstrated a new technique, temperature modulated fluorescence tomography (TM-FT), which relies on key elements: temperature sensitive ICG loaded pluronic nanocapsules and high intensity focused ultrasound (HIFU), to combine the sensitivity of fluorescence imaging with focused ultrasound resolution. While conventional fluorescence tomography measurements are acquired, the tissue is scanned by a HIFU beam and irradiated to produce a local hot spot, in which the temperature increases nearly 5K. The fluorescence emission signal measured by the optical detectors varies drastically when the hot spot overlays onto the location of the temperature dependent nanocapsules. The small size of the focal spot (~1.4 mm) up to a depth of 6 cm, allows imaging the distribution of these temperature sensitive agents with not only high spatial resolution but also high quantitative accuracy in deep tissue using a proper image reconstruction algorithm. Previously we have demonstrated this technique with a phantom study with nanocapsules sensitive to 20-25°C range. In this work, we will show the first nanocapsules optimized for in vivo animal imaging.

  18. 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.

  19. 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

  20. Endogenous synchronous fluorescence spectroscopy (SFS) of basal cell carcinoma-initial study

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

    The human skin is a complex, multilayered and inhomogeneous organ with spatially varying optical properties. Analysis of cutaneous fluorescence spectra could be a very complicated task; therefore researchers apply complex mathematical tools for data evaluation, or try to find some specific approaches, that would simplify the spectral analysis. Synchronous fluorescence spectroscopy (SFS) allows improving the spectral resolution, which could be useful for the biological tissue fluorescence characterization and could increase the tumour detection diagnostic accuracy.

  1. [Measurement of nasopharyngeal carcinoma tissue ex vivo by Raman spectroscopy].

    PubMed

    Huang, Wei; Pan, Jian-ji; Chen, Rong; Li, Yong-zeng; Feng, Shang-yuan; Xie, Shu-sen; Zeng, Hai-shan

    2009-05-01

    Raman spectroscopy has shown its potential and advantages in detecting molecular changes associated with tissue pathology, which makes it possible to diagnose with optical methods non-invasively and real-time. A compact and rapid near-infrared (NIR)Raman system was developed using 785 nm diode laser, volume phase technology (VPT)holographic grating system and NIR intensified charge-coupled device (CCD)with a specially designed Raman fibre probe which can effectively reduce the interference of fluorescence and Rayleigh scattering, maximize the ability of Raman collection as well as correct the image aberration of a planar grating diffraction. Adopting this method, signal-to-noise ratio has been greatly improved and human tissue signals can be acquired in a short time. Raman signals from fat and musculature of fresh pork were measured and referenced for further optimization, then Raman spectra of nasopharyngeal carcinoma in vitro and the effect of storage time on them were measured in 1-5 s and discussed. The sensitivities and performance of the system will be further enhanced and more Raman data will be acquired and compared between normal and cancerous nasopharyngeal tissue, expecting to discover the statistical characteristics, which will benefit the diagnosis and treatment of early nasopharyngeal carcinoma or other tumors. PMID:19650477

  2. 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.

  3. 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

  4. 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.

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

    PubMed

    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

  6. 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.

  7. 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

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

    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.

  9. 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.

  10. 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.