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1

Fluorescence Spectroscopy  

NSDL National Science Digital Library

This resource, part of the Spectroscopy Lab Suite, simulates optical transitions in a Fluorescent light. In this illustration, the transitions between bands in the phosphor coating of the light are shown. The phosphor is excited by discharge in a mercury gas. The energy levels and transitions in the phosphor material can be changed.

Zollman, Dean

2010-05-21

2

Dynamic Imaging by Fluorescence Correlation Spectroscopy Identifies Diverse Populations of Polyglutamine Oligomers Formed in Vivo*  

PubMed Central

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

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

2012-01-01

3

Special practical laboratory for training on optical biophysics: in vivo reflectance and fluorescence spectroscopy of the human skin  

NASA Astrophysics Data System (ADS)

The set of topically united practical works on in vivo reflectance and fluorescence spectroscopy of the human skin of the special training laboratory on optical biophysics for the undergraduate and postgraduate students specialized in biophysics, biochemical physics and medical physics is described.

Sinichkin, Yurii P.; Dolotov, Leonid E.; Kiseleva, Irina A.; Zimnyakov, Dmitry A.; Tuchin, Valery V.

2002-07-01

4

Changes in chlorophyll a fluorescence of glyphosate-tolerant soybean plants induced by glyphosate: in vivo analysis by laser-induced fluorescence spectroscopy.  

PubMed

A significant increase in the use of the herbicide glyphosate has generated many questions about its residual accumulation in the environment and possible damage to crops. In this study, changes in chlorophyll a (chl-a) fluorescence induced by glyphosate in three varieties of glyphosate-resistant soybean plants were determined with an in vivo analysis based on a portable laser-induced fluorescence system. Strong suppression of chl-a fluorescence was observed for all plants treated with the herbicide. Moreover, the ratio of the emission bands in the red and far-red regions (685 nm/735 nm) indicates that the application of glyphosate led to chlorophyll degradation. The results also indicated that the use of glyphosate, even at concentrations recommended by the manufacturer, suppressed chl-a fluorescence. In summary, this study shows that fluorescence spectroscopy can detect, in vivo, very early changes in the photosynthetic status of transgenic soybeans treated with this herbicide. PMID:23669766

Fernandes, Joelson; Falco, William Ferreira; Oliveira, Samuel Leite; Caires, Anderson Rodrigues Lima

2013-05-01

5

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

NASA Astrophysics Data System (ADS)

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.

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

2012-09-01

6

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

NASA Astrophysics Data System (ADS)

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 (?rad=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.

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

2003-11-01

7

In vivo detection of membrane protein expression using surface plasmon enhanced fluorescence spectroscopy (SPFS).  

PubMed

Surface plasmon enhanced fluorescence spectroscopy (SPFS) was applied for the detection of expression and functional incorporation of integral membrane proteins into plasma membranes of living cells in real time. A vesicular stomatitis virus (VSV) tagged mutant of photoreceptor bovine rhodopsin was generated for high level expression with the semliki forest virus (SFV) system. Adherent baby hamster kidney (BHK-21) cells were cultivated on fibronectin-coated gold surfaces and infected with genetically engineered virus driving the expression of rhodopsin. Using premixed fluorescently (Alexa Fluor 647) labeled anti-mouse secondary antibody and monoclonal anti-VSV primary antibody, expression of rhodopsin in BHK-21 cells was monitored by SPFS. Fluorescence enhancement by surface plasmons occurs exclusively in the close vicinity of the gold surface. Thus, only the Alexa Fluor 647 labeled antibodies binding to the VSV-tag at rhodopsin molecules exposed on the cell surface experienced fluorescence enhancement, whereas, unbound antibody molecules in the bulk solution were negligibly excited. With this novel technique, we successfully recorded an increase of fluorescence with proceeding rhodopsin expression. Thus, we were able to observe the incorporation of heterologously expressed rhodopsin in the plasma membrane of living cells in real time using a relatively simple and rapid method. We confirmed our results by comparison with conventional wide field fluorescence microscopy. PMID:16530398

Krupka, Simone S; Wiltschi, Birgit; Reuning, Ute; Hölscher, Kerstin; Hara, Masahiko; Sinner, Eva-Kathrin

2006-08-15

8

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

NASA Astrophysics Data System (ADS)

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 (?ex=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.

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

2012-03-01

9

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

NASA Astrophysics Data System (ADS)

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.

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

2013-11-01

10

In-vivo fluorescence spectroscopy to optimize the detection of early bronchial carcinoma by autoflourescene imaging  

NASA Astrophysics Data System (ADS)

Autofluorescence bronchoscopy is a promising approach to detect and characterize precancerous and early cancerous lesions. Nevertheless, many spectral features of photodetection systems remain unclear and sub-optimal at the present time. We report here a comprehensive study of the autofluorescence of the human healthy, metaplastic, dysplastic and cancerous bronchial tissues, covering a range of excitation wavelengths going from 350 nm to 480 nm. Moreover, the absolute values of these tissue autofluorescence yields were determined. These measurements were performed with a spectrally and intensity calibrated optical fiber-based spectrofluorometer which has been designed to optimize the spectroscopy conditions encountered by an endoscopic fluorescence imaging system. Our data yield information about the excitation and emission windows to be used in a bispectral detection imaging system. We found that the order of magnitude of the autofluorescence brightness is stable as the excitation varies from 350 to 495 nm (on the order of 5 nW/mW x nm). We also found that the use of backscattered red light instead of red autofluorescence enhances the lesion/normal tissues contrast. The excitation wavelengths yielding the highest contrasts are between 400 and 480 nm with a peak at 405 nm. It was finally observed that the transition wavelength for bispectral fluorescence imaging systems is around 590 nm, regardless of the excitation wavelength.

Wagnieres, Georges A.; Zellweger, Matthieu; Grosjean, Pierre; Goujon, Didier; Conde, Ramiro; Forrer, Martin; Monnier, Philippe; van den Bergh, Hubert

2002-05-01

11

Quantification of fluorophore concentration and fluorescence quantum yield in tissue simulating phantoms and in vivo by fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

The quantification of fluorophore concentration in tissue has many practical applications in the fields of pharmacokinetics, diagnosis of various diseases and the treatment of cancer. To avoid the inconveniences associated with the acquisition and processing of tissue samples (the standard method of quantitation), fast, reliable (<15% error) and non or minimally invasive methods of quantitation are desired. Measurements of fluorescence correlate well with concentration, making it a good candidate. The fluorescence signal depends on a number of factors, including the optical properties of the tissue and the local fluorophore environment (through the fluorescence quantum yield). A difference in the fluorescence quantum yield between the fluorophore in tissue and in a calibration standard, for example, would be erroneously interpreted as a change in the concentration of the fluorophore. Two approaches were taken to estimate fluorophore concentration. In the first, fluorescence was excited and detected using a single optical fibre. In this configuration, concentration estimates were obtained from small (<1 muL) volumes with minimal dependence on the optical properties. In this first study, concentration measured on the surface and the interior of tissue-simulating phantoms could be estimated with a rootmean-square error of 10.6%. The second approach incorporated the optical properties of the tissue into two analytical models of fluorescence excitation and detection based on diffusion theory. These were compared to Monte Carlo simulations and a more complicated diffusion model that required numerical solution. If the fluorescence quantum yield was known, then the fluorophore concentration could be estimated from measurements of spatially resolved reflectance and fluorescence. The better of the two diffusion models derived in this paper yielded estimates in tissue-simulating phantoms with a root-mean-square error of 11.4%. Conversely, if the concentration was known then the fluorescence quantum yield could be estimated. The quantum yield of two fluorophores, meso-tetra-(4-sulfonatophenyl)-porphine dihydrochloride (TPPS4) and aluminum phthalocyanine tetrasulfonate (AlPcS 4) was measured accurately (0.121 +/- 0.001 cf. 0.12 and 0.59 +/- 0.03 cf. 0.58 respectively) when the concentration was estimated using an absorption measurement. (Abstract shortened by UMI.)

Diamond, Kevin-Ross

2005-11-01

12

Fluorescence Correlation Spectroscopy  

NSDL National Science Digital Library

This paper, which was previously published as part of an online biophysics textbook, provides detailed information about concepts related to fluorescence correlation spectroscopy. Sections of the document include writing on experimental realization, theoretical concepts, and applications of this technology.

Haustein, Elke

13

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

NASA Astrophysics Data System (ADS)

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

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

2012-06-01

14

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

PubMed Central

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

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

2014-01-01

15

Nanosecond fluorescence spectroscopy  

SciTech Connect

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.

Leskovar, B.

1985-03-01

16

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

PubMed

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

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

2014-02-01

17

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

PubMed Central

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

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

2014-01-01

18

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

NASA Astrophysics Data System (ADS)

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.

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

19

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

PubMed Central

Abstract. 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-?m axial, 65-?m 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

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

2012-01-01

20

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

PubMed

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-?m axial, 65-?m 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

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

21

CHICKEN DISEASE CHARACTERIZATION BY FLUORESCENCE SPECTROSCOPY  

Technology Transfer Automated Retrieval System (TEKTRAN)

Fluorescence spectroscopy was used to characterize chicken carcass spectra. Spectral signatures of three different disease categories of poultry carcasses (airsacculitis, cadaver, and septicemia) were obtained from fluorescence emission measurements in the wavelength range of 360 to 600 nm with 330 ...

22

Laser-induced fluorescence spectroscopy in tissue local necrosis detection  

NASA Astrophysics Data System (ADS)

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.

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

2014-03-01

23

Non-invasive detection of oral cancer using reflectance and fluorescence spectroscopy  

E-print Network

In vivo reflectance and fluorescence spectra were collected from patients with oral lesions, as well as healthy volunteers, in order to evaluate the potential of spectroscopy to serve as a non-invasive tool for the detection ...

McGee, Sasha Alanda

2008-01-01

24

Fluorescence spectroscopy of rhodopsins: insights and approaches.  

PubMed

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

Alexiev, Ulrike; Farrens, David L

2014-05-01

25

Fluorescent Multiblock ?-Conjugated Polymer Nanoparticles for In Vivo Tumor Targeting  

E-print Network

Highly fluorescent multiblock conjugated polymer nanoparticles with folic acid surface ligands are highly effective for bioimaging and in vivo tumor targeting. The targeted nanoparticles were preferentially localized in ...

Ahmed, Eilaf

26

Spatial distribution and function of sterol regulatory element-binding protein 1a and 2 homo- and heterodimers by in vivo two-photon imaging and spectroscopy fluorescence resonance energy transfer.  

PubMed

Sterol regulatory element-binding proteins (SREBPs) are a subfamily of basic helix-loop-helix-leucine zipper proteins that regulate lipid metabolism. We show novel evidence of the in vivo occurrence and subnuclear spatial localization of both exogenously expressed SREBP-1a and -2 homodimers and heterodimers obtained by two-photon imaging and spectroscopy fluorescence resonance energy transfer. SREBP-1a homodimers localize diffusely in the nucleus, whereas SREBP-2 homodimers and the SREBP-1a/SREBP-2 heterodimer localize predominantly to nuclear speckles or foci, with some cells showing a diffuse pattern. We also used tethered SREBP dimers to demonstrate that both homo- and heterodimeric SREBPs activate transcription in vivo. Ultrastructural analysis revealed that the punctate foci containing SREBP-2 are electron-dense nuclear bodies, similar or identical to structures containing the promyelocyte (PML) protein. Immunofluorescence studies suggest that a dynamic interplay exists between PML, as well as another component of the PML-containing nuclear body, SUMO-1, and SREBP-2 within these nuclear structures. These findings provide new insight into the overall process of transcriptional activation mediated by the SREBP family. PMID:15798184

Zoumi, Aikaterini; Datta, Shrimati; Liaw, Lih-Huei L; Wu, Cristen J; Manthripragada, Gopi; Osborne, Timothy F; Lamorte, Vickie J

2005-04-01

27

Extraction of masked fluorescence peaks through synchronous fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

Fluorescence spectroscopy has been demonstrated as a viable tool for noting subtle biochemical changes that occur during early-stage cervical cancer progression. Due to multiple fluorophore contributions, the individual fluorophore activities often get masked due to overlapping spectra of neighboring fluorophores. Recently synchronous fluorescence spectroscopy has been demonstrated as an efficient technique for investigation of such non-dominant fluorophores. With synchronous fluorescence spectroscopy individual fluorophore responses are highlighted as sharp peaks by choosing appropriate offsets during signal acquisition. Such peaks may, however be missed due to absorption effects. By correcting the measured synchronous fluorescence spectrum with elastic scattering data, it was observed that the masked fluorophores are highlighted while the broader bands are sharpened. Interestingly, fluorophore activities of protoporphyrin, collagen, NADH, FAD and porphyrin can now be studied using this technique, as compared to only collagen and NADH seen earlier. The results have been verified using tissue phantoms with known fluorophores and scatterers. Use of normalized synchronous spectra has led to enhancement of several fluorophore responses. It was also observed that among the different offsets, the lower ones show sharper features, whereas the larger offsets show a broadband response. Among the different offsets 120nm is found optimal for further investigation.

Devi, Seema; Mozumder, Meghdoot; Ghosh, Nirmalya; Pradhan, Asima

2012-03-01

28

Fluorescence spectroscopy of normal and cancerous human stomach tissue  

NASA Astrophysics Data System (ADS)

The autofluorescence of cancerous and normal human stomach tissues was measured in vitro, by fluorescence spectroscopy, within three hours of surgery ablation. A new fluorescence emission band, centered at about 380 nm for the cancerous stomach tissue is reported, for 340 nm excitation. This band is practically absent for the normal tissue when this is excited at the same wavelength. For UVB excitation (between 283 and 305 nm) the emission bands are centered around 350 nm and 470 nm, for both tissues, in agreement with the literature. The ratios of the fluorescence intensities for cancerous and normal tissues are measured at the center of the three bands, the intensity for the diseased tissue being always higher than for the normal one. The presence of a new band centered at 380 nm, combined with the intensity ratios, may prove of great relevance towards early in vivo detection of stomach cancer.

Fraser Monteiro, Maria L.; Rezio, T.; Soares, Jorge; Martinho, J. M.; Liang, Dawei; Fraser Monteiro, Luis

1994-01-01

29

Dual-Color Fluorescence Cross-Correlation Spectroscopy on a  

E-print Network

Dual-Color Fluorescence Cross-Correlation Spectroscopy on a Single Plane Illumination Microscope fluorescence correlation spectroscopy (SPIM-FCS) is a new method for imaging FCS in 3D samples, providing to two-color fluorescence cross-correlation spectroscopy (SPIM-FCCS), which allows to measure molecular

Garbe, Christoph S.

30

Fluorescence spectroscopy for diagnosis of esophageal cancer  

NASA Astrophysics Data System (ADS)

Laser-induced fluorescence spectroscopy was employed to measure fluorescence emission of normal and malignant tissue during endoscopy in patients with esophageal adenocarcinoma. A nitrogen/dye laser tuned at 410 nm was used for excitation source. The fluorescence lineshape of each spectrum was determined and sampled at 15 nm intervals from 430 nm to 716 nm. A calibration set from normal and malignant spectra were selected. Using stepwise discriminate analysis, significant wavelengths that separated normal and malignant spectra were selected. The intensities at these wavelengths were used to formulate a classification model using linear discriminate analysis. The model was used to classify additional tissue spectra from 26 malignant and 108 normal sites into either normal or malignant spectra with a sensitivity of 100 percent and specificity of 98 percent.

Panjehpour, Masoud; Overholt, Bergein F.; Vo-Dinh, Tuan; Farris, Christie; Schmidhammer, James L.; Sneed, Rick E.; Buckley, Paul F., III

1994-07-01

31

Scanning fluorescence correlation spectroscopy on biomembranes.  

PubMed

Fluorescence correlation spectroscopy (FCS) is a powerful quantitative method to study dynamical properties of biophysical systems. It exploits the temporal autocorrelation of fluorescence intensity fluctuations originating from a tiny volume (~fL). A theoretical model function can be then fitted to the measured auto-correlation curve to obtain physical parameters such as local concentration and diffusion time. However, the application of FCS on membranes is coupled to several difficulties like accurate positioning and stability of the set-up. In this book chapter, we explain the theoretical framework of point FCS and Scanning FCS (SFCS), which is a variation especially suitable for membrane studies. We present a list of materials necessary for SFCS studies on Giant Unilamellar Vesicles (GUVs). Finally, we provide simple protocols for the preparation of GUVs, calibration of the microscope setup, and acquisition and analysis of SFCS data to determine diffusion coefficients and concentrations of fluorescent particles embedded in lipid membranes. PMID:25331137

Hermann, Eduard; Ries, Jonas; García-Sáez, Ana J

2015-01-01

32

In vivo absorption spectroscopy for absolute measurement  

PubMed Central

In in vivo spectroscopy, there are differences between individual subjects in parameters such as tissue scattering and sample concentration. We propose a method that can provide the absolute value of a particular substance concentration, independent of these individual differences. Thus, it is not necessary to use the typical statistical calibration curve, which assumes an average level of scattering and an averaged concentration over individual subjects. This method is expected to greatly reduce the difficulties encountered during in vivo measurements. As an example, for in vivo absorption spectroscopy, the method was applied to the reflectance measurement in retinal vessels to monitor their oxygen saturation levels. This method was then validated by applying it to the tissue phantom under a variety of absorbance values and scattering efficiencies. PMID:23082298

Furukawa, Hiromitsu; Fukuda, Takashi

2012-01-01

33

Predicting in vivo fluorescence lifetime behavior of near-infrared fluorescent contrast agents  

E-print Network

Predicting in vivo fluorescence lifetime behavior of near-infrared fluorescent contrast agents capability. In this study, we evaluate the FLTs of eight near-infrared fluorescent mo- lecular probes further demonstrate that the diffusion of a near-infrared NIR reporter from a dye-loaded gel can

Larson-Prior, Linda

34

Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent  

E-print Network

Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins Arie Krumholz1 the application of two spectrally distinct near-infrared fluorescent proteins, iRFP670 and iRFP720, engineered-tissue PAT, probes absorbing in the near-infrared (NIR) spectral range are desirable. In the NIR optical

Verkhusha, Vladislav V.

35

Plasmon-controlled fluorescence: a new paradigm in fluorescence spectroscopy  

PubMed Central

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

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

2009-01-01

36

Diagnosing breast cancer using diffuse reflectance spectroscopy and intrinsic fluorescence spectroscopy  

E-print Network

Using diffuse reflectance spectroscopy and intrinsic fluorescence spectroscopy, we have developed an algorithm that successfully classifies normal breast tissue, fibrocystic change, fibroadenoma, and infiltrating ductal ...

Fitzmaurice, Maryann

37

Fluorescent Multiblock ?-Conjugated Polymer Nanoparticles for In Vivo Tumor Targeting  

PubMed Central

Highly fluorescent multiblock conjugated polymer nanoparticles with folic acid surface ligands are highly effective for bioimaging and in vivo tumor targeting. The targeted nanoparticles were preferentially localized in tumor cells in vivo, thereby illustrating their potential for diagnostic and therapeutic applications. PMID:23794490

Ahmed, Eilaf; Morton, Stephen W.

2014-01-01

38

Trp aporepressor engineered for fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

The tryptophan repressor from Escherichia coli binds to the trp operator in the presence of L- tryptophan, thereby inhibiting the biosynthesis of L-tryptophan. Site-directed mutagenesis was used to change tryptophan-19 and tryptophan-99 to leucine and methionine, respectively. This mutant protein without tryptophan in its amino acid sequence has wild-type repressor activity and is a suitable model for fluorescence studies of corepressor binding. Both steady-state and time-resolved fluorescence spectroscopy have been used to compare the binding of L- tryptophan, indole-3-propionic acid, indole-3-butyric acid, and indole. In all cases, binding to the mutant aporepressor results in a large blue shift and a change in the intensity of the ligand fluorescence. The decay of the total fluorescence intensity from the complex indicates the presence of three distinct bound states of the ligand. The distribution of ligand binding modes is influenced by the substituent at the 3-position of the indole ring. The rotational correlation time of the complexes formed with L-tryptophan or indole-3-propionic acid indicate that the protein is present as a dimer, whereas with indole or indole-3-butyric acid the correlation times are much lower, suggesting that the protein is present as a monomer.

Millar, David P.; Hochstrasser, Remo A.; Chapman, David; Youderian, Philip

1992-04-01

39

Noninvasive skin fluorescence spectroscopy for diabetes screening.  

PubMed

The development of cost-effective, simple, and reproducible tests for diabetes screening represents a priority of modern medicine in light of the increasing prevalence of diabetes mellitus. Besides fasting plasma glucose, the oral glucose tolerance test, and glycated hemoglobin A1c, several tests have been proposed, among them the assessment of skin fluorescence spectroscopy (SFS). This article comments on the article by Olson and coauthors published in this issue of Journal of Diabetes Science and Technology and comprehensively reviews related available information. Overall, SFS seems to represent an easy-to-use, noninvasive tool that adds value to existing tests for diabetes screening. PMID:23911182

Stirban, Alin

2013-07-01

40

Two-Photon Fluorescence Correlation Spectroscopy  

NASA Technical Reports Server (NTRS)

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.

Zimmerli, Gregory A.; Fischer, David G.

2002-01-01

41

Fluorescence fluctuation spectroscopy for clinical applications  

NASA Astrophysics Data System (ADS)

Fluorescence correlation spectroscopy (FCS) and the related techniques of brightness analysis have become standard tools in biological and biophysical research. By analyzing the statistics of fluorescence emitted from a restricted volume, a number of parameters including concentrations, diffusion coefficients and chemical reaction rates can be determined. The single-molecule sensitivity, spectral selectivity, small sample volume and non-perturbative measurement mechanism of FCS make it an excellent technique for the study of molecular interactions. However, its adoption outside of the research laboratory has been limited. Potential reasons for this include the cost and complexity of the required apparatus. In this work, the application of fluorescence fluctuation analysis to several clinical problems is considered. Optical designs for FCS instruments which reduce the cost and increase alignment tolerance are presented. Brightness analysis of heterogenous systems, with application to the characterization of protein aggregates and multimer distributions, is considered. Methods for FCS-based assays of two clinically relevant proteins, von Willebrand factor and haptoglobin, are presented as well.

Olson, Eben

42

In vivo near-infrared fluorescence imaging  

Microsoft Academic Search

Photon penetration into living tissue is highly dependent on the absorption and scattering properties of tissue components. The near-infrared region of the spectrum offers certain advantages for photon penetration, and both organic and inorganic fluorescence contrast agents are now available for chemical conjugation to targeting molecules. This review focuses on those parameters that affect image signal and background during in

John V Frangioni

2003-01-01

43

[In vivo NMR spectroscopy of the liver].  

PubMed

The application of in vivo MR spectroscopy to the study of the liver is currently an expanding field of research. Owing to technical difficulties, the results obtained thus far were mainly those of animal observations. Several nuclei have been considered: hydrogen, phosphorus, carbon or fluorine. This non-traumatic method allows following and quantifying the various metabolic pathways, especially during hepatic diseases. The major metabolic pathways, i.e. neoglycogenesis, glycogenolysis, Krebs' cycle, etc., are studied, as well as their alterations during diseases such as ischemia, diabetes or alcoholism. The development of this promising technique requires the cooperation of various clinical and fundamental disciplines. PMID:2677330

Jehenson, P; Cuenod, C A; Syrota, A

1989-01-01

44

Optimizing Disinfection Pretreatment using Excitation-emission Matrix Fluorescence Spectroscopy  

Microsoft Academic Search

Excitation-emission matrix fluorescence spectroscopy (EEM) can be used to characterize organic matter present in water samples. When excited, the intensity of fluorescence emitted can be used to generate a representation of organic matter makes it possible to localize fluorescence centers related to particular groups, which can ‘fingerprint’ a sample. The technique is applicable to wastewater samples to identify contributors of

Katherine Y. Bell; Juan Sánez; Martha J. M. Wells

2012-01-01

45

Imaging cellular dynamics in vivo with multicolor fluorescent proteins  

NASA Astrophysics Data System (ADS)

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.

Hoffman, Robert M.

2005-04-01

46

Near-infrared fluorescence: application to in vivo molecular imaging.  

PubMed

Molecular imaging often relies on the use of targeted and activatable reporters to quantitate and visualize targets, biological processes, and cells in vivo. The use of optical probes with near-infrared fluorescence allows for improved photon penetration through tissue and minimizes the effects of tissue autofluorescence. There are several parameters that define the effectiveness of imaging agents in vivo. These factors include probe targeting, activation, pharmacokinetics, biocompatibility, and photophysics. Recent advances in our understanding of these variables as they pertain to the application of optical reporters for in vivo imaging are discussed in this review. PMID:19879798

Hilderbrand, Scott A; Weissleder, Ralph

2010-02-01

47

TWO-COLOR FLUORESCENCE (CROSS-)CORRELATION SPECTROSCOPY ON A SELECTIVE PLANE ILLUMINATION MICROSCOPE  

E-print Network

TWO-COLOR FLUORESCENCE (CROSS-)CORRELATION SPECTROSCOPY ON A SELECTIVE PLANE ILLUMINATION: Imaging fluorescence cross-correlation spectroscopy, selective plane illumination microscopy, quantitative imaging, EMCCD camera ABSTRACT: Fluorescence correlation spectroscopy (FCS) is a useful technology

Garbe, Christoph S.

48

Laser Induced Fluorescence Spectroscopy of Soft Tissues of the Oral Cavity  

NASA Astrophysics Data System (ADS)

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

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

2011-07-01

49

Fluorescence Spectroscopy of Neoplastic and Non-Neoplastic Tissues  

PubMed Central

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

Ramanujam, Nirmala

2000-01-01

50

Fluorescence spectroscopic analysis (FSA) detects quantitative changes in atherosclerotic plaque collagen and elastin content In Vivo  

NASA Astrophysics Data System (ADS)

In order to assess the capacity for in vivo fluorescence spectroscopi8c analysis of arterial collagen and elastin, fluorescence emission intensity was recorded form rabbit aorta after angioplasty and stent implant, and correlated with extracted elastin and collagen content. FEI from saline treated rabbits after stent implant was higher between 485 and 500 nm than after anti-inflammatory treatment. FEI was significantly decreased after implantation of shorter stents at 476-500 nm. Multiple regression analysis demonstrated an excellent correlation between FEI and elastin and HPLC- measured collagen content at 486-500 nm and 476-480 nm respectively. Conclusions: FEI recorded in vivo form arterial intimal surface, can be successfully used for quantitative assessment of compositional changes in connective tissue. Stent implant can induce changes in intimal arterial structure at discrete sites distant from the stent implant site.

Christov, Alexander M.; Dai, Erbin; Liu, Liying; Guan, Haiyan; Bernards, Mark A.; Cavers, Paul B.; Susko, David; Lucas, Alexandra

2002-05-01

51

Structured illumination fluorescence correlation spectroscopy for velocimetry in Zebrafish embryos  

NASA Astrophysics Data System (ADS)

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

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

2013-02-01

52

In vivo Raman spectroscopy of cervix cancers  

NASA Astrophysics Data System (ADS)

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.

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

2014-03-01

53

Effect of probe pressure on cervical fluorescence spectroscopy measurements  

E-print Network

Fluorescence spectroscopy is a promising technology for detection of epithelial precancers and cancers. While age and menopausal status influence measurements in the cervix, other variables do not significantly affect the ...

Nath, Audrey

54

Native fluorescence spectroscopy of thymus and fat tissues  

NASA Astrophysics Data System (ADS)

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.

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

1993-08-01

55

Quantitative Determination of DNA-Ligand Binding Using Fluorescence Spectroscopy  

ERIC Educational Resources Information Center

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.

Healy, Eamonn F.

2007-01-01

56

Fluorescent-Spectroscopic Research of in Vivo Tissues Pathological Conditions  

NASA Astrophysics Data System (ADS)

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 (?ex=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.

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

57

In vivo lipidomics using single-cell Raman spectroscopy  

PubMed Central

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

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

2011-01-01

58

Multimodal, multiplex, Raman spectroscopy of alcohol in diffuse, fluorescent media  

E-print Network

Multimodal, multiplex, Raman spectroscopy of alcohol in diffuse, fluorescent media Scott T. Mc spectrometer to detect alcohol in a lipid tissue phantom solution. Keywords: Raman Spectroscopy, Tissue in solutions of a lipid tissue phantom. At high lipid concentrations, our instrument detects Raman signatures

Pitsianis, Nikos P.

59

Validation of temperature-modulated fluorescence tomography in vivo  

NASA Astrophysics Data System (ADS)

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.

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

2014-02-01

60

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

NASA Astrophysics Data System (ADS)

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

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

2000-05-01

61

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

NASA Astrophysics Data System (ADS)

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.

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

2001-05-01

62

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

NASA Astrophysics Data System (ADS)

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.

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

63

In vivo 2D magnetic resonance spectroscopy of small animals  

Microsoft Academic Search

Localized in vivo NMR spectroscopy, chemical shift imaging or multi-voxel spectroscopy are potentially useful tools in small animals that are complementary to MRI, adding biochemical information to the mainly anatomical data provided by imaging of water protons. However the contribution of such methods remains hampered by the low spectral resolution of the in vivo 1D spectra. Two-dimensional methods widely developed

P. Méric; G. Autret; B. T. Doan; B. Gillet; C. Sébrié; J.-C. Beloeil

2004-01-01

64

Deep tissue fluorescence imaging and in vivo biological applications  

NASA Astrophysics Data System (ADS)

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

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

2012-11-01

65

Uptake of fluorescent gentamicin by vertebrate sensory cells in vivo  

PubMed Central

Aminoglycoside uptake in the inner ear remains poorly understood. We subcutaneously injected a fluorescently-conjugated aminoglycoside, gentamicin–Texas Red (GTTR), to investigate the in vivo uptake of GTTR in the inner ear of several vertebrates, and in various murine sensory cells using confocal microscopy. In bullfrogs, GTTR uptake was prominent in mature hair cells, but not in immature hair cells. Avian hair cells accrued GTTR more rapidly at the base of the basilar papilla. GTTR was associated with the hair bundle; and, in guinea pigs and mice, somatic GTTR fluorescence was initially diffuse before punctate (endosomal) fluorescence could be observed. A baso-apical gradient of intracellular GTTR uptake in guinea pig cochleae could only be detected at early time points (<3 h). In 21?28 day mice, cochlear GTTR uptake was greatly reduced compared to guinea pigs, 6-day-old mice, or mice treated with ethacrynic acid. In mice, GTTR was also rapidly taken up, and retained, in the kidney, dorsal root and trigeminal ganglia. In linguinal and vibrissal tissues rapid GTTR uptake cleared over a period of several days. The preferential uptake of GTTR by mature saccular, and proximal hair cells resembles the pattern of aminoglycoside-induced hair cell death in bullfrogs and chicks. Differences in the degree of GTTR uptake in hair cells of different species suggests variation in serum levels, clearance rates from serum, and/or the developmental and functional integrity of the blood–labyrinth barrier. GTTR uptake by hair cells in vivo suggests that GTTR has potential to elucidate aminoglycoside transport mechanisms into the inner ear, and as a bio-tracer for in vivo pharmacokinetic studies. PMID:16466873

Dai, C.F.; Mangiardi, D.; Cotanche, D.A.; Steyger, P.S.

2008-01-01

66

In Vivo Blood Characterization from Bioimpedance Spectroscopy of Blood  

E-print Network

In Vivo Blood Characterization from Bioimpedance Spectroscopy of Blood Pooling Tao Dai Department@sce.carleton.ca Abstract Characterization of blood impedance properties is important to estimate clinical diagnos- tic in the measurement field, rather than the blood individually. This paper describes a novel in vivo measurement

Adler, Andy

67

Fluorescence correlation spectroscopy and its potential for intracellular applications  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) is a time-averaging fluctuation analysis of small molecular ensembles, combining\\u000a maximum sensitivity with high statistical confidence. Among a multitude of physical parameters that are, in principle, accessible\\u000a by FCS, it most conveniently allows to determine local concentrations, mobility coefficients, and characteristic rate constants\\u000a of fast-reversible and slow-irreversible reactions of fluorescently labeled biomolecules at very low (nanomolar)

Petra Schwille

2001-01-01

68

Pancreatic tissue assessment using fluorescence and reflectance spectroscopy  

NASA Astrophysics Data System (ADS)

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.

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

2007-07-01

69

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

PubMed Central

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

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

2014-01-01

70

Single molecule fluorescence spectroscopy of mutants of the Discosoma red fluorescent protein DsRed  

NASA Astrophysics Data System (ADS)

We studied the emission of mutants of the red fluorescent protein DsRed by room temperature single molecule fluorescence spectroscopy. Bulk samples of the DsRed variant E8 show mixed green and red fluorescence of equivalent intensities individually spectrally similar to arrested green and mature red fluorescent forms of DsRed. Investigations at the single molecule level indicate that, like DsRed, E8 is not monomeric at single molecule concentrations. The entities visualized are composed of green and red emitting proteins without a fixed ratio of green to red fluorescing units. We find indications for only weak, if any, fluorescence resonance energy transfer (FRET) between red and green chromophores within one E8 entity.

Blum, Christian; Subramaniam, Vinod; Schleifenbaum, Frank; Stracke, Frank; Angres, Brigitte; Terskikh, Alexey; Meixner, A. J.

2002-08-01

71

"FluSpec": A Simulated Experiment in Fluorescence Spectroscopy  

ERIC Educational Resources Information Center

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.

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

2014-01-01

72

EMCCD-based spectrally resolved fluorescence correlation spectroscopy.  

PubMed

We present an implementation of fluorescence correlation spectroscopy with spectrally resolved detection based on a combined commercial confocal laser scanning/fluorescence correlation spectroscopy microscope. We have replaced the conventional detection scheme by a prism-based spectrometer and an electron-multiplying charge-coupled device camera used to record the photons. This allows us to read out more than 80,000 full spectra per second with a signal-to-noise ratio and a quantum efficiency high enough to allow single photon counting. We can identify up to four spectrally different quantum dots in vitro and demonstrate that spectrally resolved detection can be used to characterize photophysical properties of fluorophores by measuring the spectral dependence of quantum dot fluorescence emission intermittence. Moreover, we can confirm intracellular cross-correlation results as acquired with a conventional setup and show that spectral flexibility can help to optimize the choice of the detection windows. PMID:21164726

Bestvater, Felix; Seghiri, Zahir; Kang, Moon Sik; Gröner, Nadine; Lee, Ji Young; Im, Kang-Bin; Wachsmuth, Malte

2010-11-01

73

Laser induced fluorescence spectroscopy of boron carbide  

NASA Astrophysics Data System (ADS)

Laser induced fluorescence spectrum of boron carbide (BC) between 490 and 560 nm has been recorded and analyzed. Gas-phase BC molecule was produced by the reaction of diborane and methane in the presence of magnesium atom from laser ablation process. The (0, 0), (1, 0), and (2, 0) bands of the B 4? - - X 4? - transition were rotationally analyzed. Spectra of both isotopes: 10BC and 11BC were analyzed. Equilibrium molecular constants for the B 4? - and the X 4? - states for both isotopes were determined.

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

2011-06-01

74

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

NASA Astrophysics Data System (ADS)

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.

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

2012-02-01

75

Emerging biomedical applications of time-resolved fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

Time-resolved fluorescence spectroscopy is presently regarded as a research tool in biochemistry, biophysics, and chemical physics. Advances in laser technology, the development of long-wavelength probes, and the use of lifetime-based methods are resulting in the rapid migration of time-resolved fluorescence to the clinical chemistry lab, to the patient's bedside, to flow cytometers, to the doctor's office, and even to home health care. Additionally, time-resolved imaging is now a reality in fluorescence microscopy, and will provide chemical imaging of a variety of intracellular analytes and/or cellular phenomena. In this overview paper we attempt to describe some of the opportunities available using chemical sensing based on fluorescence lifetimes, and to predict those applications of lifetime-based sensing which are most likely in the near future.

Lakowicz, Joseph R.; Szmacinski, Henryk; Koen, Peter A.

1994-07-01

76

Laser Induced Fluorescence Spectroscopy of Scandium Monoiodide  

NASA Astrophysics Data System (ADS)

The laser induced fluorescence spectrum of scandium monoiodide (ScI) between 787 and 814 nm has been recorded. ScI molecule was produced by reacting laser vaporized Sc atoms with methyl iodide (CH3I). Spectra of eleven vibrational bands of the C1 ?+ - X1 ?+ transition of ScI were obtained and analyzed. A merged least-squares fit of the measured line positions yielded accurate molecular constants for the upper levels of the C1 ?+ state and the v = 1 - 4 levels of the X1 ?+state. One vibrational band observed at 11627 cm-1 belongs to a sub-band transition of the a3 ? state, which is found to be perturbed by the X1 ?+ state. Details of the perturbation and molecular constants obtained will be reported.

Liao, Zhenwu; Yang, Mei; Chan, Man-Chor; Xia, Ye; Cheung, A. S.-C.

2012-06-01

77

Laser induced fluorescence spectroscopy of ruthenium monoboride  

NASA Astrophysics Data System (ADS)

Laser induced fluorescence spectrum of ruthenium monoboride (RuB) in the visible region between 500 and 575 nm was studied. RuB molecule was produced by reacting laser ablated ruthenium atom with 0.5% diborane (B2H6) seeded in argon. Three transition bands of the [18.4]2.5-X2?5/2 transition were recorded and rotationally analyzed. The ground state symmetry and bond length, ro, were determined to be X2?5/2 state and 1.7099 Å, respectively, which is consistent with a 2?i state predicted from electronic configuration using a molecular orbital energy level diagram. This work represents the first experimental investigation of the spectrum of the RuB molecule.

Wang, Na; Ng, Y. W.; Cheung, A. S.-C.

2012-09-01

78

Ultrasensitive molecular fluorescence spectroscopy in levitated microdroplets  

SciTech Connect

The extreme sensitivity of fluorescence spectrophotometry results from the fact that a molecule can undergo many excitation-emission cycles before destruction by photochemical degradation. For example, Rhodamine 6G (R6G) can emit in excess of 10{sup 5} photons before photolysis takes place. The fraction of emitted photons collected and converted to countable pulses can be as high as 10{sup {minus}3}, although 10{sup {minus}4} is more readily attainable. Therefore, sufficient signal exists for single molecules to be detectable. Detection limits for molecules in solution have been limited by background signal from solvent Raman scattering and fluorescence. This background signal adds noise to the measurement and has effectively restricted the detectable concentration to about 10{sup {minus}13} M. Over the past decade, advances in detection of fewer molecules have all been made by reducing the measurement volume and/or increasing the measuring time. Given the above concentration detection limit a reduction of the measurement volume to 1 pL leads to a minimum observable quantity of {approx}1 molecule. The ability to detect a single molecule in condensed phase could have many important applications in addition to being an interesting problem. The obvious application of this approach is to situations where small quantities of material are available for analysis. The capability to reliably detect a single fluorophore might also allow the screening and/or sorting of a collection of molecules. Such abilities would have application to many biological problems such as DNA sequencing and detection of DNA adducts.

Ramsey, J.M.; Whitten, W.B. (Oak Ridge National Lab., TN (USA)); Arnold, S. (Polytechnic Univ., Brooklyn, NY (USA)); Bronk, B.V. (Chemical Research, Development and Engineering Center, Aberdeen Proving Ground, MD (USA))

1990-01-01

79

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

NASA Astrophysics Data System (ADS)

Multiphoton fluorescence recovery after photobleaching (MP-FRAP) is a laser microscopy technique used to probe the transport properties of macromolecules in biological systems. MP-FRAP utilizes two-photon fluorescence and photobleaching to produce a three-dimensionally resolved diffusion coefficient for an ensemble of molecules in the region of the two-photon focal volume. This thesis describes two fundamental improvements to the MP-FRAP technique, which are vital steps to enable MP-FRAP to be applied to the complex in vivo environment. In Chapter 1, we lay the groundwork for our discussion of these advancements by introducing the MP-FRAP technique and the physics upon which it is based. We begin with a description of fluorescence and diffusion and discuss their importance in biomedical research. Next, we describe how two-photon fluorescence and photobleaching are applied to a diffusing system to measure the diffusion coefficient via fluorescence recovery after photobleaching (FRAP). Then, we take the reader through the evolution of FRAP, which leads to the application of two- photon fluorescence and photobleaching to produce MP-FRAP. Along the way, we highlight applications and advancements of the FRAP techniques, and introduce fluorescence correlation spectroscopy, a popular complement to FRAP. In Chapter 2, we collect the experimental methods for the studies presented in Chapters 3 and 4. We begin with an in-depth discussion of our work to build and troubleshoot our MP-FRAP apparatus, followed by a detailed description of our data analysis protocol. Next, we delve into the specific methods for producing computer generated data and fits, as well as in vitro and in vivo experimental data, for our work in Chap. 3 on improving MP-FRAP to measure diffusion in the presence of convective flow. We end with a description of the Monte Carlo algorithm we developed for our work in Chap. 4 to model diffusion and multiphoton fluorescence recovery after photobleaching in the presence of reflective boundaries of various geometries. In Chapter 3, we develop an improved analytical model of multiphoton fluorescence recovery after photobleaching that includes the effects of convective flows within a system. We use computer generated data and fits to explore the effect of convective flow on the shape and speed of fluorescence recovery, and to estimate the range of diffusion coefficients and flow speeds over which this new "diffusion-convection" model yields accurate diffusion coefficients (as compared to the diffusion-only model). We then demonstrate the validity of the diffusion-convection model through in vitro experimentation in systems with known diffusion coefficients and known flow speeds, and show that the diffusion-convection model enables accurate determination of the diffusion coefficient via MP-FRAP, even when significant flows are present. We conclude by demonstrating the effectiveness of the diffusion-convection model in vivo by measuring the diffusion coefficient and flow speed within tumor vessels of 4T1 murine mammary adenocarcinomas implanted in the dorsal skinfold chamber. In Chapter 4, we present our work that allows MP-FRAP to be performed accurately near reflective boundaries of various geometries. Using Monte Carlo techniques, we first generate an initial distribution of bleached molecules, then simulate their diffusion away from the initial distribution, thereby producing fluorescence vs. time recovery curves in the region of the initial bleached distribution. These curves are then fit to the standard analytical MP-FRAP model to produce a diffusion coefficient. By introducing solid barriers into the model in the region of the initial bleached distribution, we learn how the presence of harriers of different geometries affects the measurement of diffusion via MP-FRAP. Finally, we supply ranges of barrier positions for each geometry within which MP-FR AP can confidently be employed to measure accurate diffusion coefficients.

Sullivan, Kelley Diane

80

Long-Term Retention of Fluorescent Quantum Dots In Vivo  

NASA Astrophysics Data System (ADS)

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.

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

81

Quantification of osmotic water transport in vivo using fluorescent albumin.  

PubMed

Osmotic water transport across the peritoneal membrane is applied during peritoneal dialysis to remove the excess water accumulated in patients with end-stage renal disease. The discovery of aquaporin water channels and the generation of transgenic animals have stressed the need for novel and accurate methods to unravel molecular mechanisms of water permeability in vivo. Here, we describe the use of fluorescently labeled albumin as a reliable indicator of osmotic water transport across the peritoneal membrane in a well-established mouse model of peritoneal dialysis. After detailed evaluation of intraperitoneal tracer mass kinetics, the technique was validated against direct volumetry, considered as the gold standard. The pH-insensitive dye Alexa Fluor 555-albumin was applied to quantify osmotic water transport across the mouse peritoneal membrane resulting from modulating dialysate osmolality and genetic silencing of the water channel aquaporin-1 (AQP1). Quantification of osmotic water transport using Alexa Fluor 555-albumin closely correlated with direct volumetry and with estimations based on radioiodinated ((125)I) serum albumin (RISA). The low intraperitoneal pressure probably accounts for the negligible disappearance of the tracer from the peritoneal cavity in this model. Taken together, these data demonstrate the appropriateness of pH-insensitive Alexa Fluor 555-albumin as a practical and reliable intraperitoneal volume tracer to quantify osmotic water transport in vivo. PMID:25100279

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

2014-10-15

82

Phytoplankton in Lake Tanganyika – vertical and horizontal distribution of in vivo fluorescence  

Microsoft Academic Search

Determinations of chlorophyll a and in vivo fluorescence of photosynthetic pigments were used to study vertical and horizontal distribution of phytoplankton in Lake Tanganyika (East Africa). Blue excited fluorescence (IVFb) was an approximate predictor of chlorophyll a at different depths and locations. Green excited fluorescence (IVFg), which reflects phycoerythrin in cyanobacteria, explained chlorophyll a variation equally well, and in combination

K. Salonen; J. Sarvala; M. Järvinen; V. Langenberg; M. Nuottajärvi; K. Vuorio; D. B. R. Chitamwebwa

1999-01-01

83

Laser Induced Fluorescence Spectroscopy of Cobalt Monoboride  

NASA Astrophysics Data System (ADS)

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

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

2011-06-01

84

UNCORRECTED 3 Fluorescence excitation spectroscopy of the ~AA2  

E-print Network

UNCORRECTED PROOF 3 Fluorescence excitation spectroscopy of the ~AA2 A1 ~XX2 B1 system 4 of jet-cooled NH2 in the region 2900­4300 AA 5 Ju Xin,a Haiyan Fan,b Ionela Ionescu,b Chris Annesley,b and Scott A excitation spectrum of the NH2 ~AA2 A1 ~XX2 B1 system in the region 2900­4300 AA has been measured under jet

Reid, Scott A.

85

Hybrid native phosphorescence and fluorescence spectroscopy for cancer detection  

NASA Astrophysics Data System (ADS)

Native fluorescence of tissues in the UV and visible spectral regions has been investigated for over two decades. Native fluorescence has been demonstrated to be an accurate tools for distinguish normal tissue from malignant and pre-malignant. Prior investigations have demonstrated that there are several ratio-based algorithms, which can distinguish malignant tissue from normal with high sensitivity and specificity.1 The wavelength combinations used in these ratios isolate the contributions from pairs of tissue fluorophors, one of which is frequently tryptophan (trp), the predominant tissue fluorophore with excitation in the UV (250-300 nm). In this work, algorithms using a combination of native fluorescence and trp phosphorescence were developed which show promise for providing enhanced detection accuracy. Using optical fibers to collect the emission from the specimen allowed interrogation of small regions of tissue, providing precise spatial information. Using a specially designed setup, specimens were excited in the UV and spectra were collected in the range of 300 to 700 nm. Three main emission bands were selected for analysis: 340 nm (trp fluorescence); 420 - 460 nm band (fluorescence from the extra cellular matrix); and 500 - 520 nm (trp phosphorescence). Normal specimens consistently exhibited a low ratio (<10) of 345 to 500 nm emission intensity while this same ratio was consistently high (>15) for cancer specimens. Creating intensities ratio maps from the tissue allows one to localize the malignant regions with high spatial precision. The study was performed on ex vivo human breast tissues. The ratio analysis correlated well with histopathology.

Alimova, Alexandra; Katz, A.; Sriramoju, Vidyasagar; Budansky, Yuri; Bykov, Alexei A.; Zeylikovich, Roman; Alfano, R. R.

2006-02-01

86

Fluorescence spectroscopy using indocyanine green for lymph node mapping  

NASA Astrophysics Data System (ADS)

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 ?M 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.

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

2014-02-01

87

Ultrasensitive fluorescence correlation spectroscopy of highly parallelized microfluidic devices  

NASA Astrophysics Data System (ADS)

Reducing reagent needs and costs while increasing throughput constitute important needs for assays in pharmaceutical drug discovery. We are developing an ultrasensitive, fluorescence-based detection system in highly parallel microfluidic channels with kHz readout rates in each channel. Prototype microfluidic devices with an array of >150 microchannels have been fabricated by direct femtosecond laser machining of fused silica substrates. A device is placed in a custombuilt, wide-field microscope where a line-generating red diode laser provides uniform epi-illumination just a few microns high across a 500 micron field of view. Single-molecule levels in the probe volumes can be attained by flowing suitably dilute aqueous solutions (~pM) of fluorescently labeled biomolecules through the microchannels. Fluorescence is detected with an electron-multiplying CCD camera allowing readout rates up to 7 kHz for each microchannel. Rapid initial assessment of detected fluorescence signals is performed through digital filtering derived from simulations based on experimental parameters. Fluorescence correlation spectroscopy can then provide more detailed analysis of the sample within each microchannel. Optimized microfluidic devices could be mass-produced in low-cost polymers using imprint lithography.

Canfield, Brian K.; King, Jason K.; Robinson, William N.; Hofmeister, William H.; Soper, Steven A.; Davis, Lloyd M.

2012-02-01

88

Synchronous fluorescence spectroscopy for analysis of wine and wine distillates  

NASA Astrophysics Data System (ADS)

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 (??) 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 ?? 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.

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

2015-01-01

89

Video-rate fluorescence diffuse optical tomography for in vivo sentinel lymph node imaging  

PubMed Central

We have developed a fiber-based, video-rate fluorescence diffuse optical tomography (DOT) system for noninvasive in vivo sentinel lymph node (SLN) mapping. Concurrent acquisition of fluorescence and reference signals allowed the efficient generation of ratio-metric data for 3D image reconstruction. Accurate depth localization and high sensitivity to fluorescent targets were established in to depths of >10 mm. In vivo accumulation of indocyanine green (ICG) dye was imaged in the region of the SLN following intradermal injection into the forepaw of rats. These results suggest that video-rate fluorescence DOT has significant potential as a clinical tool for noninvasive mapping of SLN. PMID:22162817

Solomon, Metasebya; White, Brian R.; Nothdruft, Ralph E.; Akers, Walter; Sudlow, Gail; Eggebrecht, Adam T.; Achilefu, Samuel; Culver, Joseph P.

2011-01-01

90

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

Microsoft Academic Search

The analysis of various varnishes from different origins, which are commonly found on historical musical instruments was carried out for the first time with both fluorescence excitation emission spectroscopy and laser-induced time-resolved fluorescence spectroscopy. Samples studied include varnishes prepared using shellac, and selected diterpenoid and triterpenoid resins from plants, and mixtures of these materials. Fluorescence excitation emission spectra have been

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

2009-01-01

91

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

NASA Astrophysics Data System (ADS)

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.

Zheng, Longjiang; Hu, Yuanting

2009-07-01

92

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

NASA Astrophysics Data System (ADS)

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.

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

2010-12-01

93

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

NASA Astrophysics Data System (ADS)

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.

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

2011-08-01

94

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

NASA Astrophysics Data System (ADS)

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 ?m diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8-7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence lifetime measurements of low quantum efficiency sub-nanosecond fluorophores.

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

2014-03-01

95

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

SciTech Connect

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 ?m diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8–7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence lifetime measurements of low quantum efficiency sub-nanosecond fluorophores.

Yankelevich, Diego R. [Department of Electrical and Computer Engineering, University of California, 3101 Kemper Hall, Davis, California 95616 (United States) [Department of Electrical and Computer Engineering, University of California, 3101 Kemper Hall, Davis, California 95616 (United States); Department of Biomedical Engineering, University of California, 451 Health Sciences Drive, Davis, California 95616 (United States); Ma, Dinglong; Liu, Jing; Sun, Yang; Sun, Yinghua; Bec, Julien; Marcu, Laura, E-mail: lmarcu@ucdavis.edu [Department of Biomedical Engineering, University of California, 451 Health Sciences Drive, Davis, California 95616 (United States)] [Department of Biomedical Engineering, University of California, 451 Health Sciences Drive, Davis, California 95616 (United States); Elson, Daniel S. [Hamlyn Centre for Robotic Surgery, Department of Surgery and Cancer, Imperial College London, Exhibition Road, London SW7 2AZ (United Kingdom)] [Hamlyn Centre for Robotic Surgery, Department of Surgery and Cancer, Imperial College London, Exhibition Road, London SW7 2AZ (United Kingdom)

2014-03-15

96

Brain cancer probed by native fluorescence and stokes shift spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

2012-12-01

97

Single-molecule fluorescence spectroscopy in (bio)catalysis  

PubMed Central

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

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

98

Investigation of asphaltene association by front-face fluorescence spectroscopy.  

PubMed

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

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

2003-07-01

99

The study of viral assembly with fluorescence fluctuation spectroscopy  

NASA Astrophysics Data System (ADS)

Enveloped viruses contain an encapsulating membrane that the virus acquires from the host cell during the budding process. The presence of the enveloping lipid membrane complicates the physical characterization of the proteins assembled within the virus considerably. Here we present a method based on fluorescence fluctuations that quantifies the copy number of proteins within an enveloped viral particles. We choose the viral protein Gag of the human immunodeficiency virus (HIV) type 1 as a model system, because Gag expressed in cells is sufficient to produce viral-like particles (VLPs) of the same size as authentic virions. VLPs harvested from cells that express fluorescently labeled Gag were investigated by two-photon fluorescence fluctuation spectroscopy. The autocorrelation functions of the fluctuations revealed a hydrodynamic size of the fluorescent VLPs consistent with previous results based on electron microscopy. Further analysis of the fluctuations revealed a copy number of Gag per virion that is inconsistent with the prevailing model of HIV assembly. We will discuss the implications of our experimental results for the assembly process of VLPs.

Mueller, Joachim; Wu, Bin; Chen, Yan

2008-03-01

100

Glycosaminoglycan-protein interaction studies using fluorescence spectroscopy.  

PubMed

Fluorescence spectroscopy is a quantitative analytical tool that has been extensively used to provide structural and dynamical information on GAG-protein complexes. It possesses major advantages including high sensitivity, relative ease of applicability, and wide range of available fluorescence labels and probes. It has been applied to practically every protein-GAG system through the use of either intrinsic (e.g., Trp) or extrinsic (e.g., a non-covalent fluorophore) probe. For studies involving GAGs, it forms the basis for measurement of dissociation constant of complexes and the stoichiometry of binding, which helps elucidate many other thermodynamic and/or mechanistic parameters. We describe the step-by-step procedure to measure the affinity of GAG-protein complexes, parse the ionic and nonionic components of the free energy of binding, and identify the site of GAG binding through competitive binding experiments. PMID:25325964

Boothello, Rio S; Al-Horani, Rami A; Desai, Umesh R

2015-01-01

101

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

PubMed

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

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

2011-01-01

102

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

PubMed Central

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

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

2011-01-01

103

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

NASA Astrophysics Data System (ADS)

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.

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

2007-07-01

104

Time gated fluorescence spectroscopy in Barrett’s oesophagus  

PubMed Central

Background and aims: Specialised intestinal metaplasia and its dysplastic transformation, which precedes cancer in Barrett’s oesophagus cannot be differentiated in standard gastroscopy. The aim of this study was to investigate whether laser induced protoporphyrin IX fluorescence permits the detection of specialised intestinal metaplasia and dysplasia during endoscopy and to take biopsy specimens in a guided rather than random manner. Methods: In 53 patients with Barrett’s oesophagus 5-aminolaevulinic acid was sprayed on the mucosa. Approximately 60 to 120 minutes later, biopsy specimens were taken based on point-like measurements of delayed fluorescence intensity ratios of protoporphyrin IX in vivo. Two independent pathologists examined the 596 biopsy specimens taken, 168 of which were selected to be investigated by a third pathologist. Among these specimens only those (n=141) with a consensus diagnosis by at least two pathologists and p53 expression as additional marker were included in the analysis. Results: The median of normalised fluorescence intensity (ratio of delayed PpIX fluorescence intensity to immediate autofluorescence intensity) in non-dysplastic specialised intestinal metaplasia (0.51, 68% CI 0.09 to 1.92) and low grade dysplasia (1.89, 68% CI 0.55 to 3.92) differed significantly (p<0.005). Dysplasia was detected at a rate 2.8-fold higher compared with screening endoscopy despite taking fewer specimens. In addition, three early cancers were detected for the first time. Moreover, this method permitted differentiation of specialised intestinal metaplasia from junctional or gastric-fundic type epithelium (p<0.013). Conclusions: For the first time it was possible to differentiate low grade dysplasia from non-dysplastic Barrett’s mucosa during endoscopy based on delayed laser induced fluorescence endoscopy of PpIX. Furthermore, the method helps to detect specialised intestinal metaplasia in short Barrett’s oesophagus. PMID:12477755

Ortner, M-A E J; Ebert, B; Hein, E; Zumbusch, K; Nolte, D; Sukowski, U; Weber-Eibel, J; Fleige, B; Dietel, M; Stolte, M; Oberhuber, G; Porschen, R; Klump, B; Hörtnagl, H; Lochs, H; Rinneberg, H

2003-01-01

105

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

PubMed Central

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

Wachsmann-Hogiu, Sebastian; Weeks, Tyler

2009-01-01

106

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

NASA Astrophysics Data System (ADS)

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.

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

1999-04-01

107

Long Fluorescence Lifetime Molecular Probes Based on Near Infrared Pyrrolopyrrole Cyanine Fluorophores for In Vivo Imaging  

E-print Network

Long Fluorescence Lifetime Molecular Probes Based on Near Infrared Pyrrolopyrrole Cyanine of organic molecules provide a new reporting strategy for molecular imaging in the near infrared (NIR of near-infrared (NIR) fluorescent molecular probes have been developed for in vivo imaging applications

Larson-Prior, Linda

108

Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging  

Microsoft Academic Search

.   A recent development in biomedical imaging is the non-invasive mapping of molecular events in intact tissues using fluorescence.\\u000a Underpinning to this development is the discovery of bio-compatible, specific fluorescent probes and proteins and the development\\u000a of highly sensitive imaging technologies for in vivo fluorescent detection. Of particular interest are fluorochromes that\\u000a emit in the near infrared (NIR), a spectral

Vasilis Ntziachristos; Christoph Bremer; Ralph Weissleder

2003-01-01

109

Monitoring the Biodegradation of Dendritic Near-Infrared Nanoprobes by in Vivo Fluorescence Imaging  

E-print Network

Monitoring the Biodegradation of Dendritic Near-Infrared Nanoprobes by in Vivo Fluorescence Imaging to monitor noninvasively their fate in vivo. Here we report multilayered nanoprobes, consisting of a near-infrared. Covalent encapsulation of the near-infrared fluorophores in the dendritic scaffold conferred enhanced

Larson-Prior, Linda

110

Fluorescent PolystyreneFe3O4 Composite Nanospheres for In Vivo Imaging and Hyperthermia  

E-print Network

Fluorescent Polystyrene­Fe3O4 Composite Nanospheres for In Vivo Imaging and Hyperthermia By Donglu hyperthermia.[16] The main purpose of this work is to present a new strategy in biomedical nanotechnology that allows simultaneous in vivo imaging and local therapy via hyperthermia. This novel concept is based

Papautsky, Ian

111

Anatomy-Based Algorithms for Detecting Oral Cancer Using Reflectance and Fluorescence Spectroscopy  

E-print Network

OBJECTIVES: We used reflectance and fluorescence spectroscopy to noninvasively and quantitatively distinguish benign from dysplastic/malignant oral lesions. We designed diagnostic algorithms to account for differences in ...

McGee, Sasha

112

Developmental changes in spatial distribution of in vivo fluorescence and epidermal UV absorbance over Quercus petraea leaves  

PubMed Central

Background and Aims Epidermal phenolic compounds (mainly flavonoids) constitute a vital screen that protects the leaf from damage by natural ultraviolet (UV) radiation. The effectiveness of epidermal UV-screening depends on leaf anatomy, the content of UV-screening compounds and their spatial uniformity over the leaf area. To investigate in vivo the spatial pattern of the epidermal UV-screen during leaf development, a fluorescence imaging method was developed to map the epidermal UV-absorbance at a microscopic scale. This study was done on oak (Quercus petraea) leaves that were used as a model of woody dicotyledonous leaves. Methods The leaf development of 2-year-old trees, grown outdoors, was monitored, at a macroscopic scale, by in vivo measurements of chlorophyll content per unit area and epidermal UV-absorbance using two optical leaf-clip meters. The distribution of pigments within leaves was assessed in vivo spectroscopically. The microscopic images of UV-induced fluorescence and UV-absorbance acquired in vivo during leaf development were interpreted from spectral characteristics of leaves. Key Results At a macroscopic scale, epidermal UV-absorbance was high on the upper leaf side during leaf development, while it increased on the lower leaf side during leaf expansion and reached the adaxial value at maturity. At a microscopic scale, in immature leaves, for both leaf sides, the spatial distribution of epidermal UV-absorbance was heterogeneous, with a pattern depending on the flavonoid content of vacuoles in developing epidermal cells. At maturity, epidermal UV-absorbance was uniform. Conclusions The spatial pattern of epidermal UV-screen over the area of oak leaves is related to leaf anatomy during development. In vivo spectroscopy and fluorescence imaging of the leaf surface showed the distribution of pigments within the leaf and hence can provide a tool to monitor optically the leaf development in nature. PMID:19561346

Meyer, S.; Louis, J.; Moise, N.; Piolot, T.; Baudin, X.; Cerovic, Z. G.

2009-01-01

113

In Vivo Dendritic Cell Tracking Using Fluorescence Lifetime Imaging and Near-Infrared-Emissive Polymersomes  

Microsoft Academic Search

Purpose  Noninvasive in vivo cell-tracking techniques are necessary to advance the field of cellular-based therapeutics as well as to elucidate mechanisms\\u000a governing in vivo cell biology. Fluorescence is commonly used for in vitro and postmortem biomedical studies but has been limited by autofluorescence at the whole-animal level.\\u000a \\u000a \\u000a \\u000a Procedures  In this report, we demonstrate the ability of in vivo fluorescent lifetime imaging to

Natalie A. Christian; Fabian Benencia; Michael C. Milone; Guizhi Li; Paul R. Frail; Michael J. Therien; George Coukos; Daniel A. Hammer

2009-01-01

114

In Vivo Imaging with Fluorescent Smart Probes to Assess Treatment Strategies for Acute Pancreatitis  

PubMed Central

Background and Aims Endoprotease activation is a key step in acute pancreatitis and early inhibition of these enzymes may protect from organ damage. In vivo models commonly used to evaluate protease inhibitors require animal sacrifice and therefore limit the assessment of dynamic processes. Here, we established a non-invasive fluorescence imaging-based biomarker assay to assess real-time protease inhibition and disease progression in a preclinical model of experimental pancreatitis. Methods Edema development and trypsin activation were imaged in a rat caerulein-injection pancreatitis model. A fluorescent “smart” probe, selectively activated by trypsin, was synthesized by labeling with Cy5.5 of a pegylated poly-L-lysine copolymer. Following injection of the probe, trypsin activation was monitored in the presence or absence of inhibitors by in vivo and ex vivo imaging. Results We established the trypsin-selectivity of the fluorescent probe in vitro using a panel of endopeptidases and specific inhibitor. In vivo, the probe accumulated in the liver and a region attributed to the pancreas by necropsy. A dose dependent decrease of total pancreatic fluorescence signal occurred upon administration of known trypsin inhibitors. The fluorescence-based method was a better predictor of trypsin inhibition than pancreatic to body weight ratio. Conclusions We established a fluorescence imaging assay to access trypsin inhibition in real-time in vivo. This method is more sensitive and dynamic than classic tissue sample readouts and could be applied to preclinically optimize trypsin inhibitors towards intrapancreatic target inhibition. PMID:23409095

Agarwal, Abhiruchi; Boettcher, Andreas; Kneuer, Rainer; Sari-Sarraf, Farid; Donovan, Adriana; Woelcke, Julian; Simic, Oliver; Brandl, Trixi; Krucker, Thomas

2013-01-01

115

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

PubMed

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

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

2011-08-16

116

Intramyocardial triglyceride quantification by magnetic resonance spectroscopy: in vivo and ex vivo correlation in human subjects  

PubMed Central

Accumulation of triglycerides (TG) in heart tissue has been associated with changes in left ventricular function. Proton magnetic resonance spectroscopy (1H-MRS) is currently the only non-invasive in vivo method to measure myocardial TG content. The primary aim of this study was to determine if these in vivo measurements are specific to myocardial TG in human subjects. Thus, in vivo 1H-MRS measurements were conducted on orthotopic heart transplant patients (n = 8) immediately before they underwent routine biopsies and ex vivo measurements were made on the endomyocardial biopsy samples. The correlation coefficient between the two measurements was 0.97, with p < 0.005, demonstrating for the first time the specificity of the in vivo measurement in human heart. From accompanying reliability experiments, the standardized typical error for the in vivo 1H-MRS method was estimated to be 7.0%, with a 95% confidence interval from 5.5 to 9.4%. These results suggest that 1H-MRS provides a specific and reliable measurement of myocardial TG content and is suitable for routine studies. PMID:21500254

O’Connor, Robert D.; Xu, Jian; Ewald, Gregory A.; Ackerman, Joseph J. H.; Peterson, Linda R.; Gropler, Robert J.; Bashir, Adil

2013-01-01

117

IN SITU PLANETARY MINERALOGY USING SIMULTANEOUS TIME RESOLVED FLUORESCENCE AND RAMAN SPECTROSCOPY. J. Blacksberg1  

E-print Network

1. Image of our pulsed 532nm laser focusedIN SITU PLANETARY MINERALOGY USING SIMULTANEOUS TIME RESOLVED FLUORESCENCE AND RAMAN SPECTROSCOPY@gps.caltech.edu Introduction: Micro-Raman spectroscopy is one of the primary methods of mineralogical analysis

Rossman. George R.

118

Remote excitation fluorescence correlation spectroscopy using silver nanowires  

NASA Astrophysics Data System (ADS)

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.

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

2014-11-01

119

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

NASA Astrophysics Data System (ADS)

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.

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

2009-02-01

120

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

PubMed

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

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

2013-11-01

121

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

PubMed Central

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

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

2013-01-01

122

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)

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)

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

123

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

NASA Astrophysics Data System (ADS)

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.

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

1997-12-01

124

Fluorescence spectroscopy: considerations for highly absorbing dissolved organic matter samples  

NASA Astrophysics Data System (ADS)

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 study by Ohno (2002), which investigated several simpler fluorescence metrics. Suwannee River fulvic acid, pH 4, absorbance at 254nm vs. loading of component Q2. At the higher absorbances the component loadings are over-predicted compared to the linear regression, estimated from data with absorbance below 0.3, where the inner-filter effect is minimal.

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

2009-12-01

125

Fluorescence Glucose Detection: Advances Toward the Ideal In Vivo Biosensor  

Microsoft Academic Search

The importance of glucose monitoring for in vivo as well as for ex vivo applications has driven a vast number of scientific groups to pursue the development of an advanced glucose sensor. Such a sensor must be robust, versatile, and capable of the long-term, accurate and reproducible detection of glucose levels in various testing media. Among the different configurations and

Elizabeth A. Moschou; Bethel V. Sharma; Sapna K. Deo; Sylvia Daunert

2004-01-01

126

In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially localized jellyfish fluorescent protein gene  

Microsoft Academic Search

We aimed to introduce foreign DNA into spermatogenic cells in the testis by injection of the DNA encoding jellyfish fluorescent proteins, green fluorescent protein (GFP) and yellow fluorescent protein (YFP) into the seminiferous tubules and in vivo electroporation. We obtained fluorescent spermatozoa only when using the gene of the YFP protein fused to a mitochondrial localization signal peptide. Intracytoplasmic injection

Zhenyong Huang; Masaru Tamura; Takayuki Sakurai; Shinichiro Chuma; Tetsuichiro Saito; Norio Nakatsuji

2000-01-01

127

Pancreatic tumor detection using hypericin-based fluorescence spectroscopy and cytology  

NASA Astrophysics Data System (ADS)

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.

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

2005-04-01

128

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

NASA Astrophysics Data System (ADS)

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.

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

2014-11-01

129

Terahertz spectroscopy for the assessment of burn injuries in vivo.  

PubMed

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

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

2013-07-01

130

A new in vivo model to test anti-tuberculosis drugs using fluorescence imaging  

PubMed Central

Objectives The current method for testing new drugs against tuberculosis in vivo is the enumeration of bacteria in organs by cfu assay. Owing to the slow growth rate of Mycobacterium tuberculosis (Mtb), these assays can take months to complete. Our aim was to develop a more efficient, fluorescence-based imaging assay to test new antibiotics in a mouse model using Mtb reporter strains. Methods A commercial IVIS Kinetic® system and a custom-built laser scanning system with fluorescence molecular tomography (FMT) capability were used to detect fluorescent Mtb in living mice and lungs ex vivo. The resulting images were analysed and the fluorescence was correlated with data from cfu assays. Results We have shown that fluorescent Mtb can be visualized in the lungs of living mice at a detection limit of ?8?×?107 cfu/lung, whilst in lungs ex vivo a detection limit of ?2?×?105 cfu/lung was found. These numbers were comparable between the two imaging systems. Ex vivo lung fluorescence correlated to numbers of bacteria in tissue, and the effect of treatment of mice with the antibiotic moxifloxacin could be visualized and quantified after only 9 days through fluorescence measurements, and was confirmed by cfu assays. Conclusions We have developed a new and efficient method for anti-tuberculosis drug testing in vivo, based on fluorescent Mtb reporter strains. Using this method instead of, or together with, cfu assays will reduce the time required to assess the preclinical efficacy of new drugs in animal models and enhance the progress of these candidates into clinical trials against human tuberculosis. PMID:22635525

Zelmer, Andrea; Carroll, Paul; Andreu, Nuria; Hagens, Kristine; Mahlo, Jacqueline; Redinger, Natalja; Robertson, Brian D.; Wiles, Siouxsie; Ward, Theresa H.; Parish, Tanya; Ripoll, Jorge; Bancroft, Gregory J.; Schaible, Ulrich E.

2012-01-01

131

Longitudinal in vivo two-photon fluorescence imaging  

PubMed Central

Fluorescence microscopy is an essential technique for the basic sciences, especially biomedical research. Since the invention of laser scanning confocal microscopy in 1980s, that enabled imaging both fixed and living biological tissue with three-dimensional precision, high-resolution fluorescence imaging has revolutionized biological research. Confocal microscopy, by its very nature, has one fundamental limitation. Due to the confocal pinhole, deep tissue fluorescence imaging is not practical. In contrast (no pun intended), two-photon fluorescence microscopy allows, in principle, the collection of all emitted photons from fluorophores in the imaged voxel, dramatically extending our ability to see deep into living tissue. Since the development of transgenic mice with genetically encoded fluorescent protein in neocortical cells in 2000, two-photon imaging has enabled the dynamics of individual synapses to be followed for up to two years. Since the initial landmark contributions to this field in 2002, the technique has been used to understand how neuronal structure are changed by experience, learning and memory and various diseases. Here we provide a basic summary of the crucial elements that are required for such studies, and discuss many applications of longitudinal two-photon fluorescence microscopy that have appeared since 2002. PMID:24214350

Crowe, Sarah E.; Ellis-Davies, Graham C.R.

2014-01-01

132

Longitudinal in vivo two-photon fluorescence imaging.  

PubMed

Fluorescence microscopy is an essential technique for the basic sciences, especially biomedical research. Since the invention of laser scanning confocal microscopy in the 1980s, which enabled imaging both fixed and living biological tissue with 3D precision, high-resolution fluorescence imaging has revolutionized biological research. Confocal microscopy, by its very nature, has one fundamental limitation. Due to the confocal pinhole, deep tissue fluorescence imaging is not practical. In contrast (no pun intended), two-photon fluorescence microscopy allows, in principle, the collection of all emitted photons from fluorophores in the imaged voxel, dramatically extending our ability to see deep into living tissue. Since the development of transgenic mice with genetically encoded fluorescent protein in neocortical cells in 2000, two-photon imaging has enabled the dynamics of individual synapses to be followed for up to 2 years. Since the initial landmark contributions to this field in 2002, the technique has been used to understand how neuronal structure are changed by experience, learning, and memory and various diseases. Here we provide a basic summary of the crucial elements that are required for such studies, and discuss many applications of longitudinal two-photon fluorescence microscopy that have appeared since 2002. PMID:24214350

Crowe, Sarah E; Ellis-Davies, Graham C R

2014-06-01

133

Upconversion fluorescence-SERS dual-mode tags for cellular and in vivo imaging.  

PubMed

Fluorescent-surface enhanced Raman scattering (F-SERS) dual mode tags showed great potential for bioimaging due to the combined advantages of intuitive, fast imaging of fluorescence and multiplex capability of SERS technique. In previously reported F-SERS tags, organic fluorescent dyes or quantum dots were generally selected to generate fluorescence signal. Herein, we reported the first proof-of-concept upconversion fluorescence (UCF)-SERS dual mode tags based on near infrared (NIR) laser (980 nm) excited upconversion nanoparticles (UCNPs) for live-cell and in vivo imaging. Three components involved in this tag: NaYF4:Yb,Er UCNPs@SiO2 serving as the fluorescent core of the tag; silver nanoparticles in situ grown on the surface of UCNPs@SiO2 for generating characteristic Raman signal; and denatured BSA coating rendering the tag's stability and biocompatibility. The UCF-SERS tags integrated the NIR imaging capability of both fluorescent UCNPs and plasmonic SERS nanoprobe, which facilitated dual mode bioimaging investigation, especially for living animals. Ex vivo experiments revealed that with 980 nm and 785 nm NIR laser irradiations, the UCF and SERS signals of the tags could be detected from 3 and 7 mm deep pork tissues, respectively. Furthermore, the in vivo imaging capabilities of UCF-SERS tags were successfully demonstrated on living mice. The developed dual modality tags held great potential for medical diagnostics and therapy. PMID:24617579

Niu, Xiaojuan; Chen, Haiyan; Wang, Yunqing; Wang, Wenhai; Sun, Xiuyan; Chen, Lingxin

2014-04-01

134

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

NASA Astrophysics Data System (ADS)

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 (HP1?) 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 HP1? as a positive regulator of active transcription in euchromatin. Intriguingly, we observed that the transcription factor CCAAT/enhancer-binding protein alpha (C/EBP?) interacts with HP1? in regions of pericentromeric heterochromatin in mouse pituitary cells. These observations prompted us to investigate the relationship between HP1? dynamic interactions in pituitary specific gene regulation.

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

2012-03-01

135

FM spectroscopy in fluorescence in laser-cooled rubidium  

NASA Astrophysics Data System (ADS)

A novel variation of the technique of two-photon frequency modulation (FM) spectroscopy has been developed, which allows signals to be obtained from small (˜10 5 atoms) and dilute (˜10 10 cm -3) samples - in this case laser-cooled rubidium atoms. This was possible by detecting and demodulating the fluorescence signal rather than the transmission through the sample. Data are presented demonstrating the dependence of the signal on phase and frequency of the imposed modulation. Initially the atoms were held in a magneto-optic trap but, in order to eliminate line broadening due to the presence of the trapping fields, data were taken with the atoms in free fall. A similar signal, slightly shifted and broadened by the trapping fields, was obtained while the trap was on and used for long-term stabilisation of the probe laser.

Snadden, M. J.; Clarke, R. B. M.; Riis, E.

1998-07-01

136

EEM Fluorescence Spectroscopy Fingerprints and Monitoring of NDMA and TTHM Formation Potentials  

Microsoft Academic Search

1. Summary Excitation-emission matrix (EEM) fluorescence spectroscopy is becoming a valuable tool in water quality monitoring, based on identifying fluorescence emitting organic substances (fluorophores) present in water systems. This study continued our effort to apply the sensitive fluorescence EEM monitoring technique for water quality management. Specifically, the EEM approach was applied to the identification of the precursors of total trihalomethane

Baolin Deng

137

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

NASA Astrophysics Data System (ADS)

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.

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

2014-03-01

138

Fluorescence correlation spectroscopy: Statistical analysis and biological applications  

NASA Astrophysics Data System (ADS)

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.

Saffarian, Saveez

2002-01-01

139

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

Microsoft Academic Search

Originally developed for applications in physics and physical chemistry, fluorescence fluctuation spectroscopy is becoming\\u000a widely used in cell biology. This review traces the development of the method and describes some of the more important applications.\\u000a Specifically, the methods discussed include fluorescence correlation spectroscopy (FCS), scanning FCS, dual color cross-correlation\\u000a FCS, the photon counting histogram and fluorescence intensity distribution analysis approaches,

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

2009-01-01

140

Fluorescence Spectroscopy Measurement for Quality Assessment of Food Systems—a Review  

Microsoft Academic Search

The present review gives an overview of the use of fluorescence spectroscopy (i.e., conventional, excitation–emission matrix,\\u000a and synchronous fluorescence) for determining changes in food products and their quality during technological process and\\u000a storage. From the present review, it was shown that fluorescence spectroscopy is able to determine several properties (functional,\\u000a composition, nutritional) without the use of chemical reagents. This is

Romdhane Karoui; Christophe Blecker

2011-01-01

141

Single molecule fluorescence correlation spectroscopy of single apoptotic cells using a red-fluorescent caspase probe.  

PubMed

The detection of single molecules in single cells has enabled biochemical analyses to be conducted with high sensitivity and high temporal resolution. In this work, detection of apoptosis was studied by single molecule fluorescence correlation spectroscopy (FCS) in single living cells. Caspase activity was assayed using a new red fluorogenic probe that avoids the spectral overlap of green fluorescent probes and cell autofluorescence. This new probe, 2SBPO-Casp, was synthesized by coupling a water-soluble Nile Blue derivative (2SBPO) to an aspartic acid residue. Upon apoptosis induction and caspase activation, free 2SBPO dye is shown to accumulate inside the cell after probe cleavage. In previous work in our lab, single molecule fluorescence in single apoptotic cells was detected 45 min after induction using a rhodamine 110-based probe. However, significant statistical analysis was needed to exclude false positives. The use of 2SBPO-Casp overcomes the autofluorescence problem and offers a steady fluorescence signal. In our single molecule FCS measurements, Ramos cells were determined apoptotic on the basis of their correlation coefficient value (R(2)). Cells that contain an R(2) ? 0.65 were identified as highly correlated and therefore determined to be apoptotic. Single apoptotic cells identified in this manner were found as early as 30 min after induction and the number of apoptotic cells reached a peak value at the 3rd hour, which is consistent with other techniques. Using single molecule techniques and a new apoptosis probe, the temporal dynamics were elucidated with better sensitivity and resolution than in previous studies. PMID:22314869

Dong, Meicong; Martinez, Michelle M; Mayer, Michael F; Pappas, Dimitri

2012-07-01

142

Fluorescent Pluronic nanodots for in vivo two-photon imaging  

NASA Astrophysics Data System (ADS)

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.

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

2009-06-01

143

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

NASA Astrophysics Data System (ADS)

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

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

2011-09-01

144

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

NASA Astrophysics Data System (ADS)

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.

Zettergren, Eric William

2011-12-01

145

Noninvasive imaging in vivo with fluorescent proteins from centimeters to micrometers  

NASA Astrophysics Data System (ADS)

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.

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

2008-02-01

146

Measurement of absolute fluorescence quantum yield of oxazine 1 perchlorate with photoacoustic spectroscopy  

SciTech Connect

The fluorescence quantum efficiency of oxazine 1 perchlorate in 1,2-dichloroethane was measured with photoacoustic spectroscopy in which fluorescence quenching was used. The measurement shows that the relation between the fluorescence quantum efficiency and the concentration of solution of oxazine 1 in 1.2-dichloroethane is obviously different from that of some conventional dyes and solid fluorescence materials. A theoretical molecular energy transfer model was derived; it is in good agreement with the experimental results.

Zengfa Li; Hong Zhou; Guangyin Zhang

1986-12-01

147

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

NASA Astrophysics Data System (ADS)

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 data sets of glucose detection. Comparison studies were demonstrated between LS-SVR and PLS. LS-SVR demonstrated significant improvements in accuracy over PLS for glucose detection, especially when a global calibration model was required. The improvements imparted by LS-SVR open up the possibility of developing an accurate prediction algorithm for Raman-based glucose sensing applicable to a large human population. Overall, these studies show the high promise held by the Raman-based sensor for the challenge of optimal glycemic control.

Ma, Ke

148

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

NASA Astrophysics Data System (ADS)

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.

Salomatina, Elena V.; Pravdin, Alexander B.

2003-10-01

149

In vivo near-infrared fluorescence imaging of osteoblastic activity  

Microsoft Academic Search

In vertebrates, the development and integrity of the skeleton requires hydroxyapatite (HA) deposition by osteoblasts. HA deposition is also a marker of, or a participant in, processes as diverse as cancer and atherosclerosis. At present, sites of osteoblastic activity can only be imaged in vivo using ?-emitting radioisotopes. The scan times required are long, and the resultant radioscintigraphic images suffer

Atif Zaheer; Robert E. Lenkinski; Ashfaq Mahmood; Alun G. Jones; Lewis C. Cantley; John V. Frangioni

2001-01-01

150

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

NASA Astrophysics Data System (ADS)

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

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

2012-10-01

151

Fluorescence spectroscopy of fulvic acids from fen peatlands  

NASA Astrophysics Data System (ADS)

Intensive cultivation and agricultural use of peatlands lead to the degradation and mineralization of peat. Fulvic acids (FA) as the most mobile part of peat organic matter can be considered as an early indicator of its changes. One of the most sensitive and simple methods for studying the structural chemistry of humic substances is fluorescence spectroscopy. The objective of this study was to analyze comparatively the fluorescence properties of FA from low-moor peats of different genesis and decomposition degree with respect to the peculiarities of their chemical structure. FA were isolated from 4 peat samples collected from different fen peatlands of Belarus. Fluorescence spectra were obtained on water solutions of FA at a concentration of 50 mg/L after adjustment to pH=2, 6 and 13 on a MSL-4800 spectrofluorimeter (Perkin Elmer, USA.) at 20 ± 2 oC. Emission spectra were obtained using an excitation wavelength of 365 nm. Excitation spectra were recorded by varying the excitation wavelength from 260 to 520 nm and measuring the fluorescence emission at a fixed wavelength of 520 nm. Elemental composition of FA and optical density at 465 nm (D465) of FA solutions in 0.1 N NaOH were determined. Emission spectra of FA are characterized by a broad featureless band of the maximum wavelengths at ?=460-475 nm. Excitation spectra of FA have three peaks localized in different wavelength regions. The maximum wavelengths and intensities of the excitation peaks depend on the pH values. The highest intensities are observed at pH=6. FA exhibit a main excitation peak at ?=355-370 nm, a minor peak at ?=395-400 nm, and a weak band at ?=430-440 nm. At pH=2, all the peaks decrease in intensity. With increasing the pH to 13, the excitation maximum at ?=355-370 nm shifts from 10 to 20 nm towards longer wavelengths compared to acidic solutions. A general decrease in fluorescence intensity is observed, the intensity decline of the peak at ?=355-370 nm being more marked than of the peak at 395-400 nm. These three peaks can be attributed to three types of fluorophore structures with different degrees of conjugation. The excitation peak at ?=355-370 nm is due to the phenolic units conjugated with carbonyl groups. The peak at ?=395-400 nm can be assigned to the structural components with relatively higher degrees of conjugation (various substituted bicyclic aromatic and heteroaromatic structural units). The excitation band at ?=430-460 nm is suggested to be ascribed to the aromatic polyconjugation systems, consisting of aromatic structural units connected via various bridges which do not break off the polyconjugation. The ratios of fluorescence intensities at bands 355-370 nm and 395-400 nm (I355/I395), 355-370 nm and 430-440 nm (I355/I430), as well as 395-400 nm and 430-440 nm (I395/I430) were calculated. These ratios may indicate to a certain extent the contributions of each type of fluorophores to the total fluorescence of the humic molecules, and, therefore, the relative contributions of the corresponding structural units to the whole molecular structure of FA. The main fluorophores contributing to the fluorescence of FA are supposed to be phenolic units, aromatic and heteroaromatic moieties of a low degree of condensation, conjugated with functional groups and double bonds extending ?-electron systems. The fluorescence properties of peat FA were found to reflect the peculiarities of their chemical structure that depend mainly on geobotanical nature of the initial peat samples. FA from reed and alder peats with the highest D465 and the lowest H/C ratios reflecting the most developed systems of polyconjugation in their molecules are characterized with the longest emission wavelength and the lowest ratios of I355/I395, I355/I430, and I395/I430. These ratios suggest the most significant contribution of the aromatic polyconjugation systems to the fluorescence of these FA. The highest I355/I395, I355/I430, and I395/I430 ratios found for sedge-peat FA confirm that the aromatic polyconjugation systems in its molecules are less developed.

Maryganova, Victoria; Wojciech Szajdak, Lech

2010-05-01

152

Biocompatible fluorescent nanoparticles for in vivo stem cell tracking.  

PubMed

Efficient application of stem cells to the treatment of neurodegenerative diseases requires safe cell tracking to follow stem cell fate over time in the host environment after transplantation. In this work, for the first time, fluorescent and biocompatible methyl methacrylate (MMA)-based nanoparticles (fluoNPs) were synthesized through a free-radical co-polymerization process with a fluorescent macromonomer obtained by linking Rhodamine B and hydroxyethyl methacrylate. We demonstrate that the fluoNPs produced by polymerization of MMA-Rhodamine complexes (1) were efficient for the labeling and tracking of multipotent human amniotic fluid cells (hAFCs); (2) did not alter the main biological features of hAFCs (such as viability, cell growth and metabolic activity); (3) enabled us to determine the longitudinal bio-distribution of hAFCs in different brain areas after graft in the brain ventricles of healthy mice by a direct fluorescence-based technique. The reliability of our approach was furthermore confirmed by magnetic resonance imaging analyses, carried out by incubating hAFCs with both superparamagnetic iron oxide nanoparticles and fluoNPs. Our data suggest that these finely tunable and biocompatible fluoNPs can be exploited for the longitudinal tracking of stem cells. PMID:23690139

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

2013-06-21

153

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

NASA Astrophysics Data System (ADS)

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.

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

2013-03-01

154

In vivo mitochondrial oxygen tension measured by a delayed fluorescence lifetime technique.  

PubMed

Mitochondrial oxygen tension (mitoPO(2)) is a key parameter for cellular function, which is considered to be affected under various pathophysiological circumstances. Although many techniques for assessing in vivo oxygenation are available, no technique for measuring mitoPO(2) in vivo exists. Here we report in vivo measurement of mitoPO(2) and the recovery of mitoPO(2) histograms in rat liver by a novel optical technique under normal and pathological circumstances. The technique is based on oxygen-dependent quenching of the delayed fluorescence lifetime of protoporphyrin IX. Application of 5-aminolevulinic acid enhanced mitochondrial protoporphyrin IX levels and induced oxygen-dependent delayed fluorescence in various tissues, without affecting mitochondrial respiration. Using fluorescence microscopy, we demonstrate in isolated hepatocytes that the signal is of mitochondrial origin. The delayed fluorescence lifetime was calibrated in isolated hepatocytes and isolated perfused livers. Ultimately, the technique was applied to measure mitoPO(2) in rat liver in vivo. The results demonstrate mitoPO(2) values of approximately 30-40 mmHg. mitoPO(2) was highly sensitive to small changes in inspired oxygen concentration around atmospheric oxygen level. Ischemia-reperfusion interventions showed altered mitoPO(2) distribution, which flattened overall compared to baseline conditions. The reported technology is scalable from microscopic to macroscopic applications, and its reliance on an endogenous compound greatly enhances its potential field of applications. PMID:18641065

Mik, Egbert G; Johannes, Tanja; Zuurbier, Coert J; Heinen, Andre; Houben-Weerts, Judith H P M; Balestra, Gianmarco M; Stap, Jan; Beek, Johan F; Ince, Can

2008-10-01

155

In Vivo Biosensing Via Tissue Localizable Near Infrared Fluorescent Single Walled Carbon Nanotubes  

PubMed Central

Single-walled carbon nanotubes (SWNT) are particularly attractive for biomedical applications, because they exhibit a fluorescent signal in a spectral region where there is minimal interference from biological media. Although SWNT have been used as highly-sensitive detectors for various molecules, their use as in vivo biosensors requires the simultaneous optimization of various parameters, including biocompatibility, molecular recognition, high fluorescence quantum efficiency and signal transduction. Here we demonstrate that a polyethylene glycol ligated copolymer stabilizes near infrared fluorescent SWNT sensors in solution, enabling intravenous injection into mice and the selective detection of local nitric oxide (NO) concentration with a detection limit of 1 ?M. The half-life for liver retention is 4 hours, with sensors clearing the lungs within 2 hours after injection, thus avoiding a dominant route of in vivo nanotoxicology. After localization within the liver, it is possible to follow the transient inflammation using NO as a marker and signalling molecule. To this end, we also report a spatial-spectral imaging algorithm to deconvolute fluorescence intensity and spatial information from measurements. Finally, we show that alginate encapsulated SWNT can function as an implantable inflammation sensor for in vivo NO detection, with no intrinsic immune reactivity or other adverse response, for more than 400 days. These results open new avenues for the use of such nanosensors in vivo for biomedical applications. PMID:24185942

Iverson, Nicole M; Barone, Paul W; Shandell, Mia; Trudel, Laura J; Sen, Selda; Sen, Fatih; Ivanov, Vsevolod; Atolia, Esha; Farias, Edgardo; McNicholas, Thomas P; Reuel, Nigel; Parry, Nicola M. A.; Wogan, Gerald N

2014-01-01

156

In vivo fluorescence lifetime detection of an activatable probe in infarcted  

E-print Network

. Continuous wave in vivo detection of these protease-activatable fluorophores in the heart, however ionizing radiation.2 In addition, fluorescence imaging is ideally suited for the imaging of enzyme-activatable fluorophore-labeled probes, such as those activated by proteases.3 Traditional continuous wave (CW

Kumar, Anand T.N.

157

Quantification of fluorophore concentration in vivo using two simple fluorescence-based measurement techniques.  

PubMed

The effect of photodynamic therapy treatments depends on the concentration of photosensitizer at the treatment site; thus a simple method to quantify concentration is desirable. This study compares the concentration of a fluorophore and sensitizer, aluminum phthalocyanine tetrasulfonate (AlPcS4), measured by two simple fluorescence-based techniques in vivo to post mortem chemical extraction and fluorometric assay of those tissues: skin, muscle, fascia, liver, and kidney (cortex and medulla). Fluorescence was excited and detected by a single optical fiber, or by an instrument that measured the ratio of the fluorescence and excitation reflectance. The in vivo measurements were compared to calibration measurements made in tissue-simulating phantoms to estimate the tissue concentrations. Reasonable agreement was observed between the concentration estimates of the two instruments in the lighter colored tissues (skin, muscle, and fascia). The in vivo measurements also agreed with the chemical extractions at low (< 0.6 microg/g) tissue concentrations, but underestimated higher tissue concentrations. Measurements of fluorescence lifetime in vivo demonstrated that AlPcS4 retains its mono-exponential decay in skin, muscle, and fascia tissues with a lifetime similar to that measured in aqueous tissue-simulating phantoms. In liver and kidney an additional short lifetime component was evident. PMID:15910081

Diamond, Kevin R; Malysz, Pawel P; Hayward, Joseph E; Patterson, Michael S

2005-01-01

158

In Vivo Mitochondrial Oxygen Tension Measured by a Delayed Fluorescence Lifetime Technique  

PubMed Central

Mitochondrial oxygen tension (mitoPO2) is a key parameter for cellular function, which is considered to be affected under various pathophysiological circumstances. Although many techniques for assessing in vivo oxygenation are available, no technique for measuring mitoPO2 in vivo exists. Here we report in vivo measurement of mitoPO2 and the recovery of mitoPO2 histograms in rat liver by a novel optical technique under normal and pathological circumstances. The technique is based on oxygen-dependent quenching of the delayed fluorescence lifetime of protoporphyrin IX. Application of 5-aminolevulinic acid enhanced mitochondrial protoporphyrin IX levels and induced oxygen-dependent delayed fluorescence in various tissues, without affecting mitochondrial respiration. Using fluorescence microscopy, we demonstrate in isolated hepatocytes that the signal is of mitochondrial origin. The delayed fluorescence lifetime was calibrated in isolated hepatocytes and isolated perfused livers. Ultimately, the technique was applied to measure mitoPO2 in rat liver in vivo. The results demonstrate mitoPO2 values of ?30–40 mmHg. mitoPO2 was highly sensitive to small changes in inspired oxygen concentration around atmospheric oxygen level. Ischemia-reperfusion interventions showed altered mitoPO2 distribution, which flattened overall compared to baseline conditions. The reported technology is scalable from microscopic to macroscopic applications, and its reliance on an endogenous compound greatly enhances its potential field of applications. PMID:18641065

Mik, Egbert G.; Johannes, Tanja; Zuurbier, Coert J.; Heinen, Andre; Houben-Weerts, Judith H. P. M.; Balestra, Gianmarco M.; Stap, Jan; Beek, Johan F.; Ince, Can

2008-01-01

159

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

PubMed Central

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

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

2011-01-01

160

In vivo Raman spectroscopy for oral cancers diagnosis  

NASA Astrophysics Data System (ADS)

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.

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

2012-01-01

161

In vivo magnetic resonance spectroscopy of liver tumors and metastases  

PubMed Central

Primary liver cancer is the fifth most common malignancy in men and the eighth in women worldwide. The liver is also the second most common site for metastatic spread of cancer. To assist in the diagnosis of these liver lesions non-invasive advanced imaging techniques are desirable. Magnetic resonance (MR) is commonly used to identify anatomical lesions, but it is a very versatile technique and also can provide specific information on tumor pathophysiology and metabolism, in particular with the application of MR spectroscopy (MRS). This may include data on the type, grade and stage of tumors, and thus assist in further management of the disease. The purpose of this review is to summarize and discuss the available literature on proton, phosphorus and carbon-13-MRS as performed on primary liver tumors and metastases, with human applications as the main perspective. Upcoming MRS approaches with potential applications to liver tumors are also included. Since knowledge of some technical background is indispensable to understand the results, a basic introduction of MRS and some technical issues of MRS as applied to tumors and metastases in the liver are described as well. In vivo MR spectroscopy of tumors in a metabolically active organ such as the liver has been demonstrated to provide important information on tumor metabolism, but it also is challenging as compared to applications on some other tissues, in particular in humans, mostly because of its abdominal location where movement may be a disturbing factor. PMID:22215937

ter Voert, EGW; Heijmen, L; van Laarhoven, HWM; Heerschap, A

2011-01-01

162

Physical and biological limitations of in vivo multiphoton fluorescence microscopy  

NASA Astrophysics Data System (ADS)

In this paper we present experimental and theoretical study of limitations on deep imaging of biological tissue structure using multiphoton fluorescence microscopy (MFM), that are imposed by scattering of short NIR pulses in optically turbid medium. The time stretching of the laser pulse and its transversal widening limit the maximum imaging depth of MFM since the magnitude of fluorescence response from the medium is the power function of energy flux density in the excitation laser beam. Increasing the imaging depth by raising the laser power may result in phototoxic damage of biological objects. We have theoretically and experimentally studied temporal structure of collimated femtosecond laser pulse scattered in an optically turbid model medium with controlled concentration of micron-sized spherical beads. In parallel we investigated phototoxic effect on biological liquid at high-intense femtosecond NIR pulse irradiation for various expositions by structural analysis of dehydrated blood plasma droplet. The irradiation regimes were found with complete recovery of the droplet structure, as well as with its slight and severe stable modification.

Korytin, Alexey I.; Sergeeva, Ekaterina A.; Shcherbatyuk, Tatiana; Parfenova, Irina

2005-03-01

163

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

NASA Astrophysics Data System (ADS)

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.

Rujihan, Suparat; Damrongsak, Badin; Kittidachachan, Pattareeya

2013-06-01

164

Fluorescence Correlation Spectroscopy Evidence for Structural Heterogeneity in Ionic Liquids  

SciTech Connect

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.

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

2011-01-01

165

Fluorescence Correlation Spectroscopy of Single-wall Carbon Nanotubes  

NASA Astrophysics Data System (ADS)

We demonstrate the application of fluorescence correlation spectroscopy (FCS) for the characterization of semiconductor type single-wall carbon nanotubes (SWCNT) in aqueous solution at low concentrations. The technique relies on the intrinsic nanotube bandgap luminescence in the near infrared (NIR) range and does not require nanotube functionalization. The nanotubes used in this study have been dispersed in solution of sodium deoxycholate and length fractionated via centrifugation in an iodixanol density gradient. The (6,5) type nanotubes were resonantly excited by focusing a circularly-polarized 568 nm laser beam into a diffraction limited spot in solution and the luminescence intensity fluctuations were detected at wavelengths above 950 nm using a custom FCS setup with enhanced NIR transmission optics. Correlation functions were analyzed using a model of segmental dynamics of weakly bending rods, modified to account for an additional fast relaxation mode associated with nanotube rotational dynamics and its polarization dependent luminescence. The accessible sample concentration range and the optimum excitation power range were identified. Sample luminescent signal stability significantly exceeding a typical measurement time of few minutes was demonstrated.

Pristinski, Denis; Prabhu, Vivek M.; Fagan, Jeffrey A.

2010-03-01

166

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy  

PubMed Central

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, ?G°H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below. PMID:21559004

Schlamadinger, Diana E.; Kim, Judy E.

2011-01-01

167

Intraoperative metastases detection by laser-induced fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

The authors studied the ability of Laser Induced Fluorescence Spectroscopy (LIFS) for the intraoperative identification of metastases using a photosensitizing agent Photofrin IIr to enhance spectroscopic detection. A He-Cd laser source (442 nm) was used to produce low-power illumination of tissue via a hand-held 400 micrometers fiberoptic probe. Through the same fiber, reflected and emitted light was returned to an optical multi-channel analyzer (OMA III) for analysis. Spectroscopic signals were displayed on a screen for immediate examination. Lobund Wistar rats, inoculated with Pollard rat adenocarcinoma cells, were used as an animal model. Photofrin IIr was administered intraperitoneal 24 or 48 hours prior to surgical exploration in doses varying from 0.75-7.5 mg/kg. Metastases detection was performed during abdominal exploration directed to ipsilateral and contralateral inguinal, iliac, para-aortic and renal lymph nodes. Nineteen tissue samples, identified as abnormal by LIFS, were removed for histologic analysis; 11 of these samples were larger than 5mm and histologic examination revealed malignancy in all cases. While LIFS signals showed malignancy in 8 tissue samples with dimensions less than 5mm, histology confirmed this in only 3. However, serial histologic sections were not performed. From the initial results, it was concluded that LIFS detection of malignant tissue is feasible and enhanced by the addition of Photofrin IIr. LIFS may be a promising technique for the intraoperative detection of primary malignant and metastatic tissue.

Vari, Sandor G.; Papazoglou, Theodore G.; van der Veen, Maurits J.; Fishbein, Michael C.; Young, J. D.; Chandra, Mudjianto; Papaioannou, Thanassis; Beeder, Clain; Shi, Wei-Qiang; Grundfest, Warren S.

1991-06-01

168

Observation Volumes and ?-Factors in Two-Photon Fluorescence Fluctuation Spectroscopy  

PubMed Central

Fluorescence fluctuation spectroscopy has become an important measurement tool for investigating molecular dynamics, molecular interactions, and chemical kinetics in biological systems. Although the basic theory of fluctuation spectroscopy is well established, it is not widely recognized that saturation of the fluorescence excitation can dramatically alter the size and profile of the fluorescence observation volume from which fluorescence fluctuations are measured, even at relatively modest excitation levels. A precise model for these changes is needed for accurate analysis and interpretation of fluctuation spectroscopy data. We here introduce a combined analytical and computational approach to characterize the observation volume under saturating conditions and demonstrate how the variation in the volume is important in two-photon fluorescence correlation spectroscopy. We introduce a simple approach for analysis of fluorescence correlation spectroscopy data that can fully account for the effects of saturation, and demonstrate its success for characterizing the observed changes in both the amplitude and relaxation timescale of measured correlation curves. We also discuss how a quantitative model for the observed phenomena may be of broader importance in fluorescence fluctuation spectroscopy. PMID:15994890

Nagy, Attila; Wu, Jianrong; Berland, Keith M.

2005-01-01

169

Early detection of tumor masses by in vivo hematoporphyrin-mediated fluorescence imaging  

NASA Astrophysics Data System (ADS)

We investigated the capability of fluorescence reflectance imaging (FRI) for the early detection of surface tumors in mice. We used a hematoporphyrin (HP) compound (HP dichlorohydrate) as a red fluorescent marker and a low noise, high sensitivity, digital CCD camera for fluorescence imaging. In this preliminary study, highly malignant anaplastic human thyroid carcinoma cells were implanted subcutaneously in one mouse and their growth was monitored daily for 5 days by FRI. The selective HP uptake by the tumor tissues was successfully observed: we observed the fluorescence of tumor only 3 days after cancer cells injection, i.e. when the tumor mass was neither visible (to the naked eye) or palpable. These measurements indicate that FRI is a suitable technique to detect minute subcutaneous tumor masses. This FRI system will be coupled to a radionuclide imaging system based on a CdTe detector for in vivo multimodal imaging in mice.

Autiero, Maddalena; Celentano, Luigi; Cozzolino, Rosanna; Laccetti, Paolo; Marotta, Marcello; Mettivier, Giovanni; Cristina Montesi, Maria; Quarto, Maria; Riccio, Patrizia; Roberti, Giuseppe; Russo, Paolo

2007-02-01

170

Characterization of flow direction in microchannels and zebrafish blood vessels by scanning fluorescence correlation spectroscopy  

E-print Network

The investigation of flow profiles in microstructures and tissues by fluorescence correlation spectroscopy (FCS) has been a challenging topic in the past decade. Due to its inherent optical configuration, a circular focused ...

Pan, Xiaotao

171

Automation of the Laguerre Expansion Technique for Analysis of Time-resolved Fluorescence Spectroscopy Data  

E-print Network

Time-resolved fluorescence spectroscopy (TRFS) is a powerful analytical tool for quantifying the biochemical composition of organic and inorganic materials. The potentials of TRFS as nondestructive clinical tool for tissue diagnosis have been...

Dabir, Aditi Sandeep

2010-07-14

172

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

PubMed

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

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

2014-10-01

173

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

NASA Astrophysics Data System (ADS)

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.

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

2014-10-01

174

Micro-Raman Spectroscopy of Algae: Composition Analysis and Fluorescence Background Behavior  

E-print Network

ARTICLE Micro-Raman Spectroscopy of Algae: Composition Analysis and Fluorescence Background performed using Stokes Raman scattering for compositional analysis of algae. Two algal species, Chlorella while acquiring Raman signals from the algae. The time dependence of fluorescence background is char

175

Analysis of protein-based binding media found in paintings using laser induced fluorescence spectroscopy  

Microsoft Academic Search

Laser induced fluorescence (LIF) spectroscopy of intrinsic fluorophores from organic media found in paintings (casein, animal glue and egg proteins) provides novel non-invasive means of characterisation of general classes of media on the basis of fluorescence emission arising from the presence of certain amino acids and their degradation byproducts. Proteins from traditionally employed binding media include collagen, casein, albumin and

Austin Nevin; Sharon Cather; Demetrios Anglos; Costas Fotakis

2006-01-01

176

Single-Molecule Fluorescence Spectroscopy: New Probes of Protein Function and Dynamics  

NSDL National Science Digital Library

Single-molecule fluorescence methods provide new tools for the study of biological systems. Single-pair fluorescence resonance energy transfer has provided detailed information about dynamics and structure of the Ca2+-signaling protein calmodulin. Single-molecule polarization modulation spectroscopy has probed the mechanism by which calmodulin activates the plasma membrane Ca2+ pump

PhD Carey K. Johnson (University of Kansas Department of Chemistry)

2005-02-01

177

Fluorescence spectroscopy of single-walled carbon nanotubes synthesized from alcohol  

E-print Network

I&EC 221 Fluorescence spectroscopy of single-walled carbon nanotubes synthesized from alcohol fluorescence measurements of single-walled carbon nanotubes (SWNTs) catalytically synthesized from alcohol, and Characterization of Single-Walled Carbon Nanotubes (SWNT's) (sponsored by Advanced Materials & Nanotechnology

Maruyama, Shigeo

178

Probing the interactions of hemoglobin with antioxidant flavonoids via fluorescence spectroscopy and molecular modeling studies  

Microsoft Academic Search

Steady state and time resolved fluorescence spectroscopy, combined with molecular modeling computations, have been used to explore the interactions of two therapeutically important flavonoids, fisetin (3,7,3?,4?-OH-flavone) and 3-hydroxyflavone (3-HF), with normal human hemoglobin (HbA). Distinctive ‘two color’ fluorescence signatures and fairly high fluorescence anisotropy (r=0.12–0.28) of fisetin and 3-HF reveal their specific interactions with HbA. Binding constants estimated from the

Sudip Chaudhuri; Sandipan Chakraborty; Pradeep K. Sengupta

2011-01-01

179

Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei.  

PubMed

Cre-responsive dual-fluorescent alleles allow in situ marking of cell lineages or genetically modified cells. Here we report a dual-fluorescent allele, ROSA (nT-nG) , which directs nuclear accumulation of tdTomato in Cre-naïve lineages. Cre converts the allele to ROSA (nG) , which drives nuclear EGFP accumulation. Conditions were established for analyzing marked nuclei by flow cytometry on the basis of red-green fluorescence and ploidy, with a particular focus on liver nuclei. Hydrodynamic delivery of a Cre-expression plasmid was used to time-stamp arbitrary hepatocytes for lineage tracing. The distinct green fluorescence of nuclei from Cre-exposed lineages facilitated analyses of ploidy transitions within clones. To assess developmental transitions in liver nuclei, ROSA (nT-nG) was combined with the hepatocyte-specific AlbCre transgene, facilitating discrimination between hepatocyte and nonhepatocyte nuclei. Nuclei extracted from postnatal day 2 (P2) livers were 41 % green and 59 % red and reached a stable level of 84 % green by P22. Until P20, green nuclei were >98 % diploid (2N); at P40 they were ~56 % 2N, 43 % 4N, and <1 % 8N; and by P70 they reached a stable distribution of ~46 % 2N, 45 % 4N, and 9 % 8N. In conclusion, ROSA (nT-nG) will facilitate in vivo and ex vivo studies on liver and will likely be valuable for studies on tissues like muscle, kidney, or brain in which cells are refractory to whole-cell flow cytometry, or like trophectoderm derivatives or cancers in which cells undergo ploidy transitions. PMID:24022199

Prigge, Justin R; Wiley, James A; Talago, Emily A; Young, Elise M; Johns, Laura L; Kundert, Jean A; Sonsteng, Katherine M; Halford, William P; Capecchi, Mario R; Schmidt, Edward E

2013-09-11

180

Fluorescent liposomes for intravital staining of Kupffer cells to aid in vivo microscopy in rats.  

PubMed

The potential of newly formulated fluorescent-labeled liposomes for the intravital staining of Kupffer cells was evaluated in rats. Fluorescently labeled phosphatidylcholine (PC) was incorporated into liposomes consisting of PC and phosphatidylserine. After intravenous injection, Kupffer cells in the rat liver were intravitally stained and were clearly delineated under the fluorescence image of both confocal laser scanning microscopy and in vivo microscopy. Specificity of the staining was confirmed by immunohistochemistry using the anti-rat macrophage antibody Ki-M2R, which suggested that the liposomes were selectively entrapped by the hepatic reticuloendothelial system. A time-course study revealed that the suitable observation window was between 16 and 24 h after the injection. Phagocytic activity of Kupffer cells after the administration of liposomes was examined by measuring the amount of hepatic uptake of intravenously administered fluorescent microspheres; no detrimental influence of the liposomes on the phagocytic activity was observed. Additionally, no histopathologic changes were found in the livers from liposome-treated rats. Therefore, the fluorescent-labeled liposomes appear to be a useful research tool for labeling Kupffer cells for in vivo microscopic observation of the liver. PMID:17805433

Watanabe, R; Munemasa, T; Matsumura, M; Fujimaki, M

2007-06-01

181

Development of an Immunologically Tolerated Combination of Fluorescent Proteins for In vivo Two-photon Imaging  

PubMed Central

Combinations of fluorescent proteins (FPs) are routinely used for multi-parameter in vivo imaging experiments to visualize tagged proteins or cell populations of interest. Studies involving FPs are often limited by spectral overlap, toxicity, relative quantum efficiency, and the potential for immunological rejection upon transfer into a non-tolerant recipient. Here we evaluate the immunologic visibility of several commonly used FPs by the murine immune system and identify a spectrally compatible, immunologically tolerated combination of FPs well suited for in vivo two-photon imaging. PMID:25322934

Gossa, Selamawit; Nayak, Debasis; Zinselmeyer, Bernd H.; McGavern, Dorian B.

2014-01-01

182

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

NASA Astrophysics Data System (ADS)

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

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

2015-01-01

183

In vivo impedance spectroscopy of deep brain stimulation electrodes  

PubMed Central

Deep brain stimulation (DBS) represents a powerful clinical technology, but a systematic characterization of the electrical interactions between the electrode and the brain are lacking. The goal of this study was to examine the in vivo changes in DBS electrode impedance that occur after implantation and during clinically-relevant stimulation. Clinical DBS devices typically apply high-frequency voltage-controlled stimulation, and as a result the injected current is directly regulated by the impedance of the electrode-tissue interface. We monitored the impedance of scaled-down clinical DBS electrodes implanted in the thalamus and subthalamic nucleus of a rhesus macaque using electrode impedance spectroscopy (EIS) measurements ranging from 0.5 Hz to 10 kHz. To further characterize our measurements, equivalent circuit models of the electrode-tissue interface were used to quantify the role of various interface components in producing the observed electrode impedance. Following implantation, DBS electrode impedance increased and a semicircular arc was observed in the high frequency range of the EIS measurements, commonly referred to as the tissue component of the impedance. Clinically-relevant stimulation produced a rapid decrease in electrode impedance with extensive changes in the tissue component. These post-operative and stimulation-induced changes in impedance could play an important role in the observed functional effects of voltage-controlled DBS and should be considered during clinical stimulation parameter selection and chronic animal research studies. PMID:19494421

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

2010-01-01

184

Autofluorescence and Photofrin-induced fluorescence imaging and spectroscopy in an animal model of oral cancer.  

PubMed

Developments in the fluorescence detection of cancer aim either to distinguish tissue autofluorescence from that of injected fluorophores or to exploit differences in autofluorescent spectra of normal versus transforming, premalignant and malignant tissue. This study evaluates the utility of autofluorescence and Photofrin-induced fluorescence imaging and spectroscopy to distinguish tissue transformation associated with early malignant change in the oral cavity. The model of tissue transformation used was that induced by the carcinogen DMBA in the hamster buccal cheek pouch. Fluorescence spectra were obtained using a high-sensitivity fiber optic spectrometer, while imaging was performed using a Multispectral Fluorescence Guidance (MFG) system designed for use in intraoperative fluorescence imaging during photodynamic therapy. The results demonstrate that Photofrin fluorescence can be used to predict the pathologic state of tissue, the fluorescence intensity being directly proportional to the degree of malignant transformation. Autofluorescence detection measured two parameters that are altered by transformation stage: the red/green fluorescence ratio and the total fluorescence intensity. The most striking feature was the change in the latter in malignant tissue. The MFG imaging device performed as well as spectroscopy: the sensitivity and specificity for the imaging system were 65% and 90% for autofluorescence and 87% and 85% with Photofrin. This indicates that either the autofluorescence intensity index of the tissue or the Photofrin-induced fluorescence may provide a good parameter for the "first approximation" characterization of the tissue. PMID:25049151

Mang, Thomas; Kost, James; Sullivan, Maureen; Wilson, Brian C

2006-09-01

185

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

PubMed Central

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

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

2014-01-01

186

[Rapid identification of potato cultivars using NIR-excited fluorescence and Raman spectroscopy].  

PubMed

Potato is one of the most important food in the world. Rapid and noninvasive identification of potato cultivars plays a important role in the better use of varieties. In this study, The identification ability of optical spectroscopy techniques, including near-infrared (NIR) Raman spectroscopy and NIR fluorescence spectroscopy, for invasive detection of potato cultivars was evaluated. A rapid NIR Raman spectroscopy system was applied to measure the composite Raman and NIR fluorescence spectroscopy of 3 different species of potatoes (98 samples in total) under 785 nm laser light excitation. Then pure Raman and NIR fluorescence spectroscopy were abstracted from the composite spectroscopy, respectively. At last, the partial least squares-discriminant analysis (PLS-DA) was utilized to analyze and classify Raman spectra of 3 different types of potatoes. All the samples were divided into two sets at random: the calibration set (74samples) and prediction set (24 samples), the model was validated using a leave-one-out, cross-validation method. The results showed that both the NIR-excited fluorescence spectra and pure Raman spectra could be used to identify three cultivars of potatoes. The fluorescence spectrum could distinguish the Favorita variety well (sensitivity: 1, specificity: 0.86 and accuracy: 0.92), but the result for Diamant (sensitivity: 0.75, specificity: 0.75 and accuracy: 0. 75) and Granola (sensitivity: 0.16, specificity: 0.89 and accuracy: 0.71) cultivars identification were a bit poorer. We demonstrated that Raman spectroscopy uncovered the main biochemical compositions contained in potato species, and provided a better classification sensitivity, specificity and accuracy (sensitivity: 1, specificity: 1 and accuracy: 1 for all 3 potato cultivars identification) among the three types of potatoes as compared to fluorescence spectroscopy. PMID:25208390

Dai, Fen; Bergholt, Mads Sylvest; Benjamin, Arnold Julian Vinoj; Hong, Tian-Sheng; Zhiwei, Huang

2014-03-01

187

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

NASA Astrophysics Data System (ADS)

Fluorescence Correlation Spectroscopy (FCS) is widely used to quantify reaction rates and concentrations of molecules in vitro and in vivo. We recently reported Fluorescence Triple Correlation Spectroscopy (F3CS), which correlates three signals together instead of two. F3CS can analyze the stoichiometries of complex mixtures and detect irreversible processes by identifying time-reversal asymmetries. Here we report the computational developments that were required for the realization of F3CS and present the results as the Triple Correlation Toolbox suite of programs. Triple Correlation Toolbox is a complete data analysis pipeline capable of acquiring, correlating and fitting large data sets. Each segment of the pipeline handles error estimates for accurate error-weighted global fitting. Data acquisition was accelerated with a combination of off-the-shelf counter-timer chips and vectorized operations on 128-bit registers. This allows desktop computers with inexpensive data acquisition cards to acquire hours of multiple-channel data with sub-microsecond time resolution. Off-line correlation integrals were implemented as a two delay time multiple-tau scheme that scales efficiently with multiple processors and provides an unprecedented view of linked dynamics. Global fitting routines are provided to fit FCS and F3CS data to models containing up to ten species. Triple Correlation Toolbox is a complete package that enables F3CS to be performed on existing microscopes. Catalogue identifier: AEOP_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEOP_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 50189 No. of bytes in distributed program, including test data, etc.: 6135283 Distribution format: tar.gz Programming language: C/Assembly. Computer: Any with GCC and library support. Operating system: Linux and OS X (data acq. for Linux only due to library availability), not tested on Windows. RAM: ?512 MB. Classification: 16.4. External routines: NIDAQmx (National Instruments), Gnu Scientific Library, GTK+, PLplot (optional) Nature of problem: Fluorescence Triple Correlation Spectroscopy required three things: data acquisition at faster speeds than were possible without expensive custom hardware, triple-correlation routines that could process 1/2 TB data sets rapidly, and fitting routines capable of handling several to a hundred fit parameters and 14,000 + data points, each with error estimates. Solution method: A novel data acquisition concept mixed signal processing with off-the-shelf hardware and data-parallel processing using 128-bit registers found in desktop CPUs. Correlation algorithms used fractal data structures and multithreading to reduce data analysis times. Global fitting was implemented with robust minimization routines and provides feedback that allows the user to critically inspect initial guesses and fits. Restrictions: Data acquisition only requires a National Instruments data acquisition card (it was tested on Linux using card PCIe-6251) and a simple home-built circuit. Unusual features: Hand-coded ×86-64 assembly for data acquisition loops (platform-independent C code also provided). Additional comments: A complete collection of tools to perform Fluorescence Triple Correlation Spectroscopy-from data acquisition to two-tau correlation of large data sets, to model fitting. Running time: 1-5 h of data analysis per hour of data collected. Varies depending on data-acquisition length, time resolution, data density and number of cores used for correlation integrals.

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

2013-04-01

188

Reliable Assessment and Quantification of the Fluorescence-Labeled Antisense Oligonucleotides In Vivo  

PubMed Central

The availability of fluorescent dyes and the advances in the optical systems for in vivo imaging have stimulated an increasing interest in developing new methodologies to study and quantify the biodistribution of labeled agents. However, despite these great achievements, we are facing significant challenges in determining if the observed fluorescence does correspond to the quantity of the dye in the tissues. In fact, although the far-red and near-infrared lights can propagate through several centimetres of tissue, they diffuse within a few millimetres as consequence of the elastic scattering of photons. In addition, when dye-labeled oligonucleotides form stable complex with cationic carriers, a large change in the fluorescence intensity of the dye is observed. Therefore, the measured fluorescence intensity is altered by the tissue heterogeneity and by the fluctuation of dye intensity. Hence, in this study a quantification strategy for fluorescence-labeled oligonucleotides was developed to solve these disadvantageous effects. Our results proved that upon efficient homogenization and dilution with chaotropic agents, such as guanidinium thiocyanate, it is possible to achieve a complete fluorescence intensity recovery. Furthermore, we demonstrated that this method has the advantage of good sensitivity and reproducibility, as well as easy handling of the tissue samples. PMID:24967340

Chiara Munisso, Maria; Yamaoka, Tetsuji

2014-01-01

189

In vivo imaging of tumors with protease-activated near-infrared fluorescent probes  

Microsoft Academic Search

We have developed a method to image tumor-associated lysosomal protease activity in a xenograft mouse model in vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous injection, the NIRF probe carrier accumulated in solid tumors due to its long circulation time and leakage

Ching-Hsuan Tung; Umar Mahmood; Alexei Bogdanov; Ralph Weissleder

1999-01-01

190

Fluorescence spectroscopy for estimation of anticancer drug sonodestruction in vitro  

NASA Astrophysics Data System (ADS)

The effect of ultrasound exposure on bleomycin fluorescence and pharmacological properties is studied. Bleomycin was treated by ultrasound for 7 min. Bleomycin fluorescence was measured during ultrasound exposure by means of fiber-optic spectrometry. Cell colony test was used to evaluate blemycin cytotoxity before and after ultrasound exposure.

Lihachev, A.; Jakovels, D.; Ferulova, I.; Spigulis, J.; Tamosiunas, M.; Satkauskas, S.; Lo, C. W.; Chen, W. S.

2013-11-01

191

Time-resolved fluorescence spectroscopy for application to PAH contaminated areas and hydrogeological research  

SciTech Connect

A mobile fiber-optical sensor system for the on-line and in situ detection of aquatic fluorophores has been developed. By the use of time-resolved laser-induced fluorescence spectroscopy the determination of contaminants i.e. polycyclic aromatic hydrocarbons (PAH) or fluorescence tracers in various environments is possible. In both cases attempts to detect these substances in water by means of fluorescence spectroscopy are complicated by the low concentrations and the overlapping and featureless fluorescence spectroscopy are complicated by the low concentrations and the overlapping and featureless fluorescence spectra in combination with background fluorescence caused by further compounds e.g. humic material. By collecting the fluorescence decay time as an additional independent dimension, the analytical information is significantly increased, and to certain extent the determination of the desired analyte in complex natural matrices is possible. At a first application, the detection of pyrene (PYR) in real samples from a contaminated former coking plant site has been realized. The system is also best suitable for hydrogeological research. Here applications spread from the investigation of the fluorescence tracer migration in an artificial aquifer system to the determination of hydrogeological parameters at a domestic waste disposal.

Kotzick, R.; Haaszio, S.; Niessner, R. [Technical Univ. of Munich (Germany). Institute for Hydrochemistry

1995-12-31

192

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

PubMed Central

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

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

2010-01-01

193

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

PubMed

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

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

2014-01-01

194

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles  

PubMed Central

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

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

2014-01-01

195

Exploiting post-transcriptional regulation to probe RNA structures in vivo via fluorescence.  

PubMed

While RNA structures have been extensively characterized in vitro, very few techniques exist to probe RNA structures inside cells. Here, we have exploited mechanisms of post-transcriptional regulation to synthesize fluorescence-based probes that assay RNA structures in vivo. Our probing system involves the co-expression of two constructs: (i) a target RNA and (ii) a reporter containing a probe complementary to a region in the target RNA attached to an RBS-sequestering hairpin and fused to a sequence encoding the green fluorescent protein (GFP). When a region of the target RNA is accessible, the area can interact with its complementary probe, resulting in fluorescence. By using this system, we observed varied patterns of structural accessibility along the length of the Tetrahymena group I intron. We performed in vivo DMS footprinting which, along with previous footprinting studies, helped to explain our probing results. Additionally, this novel approach represents a valuable tool to differentiate between RNA variants and to detect structural changes caused by subtle mutations. Our results capture some differences from traditional footprinting assays that could suggest that probing in vivo via oligonucleotide hybridization facilitates the detection of folding intermediates. Importantly, our data indicate that intracellular oligonucleotide probing can be a powerful complement to existing RNA structural probing methods. PMID:25416800

Sowa, Steven W; Vazquez-Anderson, Jorge; Clark, Chelsea A; De La Peña, Ricardo; Dunn, Kaitlin; Fung, Emily K; Khoury, Mark J; Contreras, Lydia M

2015-01-30

196

A fluorescence spectroscopy study of traditional Chinese medicine Angelica  

NASA Astrophysics Data System (ADS)

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.

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

2013-10-01

197

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

NASA Astrophysics Data System (ADS)

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

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

2007-02-01

198

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

NASA Astrophysics Data System (ADS)

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

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

2012-02-01

199

In vivo self-bio-imaging of tumors through in situ biosynthesized fluorescent gold nanoclusters  

PubMed Central

Fluorescence imaging in vivo allows non-invasive tumor diagnostic thus permitting a direct monitoring of cancer therapies progresses. It is established herein that fluorescent gold nanoclusters are spontaneously biosynthesized by cancerous cell (i.e., HepG2, human hepatocarcinoma cell line; K562, leukemia cell line) incubated with micromolar chloroauric acid solutions, a biocompatible molecular Au(III) species. Gold nanoparticles form by Au(III) reduction inside cells cytoplasms and ultimately concentrate around their nucleoli, thus affording precise cell imaging. Importantly, this does not occur in non-cancerous cells, as evidenced with human embryo liver cells (L02) used as controls. This dichotomy is exploited for a new strategy for in vivo self-bio-imaging of tumors. Subcutaneous injections of millimolar chloroauric acid solution near xenograft tumors of the nude mouse model of hepatocellular carcinoma or chronic myeloid leukemia led to efficient biosynthesis of fluorescent gold nanoclusters without significant dissemination to the surrounding normal tissues, hence allowing specific fluorescent self-bio-marking of the tumors. PMID:23362457

Wang, Jianling; Zhang, Gen; Li, Qiwei; Jiang, Hui; Liu, Chongyang; Amatore, Christian; Wang, Xuemei

2013-01-01

200

In vivo self-bio-imaging of tumors through in situ biosynthesized fluorescent gold nanoclusters  

NASA Astrophysics Data System (ADS)

Fluorescence imaging in vivo allows non-invasive tumor diagnostic thus permitting a direct monitoring of cancer therapies progresses. It is established herein that fluorescent gold nanoclusters are spontaneously biosynthesized by cancerous cell (i.e., HepG2, human hepatocarcinoma cell line; K562, leukemia cell line) incubated with micromolar chloroauric acid solutions, a biocompatible molecular Au(III) species. Gold nanoparticles form by Au(III) reduction inside cells cytoplasms and ultimately concentrate around their nucleoli, thus affording precise cell imaging. Importantly, this does not occur in non-cancerous cells, as evidenced with human embryo liver cells (L02) used as controls. This dichotomy is exploited for a new strategy for in vivo self-bio-imaging of tumors. Subcutaneous injections of millimolar chloroauric acid solution near xenograft tumors of the nude mouse model of hepatocellular carcinoma or chronic myeloid leukemia led to efficient biosynthesis of fluorescent gold nanoclusters without significant dissemination to the surrounding normal tissues, hence allowing specific fluorescent self-bio-marking of the tumors.

Wang, Jianling; Zhang, Gen; Li, Qiwei; Jiang, Hui; Liu, Chongyang; Amatore, Christian; Wang, Xuemei

2013-01-01

201

MR and fluorescence imaging of doxorubicin loaded nanoparticles using a novel in vivo model  

PubMed Central

We report here the in vivo combined-modality imaging of multifunctional drug delivery nanoparticles. These dextran core-based stealth liposomal nanoparticles (nanosomes) contained doxorubicin, iron oxide for MRI contrast, and Bodipy for fluorescence. The particles were long-lived in vivo due to surface decoration with polyethylene glycol (PEG) and the incorporation of acetylated lipids which were UV cross-linked for physical stability. We developed a rodent dorsal skinfold window chamber which facilitated both MRI and non-destructive optical imaging of nanoparticle accumulation in the same tumors. Chamber tumors were genetically labeled with DsRed-2 that enabled the MR images, the red fluorescence of the tumor, and the blue fluorescence of the nanoparticles to be co-localized. The nanoparticle design and MR imaging developed with the window chamber were then extended to orthotopic pancreatic tumors expressing DsRed-2. The tumors were MR imaged using iron oxide-dextran liposomes and by fluorescence to demonstrate the deep imaging capability of these nanoparticles. PMID:20599526

Erten, Ahmet; Wrasidlo, Wolf; Scadeng, Miriam; Esener, Sadik; Hoffman, Robert; Bouvet, Michael; Makale, Milan

2010-01-01

202

Toward ultra-stable fluorescent dyes for single-molecule spectroscopy  

NASA Astrophysics Data System (ADS)

The wide-spread use of fluorescent dyes in molecular diagnostics and fluorescence microscopy together with new developments such as single-molecule fluorescence spectroscopy provide researchers from various disciplines with an ever expanding toolbox. Single-molecule fluorescence spectroscopy relies to a large extent on extraordinary bright and photostable organic fluorescent dyes such as rhodamine- or cyanine- derivatives. While in the last decade singlemolecule equipment and methodology have significantly advanced and in some cases reached theoretical limits (e.g. detectors approaching unity quantum yields), instable emission ("blinking") and photobleaching become more and more the bottleneck of further development and spreading of single-molecule fluorescence studies. In recent years, agents and recipes have been developed to increase the photostability of conventional fluorescent dyes. Here, we investigate some of these strategies at the single-molecule level. In particular, we focus on the dye selection criteria for multi-color applications. We investigate fluorescent dyes from the rhodamine, carborhodamine, cyanine, and oxazine family and show that within one dye class the photophysical properties are very similar but that dyes from different classes show strikingly different properties. These findings facilitate dye selection and provide improved chemical environment for demanding fluorescence microscopic applications.

Kasper, Robert; Heilemann, Mike; Tinnefeld, Philip; Sauer, Markus

2007-07-01

203

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy  

PubMed Central

The self-assembly of bacterial 30S ribosomes involves a large number of RNA folding and RNA-protein binding steps. The sequence of steps determines the overall assembly mechanism and the structure of the mechanism has ramifications for the robustness of biogenesis and resilience against kinetic traps. Thermodynamic interdependencies of protein binding inferred from omission-reconstitution experiments are thought to preclude certain assembly pathways and thus enforce ordered assembly, but this concept is at odds with kinetic data suggesting a more parallel assembly landscape. A major challenge is deconvolution of the statistical distribution of intermediates that are populated during assembly at high concentrations approaching in vivo assembly conditions. To specifically resolve the intermediates formed by binding of three ribosomal proteins to the full length 16S rRNA, we introduce Fluorescence Triple-Correlation Spectroscopy (F3CS). F3CS identifies specific ternary complexes by detecting coincident fluctuations in three-color fluorescence data. Triple correlation integrals quantify concentrations and diffusion kinetics of triply labeled species, and F3CS data can be fit alongside auto-correlation and cross-correlation data to quantify the populations of 10 specific ribosome assembly intermediates. The distribution of intermediates generated by binding three ribosomal proteins to the entire native 16S rRNA included significant populations of species that were not previously thought to be thermodynamically accessible, questioning the current interpretation of the classic omission-reconstitution experiments. F3CS is a general approach for analyzing assembly and function of macromolecular complexes, especially those too large for traditional biophysical methods. PMID:22869699

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

2012-01-01

204

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

PubMed Central

We report the development of a compact point-detection fluorescence spectroscopy system and two data analysis methods to quantify the intrinsic fluorescence redox ratio and diagnose brain cancer in an orthotopic brain tumor rat model. Our system employs one compact cw diode laser (407 nm) to excite two primary endogenous fluorophores, reduced nicotinamide adenine dinucleotide, and flavin adenine dinucleotide. The spectra were first analyzed using a spectral filtering modulation method developed previously to derive the intrinsic fluorescence redox ratio, which has the advantages of insensitivty to optical coupling and rapid data acquisition and analysis. This method represents a convenient and rapid alternative for achieving intrinsic fluorescence-based redox measurements as compared to those complicated model-based methods. It is worth noting that the method can also extract total hemoglobin concentration at the same time but only if the emission path length of fluorescence light, which depends on the illumination and collection geometry of the optical probe, is long enough so that the effect of absorption on fluorescence intensity due to hemoglobin is significant. Then a multivariate method was used to statistically classify normal tissues and tumors. Although the first method offers quantitative tissue metabolism information, the second method provides high overall classification accuracy. The two methods provide complementary capabilities for understanding cancer development and noninvasively diagnosing brain cancer. The results of our study suggest that this portable system can be potentially used to demarcate the elusive boundary between a brain tumor and the surrounding normal tissue during surgical resection. PMID:21456877

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

2011-01-01

205

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

NASA Astrophysics Data System (ADS)

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

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

2011-03-01

206

Quantitative Mueller matrix fluorescence spectroscopy for precancer detection.  

PubMed

Quantitative fluorescence spectroscopic Mueller matrix measurements from the connective tissue regions of human cervical tissue reveal intriguing fluorescence diattenuation and polarizance effects. Interestingly, the estimated fluorescence linear diattenuation and polarizance parameters were considerably reduced in the precancerous tissues as compared to the normal ones. These polarimetry effects of the autofluorescence were found to originate from anisotropically organized collagen molecular structures present in the connective tissues. Consequently, the reduction of the magnitude of these polarimetric parameters at higher grades of precancer was attributed to the loss of anisotropic organization of collagen, which was also confirmed by control experiments. These results indicate that fluorescence spectral diattenuation and polarizance parameters may serve as potentially useful diagnostic metrics. PMID:24562117

Jagtap, J; Chandel, S; Das, N; Soni, J; Chatterjee, S; Pradhan, A; Ghosh, N

2014-01-15

207

Ultrafast Fluorescence Spectroscopy via Upconversion: Applications to Biophysics  

PubMed Central

This chapter reviews basic concepts of nonlinear fluorescence upconversion, a technique whose temporal resolution is essentially limited only by the pulse width of the ultrafast laser. Design aspects for upconversion spectrophotofluorometers are discussed, and a recently developed system is described. We discuss applications in biophysics, particularly the measurement of time-resolved fluorescence spectra of proteins (with subpicosecond time resolution). Application of this technique to biophysical problems such as dynamics of tryptophan, peptides, proteins, and nucleic acids is reviewed. PMID:19152860

Xu, Jianhua; Knutson, Jay R.

2012-01-01

208

Fluorescence spectroscopy of anisole at elevated temperatures and pressures  

NASA Astrophysics Data System (ADS)

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

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

2014-06-01

209

Two-dimensional fluorescence correlation spectroscopy IV: Resolution of fluorescence of tryptophan residues in alcohol dehydrogenase and lysozyme  

NASA Astrophysics Data System (ADS)

Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve the fluorescence spectra of two tryptophan (Trp) residues in alcohol dehydrogenase and lysozyme. In each protein, one Trp residue is buried in a hydrophobic domain of the protein matrix and the other Trp residue is located at a hydrophilic domain close to the protein-water interface. Fluorescence quenching by iodide ion, a hydrophilic quencher, was employed as a perturbation to induce the intensity change in the spectra. The Trp residue which is located at the hydrophilic domain is effectively quenched by the quencher, while the Trp residue located at the hydrophobic domain is protected from the quenching. Therefore, the fluorescence of these two Trp residues have a different sensitivity to the quenching, showing a different response to the concentration of the quencher. Fluorescence spectra of the two Trp residues in alcohol dehydrogenase, which are heavily overlapped in conventional one-dimensional spectra, have been successfully resolved by the 2D correlation technique. From the asynchronous correlation map, it was revealed that the quenching of Trp located at the hydrophobic part was brought about after that of Trp located at the hydrophilic part. In contrast, the fluorescence spectra of the two Trp residues could not be resolved after the alcohol dehydrogenase was denatured with guanidine hydrochloride. These results are consistent with the well-known structure of alcohol dehydrogenase. Furthermore, it was elucidated that the present 2D analysis is not interfered by Raman bands of the solvent, which sometimes bring difficulty into the conventional fluorescence analysis. Fluorescence spectra of the Trp residues in lysozyme could not be resolved by the 2D correlation technique. The differences between the two proteins are attributed to the fact that the Trp residue in the hydrophobic site of lysozyme is not sufficiently protected from the quenching.

Fukuma, Hiroaki; Nakashima, Kenichi; Ozaki, Yukihiro; Noda, Isao

2006-11-01

210

Probing the extracellular diffusion of antibodies in brain using in vivo integrative optical imaging and ex vivo fluorescence imaging.  

PubMed

Antibody-based therapeutics exhibit great promise in the treatment of central nervous system (CNS) disorders given their unique customizable properties. Although several clinical trials have evaluated therapeutic antibodies for treatment of CNS disorders, success to date has likely been limited in part due to complex issues associated with antibody delivery to the brain and antibody distribution within the CNS compartment. Major obstacles to effective CNS delivery of full length immunoglobulin G (IgG) antibodies include transport across the blood-brain and blood-cerebrospinal fluid barriers. IgG diffusion within brain extracellular space (ECS) may also play a role in limiting central antibody distribution; however, IgG transport in brain ECS has not yet been explored using established in vivo methods. Here, we used real-time integrative optical imaging to measure the diffusion properties of fluorescently labeled, non-targeted IgG after pressure injection in both free solution and in adult rat neocortex in vivo, revealing IgG diffusion in free medium is ~10-fold greater than in brain ECS. The pronounced hindered diffusion of IgG in brain ECS is likely due to a number of general factors associated with the brain microenvironment (e.g. ECS volume fraction and geometry/width) but also molecule-specific factors such as IgG size, shape, charge and specific binding interactions with ECS components. Co-injection of labeled IgG with an excess of unlabeled Fc fragment yielded a small yet significant increase in the IgG effective diffusion coefficient in brain, suggesting that binding between the IgG Fc domain and endogenous Fc-specific receptors may contribute to the hindered mobility of IgG in brain ECS. Importantly, local IgG diffusion coefficients from integrative optical imaging were similar to those obtained from ex vivo fluorescence imaging of transport gradients across the pial brain surface following controlled intracisternal infusions in anesthetized animals. Taken together, our results confirm the importance of diffusive transport in the generation of whole brain distribution profiles after infusion into the cerebrospinal fluid, although convective transport in the perivascular spaces of cerebral blood vessels was also evident. Our quantitative in vivo diffusion measurements may allow for more accurate prediction of IgG brain distribution after intrathecal or intracerebroventricular infusion into the cerebrospinal fluid across different species, facilitating the evaluation of both new and existing strategies for CNS immunotherapy. PMID:25449807

Wolak, Daniel J; Pizzo, Michelle E; Thorne, Robert G

2015-01-10

211

In vivo targeting of colonic dysplasia on fluorescence endoscopy with near-infrared octapeptide  

PubMed Central

Objective To demonstrate a near-infrared peptide that is highly specific for colonic adenomas on fluorescence endoscopy in vivo. Design A 3 mm diameter endoscope was adapted to deliver 671 nm illumination and collect NIR fluorescence (696–736 nm). Target (QPIHPNNM) and control (YTTNKH) peptides were labeled with Cy5.5, a near-infrared dye, and characterized by mass spectra. The peptides were topically administered separately (100 µM) through the endoscope’s instrument channel into the distal colon of CPC;Apc mice, genetically-engineered to spontaneously develop adenomas. After 5 minutes for incubation, the unbound peptides were rinsed off, and images were collected at a rate of 10 per second. Regions-of-interest were identified around the adenoma and adjacent normal-appearing mucosa on white light. Intensity measurements were made from these same regions on fluorescence, and the target-to-background ratio (TBR) was calculated. Results We achieved an image resolution of 9.8 µm and field-of-view of 3.6 mm at a distance of 2.5 mm between the distal end of the instrument and the tissue surface. On mass spectra, the experimental mass-to-charge ratio for the Cy5.5-labeled target and control peptides agreed with expected values. The near-infrared fluorescence images of adenomas revealed individual dysplastic crypts with distorted morphology. By comparison, only amorphous surface features could be visualized from reflected near-infrared light. The average TBR for adenomas was found to be 3.42±1.30 and 1.88±0.38 for the target and control peptides, respectively, p=0.007. Conclusion We demonstrate a near-infrared peptide that is highly specific for colonic adenomas on fluorescence endoscopy in vivo and achieved sub-cellular resolution images. PMID:22427239

Liu, Zhongyao; Miller, Sharon J.; Joshi, Bishnu P.; Wang, Thomas D.

2012-01-01

212

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

PubMed

Single-molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as Förster fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) are limited by diffraction to the nanomolar concentration range, far below the physiological micromolar concentration range where most biological reaction occur. To breach the diffraction limit, zero-mode waveguides (ZMW) and plasmonic antennas exploit the surface plasmon resonances to confine and enhance light down to the nanometer scale. The ability of plasmonics to achieve extreme light concentration unlocks an enormous potential to enhance fluorescence detection, FRET, and FCS. Single molecule spectroscopy techniques greatly benefit from ZMW and plasmonic antennas to enter a new dimension of molecular concentration reaching physiological conditions. The application of nano-optics to biological problems with FRET and FCS is an emerging and exciting field, and is promising to reveal new insights on biological functions and dynamics. PMID:24616447

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

2014-01-01

213

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

NASA Astrophysics Data System (ADS)

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

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

2014-09-01

214

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

SciTech Connect

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

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

2003-11-15

215

In vitro and in vivo demonstrations of Fluorescence by Unbound Excitation from Luminescence (FUEL).  

PubMed

Bioluminescence imaging is a powerful technique that allows for deep-tissue analysis in living, intact organisms. However, in vivo optical imaging is compounded by difficulties due to light scattering and absorption. While light scattering is relatively difficult to overcome and compensate, light absorption by biological tissue is strongly dependent upon wavelength. For example, light absorption by mammalian tissue is highest in the blue-yellow part of the visible energy spectrum. Many natural bioluminescent molecules emit photonic energy in this range, thus in vivo optical detection of these molecules is primarily limited by absorption. This has driven efforts for probe development aimed to enhance photonic emission of red light that is absorbed much less by mammalian tissue using either direct genetic manipulation, and/or resonance energy transfer methods. Here we describe a recently identified alternative approach termed Fluorescence by Unbound Excitation from Luminescence (FUEL), where bioluminescent molecules are able to induce a fluorescent response from fluorescent nanoparticles through an epifluorescence mechanism, thereby significantly increasing both the total number of detectable photons as well as the number of red photons produced. PMID:24166383

Dragavon, Joe; Rekiki, Abdessalem; Theodorou, Ioanna; Samson, Chelsea; Blazquez, Samantha; Rogers, Kelly L; Tournebize, Régis; Shorte, Spencer

2014-01-01

216

A computer vision approach to rare cell in vivo fluorescence flow cytometry  

PubMed Central

Non-invasive enumeration of rare circulating cell populations in small animals is of great importance in many areas of biomedical research. In this work we describe a macroscopic fluorescence imaging system and automated computer vision algorithm that allows in vivo detection, enumeration and tracking of circulating fluorescently-labeled cells from multiple large blood vessels in the ear of a mouse. This imaging system uses a 660 nm laser and a high sensitivity electron-multiplied charge coupled device camera (EMCCD) to acquire fluorescence image sequences from relatively large (~5 × 5 mm2) imaging areas. The primary technical challenge was developing an automated method for identifying and tracking rare cell events in image sequences with substantial autofluorescence and noise content. To achieve this, we developed a two-step image analysis algorithm that first identified cell candidates in individual frames, and then merged cell candidates into tracks by dynamic analysis of image sequences. The second step was critical since it allowed rejection of >97% of false positive cell counts. Overall, our computer vision IVFC (CV-IVFC) approach allows single-cell detection sensitivity at estimated concentrations of 20 cells per mL of peripheral blood. In addition to simple enumeration, the technique recovers the cell’s trajectory, which in the future could be used to automatically identify, for example, in vivo homing and docking events. PMID:24273157

Markovic, Stacey; Li, Binlong; Pera, Vivian; Sznaier, Mario; Camps, Octavia; Niedre, Mark

2014-01-01

217

A computer vision approach to rare cell in vivo fluorescence flow cytometry.  

PubMed

Noninvasive enumeration of rare circulating cell populations in small animals is of great importance in many areas of biomedical research. In this work, we describe a macroscopic fluorescence imaging system and automated computer vision algorithm that allows in vivo detection, enumeration and tracking of circulating fluorescently-labeled cells from multiple large blood vessels in the ear of a mouse. This imaging system uses a 660 nm laser and a high sensitivity electron-multiplied charge coupled device camera (EMCCD) to acquire fluorescence image sequences from relatively large (?5 × 5 mm(2) ) imaging areas. The primary technical challenge was developing an automated method for identifying and tracking rare cell events in image sequences with substantial autofluorescence and noise content. To achieve this, we developed a two-step image analysis algorithm that first identified cell candidates in individual frames, and then merged cell candidates into tracks by dynamic analysis of image sequences. The second step was critical since it allowed rejection of >97% of false positive cell counts. Overall, our computer vision IVFC (CV-IVFC) approach allows single-cell detection sensitivity at estimated concentrations of 20 cells/mL of peripheral blood. In addition to simple enumeration, the technique recovers the cell's trajectory, which in the future could be used to automatically identify, for example, in vivo homing and docking events. PMID:24273157

Markovic, Stacey; Li, Binlong; Pera, Vivian; Sznaier, Mario; Camps, Octavia; Niedre, Mark

2013-12-01

218

In vivo X-ray fluorescence of lead in bone: review and current issues.  

PubMed Central

Bone lead measurements can assess long-term lead dosimetry because the residence time of lead in bone is long. Bone lead measurements thus complement blood and plasma lead measurements, which reflect more short-term exposure. Although the noninvasive, in vivo measurement of lead in bone by X-ray fluorescence (XRF) has been under development since the 1970s, its use is still largely confined to research institutions. There are three principal methods used that vary both in the how lead X-rays are fluoresced and in which lead X-rays are fluoresced. Several groups have reported the independent development of in vivo measurement systems, the majority adopting the 109Cd K XRF method because of its advantages: a robust measurement, a lower detection limit (compared to 57Co K XRF), and a lower effective (radiation) dose (compared to L XRF) when calculated according to the most recent guidelines. These advantages, and the subsequent widespread adoption of the 109Cd method, are primarily consequences of the physics principles of the technique. This paper presents an explanation of the principles of XRF, a description of the practical measurement systems, a review of the human bone lead studies performed to date; and a discussion of some issues surrounding future application of the methods. Images p172-a PMID:8033846

Todd, A C; Chettle, D R

1994-01-01

219

Combining chemical sequential extractions with 3D fluorescence spectroscopy to characterize sludge organic matter.  

PubMed

The design and management of anaerobic digestion of sewage sludge (SS) require a relevant characterisation of the sludge organic matter (OM). Methods currently used are time-consuming and often insufficiently informative. A new method combining chemical sequential extractions (CSE) with 3D fluorescence spectroscopy was developed to provide a relevant SS characterisation to assess both OM bioaccessibility and complexity which govern SS biodegradability. CSE fractionates the sludge OM into 5 compartments of decreasing accessibility. First applied on three SS samples with different OM stability, fractionation profiles obtained were in accordance with the latter. 3D fluorescence spectroscopy revealed that the bioaccessible compartments were mainly constituted of simple and easily biodegradable OM while the unaccessible ones were largely made of complex and refractory OM. Then, primary, secondary and anaerobically digested sludge with different biodegradabilities were tested. Complexity revealed by 3D fluorescence spectroscopy was linked with biodegradability and chemical accessibility was correlated with sludge bioaccessibility. PMID:25223440

Muller, Mathieu; Jimenez, Julie; Antonini, Maxime; Dudal, Yves; Latrille, Eric; Vedrenne, Fabien; Steyer, Jean-Philippe; Patureau, Dominique

2014-12-01

220

In vivo metabolic imaging of insulin with multiphoton fluorescence of human insulin-Au nanodots.  

PubMed

Functional human insulin-Au nanodots (NDs) are synthesized for the in vivo imaging of insulin metabolism. Benefiting from its efficient red to near infrared fluorescence, deep tissue subcellular uptake of insulin-Au NDs can be clearly resolved through a least-invasive harmonic generation and two-photon fluorescence (TPF) microscope. In vivo investigations on mice ear and ex vivo assays on human fat tissues conclude that cells with rich insulin receptors have higher uptake of administrated insulin. Interestingly, the insulin-Au NDs can even permeate into lipid droplets (LDs) of adipocytes. Using this newly discovered metabolic phenomenon of insulin, it is found that enlarged adipocytes in type II diabetes mice have higher adjacent/LD concentration contrast with small-sized ones in wild type mice. For human clinical samples, the epicardial adipocytes of patients with diabetes and coronary artery disease (CAD) also show elevated adjacent/LD concentration contrast. As a result, human insulin-Au nanodots provide a new approach to explore subcellular insulin metabolism in model animals or patients with metabolic or cardiovascular diseases. PMID:23172627

Liu, Chien-Liang; Liu, Tzu-Ming; Hsieh, Tsung-Yuan; Liu, Han-Wen; Chen, Yu-Shing; Tsai, Cheng-Kun; Chen, Hsieh-Chih; Lin, Jong-Wei; Hsu, Ron-Bin; Wang, Tzung-Dau; Chen, Chien-Cheng; Sun, Chi-Kuang; Chou, Pi-Tai

2013-06-24

221

(Compensation for peak shifts and variable background responses in fluorescence spectroscopy)  

SciTech Connect

In the past year, we have made significant progress in several areas. Most of our research has focused on improvements in data analysis methodologies for fluorescence spectroscopic detection in thin-layer and high performance liquid chromatographies, although some experiments have extended the applicability of uv-visible detection methods on thin-layer chromatographic plates. One area of research has focused on the development and evaluation of methods for background correction in fluorescence spectroscopy.

Rutan, S.C.

1989-01-01

222

Surface intrinsic fluorescence spectroscopy of proteins using a UV linearly polarized pulsed laser beam  

NASA Astrophysics Data System (ADS)

The proposed new interfacial spectroscopic method allows to measure the fluorescence emission spectra of the two tryptophans of the Bovine Serum Albumin (BSA) during its adsorption at the air/water and egg-lecithin (egg-PC)/water interfaces, using an UV excimer laser. Surface fluorescence spectroscopy shows changes in the spectroscopic properties of adsorbed BSA, spread BSA and mixed egg-PC/BSA films. These results are related to the surface pressure measurements which characterize the different BSA surface organizations.

Yapoudjian, S.; Ivanova, M.; Uteza, Olivier P.; Marine, Vladimir I.; Sentis, Marc L.

2000-04-01

223

Quantifying Interactions of a Membrane Protein Embedded in a Lipid Nanodisc using Fluorescence Correlation Spectroscopy  

PubMed Central

Using fluorescence correlation spectroscopy, we measured a dissociation constant of 20 nM between EGFP-labeled LcrV from Yersinia pestis and its cognate membrane-bound protein YopB inserted into a lipid nanodisc. The combination of fluorescence correlation spectroscopy and nanodisc technologies provides a powerful approach to accurately measure binding constants of interactions between membrane bound and soluble proteins in solution. Straightforward sample preparation, acquisition, and analysis procedures make this combined technology attractive for accurately measuring binding kinetics for this important class of protein-protein interactions. PMID:24461026

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

2014-01-01

224

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

NASA Astrophysics Data System (ADS)

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

Cerussi, Albert Edward

1999-09-01

225

Monitoring of photobleaching in photodynamic therapy using fluorescence spectroscopy.  

PubMed

Photodynamic therapy (PDT) utilizing aminolevulinic acid (ALA) as a photosensitiser has been used to ablate premalignant/malignant skin conditions including superficial basal cell carcinoma (BCC) with acceptable cosmetic outcome. Clinicians continue however to face difficulties in determining the exact dose that is sufficient to achieve a complete healing from the condition where the experience of the clinician remains the only determinant factor. This inaccuracy sometimes leads to undertreating these lesions. Here, we have clinically evaluated the use of fluorescence imaging system as monitor of protoporphyrin IX (PpIX) photobleaching in patients with BCC and compares this to the clinical outcome. Four readings were acquired from each patient (n=14) "pre-PDT", "peri-PDT" (333secs), "peri-PDT" (660secs) and post-PDT. It was found that red fluorescence values decreased markedly during PDT, and considered to be significant when compared to pre-PDT (P = 0.0018) and post- PDT (P = 0.0025). The red fluorescence value in pre-PDT was found to be higher in BCC of cheek and scalp (where the response rate, RR = 100%) in comparison to the temple (RR = 75%) and nose (RR = 33%). By comparison, the post- PDT red fluorescence values in cheek and scalp were lower than that of the temple and nose, respectively; this may be a useful indicative of the response rate of tissue to therapy. PMID:25316396

Sharwani, A; Alharbi, F A

2014-07-01

226

Bio-imaging using high vertical resolution fluorescence spectroscopy  

E-print Network

of a single layer of streptavidin. Applications of this technique include intracellular imaging and screening for specific bacteria, virus or proteins, where discrimination between raised fluorescently labeled and evaluate the potential of this method for deconvoluting a spectral signal obtained simultaneously from

227

Fluorescence Spectroscopy of Conformational Changes of Single LH2 Complexes  

PubMed Central

We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope. The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between long-lived quasi-stable levels differing by up to 30 nm. The frequency and size of these fluorescence peak movements were found to increase linearly with the excitation intensity. These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum. The probability for a particle to undergo significant spectral shift in either direction was found to be roughly the same. Using the modified Redfield theory, the observed changes in spectral shape and intensity were accounted for by changes in the realization of the static disorder. Long lifetimes of the quasi-stable states suggest large energetic barriers between the states characterized by different emission spectra. PMID:15501944

Rutkauskas, Danielis; Novoderezhkin, Vladimir; Cogdell, Richard J.; van Grondelle, Rienk

2005-01-01

228

In vivo fluorescence imaging of primate retinal ganglion cells and retinal pigment epithelial cells  

NASA Astrophysics Data System (ADS)

The ability to resolve single cells noninvasively in the living retina has important applications for the study of normal retina, diseased retina, and the efficacy of therapies for retinal disease. We describe a new instrument for high-resolution, in vivo imaging of the mammalian retina that combines the benefits of confocal detection, adaptive optics, multispectral, and fluorescence imaging. The instrument is capable of imaging single ganglion cells and their axons through retrograde transport in ganglion cells of fluorescent dyes injected into the monkey lateral geniculate nucleus (LGN). In addition, we demonstrate a method involving simultaneous imaging in two spectral bands that allows the integration of very weak signals across many frames despite inter-frame movement of the eye. With this method, we are also able to resolve the smallest retinal capillaries in fluorescein angiography and the mosaic of retinal pigment epithelium (RPE) cells with lipofuscin autofluorescence.

Gray, Daniel C.; Merigan, William; Wolfing, Jessica I.; Gee, Bernard P.; Porter, Jason; Dubra, Alfredo; Twietmeyer, Ted H.; Ahamd, Kamran; Tumbar, Remy; Reinholz, Fred; Williams, David R.

2006-08-01

229

In vivo tomographic imaging with fluorescence and MRI using tumor-targeted dual-labeled nanoparticles.  

PubMed

Dual-modality imaging combines the complementary advantages of different modalities, and offers the prospect of improved preclinical research. The combination of fluorescence imaging and magnetic resonance imaging (MRI) provides cross-validated information and direct comparison between these modalities. Here, we report on the application of a novel tumor-targeted, dual-labeled nanoparticle (NP), utilizing iron oxide as the MRI contrast agent and near infrared (NIR) dye Cy5.5 as the fluorescent agent. Results of in vitro experiments verified the specificity of the NP to tumor cells. In vivo tumor targeting and uptake of the NPs in a mouse model were visualized by fluorescence and MR imaging collected at different time points. Quantitative analysis was carried out to evaluate the efficacy of MRI contrast enhancement. Furthermore, tomographic images were also acquired using both imaging modalities and cross-validated information of tumor location and size between these two modalities was revealed. The results demonstrate that the use of dual-labeled NPs can facilitate the dual-modal detection of tumors, information cross-validation, and direct comparison by combing fluorescence molecular tomography (FMT) and MRI. PMID:24368885

Zhang, Yue; Zhang, Bin; Liu, Fei; Luo, Jianwen; Bai, Jing

2014-01-01

230

In Vivo Multiphoton NADH Fluorescence Reveals Depth-Dependent Keratinocyte Metabolism in Human Skin  

PubMed Central

We employ a clinical multiphoton microscope to monitor in vivo and noninvasively the changes in reduced nicotinamide adenine dinucleotide (NADH) fluorescence of human epidermal cells during arterial occlusion. We correlate these results with measurements of tissue oxy- and deoxyhemoglobin concentration during oxygen deprivation using spatial frequency domain imaging. During arterial occlusion, a decrease in oxyhemoglobin corresponds to an increase in NADH fluorescence in the basal epidermal cells, implying a reduction in basal cell oxidative phosphorylation. The ischemia-induced oxygen deprivation is associated with a strong increase in NADH fluorescence of keratinocytes in layers close to the stratum basale, whereas keratinocytes from epidermal layers closer to the skin surface are not affected. Spatial frequency domain imaging optical property measurements, combined with a multilayer Monte Carlo-based radiative transport model of multiphoton microscopy signal collection in skin, establish that localized tissue optical property changes during occlusion do not impact the observed NADH signal increase. This outcome supports the hypothesis that the vascular contribution to the basal layer oxygen supply is significant and these cells engage in oxidative metabolism. Keratinocytes in the more superficial stratum granulosum are either supplied by atmospheric oxygen or are functionally anaerobic. Based on combined hemodynamic and two-photon excited fluorescence data, the oxygen consumption rate in the stratum basale is estimated to be ?0.035 ?moles/106 cells/h. PMID:23332078

Balu, Mihaela; Mazhar, Amaan; Hayakawa, Carole K.; Mittal, Richa; Krasieva, Tatiana B.; König, Karsten; Venugopalan, Vasan; Tromberg, Bruce J.

2013-01-01

231

Label-free in vivo imaging of human leukocytes using two-photon excited endogenous fluorescence  

NASA Astrophysics Data System (ADS)

We demonstrate that two-photon excited endogenous fluorescence enables label-free morphological and functional imaging of various human blood cells. Specifically, we achieved distinctive morphological contrast to visualize morphology of important leukocytes, such as polymorphonuclear structure of granulocyte and mononuclear feature of agranulocyte, through the employment of the reduced nicotinamide adenine dinucleotide (NADH) fluorescence signals. In addition, NADH fluorescence images clearly reveal the morphological transformation process of neutrophils during disease-causing bacterial infection. Our findings also show that time-resolved NADH fluorescence can be potentially used for functional imaging of the phagocytosis of pathogens by leukocytes (neutrophils) in vivo. In particular, we found that free-to-bound NADH ratios measured in infected neutrophils increased significantly, which is consistent with a previous study that the energy consumed in the phagocytosis of neutrophils is mainly generated through the glycolysis pathway that leads to the accumulation of free NADH. Future work will focus on further developing and applying label-free imaging technology to investigate leukocyte-related diseases and disorders.

Zeng, Yan; Yan, Bo; Sun, Qiqi; Teh, Seng Khoon; Zhang, Wei; Wen, Zilong; Qu, Jianan Y.

2013-04-01

232

Embryonic lineage analysis using three-dimensional, time-lapse in-vivo fluorescent microscopy  

NASA Astrophysics Data System (ADS)

Drosophila melanogaster has become one of the most extensively studied organisms because of its amenability to genetic analysis. Unfortunately, the biochemistry and cell biology ofDrosophila has lagged behind. To this end we have been microinjecting fluorescently labelled proteins into the living embryo and observing the behavior of these proteins to determine their role in the cell cycle and development. Imaging of these fluorescent probes is an extremely important element to this form of analysis. We have taken advantage of the sensitivity and well behaved characteristics of the charge coupled device (CCD) camera in conjunction with digital image enhancement schemes to produce highly accurate images of these fluorescent probes in vivo. One of our major goals is to produce a detailed map of cell fate so that we can understand how fate is determined and maintained. In order produce such a detailed map, protocols for following the movements and mitotic behavior of a large number of cells in three dimensions over relatively long periods of time were developed. We will present our results using fluorescently labelled histone proteins as a marker for nuclear location1. In addition, we will also present our initial results using a photoactivatable analog of fluorescein to mark single cells so that their long range fate can be unambiguously determined.

Minden, Jonathan; Kam, Zvi; Agard, David A.; Sedat, John W.; Alberts, Bruce

1990-08-01

233

Tomographic sensing and localization of fluorescently labeled circulating cells in mice in vivo.  

PubMed

Sensing and enumeration of specific types of circulating cells in small animals is an important problem in many areas of biomedical research. Microscopy-based fluorescence in vivo flow cytometry methods have been developed previously, but these are typically limited to sampling of very small blood volumes, so that very rare circulating cells may escape detection. Recently, we described the development of a 'diffuse fluorescence flow cytometer' (DFFC) that allows sampling of much larger blood vessels and therefore circulating blood volumes in the hindlimb, forelimb or tail of a mouse. In this work, we extend this concept by developing and validating a method to tomographically localize circulating fluorescently labeled cells in the cross section of a tissue simulating optical flow phantom and mouse limb. This was achieved using two modulated light sources and an array of six fiber-coupled detectors that allowed rapid, high-sensitivity acquisition of full tomographic data sets at 10 Hz. These were reconstructed into two-dimensional cross-sectional images using Monte Carlo models of light propagation and the randomized algebraic reconstruction technique. We were able to obtain continuous images of moving cells in the sample cross section with 0.5 mm accuracy or better. We first demonstrated this concept in limb-mimicking optical flow photons with up to four flow channels, and then in the tails of mice with fluorescently labeled multiple myeloma cells. This approach increases the overall diagnostic utility of our DFFC instrument. PMID:22750660

Zettergren, Eric; Swamy, Tushar; Runnels, Judith; Lin, Charles P; Niedre, Mark

2012-07-21

234

Tomographic sensing and localization of fluorescently labeled circulating cells in mice in vivo  

NASA Astrophysics Data System (ADS)

Sensing and enumeration of specific types of circulating cells in small animals is an important problem in many areas of biomedical research. Microscopy-based fluorescence in vivo flow cytometry methods have been developed previously, but these are typically limited to sampling of very small blood volumes, so that very rare circulating cells may escape detection. Recently, we described the development of a ‘diffuse fluorescence flow cytometer’ (DFFC) that allows sampling of much larger blood vessels and therefore circulating blood volumes in the hindlimb, forelimb or tail of a mouse. In this work, we extend this concept by developing and validating a method to tomographically localize circulating fluorescently labeled cells in the cross section of a tissue simulating optical flow phantom and mouse limb. This was achieved using two modulated light sources and an array of six fiber-coupled detectors that allowed rapid, high-sensitivity acquisition of full tomographic data sets at 10 Hz. These were reconstructed into two-dimensional cross-sectional images using Monte Carlo models of light propagation and the randomized algebraic reconstruction technique. We were able to obtain continuous images of moving cells in the sample cross section with 0.5 mm accuracy or better. We first demonstrated this concept in limb-mimicking optical flow photons with up to four flow channels, and then in the tails of mice with fluorescently labeled multiple myeloma cells. This approach increases the overall diagnostic utility of our DFFC instrument.

Zettergren, Eric; Swamy, Tushar; Runnels, Judith; Lin, Charles P.; Niedre, Mark

2012-07-01

235

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

NASA Astrophysics Data System (ADS)

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

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

2014-09-01

236

Fluorescence Instrument Response Standards in Two-Photon Time-Resolved Spectroscopy  

PubMed Central

We studied the fluorescence properties of several potential picosecond lifetime standards suitable for two-photon excitation from a Ti : sapphire femtosecond laser. The fluorescence emission of the selected fluorophores (rose bengal, pyridine 1, and LDS 798) covered the visible to near-infrared wavelength range from 550 to 850 nm. We suggest that these compounds can be used to measure the appropriate instrument response functions needed for accurate deconvolution of fluorescence lifetime data. Lifetime measurements with multiphoton excitation that use scatterers as a reference may fail to properly resolve fluorescence intensity decays. This is because of the different sensitivities of photodetectors in different spectral regions. Also, detectors often lose sensitivity in the near-infrared region. We demonstrate that the proposed references allow a proper reconvolution of measured lifetimes. We believe that picosecond lifetime standards for two-photon excitation will find broad applications in multiphoton spectroscopy and in fluorescence lifetime imaging microscopy (FLIM). PMID:20719056

LUCHOWSKI, RAFAL; SZABELSKI, MARIUSZ; SARKAR, PABAK; APICELLA, ELISA; MIDDE, KRISHNA; RAUT, SANGRAM; BOREJDO, JULIAN; GRYCZYNSKI, ZYGMUNT; GRYCZYNSKI, IGNACY

2011-01-01

237

Fluorescence instrument response standards in two-photon time-resolved spectroscopy.  

PubMed

We studied the fluorescence properties of several potential picosecond lifetime standards suitable for two-photon excitation from a Ti:sapphire femtosecond laser. The fluorescence emission of the selected fluorophores (rose bengal, pyridine 1, and LDS 798) covered the visible to near-infrared wavelength range from 550 to 850 nm. We suggest that these compounds can be used to measure the appropriate instrument response functions needed for accurate deconvolution of fluorescence lifetime data. Lifetime measurements with multiphoton excitation that use scatterers as a reference may fail to properly resolve fluorescence intensity decays. This is because of the different sensitivities of photodetectors in different spectral regions. Also, detectors often lose sensitivity in the near-infrared region. We demonstrate that the proposed references allow a proper reconvolution of measured lifetimes. We believe that picosecond lifetime standards for two-photon excitation will find broad applications in multiphoton spectroscopy and in fluorescence lifetime imaging microscopy (FLIM). PMID:20719056

Luchowski, Rafal; Szabelski, Mariusz; Sarkar, Pabak; Apicella, Elisa; Midde, Krishna; Raut, Sangram; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, Ignacy

2010-08-01

238

Development of a homogeneous assay format for p53 antibodies using fluorescence correlation spectroscopy  

NASA Astrophysics Data System (ADS)

The development of reliable methods for the detection of minute amounts of antibodies directly in homogeneous solution represents one of the major tasks in the current research field of molecular diagnostics. We demonstrate the potential of fluorescence correlation spectroscopy (FCS) in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing two parameters simultaneously: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Our data demonstrate that FCS in combination with fluorescence-quenched peptide epitopes opens new possibilities for the reliable detection of antibody binding events in homogeneous solution.

Neuweiler, Hannes; Scheffler, Silvia; Sauer, Markus

2005-08-01

239

Monitoring cellular movement in vivo with photoconvertible fluorescence protein "Kaede" transgenic mice.  

PubMed

Kaede is a photoconvertible fluorescence protein that changes from green to red upon exposure to violet light. The photoconversion of intracellular Kaede has no effect on cellular function. Using transgenic mice expressing the Kaede protein, we demonstrated that movement of cells with the photoconverted Kaede protein could be monitored from lymphoid organs to other tissues as well as from skin to the draining lymph node. Analysis of the kinetics of cellular movement revealed that each subset of cells in the lymph node, such as CD4(+) T, CD8(+) T, B, and dendritic cells, has a distinct migration pattern in vivo. Thus, the Kaede transgenic mouse system would be an ideal tool to monitor precise cellular movement in vivo at different stages of immune response to pathogens as well as in autoimmune diseases. PMID:18663225

Tomura, Michio; Yoshida, Naoki; Tanaka, Junko; Karasawa, Satoshi; Miwa, Yoshihiro; Miyawaki, Atsushi; Kanagawa, Osami

2008-08-01

240

An individually coated near-infrared fluorescent protein as a safe and robust nanoprobe for in vivo imaging  

NASA Astrophysics Data System (ADS)

A prerequisite for in vivo fluorescence imaging is the safety of fluorescent probes. Among all fluorescent probes, fluorescent proteins (FPs) might be the safest ones, which have been widely used in biological sciences at the gene level. But FPs have not been used in vivo in the purified form yet due to the instability of proteins. Here, we individually coat near-infrared (NIR) FPs (NIRFPs) with a silica nanoshell, resulting in NIRFP@silica, one of the safest and brightest NIR fluorescent nanoprobes with a quantum yield of 0.33 for in vivo imaging. The silica shell not only protects NIRFPs from denaturation and metabolic digestion, but also enhances the quantum yield and photostability of the coated NIRFPs. When injected via the tail vein, NIRFP@silica NPs can distribute all over the mouse body, and then can be efficiently eliminated through urine in 24 h, demonstrating its potential applications as a safe and robust NIR fluorescence probe for whole body imaging.A prerequisite for in vivo fluorescence imaging is the safety of fluorescent probes. Among all fluorescent probes, fluorescent proteins (FPs) might be the safest ones, which have been widely used in biological sciences at the gene level. But FPs have not been used in vivo in the purified form yet due to the instability of proteins. Here, we individually coat near-infrared (NIR) FPs (NIRFPs) with a silica nanoshell, resulting in NIRFP@silica, one of the safest and brightest NIR fluorescent nanoprobes with a quantum yield of 0.33 for in vivo imaging. The silica shell not only protects NIRFPs from denaturation and metabolic digestion, but also enhances the quantum yield and photostability of the coated NIRFPs. When injected via the tail vein, NIRFP@silica NPs can distribute all over the mouse body, and then can be efficiently eliminated through urine in 24 h, demonstrating its potential applications as a safe and robust NIR fluorescence probe for whole body imaging. Electronic supplementary information (ESI) available: A chromatogram of APTS-NIRFP, a TEM image of 40 nm NIRFP@silica, dispersion stability of NIRFP@silica, more whole body fluorescent images, serum biochemical parameters, and optical images of HE stained organ slices. See DOI: 10.1039/c3nr02508j

Yang, Yu; Xiang, Kun; Yang, Yi-Xin; Wang, Yan-Wen; Zhang, Xin; Cui, Yangdong; Wang, Haifang; Zhu, Qing-Qing; Fan, Liqiang; Liu, Yuanfang; Cao, Aoneng

2013-10-01

241

MoI density measurements by laser induced fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

The density of molybdenum atoms produced by sputtering of a TZM (molybdenum) target by Ar+ ions is measured by laser induced fluorescence (LIF) using tunable dye laser. The excitation transition involved is a7S3-z5P30 at 345.64 nm, while the fluorescence signal is from the decay z5P30-a5S2 at 550.6 nm. The LIF measurements are carried out by varying the laser power incident on Mo atoms by means of neutral density filters. An absolute calibration of the detection system together with the realization of a well defined optical probe volume allows for the determination of the density of the emitting atoms. An evaluation of LIF diagnostic performance on Frascati Tokamak upgrade put a lower limit of 2.5×1014 atoms/m3 on the detectable local density of MoI close to the toroidal limiter.

Orsitto, F.; Borra, M.; Coppotelli, F.; Gatti, G.; Neri, E.

1999-01-01

242

Tracking dynamics of muscle engraftment in small animals by in vivo fluorescent imaging.  

PubMed

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently. In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 10(5) cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells. PMID:19770816

Yang, Zhong; Zeng, Qing; Ma, Zhiyuan; Wang, Yaming; Xu, Xiaoyin

2009-01-01

243

Fluorescence polarization standard for near infrared spectroscopy and microscopy.  

PubMed

We present studies of polarized absorption [linear dichroism (LD)] and fluorescence polarization of the styryl derivative (LDS 798) embedded in oriented poly(vinyl alcohol) (PVA) films. These films were oriented by progressive stretching up to eight folds. Both vertical and horizontal components of absorptions and fluorescence were measured and dichroic ratios were determined for different film stretching ratios. The dichroic ratio and fluorescence anisotropy values were analyzed as a function of PVA film stretching ratio by fitting according to the previously developed theory. For maximum stretching ratios, exceptionally high anisotropy (approximately 0.8) and polarization (approximately 0.9) values have been measured. The stretched films have high polarization values also for isotropic excitation in a wide spectral range (500-700 nm). Such films can be conveniently used as high polarization standards and we envision they will also have applications in near infrared (NIR) imaging microscopy, where they can be used for correcting an instrumental factor in polarization measurements. PMID:19023392

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

2008-11-20

244

Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy  

PubMed Central

Frequency domain fluorescence lifetime imaging is a powerful technique that enables the observation of subtle changes in the molecular environment of a fluorescent probe. This technique works by measuring the phase delay between the optical emission and excitation of fluorophores as a function of modulation frequency. However, high-resolution measurements are time consuming, as the excitation modulation frequency must be swept, and faster low-resolution measurements at a single frequency are prone to large errors. Here, we present a low cost optical system for applications in real-time confocal lifetime imaging, which measures the phase vs. frequency spectrum without sweeping. Deemed Lifetime Imaging using Frequency-multiplexed Excitation (LIFE), this technique uses a digitally-synthesized radio frequency comb to drive an acousto-optic deflector, operated in a cat’s-eye configuration, to produce a single laser excitation beam modulated at multiple beat frequencies. We demonstrate simultaneous fluorescence lifetime measurements at 10 frequencies over a bandwidth of 48 MHz, enabling high speed frequency domain lifetime analysis of single- and multi-component sample mixtures. PMID:25574449

Chan, Jacky C. K.; Diebold, Eric D.; Buckley, Brandon W.; Mao, Sien; Akbari, Najva; Jalali, Bahram

2014-01-01

245

Classification of aortic atherosclerotic lesions with time-resolved fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

1999-07-01

246

Hyperspectral Imaging and Spectroscopy of Fluorescently Coupled Acyl-CoA: Cholesterol Acyltransferase in Insect Cells  

NASA Technical Reports Server (NTRS)

A high-performance hyperspectral imaging module with high throughput of light suitable for low-intensity fluorescence microscopic imaging and subsequent analysis, including single-pixel-defined emission spectroscopy, was tested on Sf21 insect cells expressing green fluorescence associated with recombinant green fluorescent protein linked or not with the membrane protein acyl-CoA:cholesterol acyltransferase. The imager utilized the phenomenon of optical activity as a new technique providing information over a spectral range of 220-1400 nm, and was inserted between the microscope and an 8-bit CCD video-rate camera. The resulting fluorescence image did not introduce observable image aberrations. The images provided parallel acquisition of well resolved concurrent spatial and spectral information such that fluorescence associated with green fluorescent protein alone was demonstrated to be diffuse within the Sf21 insect cell, and that green fluorescence associated with the membrane protein was shown to be specifically concentrated within regions of the cell cytoplasm. Emission spectra analyzed from different regions of the fluorescence image showed blue shift specific for the regions of concentration associated with the membrane protein.

Malak, H.; Mahtani, H.; Herman, P.; Vecer, J.; Lu, X.; Chang, T. Y.; Richmond, Robert C.; Whitaker, Ann F. (Technical Monitor)

2001-01-01

247

Capillary Electrophoresis and Fluorescence Excitation-Emission Matrix Spectroscopy for Characterization of Humic Substances  

Technology Transfer Automated Retrieval System (TEKTRAN)

Capillary electrophoresis (CE) and fluorescence spectroscopy have been used in natural organic matter (NOM) studies. In this study, we characterized five fulvic acids, six humic acids and two unprocessed NOM samples obtained from the International Humic Substances Society (IHSS) using these two ana...

248

Fluorescence spectroscopy and molecular weight distribution of extracellular polymers from full-scale activated sludge biomass  

Microsoft Academic Search

Two fractions of extracellular polymer substances (EPSs), soluble and readily extractable (RE), were characterised in terms of their molecular weight distributions (MWD) and 3-D excitation-emission- matrix (EEM) fluorescence spectroscopy signatures. The EPS fractions were different: the soluble EPSs were composed mainly of high molecular weight compounds, while the RE EPSs were composed of small molecular weight compounds. Contrary to previous

M. Esparza-Soto; P. K. Westerhoff

2001-01-01

249

Studies of multifrequency phase-resolved fluorescence spectroscopy for spectral fingerprinting  

SciTech Connect

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

McGown, L.B.

1990-01-01

250

Room-temperature fluorescence spectroscopy of monohydroxy metabolites of polycyclic aromatic hydrocarbons on octadecyl extraction membranes  

Microsoft Academic Search

Urine analysis of monohydroxy metabolites is recognized as an accurate assessment of human exposure to polycyclic aromatic hydrocarbons. Despite the sophisticated arsenal of analytical tools, monitoring of monohydroxy metabolites via simple, cost effective and direct methods of analysis still remains a challenge. This article evaluates the analytical potential of solid-phase extraction room-temperature fluorescence spectroscopy for the problem at hand. Extraction

Korina Calimag-Williams; Hector C. Goicoechea; Andres D. Campiglia

2011-01-01

251

A quantitative study of the intracellular dynamics of fluorescently labelled glyco-gold nanoparticles via fluorescence correlation spectroscopy.  

PubMed

The dynamic behaviour of gold nanoparticles functionalised with glucose (Glc-Au NPs) has been studied here by means of fluorescence correlation spectroscopy (FCS). Meaningful data on the state of aggregation and dynamics of Glc-Au NPs fluorescently-labelled with HiLyte Fluor647 (Glc-Au-Hi NPs) in the intracellular environment were obtained. Moreover, the work presented here shows that FCS can be used to visualise the presence of single NPs or NP aggregates following uptake and to estimate, locally, NP concentrations within the cell. FCS measurements become possible after applying a "prebleaching" methodology, when the immobile NP fraction has been effectively removed and thus significant FCS data has been recorded. In this study, Glc-Au-Hi NPs have been incubated with HepG2 cells and their diffusion time in the intracellular environment has been measured and compared with their diffusion value in water and cell media. PMID:24639360

Murray, Richard A; Qiu, Yuan; Chiodo, Fabrizio; Marradi, Marco; Penadés, Soledad; Moya, Sergio E

2014-07-01

252

Ultrafast fluorescence imaging in vivo with conjugated polymer fluorophores in the second near-infrared window.  

PubMed

In vivo fluorescence imaging in the second near-infrared window (1.0-1.7??m) can afford deep tissue penetration and high spatial resolution, owing to the reduced scattering of long-wavelength photons. Here we synthesize a series of low-bandgap donor/acceptor copolymers with tunable emission wavelengths of 1,050-1,350?nm in this window. Non-covalent functionalization with phospholipid-polyethylene glycol results in water-soluble and biocompatible polymeric nanoparticles, allowing for live cell molecular imaging at >1,000?nm with polymer fluorophores for the first time. Importantly, the high quantum yield of the polymer allows for in vivo, deep-tissue and ultrafast imaging of mouse arterial blood flow with an unprecedented frame rate of >25 frames per second. The high time-resolution results in spatially and time resolved imaging of the blood flow pattern in cardiogram waveform over a single cardiac cycle (~200?ms) of a mouse, which has not been observed with fluorescence imaging in this window before. PMID:24947309

Hong, Guosong; Zou, Yingping; Antaris, Alexander L; Diao, Shuo; Wu, Di; Cheng, Kai; Zhang, Xiaodong; Chen, Changxin; Liu, Bo; He, Yuehui; Wu, Justin Z; Yuan, Jun; Zhang, Bo; Tao, Zhimin; Fukunaga, Chihiro; Dai, Hongjie

2014-01-01

253

In vivo fluorescence confocal microscopy: indocyanine green enhances the contrast of epidermal and dermal structures  

NASA Astrophysics Data System (ADS)

In recent years, in vivo skin imaging devices have been successfully implemented in skin research as well as in clinical routine. Of particular importance is the use of reflectance confocal microscopy (RCM) and fluorescence confocal microscopy (FCM) that enable visualization of the tissue with a resolution comparable to histology. A newly developed commercially available multi-laser device in which both technologies are integrated now offers the possibility to directly compare RCM with FCM. The fluorophore indocyanine green (ICG) was intradermally injected into healthy forearm skin of 10 volunteers followed by in vivo imaging at various time points. In the epidermis, accurate assessment of cell morphology with FCM was supplemented by identification of pigmented cells and structures with RCM. In dermal layers, only with FCM connective tissue fibers were clearly contoured down to a depth of more than 100 ?m. The fluorescent signal still provided a favorable image contrast 24 and 48 hours after injection. Subsequently, ICG was applied to different types of skin diseases (basal cell carcinoma, actinic keratosis, seborrhoeic keratosis, and psoriasis) in order to demonstrate the diagnostic benefit of FCM when directly compared with RCM. Our data suggest a great impact of FCM in combination with ICG on clinical and experimental dermatology in the future.

Skvara, Hans; Kittler, Harald; Schmid, Johannes A.; Plut, Ulrike; Jonak, Constanze

2011-09-01

254

Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins  

NASA Astrophysics Data System (ADS)

Non-invasive imaging of biological processes in vivo is invaluable in advancing biology. Photoacoustic tomography is a scalable imaging technique that provides higher resolution at greater depths in tissue than achievable by purely optical methods. Here we report the application of two spectrally distinct near-infrared fluorescent proteins, iRFP670 and iRFP720, engineered from bacterial phytochromes, as photoacoustic contrast agents. iRFPs provide tissue-specific contrast without the need for delivery of any additional substances. Compared to conventional GFP-like red-shifted fluorescent proteins, iRFP670 and iRFP720 demonstrate stronger photoacoustic signals at longer wavelengths, and can be spectrally resolved from each other and hemoglobin. We simultaneously visualized two differently labeled tumors, one with iRFP670 and the other with iRFP720, as well as blood vessels. We acquired images of a mouse as 2D sections of a whole animal, and as localized 3D volumetric images with high contrast and sub-millimeter resolution at depths up to 8 mm. Our results suggest iRFPs are genetically-encoded probes of choice for simultaneous photoacoustic imaging of several tissues or processes in vivo.

Krumholz, Arie; Shcherbakova, Daria M.; Xia, Jun; Wang, Lihong V.; Verkhusha, Vladislav V.

2014-02-01

255

Ultrafast fluorescence imaging in vivo with conjugated polymer fluorophores in the second near-infrared window  

NASA Astrophysics Data System (ADS)

In vivo fluorescence imaging in the second near-infrared window (1.0-1.7??m) can afford deep tissue penetration and high spatial resolution, owing to the reduced scattering of long-wavelength photons. Here we synthesize a series of low-bandgap donor/acceptor copolymers with tunable emission wavelengths of 1,050-1,350?nm in this window. Non-covalent functionalization with phospholipid-polyethylene glycol results in water-soluble and biocompatible polymeric nanoparticles, allowing for live cell molecular imaging at >1,000?nm with polymer fluorophores for the first time. Importantly, the high quantum yield of the polymer allows for in vivo, deep-tissue and ultrafast imaging of mouse arterial blood flow with an unprecedented frame rate of >25 frames per second. The high time-resolution results in spatially and time resolved imaging of the blood flow pattern in cardiogram waveform over a single cardiac cycle (~200?ms) of a mouse, which has not been observed with fluorescence imaging in this window before.

Hong, Guosong; Zou, Yingping; Antaris, Alexander L.; Diao, Shuo; Wu, Di; Cheng, Kai; Zhang, Xiaodong; Chen, Changxin; Liu, Bo; He, Yuehui; Wu, Justin Z.; Yuan, Jun; Zhang, Bo; Tao, Zhimin; Fukunaga, Chihiro; Dai, Hongjie

2014-06-01

256

High-throughput single-molecule fluorescence spectroscopy using parallel detection  

PubMed Central

Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. The basic concept of this technique is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements obtained with different novel multipixel single-photon counting detectors. PMID:21625288

Michalet, X.; Colyer, R. A.; Scalia, G.; Kim, T.; Levi, Moran; Aharoni, Daniel; Cheng, Adrian; Guerrieri, F.; Arisaka, Katsushi; Millaud, Jacques; Rech, I.; Resnati, D.; Marangoni, S.; Gulinatti, A.; Ghioni, M.; Tisa, S.; Zappa, F.; Cova, S.; Weiss, S.

2011-01-01

257

Determination of changes in wastewater quality through a treatment works using fluorescence spectroscopy.  

PubMed

Fluorescence spectroscopy was used to characterize municipal wastewater at various stages of treatment in order to understand how its fluorescence signature changes with treatment and how the signal relates to biochemical oxygen demand (BOD) and chemical oxygen demand (COD). The impact of size fractionation on the fluorescence signal was also investigated. Fluorescence measurements were taken for unfiltered and filtered (0.45 and 0.20 microm) samples of crude, settled and secondary treated wastewater (activated sludge and trickling filter), and final effluent. Good correlations were observed for unfiltered, diluted wastewater samples between BOD and fluorescence intensity at excitation 280 nm, emission 350 nm (Peak T1) (r = 0.92) and between COD and Peak T1 intensity (r = 0.85). The majority of the T1 and T2 signal was found to be derived from the <0.20 microm fraction. Initial results indicate that fluorescence spectroscopy, and changes in Peak T1 intensity in particular, could be used for continuous, real-time wastewater quality assessment and process control of wastewater treatment works. PMID:24617065

Bridgeman, John; Baker, Andy; Carliell-Marquet, Cynthia; Carstea, Elfrida

2013-01-01

258

Whole Cell-SELEX Aptamers for Highly Specific Fluorescence Molecular Imaging of Carcinomas In Vivo  

PubMed Central

Background Carcinomas make up the majority of cancers. Their accurate and specific diagnoses are of great significance for the improvement of patients' curability. Methodology/Principal Findings In this paper, we report an effectual example of the in vivo fluorescence molecular imaging of carcinomas with extremely high specificity based on whole cell-SELEX aptamers. Firstly, S6, an aptamer against A549 lung carcinoma cells, was adopted and labeled with Cy5 to serve as a molecular imaging probe. Flow cytometry assays revealed that Cy5-S6 could not only specifically label in vitro cultured A549 cells in buffer, but also successfully achieve the detection of ex vivo cultured target cells in serum. When applied to in vivo imaging, Cy5-S6 was demonstrated to possess high specificity in identifying A549 carcinoma through a systematic comparison investigation. Particularly, after Cy5-S6 was intravenously injected into nude mice which were simultaneously grafted with A549 lung carcinoma and Tca8113 tongue carcinoma, a much longer retention time of Cy5-S6 in A549 tumor was observed and a clear targeted cancer imaging result was presented. On this basis, to further promote the application to imaging other carcinomas, LS2 and ZY8, which are two aptamers selected by our group against Bel-7404 and SMMC-7721 liver carcinoma cells respectively, were tested in a similar way, both in vitro and in vivo. Results showed that these aptamers were even effective in differentiating liver carcinomas of different subtypes in the same body. Conclusions/Significance This work might greatly advance the application of whole cell-SELEX aptamers to carcinomas-related in vivo researches. PMID:23950940

He, Xiaoxiao; Guo, Qiuping; Wang, Kemin; Ye, Xiaosheng; Tang, Jinlu

2013-01-01

259

Classification evaluation of tobaccos using LED-induced fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

Tobacco is one of the most important economic crops in the world, assessment of its quality has a very important business significance. A compact, low-cost, and maneuverable optical sensor system for classification evaluation of different tobaccos was described in this paper using light-emitting-diodes (LEDs)-induced fluorescence. The principal components analysis (PCA) method is used to extract the dominant features of the tobaccos for identifying the classification of tobaccos. The technique is suitable for practical identification due to the use of a straightforward data evaluation method and compact system.

Zhong, Weijia; Dong, Yongjiang; Liu, Xuan; Lin, Hongze; Mei, Liang; Yan, Chunsheng

2014-02-01

260

Vibrationally Resolved Photoabsorption Spectroscopy of Red Fluorescent Protein Chromophore Anions  

NASA Astrophysics Data System (ADS)

Photoabsorption studies of red fluorescent protein chromophore anions have been performed at the ELISA electrostatic heavy-ion storage ring. The broad absorption band due to electronic excitation of the chromophores is tuned to a longer wavelength (redshifted) by extending the electronic conjugation of the molecule. A clear vibrational progression is resolved with Evib˜380 and 520 cm-1 for two different forms of the chromophore. The vibrational modes correspond to collective motions of the entire molecular structure. It is argued that the excited electronic state has an equilibrium configuration far from that of the electronic ground state, i.e., poor Franck Condon overlap.

Boyé, S.; Krogh, H.; Nielsen, I. B.; Nielsen, S. B.; Pedersen, S. U.; Pedersen, U. V.; Andersen, L. H.; Bell, A. F.; He, X.; Tonge, P. J.

2003-03-01

261

Pattern recognition analysis of in vivo enzyme-substrate fluorescence velocities in microorganism detection and identification.  

PubMed Central

A spectrometric technique is presented that combines most of the important criteria necessary for efficient detection and identification of microorganisms. These criteria include simplicity of experimental design, various degrees of sensitivity and selectivity, convenience, and total reaction times of less than 15 min. The study takes advantage of the inherent extracellular enzymes present in living as opposed to dead, non-enzyme-producing organisms. Sequentially these are harnessed in in vivo reactions with a substrate containing a select organic functional group that is known to be cleaved or hydrolyzed by a certain enzyme. The substrate is tailored so that one of the products can be induced to fluoresce, and by using a conventional spectrofluorimeter the rate at which the fluorescence appears can be recorded. By subjecting the same bacterial sample to a number of different enzyme substrates, a pattern of fluorescence response rates emerges from a 7 by 7 microorganism-substrate matrix. Detection limits ranged from 3.6 X 10(2) to 3.5 X 10(8) cells per ml for the Bacillus globigii-indoxyl acetate and Escherichia coli-diacetylfluorescein pairs, respectively. The specificity and versatility of the method for bacterial determination is demonstrated in probing different bacterial enzymes through their spectrally active metabolic products. PMID:3089149

Snyder, A P; Wang, T T; Greenberg, D B

1986-01-01

262

Development of a noncontact 3-D fluorescence tomography system for small animal in vivo imaging  

NASA Astrophysics Data System (ADS)

Fluorescence imaging is an important tool for tracking molecular-targeting probes in preclinical studies. It offers high sensitivity, but nonetheless low spatial resolution compared to other leading imaging methods such CT and MRI. We demonstrate our methodological development in small animal in vivo whole-body imaging using fluorescence tomography. We have implemented a noncontact fluid-free fluorescence diffuse optical tomography system that uses a raster-scanned continuous-wave diode laser as the light source and an intensified CCD camera as the photodetector. The specimen is positioned on a motorized rotation stage. Laser scanning, data acquisition, and stage rotation are controlled via LabVIEW applications. The forward problem in the heterogeneous medium is based on a normalized Born method, and the sensitivity function is determined using a Monte Carlo method. The inverse problem (image reconstruction) is performed using a regularized iterative algorithm, in which the cost function is defined as a weighted sum of the L-2 norms of the solution image, the residual error, and the image gradient. The relative weights are adjusted by two independent regularization parameters. Our initial tests of this imaging system were performed with an imaging phantom that consists of a translucent plastic cylinder filled with tissue-simulating liquid and two thin-wall glass tubes containing indocyanine green. The reconstruction is compared to the output of a finite element method-based software package NIRFAST and has produced promising results.

Zhang, Xiaofeng; Badea, Cristian; Jacob, Mathews; Johnson, G. Allan

2009-02-01

263

Bacteriophage ?6--structure investigated by fluorescence Stokes shift spectroscopy.  

PubMed

The Stokes shift of tryptophan (Trp) fluorescence from layers of the lipid-containing bacteriophage ?6 is compared to determine the relative effect of the layers on virus hydrophobicity. In the inner most layer, the empty procapsid (PC) which contains 80-90% of the virion Trp residues, ?(max) = 339.8 nm. The PC emission is substantially more redshifted than the other ?6 layers and nearer to that of the Pseudomonad host cell than the other ?6 layers. The Trp emission from the nucleocapsid (NC) with ?(max) ?= 337.4 nm, is blueshifted by 2.4 nm relative to the PC although the number of Trp in the NC is identical to the PC. This shift represents an increase in Trp hydrophobicity, likely a requirement for the maintenance of A-form doubled-stranded RNA. Fluorescence from the completely assembled virion indicates it is in a considerably more hydrophobic environment with ?(max) ?= 330.9 nm. Density measurements show that the water content in the NC does not change during envelope assembly, therefore the blueshifted ?6 emission suggests that the envelope changes the PC environment, probably via the P8 layer. This change in hydrophobicity likely arises from charge redistribution or envelope-induced structural changes in the PC proteins. PMID:22181691

Katz, Alvin; Alimova, Alexandra; Futerman, Elina; Katz, Garrett; Wei, Hui; Gottlieb, Paul

2012-01-01

264

In-Vivo NMR Spectroscopy of the Brain at High Fields  

Microsoft Academic Search

Increased magnetic fields in principle provide increased sensitivity and specificity. In vivo, however, the increase in magnetic\\u000a field alone does not automatically result in obvious improvements. Among the factors that are set to impede the improvements\\u000a in sensitivity for in-vivo NMR spectroscopy are the increased challenges in eliminating the macroscopic inhomogeneities caused\\u000a by mainly the air- tissue interface and increased

Rolf Gruetter; Pierre-Gilles Henry; Hongxia Lei; Silvia Mangia; Gülin Öz; Melissa Terpstra; Ivan Tkac

265

Uptake of trivalent actinides (curium(III)) by hardened cement paste: a time-resolved laser fluorescence spectroscopy study  

Microsoft Academic Search

The curium(III) interaction with cement was investigated using time-resolved laser fluorescence spectroscopy at trace concentrations. Four different Cm(III) species were identified: a nonfluorescing species which corresponds to curium hydroxide real colloids, which were characterized in detail by laser-induced breakdown detection (LIBD), a fluorescing Cm(III)\\/portlandite sorption species, and two fluorescing Cm(III)\\/calcium silicate hydrate (CSH) species. From the fluorescence emission lifetimes it

Thorsten Stumpf; Clemens Walther; Erich Wieland; Thomas Fanghänel

2004-01-01

266

Study of the interaction between icariin and human serum albumin by fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

The interaction between icariin and human serum albumin (HSA) in physiological buffer (pH 7.4) was investigated by fluorescence and UV-Vis absorption spectroscopy. Results obtained from analysis of fluorescence spectrum and fluorescence intensity indicated that icariin has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The thermodynamic parameters, ? H ? and ? S ?, were calculated to be 12.29 kJ mol -1 > 0, and 47.08 J mol -1 K -1 > 0, respectively, which suggested that hydrophobic force plays a major role in the reaction of icariin with HSA. The binding constants of icariin with HSA were determined at different temperatures by fluorescence quenching method. The distance r between donor (HSA) and acceptor (icariin) was calculated to be 4.18 nm based on Förster's non-radiative energy transfer theory. The results of synchronous fluorescence spectra and three-dimensional fluorescence spectra showed that binding of icariin to HSA can induce conformational changes in HSA.

Zhang, Guowen; Que, Qingmin; Pan, Junhui; Guo, Jinbao

2008-06-01

267

Doppler-free Yb spectroscopy with the fluorescence spot technique  

SciTech Connect

We demonstrate a simple technique to measure the resonant frequency of the 398.9-nm {sup 1}S{sub 0}{leftrightarrow}{sup 1}P{sub 1} transition for the different Yb isotopes. The technique, which works by observing and aligning fluorescence spots, has enabled us to measure transition frequencies and isotope shifts with an accuracy of 60 MHz. We provide wavelength measurements for the transition that differ from previously published work. Our technique also allows for the determination of Doppler-shifted transition frequencies for photoionization experiments when the atomic beam and the laser beam are not perpendicular and furthermore allows us to determine the average velocity of the atoms along the direction of the atomic beam.

Nizamani, Altaf H.; McLoughlin, James J.; Hensinger, Winfried K. [Department of Physics and Astronomy, University of Sussex, Falmer, Brighton, East-Sussex, BN1 9QH (United Kingdom)

2010-10-15

268

Fluorescence spectroscopy determination of fluoroquinolones by charge-transfer reaction.  

PubMed

The charge-transfer (CT) reaction between chloranilic acid (CL) as a pi-electron acceptor and lomefloxacin (LOM), fleroxacin (FLX), ciprofloxacin (CPFX), norfloxacin (NOR) as electron donor have been studied by fluorimetry. The CT complexes have stable purple color in acetone solution and the fluorescence intensity of the CT complexes was enhanced in 4-14 fold higher than that of the four fluoroquinolones (FQS) itself, therefore a new spectrofluorimetric method with simple, rapid, accurate, high sensitivity and good selectivity for determination of the four FQS has been developed. The method was applied for determination of drugs (LOM, FLX, CPFX and NOR) in tablets with mean percentage accuracies 99.80+/-1.12, 99.93+/-0.92, 99.23+/-1.36 and 99.87+/-0.81, respectively. PMID:14623595

Liming, Du; Qingqin, Xu; Jianmei, Yuan

2003-11-24

269

Multicolored two-photon fluorescent microscopy and localized two-photon fluorescent spectroscopy in living cells  

NASA Astrophysics Data System (ADS)

Imaging in biological systems has become one of the most relied upon tools in the study of human disease. Two-photon excitation methodology in laser scanning microscopy has resulted in 3D-imaging capability not easily achieved in one- photon systems. Our Institute, in conjunction with Andrew Schally (Noble Laureate, Tulane University), has used two- photon laser scanning microscopy (TPLSM) to understand the real time cellular transport of the chemotherapeutic agent, Luteinizing Hormone-Releasing Hormone-Doxorubicin (AN152) covalently coupled to a novel two-photon fluorophore (C625). At the Institute, new and highly efficient two-photon fluorophores that fluoresce at different wavelengths have been developed. The coupling of LH-RH and AN152 with two-photon fluorophores having different spectroscopic profiles allows for the simultaneous determination of their cellular compartmentalization. Coupled with the two-photon microspectrofluorometer, we acquired localized fluorescence spectra from the inside of living cells to differentiate the cytoplasmic and nuclear localization of the LH-RH and AN152 respectively. The ability of these new dyes to fluoresce at different wavelengths using the same excitation wavelength provides a major advantage over single photon dyes. This technology has great promise in imaging the dynamic changes or events occurring in living cells over short periods of time. Another approach to bioimaging at the Institute is the integration of two-photon and nanosized technologies. Nanoclinics (20 - 30 nm silica bubbles) can be fabricated to contain these two photon fluorophores and the surface functionalized with biological agents which can target specific cells. These highly fluorescent nanoclinics are sufficiently small in size to allow for tissue penetration, allowing for the multiple probing for different cellular functions in normal and cancerous tissues.

Bergey, Earl J.; Wang, Xiaopeng; Krebs, Linda J.; Pudavar, Haridas E.; Kapoor, Rakesh; Friend, Christopher S.; Liebow, Charles; Prasad, Paras N.

2001-04-01

270

AZIDE-SPECIFIC LABELLING OF BIOMOLECULES BY STAUDINGER-BERTOZZI LIGATION: PHOSPHINE DERIVATIVES OF FLUORESCENT PROBES SUITABLE FOR SINGLE-MOLECULE FLUORESCENCE SPECTROSCOPY  

PubMed Central

We describe the synthesis of phosphine derivatives of three fluorescent probes that have brightness and photostability suitable for single-molecule fluorescence spectroscopy and microscopy: Alexa488, Cy3B, and Alexa647. In addition, we describe procedures for use of these reagents in azide-specific, bioorthogonal labelling through use of the Staudinger-Bertozzi ligation and procedures for quantitation of labelling specificity and labelling efficiency. The reagents and procedures of this report enable chemoselective, site-selective labelling of azide-containing biomolecules for single-molecule fluorescence spectroscopy and microscopy. PMID:20580957

Chakraborty, Anirban; Wang, Dongye; Ebright, Yon W.; Ebright, Richard H.

2010-01-01

271

Application of fluorescence spectroscopy using a novel fluoroionophore for quantification of zinc in urban runoff.  

PubMed

Fluorescence spectroscopy has great potential for on-site and real-time monitoring of pollutants in aquatic environments; however, its application to environmental aquatic samples has been extremely limited. In this study, a novel fluoroionophore based on a BODIPY-terpyridine conjugate was developed and applied to determine Zn concentrations in urban runoff. The fluoroionophore selectively bound to Zn(2+) in water, which led to an instant red-shift of the fluorescence peak of the fluoroionophore from 539 nm to 567 nm that could be seen by the naked eye. Zn concentrations could be quantified using the ratio of fluorescence intensities, and the detection limit was 9 ?g/L, which is sufficiently low for environmental aquatic samples. To demonstrate applicability of the method to environmental samples, we measured Zn concentrations in urban runoff samples with a complex matrix (?60 mg/L dissolved organic carbon and ?20 mS/cm electrical conductivity). The total and dissolved fractions of Zn in the samples could be determined by fluorescence spectroscopy and its relative error was estimated to be less than 30% by inductively coupled plasma-atomic emission spectroscopy analysis. The proposed method is rapid and easy-to-use with simple pretreatment for Zn determination in environmental aquatic samples with complex matrices. PMID:24531076

Hafuka, Akira; Yoshikawa, Hiroaki; Yamada, Koji; Kato, Tsuyoshi; Takahashi, Masahiro; Okabe, Satoshi; Satoh, Hisashi

2014-05-01

272

TOPICAL REVIEW: Prospects for in vivo Raman spectroscopy  

NASA Astrophysics Data System (ADS)

Raman spectroscopy is a potentially important clinical tool for real-time diagnosis of disease and in situ evaluation of living tissue. The purpose of this article is to review the biological and physical basis of Raman spectroscopy of tissue, to assess the current status of the field and to explore future directions. The principles of Raman spectroscopy and the molecular level information it provides are explained. An overview of the evolution of Raman spectroscopic techniques in biology and medicine, from early investigations using visible laser excitation to present-day technology based on near-infrared laser excitation and charge-coupled device array detection, is presented. State-of-the-art Raman spectrometer systems for research laboratory and clinical settings are described. Modern methods of multivariate spectral analysis for extracting diagnostic, chemical and morphological information are reviewed. Several in-depth applications are presented to illustrate the methods of collecting, processing and analysing data, as well as the range of medical applications under study. Finally, the issues to be addressed in implementing Raman spectroscopy in various clinical applications, as well as some long-term directions for future study, are discussed.

Hanlon, E. B.; Manoharan, R.; Koo, T.-W.; Shafer, K. E.; Motz, J. T.; Fitzmaurice, M.; Kramer, J. R.; Itzkan, I.; Dasari, R. R.; Feld, M. S.

2000-02-01

273

Objective Assessment of Endogenous Collagen In Vivo during Tissue Repair by Laser Induced Fluorescence  

PubMed Central

Collagen, a triple helical protein with the primary role of mechanical function, provides tensile strength to the skin, and plays a pivotal task in tissue repair. During tissue regeneration, collagen level increases gradually and therefore, monitoring of such changes in vivo by laser induced fluorescence was the main objective behind the present study. In order to accomplish this, 15 mm diameter excisional wounds were created on six to eight week old Swiss albino mice. The collagen deposition accelerated upon irradiation of single exposure of 2 J/cm2 He-Ne laser dose immediately after wounding was recorded by laser induced autofluorescence in vivo along with un-illuminated and un-wounded controls. Autofluorescence spectra were recorded for each animal of the experimental groups on 0, 5, 10, 30, 45 and 60 days post-wounding, by exciting the granulation tissue/skin with 325 nm He-Cd laser. The variations in the average collagen intensities from the granulation tissue/skin of mice were inspected as a function of age and gender. Further, the spectral findings of the collagen synthesis in wound granulation tissue/un-wounded skin tissues were validated by Picro-Sirius red- polarized light microscopy in a blinded manner through image analysis of the respective collagen birefringence. The in vivo autofluorescence studies have shown a significant increase in collagen synthesis in laser treated animals as compared to the un-illuminated controls. Image analysis of the collagen birefringence further authenticated the ability of autofluorescence in the objective monitoring of collagen in vivo. Our results clearly demonstrate the potential of laser induced autofluorescence in the monitoring of collegen synthesis during tissue regeneration, which may have clinical implications. PMID:24874229

Prabhu, Vijendra; Rao, Satish B. S.; Fernandes, Edward Mark; Rao, Anuradha C. K.; Prasad, Keerthana; Mahato, Krishna K.

2014-01-01

274

Applicability of Fluorescence and Absorbance Spectroscopy to Estimate Organic Pollution in Rivers.  

PubMed

This article explores the applicability of fluorescence and absorbance spectroscopy for estimating organic pollution in polluted rivers. The relationship between absorbance, fluorescence intensity, dissolved organic carbon, biochemical oxygen demand (BOD), chemical oxygen demand (COD), and other water quality parameters were used to characterize and identify the origin and the spatial variability of the organic pollution in a highly polluted watershed. Analyses were performed for the Iguassu River, located in southern Brazil, with area about 2,700?km(2) and ?3 million inhabitants. Samples were collect at six monitoring sites covering 107?km of the main river. BOD, COD, nitrogen, and phosphorus concentration indicates a high input of sewage to the river. Specific absorbance at 254 and 285?nm (SUVA254 and A285/COD) did not show significant variation between sites monitored, indicating the presence of both dissolved compounds found in domestic effluents and humic and fulvic compounds derived from allochthonous organic matter. Correlations between BOD and tryptophan-like fluorescence peak (peak T2, r=0.7560, and peak T1, r=0.6949) and tyrosine-like fluorescence peak (peak B, r=0.7321) indicated the presence of labile organic matter and thus confirmed the presence of sewage in the river. Results showed that fluorescence and absorbance spectroscopy provide useful information on pollution in rivers from critical watersheds and together are a robust method that is simpler and more rapid than traditional methods employed by regulatory agencies. PMID:25469076

Knapik, Heloise Garcia; Fernandes, Cristovão Vicente Scapulatempo; de Azevedo, Júlio Cesar Rodrigues; do Amaral Porto, Monica Ferreira

2014-12-01

275

Strengths and Weaknesses of Recently Engineered Red Fluorescent Proteins Evaluated in Live Cells Using Fluorescence Correlation Spectroscopy  

PubMed Central

The scientific community is still looking for a bright, stable red fluorescent protein (FP) as functional as the current best derivatives of green fluorescent protein (GFP). The red FPs exploit the reduced background of cells imaged in the red region of the visible spectrum, but photophysical short comings have limited their use for some spectroscopic approaches. Introduced nearly a decade ago, mCherry remains the most often used red FP for fluorescence correlation spectroscopy (FCS) and other single molecule techniques, despite the advent of many newer red FPs. All red FPs suffer from complex photophysics involving reversible conversions to a dark state (flickering), a property that results in fairly low red FP quantum yields and potential interference with spectroscopic analyses including FCS. The current report describes assays developed to determine the best working conditions for, and to uncover the shortcoming of, four recently engineered red FPs for use in FCS and other diffusion and spectroscopic studies. All five red FPs assayed had potential shortcomings leading to the conclusion that the current best red FP for FCS is still mCherry. The assays developed here aim to enable the rapid evaluation of new red FPs and their smooth adaptation to live cell spectroscopic microscopy and nanoscopy. PMID:24129172

Siegel, Amanda P.; Baird, Michelle A.; Davidson, Michael W.; Day, Richard N.

2013-01-01

276

Integrated fluorescence correlation spectroscopy device for point-of-care clinical applications  

PubMed Central

We describe an optical system which reduces the cost and complexity of fluorescence correlation spectroscopy (FCS), intended to increase the suitability of the technique for clinical use. Integration of the focusing optics and sample chamber into a plastic component produces a design which is simple to align and operate. We validate the system by measurements on fluorescent dye, and compare the results to a commercial instrument. In addition, we demonstrate its application to measurements of concentration and multimerization of the clinically relevant protein von Willebrand factor (vWF) in human plasma. PMID:23847733

Olson, Eben; Torres, Richard; Levene, Michael J.

2013-01-01

277

Portable fluorescence spectroscopy platform for Huanglongbing (HLB) citrus disease in situ detection  

NASA Astrophysics Data System (ADS)

In this work, the development of a portable fluorescence spectroscopy platform for Huanglongbing (HLB) citrus disease in situ detection is presented. The equipment consists of an excitation blue LED light source, a commercial miniature spectrometer and embedded software. Measurements of healthy, HLB-symptomatic and HLB-asymptomatic citrus leafs were performed. Leafs were excited with the blue LED and their fluorescence spectra collected. Embedded electronics and software were responsible for the spectrum processing and classification via partial least squares regression. Global success rates above 80% and 100% distinction of healthy and HLB-symptomatic leafs were obtained.

Mota, Alessandro D.; Rossi, Giuliano; de Castro, Guilherme Cunha; Ortega, Tiago A.; de Castro N., Jarbas C.

2014-02-01

278

Analysis of protein-based binding media found in paintings using laser induced fluorescence spectroscopy.  

PubMed

Laser induced fluorescence (LIF) spectroscopy of intrinsic fluorophores from organic media found in paintings (casein, animal glue and egg proteins) provides novel non-invasive means of characterisation of general classes of media on the basis of fluorescence emission arising from the presence of certain amino acids and their degradation byproducts. Proteins from traditionally employed binding media include collagen, casein, albumin and other egg proteins, of animal sources (skins, milk and egg respectively). Wavelength dependence of the spectra is presented for analyses of thin films of protein-based binding media. PMID:17723543

Nevin, Austin; Cather, Sharon; Anglos, Demetrios; Fotakis, Costas

2006-07-28

279

Fluorescence correlation spectroscopy with visible-wavelength superconducting nanowire single-photon detector.  

PubMed

We present the first demonstration of fluorescence correlation spectroscopy (FCS) using superconducting nanowire single-photon detectors (SSPDs) which are free of afterpulses unlike the avalanche photodiode (APD). Multimode-fiber-coupled SSPDs with high system detection efficiency for visible wavelengths were developed and implemented in the FCS system. We performed FCS measurements for Rhodamine B and 6G as fluorescent samples, and found that autocorrelation functions obtained by the SSPDs showed a noise-free curve in the short correlation time region of sub microseconds where the afterpulse effect was dominant using the APD. The obtained results clearly indicate the advantage of SSPDs for the FCS system. PMID:25402117

Yamashita, Taro; Liu, Dengkuan; Miki, Shigehito; Yamamoto, Johtaro; Haraguchi, Tokuko; Kinjo, Masataka; Hiraoka, Yasushi; Wang, Zhen; Terai, Hirotaka

2014-11-17

280

Two-photon-excited fluorescence spectroscopy of atomic fluorine at 170 nm  

NASA Technical Reports Server (NTRS)

Two-photon-excited fluorescence spectroscopy of atomic fluorine is reported. A doubled dye laser at 286-nm is Raman shifted in H2 to 170 nm (sixth anti-Stokes order) to excite ground-state 2P(0)J fluorine atoms to the 2D(0)J level. The fluorine atoms are detected by one of two methods: observing the fluorescence decay to the 2PJ level or observing F(+) production through the absorption of an additional photon by the excited atoms. Relative two-photon absorption cross sections to and the radiative lifetimes of the 2D(0)J states are measured.

Herring, G. C.; Dyer, Mark J.; Jusinski, Leonard E.; Bischel, William K.

1988-01-01

281

Electron multiplying charge-coupled device-based fluorescence cross-correlation spectroscopy for blood velocimetry on zebrafish embryos  

NASA Astrophysics Data System (ADS)

Biomedical issues in vasculogenesis and cardiogenesis require methods to follow hemodynamics with high spatial (micrometers) and time (milliseconds) resolution. At the same time, we need to follow relevant morphogenetic processes on large fields of view. Fluorescence cross-correlation spectroscopy coupled to scanning or wide-field microscopy meets these needs but has limited flexibility in the excitation pattern. To overcome this limitation, we develop here a two-photon two-spots setup coupled to an all-reflective near-infrared (NIR) optimized scanning system and to an electron multiplying charge-coupled device. Two NIR laser spots are spaced at adjustable micron-size distances (1 to 50 ?m) by means of a Twyman-Green interferometer and repeatedly scanned on the sample, allowing acquisition of information on flows at 4 ms-3 ?m time-space resolution in parallel on an extended field of view. We analyze the effect of nonhomogeneous and variable flow on the cross-correlation function by numerical simulations and show exemplary application of this setup in studies of blood flow in zebrafish embryos in vivo. By coupling the interferometer with the scanning mirrors and by computing the cross-correlation function of fluorescent red blood cells, we are able to map speed patterns in embryos' vessels.

Pozzi, Paolo; Sironi, Laura; D'Alfonso, Laura; Bouzin, Margaux; Collini, Maddalena; Chirico, Giuseppe; Pallavicini, Piersandro; Cotelli, Franco; Foglia, Efrem A.

2014-06-01

282

Electron multiplying charge-coupled device-based fluorescence cross-correlation spectroscopy for blood velocimetry on zebrafish embryos.  

PubMed

Biomedical issues in vasculogenesis and cardiogenesis require methods to follow hemodynamics with high spatial (micrometers) and time (milliseconds) resolution. At the same time, we need to follow relevant morphogenetic processes on large fields of view. Fluorescence cross-correlation spectroscopy coupled to scanning or wide-field microscopy meets these needs but has limited flexibility in the excitation pattern. To overcome this limitation, we develop here a two-photon two-spots setup coupled to an all-reflective near-infrared (NIR) optimized scanning system and to an electron multiplying charge-coupled device. Two NIR laser spots are spaced at adjustable micron-size distances (1 to 50 ?m) by means of a Twyman-Green interferometer and repeatedly scanned on the sample, allowing acquisition of information on flows at 4 ms-3 ?m time-space resolution in parallel on an extended field of view. We analyze the effect of nonhomogeneous and variable flow on the cross-correlation function by numerical simulations and show exemplary application of this setup in studies of blood flow in zebrafish embryos in vivo. By coupling the interferometer with the scanning mirrors and by computing the cross-correlation function of fluorescent red blood cells, we are able to map speed patterns in embryos' vessels. PMID:24946713

Pozzi, Paolo; Sironi, Laura; D'Alfonso, Laura; Bouzin, Margaux; Collini, Maddalena; Chirico, Giuseppe; Pallavicini, Piersandro; Cotelli, Franco; Foglia, Efrem A

2014-06-01

283

Laser-induced fluorescence spectroscopy of benign and malignant cutaneous lesions  

NASA Astrophysics Data System (ADS)

The goals of this work were investigation of pigmented skin lesions by the method of laser-induced fluorescence spectroscopy. Fluorescence spectra were obtained from malignant and benign skin lesions after excitation with nitrogen laser at 337 nm, namely: benign nevi, dysplastic nevi, malignant melanoma (MM), keratopapilloma, base-cell papilloma and base-cell carcinoma, as well as from healthy skin areas near to the lesion that were used posteriori to reveal changes between healthy and lesion skin spectra. Initially lesions were classified by ABCD-dermatscopic method. All suspicious lesions were excised and were investigated histologically. Spectrum of healthy skin consists of one main maximum at 470-500 nm spectral region and secondary maxima at in the regions round 400 and 440 nm. In the cases of nevi and melanoma significant decrease of fluorescence intensity, which correlated with the type of pigment lesion was observed. This reduction of the signal is related to the accumulation of melanin in the lesions that re-absorb strongly the fluorescence from native skin fluorophores in whole visible spectral region. In cases of papilloma and base-cell carcinoma an intensity decrease was also observed, related to accumulation of pigments in these cutaneous lesions. An relative increase of the fluorescence peak at 440 nm were registered in the case of base-cell carcinoma, and appearance of green fluorescence, related to increase of keratin content in benign papilloma lesions were detected. The results, obtained in this investigation of the different pigment lesions could be used for better comprehension of the skin optical properties. The fluorescence spectroscopy of the human skin are very prominent for early diagnosis and differentiation of cutaneous diseases and gives a wide range of possibilities related to real-time determination of existing pathological condition.

Borisova, Ekaterina G.; Troyanova, P. P.; Stoyanova, V. P.; Avramov, Lachezar A.

2005-04-01

284

In Vivo X-Ray Fluorescence Microtomographic Imaging of Elements in Single-Celled Fern Spores  

NASA Astrophysics Data System (ADS)

We have observed in vivo three-dimensional distributions of constituent elements of single-celled spores of the fern Adiantum capillus-veneris using an X-ray fluorescence computed microtomography method. The images of these distributions are generated from a series of slice data, each of which is acquired by a sample translation-rotation method. An incident X-ray microbeam irradiates the sample with a spot size of 1 ?m. The high Ca concentration in the testa and the localized and overlapping Fe and Zn concentrations inside the spore are shown in three-dimensional images. The K concentration is high throughout the cell, and there are localized regions of higher density. The atomic number densities of these elements in the testa and inside the cell in a tomographic slice are estimated with a resolution of about 1 ?m.

Hirai, Yasuharu; Yoneyama, Akio; Hisada, Akiko; Uchida, Kenko

2007-01-01

285

In Vivo X-Ray Fluorescence Microtomographic Imaging of Elements in Single-Celled Fern Spores  

SciTech Connect

We have observed in vivo three-dimensional distributions of constituent elements of single-celled spores of the fern Adiantum capillus-veneris using an X-ray fluorescence computed microtomography method. The images of these distributions are generated from a series of slice data, each of which is acquired by a sample translation-rotation method. An incident X-ray microbeam irradiates the sample with a spot size of 1 {mu}m. The high Ca concentration in the testa and the localized and overlapping Fe and Zn concentrations inside the spore are shown in three-dimensional images. The K concentration is high throughout the cell, and there are localized regions of higher density. The atomic number densities of these elements in the testa and inside the cell in a tomographic slice are estimated with a resolution of about 1 {mu}m.

Hirai, Yasuharu; Yoneyama, Akio; Hisada, Akiko [Advanced Research Laboratory, Hitachi, Ltd., Hatoyama, Saitama 350-0395 (Japan); Uchida, Kenko [Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo 185-8601 (Japan)

2007-01-19

286

Subcellular In Vivo 1 HM R Spectroscopy ofXenopus laevis Oocytes  

Microsoft Academic Search

In vivo magnetic resonance (MR) spectra are typically obtained from voxels whose spatial dimensions far exceed those of the cells they contain. This study was designed to evaluate the potential of localized MR spectroscopy to investigate subcellular phenomena. Using a high magnetic field and a home-built microscopy probe with large gradient field strengths, we achieved voxel sizes of (180 mm)3.

Seung-Cheol Lee; Jee-Hyun Cho; Daniel Mietchen; Young-Sook Kim; Kwan Soo Hong; Chulhyun Lee; Dongmin Kang; Byong-Seok Choi; Chaejoon Cheong

2006-01-01

287

Subcellular In Vivo 1 H MR Spectroscopy of Xenopus laevis Oocytes  

E-print Network

Subcellular In Vivo 1 H MR Spectroscopy of Xenopus laevis Oocytes Seung-Cheol Lee,* Jee-Hyun Cho voxel sizes of (180 mm)3 . In the large oocytes of the frog Xenopus laevis, this was small enough) microscopic imaging of a large single cell, the Xenopus laevis oocyte whose di- ameter is ;1.2 mm. Since then

Hammerton, James

288

The use of fluorescent target arrays for assessment of T cell responses in vivo.  

PubMed

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into > 250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8(+) and CD4(+) T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8(+) T cell-mediated killing of FTA target cells and CD4(+) T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since > 250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g. the cumulative magnitude of the response) and a qualitative (e.g. functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine. PMID:24998253

Quah, Benjamin J C; Wijesundara, Danushka K; Ranasinghe, Charani; Parish, Christopher R

2014-01-01

289

Quantifying receptor density in vivo using a dual probe approach with fluorescence molecular imaging  

NASA Astrophysics Data System (ADS)

Molecular imaging technologies are advancing rapidly and optical techniques in particular are set to play a large role in preclinical pharmaceutical testing. These approaches, however, are generally unable to quantify the level of expression of imaging probe reporters. In this study a novel method of quantification is presented using dual-probe fluorescence imaging, where an endothelial growth factor receptor (EGFR) fluorescent probe was paired with a non-targeted probe before being injected, and tracer kinetic compartmental modeling was used to determine EGFR expression in a region of interest from the uptake curves of the two drugs in that region. The approach was tested out in a simulation experiment and then applied in an in vivo study in one mouse to investigate EGFR expression in various tissue types (pancreas, pancreas tumor, and leg). The binding potentials (a unitless correlate of target availability) of EGFR expression in the various tissue types were 8.57, 25.64, and 0.11 for the pancreas, pancreas tumor, respectively. For the pancreas and leg, these results correlate well with expected levels of EGFR expression, with the pancreas demonstrating a much higher expression than the skin and also as expected, the tumor expressed much more EGFR than either healthy tissue.

Tichauer, Kenneth M.; Samkoe, Kimberley S.; O'Hara, Julia; Sexton, Kristian J.; Davis, Scott C.; Pogue, Brian W.

2011-03-01

290

NOTE In vivo quantification of lead in bone with a portable x-ray fluorescence system--methodology and feasibility  

Microsoft Academic Search

This study was conducted to investigate the methodology and feasibility of developing a portable x-ray fluorescence (XRF) technology to quantify lead (Pb) in bone in vivo. A portable XRF device was set up and optimal settings of voltage, current, and filter combination for bone lead quantification were selected to achieve the lowest detection limit. The minimum radiation dose delivered to

L. H. Nie; S. Sanchez; K. Newton; L. Grodzins; R. O. Cleveland; M. G. Weisskopf

2011-01-01

291

In vivo quantification of lead in bone with a portable x-ray fluorescence system---methodology and feasibility  

Microsoft Academic Search

This study was conducted to investigate the methodology and feasibility of developing a portable x-ray fluorescence (XRF) technology to quantify lead (Pb) in bone in vivo. A portable XRF device was set up and optimal settings of voltage, current, and filter combination for bone lead quantification were selected to achieve the lowest detection limit. The minimum radiation dose delivered to

L. H. Nie; S. Sanchez; K. Newton; L. Grodzins; R. O. Cleveland; M. G. Weisskopf

2011-01-01

292

View of Normal Human Skin In Vivo as Observed Using Fluorescent Fiber-Optic Confocal Microscopic Imaging  

Microsoft Academic Search

Fluorescence confocal scanning laser microscopy, using a miniaturized handheld scanner, was performed to visualize the microscopic architecture of normal human epidermis in vivo. Fluorescein sodium (?20 ?L of 0.2% wt\\/vol) was administered via intradermal injection to normal skin on the volar forearm of 22 patients. The skin was imaged continuously from 1 to 15 min after injection. Fluorescein was excited

Lucinda D. Swindle; Steven G. Thomas; Michael Freeman; Peter M. Delaney

2003-01-01

293

In vivo optical imaging of dihydroethidium oxidation in the mouse brain employing fluorescence intensity and lifetime contrast  

NASA Astrophysics Data System (ADS)

Reactive oxygen species (ROS) are believed to be involved in many diseases and injuries to the brain, but the molecular processes are not well understood due to a lack of in vivo imaging techniques to evaluate ROS. The fluorescent oxidation products of dihydroethidium (DHE) can monitor ROS production in vivo. Here we demonstrate the novel optical imaging of brain in live mice to measure ROS production via generation of fluorescent DHE oxidation products (ox-DHE) by ROS. We show that in Sod2+/- mice, which have partial loss of a key antioxidant enzyme, superoxide dismutase-2, that ox-DHE fluorescence intensity was significantly higher than in hSOD1 mice, which have four-fold overexpression of superoxide dismutase-1 activity, which had almost no ox-DHE fluorescence, confirming specificity of ox-DHE to ROS production. The DHE oxidation products were also confirmed by detecting a characteristic fluorescence lifetime of the oxidation product, which was validated with ex vivo measurements.

Hall, David J.; Han, Sung-Ho; Dugan, Laura

2009-02-01

294

In vivo confocal scanning laser microscopy: comparison of the reflectance and fluorescence mode by imaging human skin  

NASA Astrophysics Data System (ADS)

Optical, noninvasive methods have become efficient in vivo tools in dermatological diagnosis and research. From these promising imaging techniques, only the confocal scanning laser microscopy (CSLM) provides visualization of subsurface skin structures with resolutions similar to those of light microscopy. Skin annexes, as well as cutaneous cells from different epidermal layers, can be distinguished excellently. Currently, two forms of application have been established in dermatological practice: the reflectance mode, predominantly in the clinical field, and the fluorescence mode in dermatological research. Differences in both methods exist in the preparative protocol, in maximum imaging depth and, particularly, in the gain of contrast extraction. The reflectance mode demonstrates naturally occurring tissue components, whereas the fluorescent CSLM achieves contrast by administering fluorescence dye, representing the dynamic distribution pattern of the dye's fluorescent emission. Therefore, the reflectance and fluorescent modes highlight various skin microstructures, providing dissimilar in vivo confocal images of the skin. This permits different predications and information on the state of the tissue. We report the advantages and disadvantages of both optical imaging modes. The comparison was drawn by scanning human skin in vivo. Representative images in varying depths were obtained and analyzed; preparation procedures are shown and discussed.

Meyer, Lars E.; Otberg, Nina; Sterry, Wolfram; Lademann, Jürgen

2006-07-01

295

Spot Variation Fluorescence Correlation Spectroscopy Allows for Superresolution Chronoscopy of Confinement Times in Membranes  

PubMed Central

Resolving the dynamical interplay of proteins and lipids in the live-cell plasma membrane represents a central goal in current cell biology. Superresolution concepts have introduced a means of capturing spatial heterogeneity at a nanoscopic length scale. Similar concepts for detecting dynamical transitions (superresolution chronoscopy) are still lacking. Here, we show that recently introduced spot-variation fluorescence correlation spectroscopy allows for sensing transient confinement times of membrane constituents at dramatically improved resolution. Using standard diffraction-limited optics, spot-variation fluorescence correlation spectroscopy captures signatures of single retardation events far below the transit time of the tracer through the focal spot. We provide an analytical description of special cases of transient binding of a tracer to pointlike traps, or association of a tracer with nanodomains. The influence of trap mobility and the underlying binding kinetics are quantified. Experimental approaches are suggested that allow for gaining quantitative mechanistic insights into the interaction processes of membrane constituents. PMID:21641330

Ruprecht, Verena; Wieser, Stefan; Marguet, Didier; Schütz, Gerhard J.

2011-01-01

296

Combined fluorescence and X-Ray tomography for quantitative in vivo detection of fluorophore.  

PubMed

Initial results from a novel dual modality preclinical imager which combines non-contact fluorescence tomography (FT) and x-ray computed tomography (CT) for preclinical functional and anatomical in vivo imaging are presented. The anatomical data from CT provides a priori information to the FT reconstruction to create overlaid functional and anatomical images with accurate localization and quantification of fluorophore distribution. Phantoms with inclusions containing Indocyanine-Green (ICG), and with heterogeneous backgrounds including iodine in compartments at different concentrations for CT contrast, have been imaged with the dual modality FT/CT system. Anatomical information from attenuation maps and optical morphological information from absorption and scattering maps are used as a priori information in the FT reconstruction. Although ICG inclusions can be located without the a priori information, the recovered ICG concentration shows 75% error. When the a priori information is utilized, the ICG concentration can be recovered with only 15% error. Developing the ability to accurately quantify fluorophore concentration in anatomical regions of interest may provide a powerful tool for in vivo small animal imaging. PMID:20082529

Barber, W C; Lin, Y; Nalcioglu, O; Iwanczyk, J S; Hartsough, N E; Gulsen, G

2010-02-01

297

Combined Fluorescence and X-Ray Tomography for Quantitative In Vivo Detection of Fluorophore  

PubMed Central

Initial results from a novel dual modality preclinical imager which combines non-contact fluorescence tomography (FT) and x-ray computed tomography (CT) for preclinical functional and anatomical in vivo imaging are presented. The anatomical data from CT provides a priori information to the FT reconstruction to create overlaid functional and anatomical images with accurate localization and quantification of fluorophore distribution. Phantoms with inclusions containing Indocyanine-Green (ICG), and with heterogeneous backgrounds including iodine in compartments at different concentrations for CT contrast, have been imaged with the dual modality FT/CT system. Anatomical information from attenuation maps and optical morphological information from absorption and scattering maps are used as a priori information in the FT reconstruction. Although ICG inclusions can be located without the a priori information, the recovered ICG concentration shows 75% error. When the a priori information is utilized, the ICG concentration can be recovered with only 15% error. Developing the ability to accurately quantify fluorophore concentration in anatomical regions of interest may provide a powerful tool for in vivo small animal imaging. PMID:20082529

Barber, W. C.; Lin, Y.; Nalcioglu, O.; Iwanczyk, J. S.; Hartsough, N. E.; Gulsen, G.

2010-01-01

298

Assessment of phytoplankton class abundance using in vivo synchronous fluorescence spectra.  

PubMed

In this study the feasibility of using the in vivo synchronous fluorescence spectra (SFS) of phytoplankton samples for determining the relative abundance of specific classes of phytoplankton was investigated. In total, 405 SFS of nine phytoplankton species cultivated under different conditions were measured and evaluated. First, principal component analysis (PCA) was used to obtain nine representative spectra, and three feature spectrum bands at 200-235, 310-335, and 355-585 nm of SFS were found to have a better discriminatory capability. The nine phytoplankton species were spectrographically sorted into six classes. Second, a relationship between the chlorophyll a (chla) concentration of phytoplankton extracts and the in vivo SFS intensity was found; thus the corresponding phytoplankton class abundance can be expressed by the concentration of standard chla. For this data set, the detection limit ranged 1.02-6.89 microg/L chla for different classes. Finally, qualitative and quantitative analyses of 30 mixtures of phytoplankton were carried out using the nonnegative least square regression (NNLS) method. The dominant phytoplankton classes could be identified while the qualitative recognition correctness rate was 88% and the quantitative standard deviation with fluorometrically determined chla was -0.14-0.04. Hence, it was possible to estimate the abundance of dominant phytoplankton classes. PMID:18328250

Li, Hongyu; Zhang, Qianqian; Zhu, Chenjian; Wang, Xiulin

2008-06-01

299

Fluorescence Lifetime Imaging and Spectroscopy as Tools for Nondestructive Analysis of Works of Art  

NASA Astrophysics Data System (ADS)

A system for advanced fluorescence investigation of works of art has been assembled and integrated in a characterization procedure that allows one to localize and identify organic compounds that are present in artworks. At the beginning of the investigation, fluorescence lifetime imaging and spectroscopy address a selective microsampling of the artwork. Then analytical measurements of microsamples identify the chemical composition of the materials under investigation. Finally, on the basis of fluorescence lifetime and amplitude maps, analytical data are extended to the whole artwork. In such a way, information on the spatial distribution of organic materials can be inferred. These concepts have been successfully applied in an extensive campaign for analysis of Renaissance fresco paintings in Castiglione Olona, Italy. Residue of various types of glue and stucco left from a restoration carried out in the early 1970s was localized and classified. Insight into the technique used by the painter to make gilded reliefs was also obtained.

Comelli, Daniela; D'Andrea, Cosimo; Valentini, Gianluca; Cubeddu, Rinaldo; Colombo, Chiara; Toniolo, Lucia

2004-04-01

300

Excited state processes of nitrobenzaldehydes probed by ultrafast fluorescence and absorption spectroscopy  

Microsoft Academic Search

We have investigated the isomers ortho-, meta-, and para-nitro-benzaldehyde (NBA) by means of ultrafast emission and absorption spectroscopy. The fluorescence dynamics of all three molecules are very similar and feature bi-phasic emission decays with time constants of <100fs and ?1ps. The first process goes along with a strong loss of oscillator strength, which we assume is the result of a

B. Heinz; T. Schmierer; S. Laimgruber; P. Gilch

2008-01-01

301

TOTAL INTERNAL REFLECTION WITH FLUORESCENCE CORRELATION SPECTROSCOPY: APPLICATIONS TO SUBSTRATE-SUPPORTED PLANAR MEMBRANES  

PubMed Central

In this review paper, the conceptual basis and experimental design of total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) is described. The few applications to date of TIR-FCS to supported membranes are discussed, in addition to a variety of applications not directly involving supported membranes. Methods related, but not technically equivalent, to TIR-FCS are also summarized. Future directions for TIR-FCS are outlined. PMID:19269331

Thompson, Nancy L.; Wang, Xiang; Navaratnarajah, Punya

2009-01-01

302

New energy levels of atomic niobium by laser induced fluorescence spectroscopy in the near infrared  

NASA Astrophysics Data System (ADS)

Laser-induced fluorescence spectroscopy was applied in order to find new energy levels of the niobium atom. A continuous wave tuneable titanium–sapphire laser in the wavelength range from 750 to 865 nm and a hollow-cathode lamp were used. We discovered four energy levels of even parity, three lying levels below 19 000 cm?1 and one at much higher energy. Additionally hyperfine structure data of six levels of odd parity were determined.

Öztürk, I. K.; Ba?ar, Gö; Er, A.; Güzelçimen, F.; Ba?ar, Gü; Kröger, S.

2015-01-01

303

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo  

PubMed Central

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS. The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation. PMID:23928906

Lyons-Warren, Ariel M.; Kohashi, Tsunehiko; Mennerick, Steven; Carlson, Bruce A.

2013-01-01

304

Real time in vivo investigation of superoxide dynamics in zebrafish liver using a single-fiber fluorescent probe  

PubMed Central

Superoxide anion is the key radical 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 yellow fluorescent proteins, a reversible superoxide-specific indicator, in the liver and used a fiber-optic fluorescent probe to noninvasively monitor the superoxide concentration in real time. Several superoxide-inducing and scavenging reagents were administrated onto the fish to alter superoxide concentrations. The distinct biochemical pathways of the reagents can be discerned from the transient behaviors of fluorescence time courses. These results demonstrate the feasibility of this method for analyzing superoxide dynamics and its potential as an in vivo pharmaceutical screening platform. PMID:24049691

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

2013-01-01

305

Small molecule fluorophore and copolymer RGD peptide conjugates for ex vivo two-photon fluorescence tumor vasculature imaging  

PubMed Central

We report the use of small molecule and block copolymer RGD peptide conjugates for deep ex vivo imaging of tumor vasculature in “whole” excised tumors using two-photon fluorescence microscopy (2PFM). The fluorescent probes were administered to mice via tail-vein injection, after which the tumors were excised, fixed, and imaged without further sample preparation. Both RGD conjugates demonstrated specific targeting to tumor blood vessels, and this selectivity imparted excellent contrast in 2PFM micrographs that captured high-resolution 3-D images of the tumor vasculature up to depths of 830 ?m in Lewis Lung Carcinoma (LLC) tumors. 2PFM ex vivo fluorescence micrographs clearly revealed tumor vessels, while differences in the sensitivity of tumor vessel imaging were apparent between the small molecule and block copolymer conjugates. Both the small molecule and polymer-based two-photon absorbing probe conjugate are valuable for deep tissue tumor microvasculature imaging. PMID:22940216

Morales, Alma R.; Yanez, Ciceron O.; Zhang, Yuanwei; Wang, Xuhua; Biswas, Sanchita; Urakami, Takeo; Komatsu, Masanobu; Belfield, Kevin D.

2012-01-01

306

Exploring the binding mechanism of Guaijaverin to human serum albumin: fluorescence spectroscopy and computational approach.  

PubMed

The Guaijaverin (Gua) is a polyphenolic substance which exhibits some pharmacological activities such as antibacterial and antioxidant activities. Here we have investigated the binding of Gua with human serum albumin (HSA) at physiological pH 7.0. In this study, the fluorescence spectroscopy, ab initio and molecular modeling calculations were applied. The Stern-Volmer quenching constant (K(SV)) and its modified form (K(a)) were calculated at 298, 303 and 308 K, with the corresponding thermodynamic parameters ?H, ?G and ?S as well. The fluorescence quenching method was used to determine the number of binding sites (n) and binding constants (K(b)) values at 298, 303 and 308 K. The distance between donor (HSA) and acceptor (Gua) was estimated according to fluorescence resonance energy transfer. The geometry optimization of Gua was performed in its ground state by using ab initio DFT/B3LYP functional with a 6-31G(d,p) basis set used in calculations. Molecular modeling calculation indicated that the Gua is located within the hydrophobic pocket of the subdomain IIA of HSA. The theoretical results obtained by molecular modeling were corroborated by fluorescence spectroscopy data. PMID:22820048

Caruso, Icaro P; Vilegas, Wagner; Fossey, Marcelo A; Cornélio, Marinônio L

2012-11-01

307

Study of the potential of stratum corneum lipids and exogenous molecules interaction by fluorescence spectroscopy for the estimation of percutaneous penetration.  

PubMed

Considering that the skin barrier properties are closely linked to the ceramides composition and conformation within the SC, our work focused on developing a new evaluation criterion in complement of the Log Pow and MW: lipids retentive role within the SC. We developed an in vitro model to study exogenous molecules (Mol) and SC lipids interaction by fluorescence spectroscopy. As ceramides do not fluoresce, fluorescence probes that emit a fluorescence signal in contact with lipidic chains were selected for the study. A protocol was developed based on the exogenous molecule (cosmetic actives) affinity for the SC lipids. A fluorescence criterion (?I) was calculated from our results and compared to ex vivo skin penetration measurements realized with a Franz cell device. Our results indicated that polarity seems to be very representative of the ceramide and exogenous molecule interaction for most of the molecules tested. However, the ?I calculated highlighted the particular interaction of some exogenous molecules with ceramides and their skin distribution. This particular behavior was not initially possible to estimate with the Log Pow and MW. This work aimed to develop a new alternative method to enhance the percutaneous penetration estimation of exogenous molecules for the risk analysis. PMID:22659149

Jungman, Elsa; Laugel, Cécile; Kasselouri, Athéna; Baillet-Guffroy, Arlette

2012-09-15

308

Multimodal Mn-doped I-III-VI quantum dots for near infrared fluorescence and magnetic resonance imaging: from synthesis to in vivo application  

NASA Astrophysics Data System (ADS)

The development of sensitive multimodal contrast agents is a key issue to provide better global, multi-scale images for diagnostic or therapeutic purposes. Here we present the synthesis of Zn-Cu-In-(S, Se)/Zn1-xMnxS core-shell quantum dots (QDs) that can be used as markers for both near-infrared fluorescence imaging and magnetic resonance imaging (MRI). We first present the synthesis of Zn-Cu-In-(S, Se) cores coated with a thick ZnS shell doped with various proportions of Mn. Their emission wavelengths can be tuned over the NIR optical window suitable for deep tissue imaging. The incorporation of manganese ions (up to a few thousand ions per QD) confers them a paramagnetic character, as demonstrated by structural analysis and electron paramagnetic resonance spectroscopy. These QDs maintain their optical properties after transfer to water using ligand exchange. They exhibit T1-relaxivities up to 1400 mM-1 [QD] s-1 at 7 T and 300 K. We finally show that these QDs are suitable multimodal in vivo probes and demonstrate MRI and NIR fluorescence detection of regional lymph nodes in mice.The development of sensitive multimodal contrast agents is a key issue to provide better global, multi-scale images for diagnostic or therapeutic purposes. Here we present the synthesis of Zn-Cu-In-(S, Se)/Zn1-xMnxS core-shell quantum dots (QDs) that can be used as markers for both near-infrared fluorescence imaging and magnetic resonance imaging (MRI). We first present the synthesis of Zn-Cu-In-(S, Se) cores coated with a thick ZnS shell doped with various proportions of Mn. Their emission wavelengths can be tuned over the NIR optical window suitable for deep tissue imaging. The incorporation of manganese ions (up to a few thousand ions per QD) confers them a paramagnetic character, as demonstrated by structural analysis and electron paramagnetic resonance spectroscopy. These QDs maintain their optical properties after transfer to water using ligand exchange. They exhibit T1-relaxivities up to 1400 mM-1 [QD] s-1 at 7 T and 300 K. We finally show that these QDs are suitable multimodal in vivo probes and demonstrate MRI and NIR fluorescence detection of regional lymph nodes in mice. Electronic supplementary information (ESI) available: Determination of Mn content; magnetization measurements; additional TEM and spectroscopic data; additional NIR fluorescence image; MTT assay results. See DOI: 10.1039/c4nr02239d

Sitbon, Gary; Bouccara, Sophie; Tasso, Mariana; Francois, Aurélie; Bezdetnaya, Lina; Marchal, Frédéric; Beaumont, Marine; Pons, Thomas

2014-07-01

309

DETECTION OF MERCURIC BROMIDE IN A GAS PHASE FLOW CELL BY LASER PHOTOFRAGMENT FLUORESCENCE SPECTROSCOPY. (R825380)  

EPA Science Inventory

Photofragment fluorescence (PFF) spectroscopy offers real-time monitoring capability with high-analytical sensitivity and selectivity for volatile mercury compounds found in process gas streams, such as incinerator stacks. In this work, low concentrations (6 ppb to...

310

Assessment of In Vivo Mitochondrial Metabolism by Magnetic Resonance Spectroscopy  

PubMed Central

Magnetic resonance spectroscopy (MRS), a companion technique to the more familiar MRI scan, has emerged as a powerful technique for studying metabolism noninvasively in a variety of tissues. In this article, we review two techniques that we have developed which take advantage of the unique characteristics of 31P and 13C MRS to investigate two distinct parameters of muscle mitochondrial metabolism; ATP production can be estimated by using the 31P saturation-transfer technique, and oxidation via the TCA cycle can be modeled from 13C MRS data obtained during the metabolism of a 13C-labeled substrate. We will also examine applications of the techniques to investigate how these parameters of muscle mitochondrial metabolism are modulated in insulin resistant and endurance trained individuals. PMID:19426879

Befroy, Douglas E.; Petersen, Kitt Falk; Rothman, Douglas L.; Shulman, Gerald I.

2011-01-01

311

On-chip integrated lensless fluorescence microscopy/spectroscopy module for cell-based sensors  

NASA Astrophysics Data System (ADS)

The integration of a fluorescence microscopy/spectroscopy module in cell-based lab-on-a-chip systems is of high interest for applications in cell-based diagnostics and substance evaluation in situ. We present an on-chip integrated lensless fluorescence imaging module applying the principle of contact/proximate optical lithography. The pixel resolution is comparable with a 4 x objective microscope. The module can be used for morphology and fluorescence imaging of mammalian cells (15 - 20 ?m) as well as for testing the concentration of a fluorescent substance. The biological samples or solutions are sustained in disposable sterilized microfluidic chips with 1 ?m thick silicon nitride (Si3N4) membranes. These chips are assembled on the surface of a 5 megapixel colored CMOS image sensor array with 1.75 ?m pixel size, which is coated with an additional interference filter. Each culturing chip consists of a MEMS cavity chip and a PDMS microfluidic interface. The surface of the CMOS image sensor is smoothened using SU-8 photoresist spin-coating for a commercial grade interference filter (optical density >= 5) coating by Plasma-Ion Assisted Deposition thereafter. The function is demonstrated by primary imaging results of the non-/fluorescent mammalian cells/microspheres as well as by differentiating different concentrations of FITC solutions.

Li, Wei; Knoll, Thorsten; Sossalla, Adam; Bueth, Heiko; Thielecke, Hagen

2011-03-01

312

Construction, figures of merit, and testing of a single-cell fluorescence excitation spectroscopy system  

PubMed Central

Characterization of phytoplankton community composition is critical to understanding the ecology and biogeochemistry of the oceans. One approach to taxonomic characterization takes advantage of differing pigmentation between algal taxa and thus differences in fluorescence excitation spectra. Analyses of bulk water samples, however, may be confounded by interference from chromophoric dissolved organic matter or suspended particulate matter. Here, we describe an instrument that uses a laser trap based on a Nikon TE2000-U microscope to position individual phytoplankton cells for confocal fluorescence excitation spectroscopy, thus avoiding interference from the surrounding medium. Quantitative measurements of optical power give data in the form of photons emitted per photon of exposure for an individual phytoplankton cell. Residence times for individual phytoplankton in the instrument can be as long as several minutes with no substantial change in their fluorescence excitation spectra. The laser trap was found to generate two-photon fluorescence from the organisms so a modification was made to release the trap momentarily during data acquisition. Typical signal levels for an individual cell are in the range of 106 photons?s of fluorescence using a monochromated 75 W Xe arc lamp excitation source with a 2% transmission neutral density filter. PMID:20113077

Hill, Laura S.; Richardson, Tammi L.; Profeta, Luisa T. M.; Shaw, Timothy J.; Hintz, Christopher J.; Twining, Benjamin S.; Lawrenz, Evelyn; Myrick, Michael L.

2010-01-01

313

Applications of fluorescence spectroscopy for predicting percent wastewater in an urban stream  

USGS Publications Warehouse

Dissolved organic carbon (DOC) is a significant organic carbon reservoir in many ecosystems, and its characteristics and sources determine many aspects of ecosystem health and water quality. Fluorescence spectroscopy methods can quantify and characterize the subset of the DOC pool that can absorb and re-emit electromagnetic energy as fluorescence and thus provide a rapid technique for environmental monitoring of DOC in lakes and rivers. Using high resolution fluorescence techniques, we characterized DOC in the Tualatin River watershed near Portland, Oregon, and identified fluorescence parameters associated with effluent from two wastewater treatment plants and samples from sites within and outside the urban region. Using a variety of statistical approaches, we developed and validated a multivariate linear regression model to predict the amount of wastewater in the river as a function of the relative abundance of specific fluorescence excitation/emission pairs. The model was tested with independent data and predicts the percentage of wastewater in a sample within 80% confidence. Model results can be used to develop in situ instrumentation, inform monitoring programs, and develop additional water quality indicators for aquatic systems.

Goldman, Jami H.; Rounds, Stewart A.; Needoba, Joseph A.

2012-01-01

314

Biodistribution of benzoporphyrin derivative in tumor-bearing rats by laser-induced fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

The goal of this study was to detect the presence of benzoporphyrin derivative-monoacid (BPD-MA) in tissues of a tumor bearing animal model. Eighty one Lobund-Wistar rats, inoculated with Pollard rat adenocarcinoma cells, were used. This animal model exhibits unique predictable, unilateral, metastatic spread. The animals were injected intravenously with 0.75 mg/kg of BPD-MA. A Helium-Cadmium (He-Cd) laser (442 nm, 17 mW) was used as an excitation source and coupled to a 400 micrometers core diameter fiber. Following laparotomy, exploration of the abdominal and inguinal area was performed with laser induced fluorescence. Fluorescence spectra of the primary tumor, bilateral lymph nodes, and various organs were recorded. Fluorescence measurements were conducted four hours post injection. The spectra obtained were characterized by a broadband autofluorescence (approximately 540 nm) and a characteristic peak of BPD-MA (approximately 690 nm). Overall, the BPD-MA concentration was higher in lymph nodes than in the skin, kidney, large bowel, muscle or spleen. Skin exhibited the lowest fluorescence intensity ratio, indicative of a lower drug concentration in this tissue. In summary, our results suggest that laser induced fluorescence spectroscopy may provide an alternative way of assessing the biodistribution of BPD-MA or other photosensitizers.

Vari, Sandor G.; Stavridi, Marigo; Papaioannou, Thanassis; Papazoglou, Theodore G.; Pergadia, Vani R.; Fishbein, Michael C.; Wolfson, David; Grundfest, Warren S.

1993-06-01

315

Applications of fluorescence spectroscopy for predicting percent wastewater in an urban stream.  

PubMed

Dissolved organic carbon (DOC) is a significant organic carbon reservoir in many ecosystems, and its characteristics and sources determine many aspects of ecosystem health and water quality. Fluorescence spectroscopy methods can quantify and characterize the subset of the DOC pool that can absorb and re-emit electromagnetic energy as fluorescence and thus provide a rapid technique for environmental monitoring of DOC in lakes and rivers. Using high resolution fluorescence techniques, we characterized DOC in the Tualatin River watershed near Portland, Oregon, and identified fluorescence parameters associated with effluent from two wastewater treatment plants and samples from sites within and outside the urban region. Using a variety of statistical approaches, we developed and validated a multivariate linear regression model to predict the amount of wastewater in the river as a function of the relative abundance of specific fluorescence excitation/emission pairs. The model was tested with independent data and predicts the percentage of wastewater in a sample within 80% confidence. Model results can be used to develop in situ instrumentation, inform monitoring programs, and develop additional water quality indicators for aquatic systems. PMID:22435681

Goldman, Jami H; Rounds, Stewart A; Needoba, Joseph A

2012-04-17

316

A high-resolution large-acceptance analyzer for X-ray fluorescence and Raman spectroscopy  

SciTech Connect

A newly designed multi-crystal X-ray spectrometer and its applications in the fields of X-ray fluorescence and X-ray Raman spectroscopy are described. The instrument is based on 8 spherically curved Si crystals, each with a 3.5 inch diameter form bent to a radius of 86 cm. The crystals are individually aligned in the Rowland geometry capturing a total solid angle of 0.07 sr. The array is arranged in a way that energy scans can be performed by moving the whole instrument, rather than scanning each crystal by itself. At angles close to back scattering the energy resolution is between 0.3 and 1 eV depending on the beam dimensions at the sample. The instrument is mainly designed for X-ray absorption and fluorescence spectroscopy of transition metals in dilute systems such as metalloproteins. First results of the Mn K{beta} (3p -> 1s) emission in photosystem II are shown. An independent application of the instrument is the technique of X-ray Raman spectroscopy which can address problems similar to those in traditional soft X-ray absorption spectroscopies, and initial results are presented.

Bergmann, Uwe; Cramer, Stephen P.

2001-08-02

317

Fluorescence spectroscopy for the detection of potentially malignant disorders of the oral cavity: analysis of 30 cases  

NASA Astrophysics Data System (ADS)

Oral cancer is a major health problem worldwide and although early diagnosis of potentially malignant and malignant diseases is associated with better treatment results, a large number of cancers are initially misdiagnosed, with unfortunate consequences for long-term survival. Fluorescence spectroscopy is a noninvasive modality of diagnostic approach using induced fluorescence emission in tumors that can improve diagnostic accuracy. The objective of this study was to determine the ability to discriminate between normal oral mucosa and potentially malignant disorders by fluorescence spectroscopy. Fluorescence investigation under 408 and 532 nm excitation wavelengths was performed on 60 subjects, 30 with potentially malignant disorders and 30 volunteers with normal mucosa. Data was analyzed to correlate fluorescence patterns with clinical and histopathological diagnostics. Fluorescence spectroscopy used as a point measurement technique resulted in a great variety of spectral information. In a qualitative analysis of the fluorescence spectral characteristics of each type of injury evaluated, it was possible to discriminate between normal and abnormal oral mucosa. The results show the potential use of fluorescence spectroscopy for an improved discrimination of oral disorders.

Francisco, A. L. N.; Correr, W. R.; Azevedo, L. H.; Galletta, V. K.; Pinto, C. A. L.; Kowalski, L. P.; Kurachi, C.

2014-01-01

318

Using fluorescence spectroscopy to trace seasonal DOM dynamics, disturbance effects, and hydrologic transport in the Florida Everglades  

Microsoft Academic Search

Dissolved organic matter (DOM) quality reflects numerous environmental processes, including primary production and decomposition, redox gradients, hydrologic transport, and photochemistry. Fluorescence spectroscopy can detect groups of DOM compounds sensitive to these processes. However, different environmental gradients (e.g., redox, DOM provenance) can have confounding effects on DOM fluorescence spectra. This study shows how these confounding effects can be removed through discriminant

Laurel G. Larsen; George R. Aiken; Judson W. Harvey; Gregory B. Noe; John P. Crimaldi

2010-01-01

319

Complexation of curium(III) with hydroxamic acids investigated by time-resolved laser-induced fluorescence spectroscopy  

Microsoft Academic Search

The unknown complex formation of Cm(III) with two hydroxamic acids, salicylhydroxamic (SHA) and benzohydroxamic acid (BHA) was studied by time-resolved laser-induced fluorescence spectroscopy (TRLFS). Hydroxamate containing chelating substances have the potential to enhance the solubility and mobility of metals and radionuclides by forming complexes. We explored the fluorescence properties, lifetimes and individual fluorescence emission spectra of the formed Cm(III) hydroxamate

M. Glorius; H. Moll; G. Bernhard

2008-01-01

320

Characterization of extracellular polymeric substances of aerobic and anaerobic sludge using three-dimensional excitation and emission matrix fluorescence spectroscopy  

Microsoft Academic Search

In this study three-dimensional excitation–emission matrix (EEM) fluorescence spectroscopy was applied to characterize the extracellular polymeric substances (EPS) extracted from aerobic and anaerobic sludge in wastewater treatment. Three fluorescence peaks were identified in EEM fluorescence spectra of the EPS samples. Two peaks were attributed to the protein-like fluorophores, and the third to the humic-like fluorophores. The effects of both pH

Guo-Ping Sheng; Han-Qing Yu

2006-01-01

321

Evaluation of the uncertainties associated with in vivo X-ray fluorescence bone lead calibrations  

NASA Astrophysics Data System (ADS)

An anthropometric leg phantom developed at the University of Cincinnati (UC) was used to evaluate the effects that changes in leg position and variation between subjects has on in vivo x-ray fluorescence (XRF) measurements of stable lead in bone. The changes in leg position that were evaluated include changes in source-phantom distance ranging between 0.0 mm and 30.0 mm and phantom rotation over 40 degrees. Source-phantom distance was determined to have a significant effect on XRF measurement results particularly at source-phantom distances greater than 10.0 mm. Rotation of the leg phantom through 40 degrees was determined to have no significant effect on XRF measurement results. Between subject factors that were evaluated include bone calcium content and overlying tissue thickness. Bone calcium content was determined to have a significant effect on XRF measurements when measuring lead in micrograms per gram bone material. However, if measurement results of micrograms of lead per gram calcium (or per gram bone mineral) is used the normalization method makes the change in calcium content not significant. Overlying tissue thickness was determined to have no significant effect on XRF measurement results with tissue thickness ranging between 5.7 and 11.62 mm. The UC leg phantom was modified to include a fibula bone phantom so that the effect that the fibula has on XRF measurement results could be evaluated. The fibula was determined to have no significant effect on XRF measurement results and in the future need not be incorporated into in vivo XRF calibration phantoms. A knee phantom was also developed for purposes of calibrations of in vivo XRF measurement of lead in the patella. XRF measurement results using this phantom were compared to results of XRF measurements made using the plaster-of-Paris (POP) phantoms. A significant difference was observed between the normalized count rates of the two phantom types when either micrograms of lead per gram of bone material or micrograms of lead per gram calcium (bone mineral) is used as the lead content. This difference is consistent with what is observed in real in vivo XRF measurements and indicates the need for the correction factors that are used.

Lodwick, Jeffrey C.

322

In vivo Molecular Evaluation of Guinea Pig Skin Incisions Healing after Surgical Suture and Laser Tissue Welding Using Raman Spectroscopy  

PubMed Central

The healing process in guinea pig skin following surgical incisions was evaluated at the molecular level, in vivo, by the use of Raman spectroscopy. After the incisions were closed either by suturing or by laser tissue welding (LTW), differences in the respective Raman spectra were identified. The study determined that the ratio of the Raman peaks of the amide III (1247 cm?1) band to a peak at 1326 cm?1 (the superposition of elastin and keratin bands) can be used to evaluate the progression of wound healing. Conformational changes in the amide I band (1633 cm?1 to 1682 cm?1) and spectrum changes in the range of 1450 cm?1 to 1520 cm?1 were observed in LTW and sutured skin. The stages of the healing process of the guinea pig skin following LTW and suturing were evaluated by Raman spectroscopy, using histopathology as the gold standard. LTW skin demonstrated better healing than sutured skin, exhibiting minimal hyperkeratosis, minimal collagen deposition, near-normal surface contour, and minimal loss of dermal appendages. A wavelet decomposition-reconstruction baseline correction algorithm was employed to remove the fluorescence wing from the Raman spectra. PMID:19581109

Alimova, A.; Chakraverty, R.; Muthukattil, R.; Elder, S.; Katz, A.; Sriramoju, V.; Lipper, Stanley; Alfano, R. R.

2009-01-01

323

In vivo molecular evaluation of guinea pig skin incisions healing after surgical suture and laser tissue welding using Raman spectroscopy.  

PubMed

The healing process in guinea pig skin following surgical incisions was evaluated at the molecular level, in vivo, by the use of Raman spectroscopy. After the incisions were closed either by suturing or by laser tissue welding (LTW), differences in the respective Raman spectra were identified. The study determined that the ratio of the Raman peaks of the amide III (1247 cm(-1)) band to a peak at 1326 cm(-1) (the superposition of elastin and keratin bands) can be used to evaluate the progression of wound healing. Conformational changes in the amide I band (1633-1682 cm(-1)) and spectrum changes in the range of 1450-1520 cm(-1) were observed in LTW and sutured skin. The stages of the healing process of the guinea pig skin following LTW and suturing were evaluated by Raman spectroscopy, using histopathology as the gold standard. LTW skin demonstrated better healing than sutured skin, exhibiting minimal hyperkeratosis, minimal collagen deposition, near-normal surface contour, and minimal loss of dermal appendages. A wavelet decomposition-reconstruction baseline correction algorithm was employed to remove the fluorescence wing from the Raman spectra. PMID:19581109

Alimova, A; Chakraverty, R; Muthukattil, R; Elder, S; Katz, A; Sriramoju, V; Lipper, Stanley; Alfano, R R

2009-09-01

324

Fluorescent magnetic nanoparticle-labeled mesenchymal stem cells for targeted imaging and hyperthermia therapy of in vivo gastric cancer  

NASA Astrophysics Data System (ADS)

How to find early gastric cancer cells in vivo is a great challenge for the diagnosis and therapy of gastric cancer. This study is aimed at investigating the feasibility of using fluorescent magnetic nanoparticle (FMNP)-labeled mesenchymal stem cells (MSCs) to realize targeted imaging and hyperthermia therapy of in vivo gastric cancer. The primary cultured mouse marrow MSCs were labeled with amino-modified FMNPs then intravenously injected into mouse model with subcutaneous gastric tumor, and then, the in vivo distribution of FMNP-labeled MSCs was observed by using fluorescence imaging system and magnetic resonance imaging system. After FMNP-labeled MSCs arrived in local tumor tissues, subcutaneous tumor tissues in nude mice were treated under external alternating magnetic field. The possible mechanism of MSCs targeting gastric cancer was investigated by using a micro-multiwell chemotaxis chamber assay. Results show that MSCs were labeled with FMNPs efficiently and kept stable fluorescent signal and magnetic properties within 14 days, FMNP-labeled MSCs could target and image in vivo gastric cancer cells after being intravenously injected for 14 days, FMNP-labeled MSCs could significantly inhibit the growth of in vivo gastric cancer because of hyperthermia effects, and CCL19/CCR7 and CXCL12/CXCR4 axis loops may play key roles in the targeting of MSCs to in vivo gastric cancer. In conclusion, FMNP-labeled MSCs could target in vivo gastric cancer cells and have great potential in applications such as imaging, diagnosis, and hyperthermia therapy of early gastric cancer in the near future.

Ruan, Jing; Ji, Jiajia; Song, Hua; Qian, Qirong; Wang, Kan; Wang, Can; Cui, Daxiang

2012-06-01

325

Characterization of dissolved organic matter in fogwater by excitation-emission matrix fluorescence spectroscopy  

USGS Publications Warehouse

Dissolved organic matter (DOM) present in fogwater samples collected in southeastern Louisiana and central-eastern China has been characterized using excitation-emission matrix fluorescence spectroscopy. The goal of the study was to illustrate the utility of fluorescence for obtaining information on the large fraction of organic carbon in fogwaters (typically >40% by weight) that defies characterization in terms of specific chemical compounds without the difficulty inherent in obtaining sufficient fogwater volume to isolate DOM for assessment using other spectroscopic and chemical analyses. Based on the findings of previous studies using other characterization methods, it was anticipated that the unidentified organic carbon fraction would have characteristic peaks associated with humic substances and fluorescent amino acids. Both humic- and protein-like fluorophores were observed in the fogwater spectra and fluorescence-derived indices for the fogwater had similar values to those of soil and sediment porewater. Greater biological character was observed in samples with higher organic carbon concentrations. Fogwaters are shown to contain a mixture of terrestrially- and microbially-derived fluorescent organic material, which is expected to be derived from an array of different sources, such as suspended soil and dust particles, biogenic emissions and organic substances generated by atmospheric processes. The fluorescence results indicate that much of the unidentified organic carbon present in fogwater can be represented by humic-like and biologically-derived substances similar to those present in other aquatic systems, though it should be noted that fluorescent signatures representative of DOM produced by atmospheric processing of organic aerosols may be contributing to or masked by humic-like fluorophores. ?? 2010.

Birdwell, J.E.; Valsaraj, K.T.

2010-01-01

326

Feedback control of Brownian motion for single-particle fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

The stochastic Brownian motion of individual particles in solution constrains the utility of single-particle fluorescence microscopy both by limiting the dwell time of particles in the observation volume and by convolving their internal degrees of freedom with their random spatial trajectories. This thesis describes the use of active feedback control to eliminate these undesirable effects. We designed and implemented a feedback tracking system capable of locking the position of a fluorescent particle to the optic axis of our microscope, i.e., capable of tracking the two-dimensional, planar Brownian motion of a free particle in solution. A full theoretical description of the experiment is given in the language of linear stochastic control theory. The model describes both the statistics of the tracking system and provides a generalization of the theory of open-loop Fluorescence Correlation Spectroscopy (FCS) that accounts for fluctuations in fluorescence arising from competition between diffusion and damping. We find excellent agreement between theory and experiment. Using fluorescent polymer microspheres as test particles, we find that the observation time for these particles can be increased by 2-3 orders of magnitude over the open-loop scenario. The system achieves nearly optimal performance for moderately fast-moving particles at very low fluorescent count rates, comparable to those of a single fluorescent protein molecule. The system can classify particles in a binary mixture based on a real-time estimate of their diffusion coefficients (differing by a factor of ˜4), achieving 90% success using fewer than 600 photons detected over 120 ms. Future directions for both the experimental and theoretical techniques are briefly discussed.

Berglund, Andrew J.

327

In vivo Raman spectroscopy for breast cancer: diagnosis in animal model  

NASA Astrophysics Data System (ADS)

Raman spectroscopy has been well established as a powerful method for studying biological tissues and diagnosing diseases. In this study we have developed a breast cancer animal model and collected in vivo Raman spectra of mammary glands of 27 Sprague-Dawley female rats treated with DMBA and 5 non-treated used as control group. A dispersive Raman spectrometer with a @785 nm laser excitation coupled a fiber optic probe and a CCD detector was used to obtain the spectra. The obtained in vivo transcutaneous Raman spectra have shown important differences between normal and abnormal tissues when acquired from one side to the other side of the lesion.

Bitar, R.; Martins, M. A.; Ribeiro, D.; Carvalho, C.; Santos, E. A. P.; Ramalho, L. N. Z.; Ramalho, F.; Martinho, H.; Martin, A. A.

2008-02-01

328

Physicochemical characterisation of cationic polybutylcyanoacrylate-nanoparticles by fluorescence correlation spectroscopy.  

PubMed

The aim of this study was to compare different physical and chemical methods with fluorescence correlation spectroscopy (FCS) in order to characterise cationic acrylate nanoparticles (NP), which can deliver oligonucleotides (ON) into mammalian cells. These positively charged nanoparticles were prepared from diethylaminoethyl dextran (DEAE-dextran) and poly(n-butyl-2-cyanoacrylate) (PBCA). NP consists of PBCA oligochains with an average size of PBCA 9 mer and were formed by entrapping DEAE-dextran and dextran 70,000 in high amounts into the particle matrix. The oligochain length of PBCA was investigated by mass-spectroscopy (MALDI TOF). The molecular weight of a particle with d = 108 nm was estimated to be approximately 3.6 x 10(8) Da. The mean size of the nanoparticles were in a range of dh = 130-140 nm, as determined independently by FCS and dynamic light scattering. Atomic force microscopy and scanning electron microscopy images confirm this size range. Furthermore, the particle mass of the PBCA-NP was estimated by FCS measurements. For this approach two new methods for fluorescence labelling of cationic particles were developed. Fluorescent labelled dextran 70,000 was entrapped into the particle matrix; in addition, the derivatisation of hydroxyl groups of the NP was achieved with 5-([4,6-dichlorotriazin-2-yl]amino) fluorescein (DTAF). ON can be localised in a complex with the NP by dual-colour fluorescence cross correlation spectroscopy measurements. The zetapotential of the unloaded NP was positively charged with about +39 mV and decreased down to -40 mV on addition of excess ON. After centrifugation quantification of the ON loading onto the particles by strong anion exchange high performance liquid chromatography (SAX HPLC) and FCS showed that approximately 20 microg ON per 100 g NP was adsorbed. The FCS measurements of the ON adsorption in situ was found to be much higher with approximately 95 microg ON per 100 g NP. PMID:15207534

Weyermann, Jörg; Lochmann, Dirk; Georgens, Christiane; Rais, Isam; Kreuter, Jörg; Karas, Michael; Wolkenhauer, Markus; Zimmer, Andreas

2004-07-01

329

Detection of macrophage activity in atherosclerosis in vivo using multichannel, high-resolution laser scanning fluorescence microscopy  

NASA Astrophysics Data System (ADS)

Molecular and cellular mechanisms of atherogenesis and its treatment are largely being unraveled by in vitro techniques. We describe methodology to directly image macrophage cell activity in vivo in a murine model of atherosclerosis using laser scanning fluorescence microscopy (LSFM) and a macrophage-targeted, near-infrared fluorescent (NIRF) magnetofluorescent nanoparticle (MFNP). Atherosclerotic apolipoprotein E deficient (apoE -/-) mice (n=10) are injected with MFNP or 0.9% saline, and wild-type mice (n=4) are injected with MFNP as additional controls. After 24 h, common carotid arteries are surgically exposed and prepared for LSFM. Multichannel LSFM of MFNP-enhanced carotid atheroma (5×5-µm in-plane resolution) shows a strong focal NIRF signal, with a plaque target-to-background ratio of 3.9+/-1.8. Minimal NIRF signal is observed in control mice. Spectrally resolved indocyanine green (ICG) fluorescence angiograms confirm the intravascular location of atheroma. On ex vivo fluorescence reflectance imaging, greater NIRF plaque signal is seen in apoE -/- MFNP mice compared to controls (p<0.01). The NIRF signal correlates well with immunostained macrophages, both by stained surface area (r=0.77) and macrophage number (r=0.86). The validated experimental methodology thus establishes a platform for investigating macrophage activity in atherosclerosis in vivo, and has implications for the detection of clinical vulnerable plaques.

Pande, Ashvin N.; Kohler, Rainer; Aikawa, Elena; Weissleder, Ralph; Jaffer, Farouc

2006-03-01

330

In vivo imaging and detection of nitroreductase in zebrafish by a new near-infrared fluorescence off-on probe.  

PubMed

A new near-infrared fluorescence off-on probe is developed and applied to fluorescence imaging of nitroreductase in zebrafish in vivo. The probe is readily prepared by connecting 4-nitrobenzene as a quenching and recognizing moiety to a stable hemicyanine skeleton that can be formed via the decomposition of IR 780. The fluorescence off-on response of the probe to nitroreductase is based on the enzyme-catalyzed reduction of the 4-nitrobenzene moiety, followed by the 1,6-rearrangement-elimination and the fluorophore release. Compared with the existing nitroreductase probes, the proposed probe exhibits superior analytical performance such as near-infrared fluorescence emission over 700 nm as well as high selectivity and sensitivity, with a detection limit of 14 ng/mL. More importantly, the probe has been successfully applied to visualize the distribution of nitroreductase in living zebrafish in vivo, revealing that nitroreductase might mainly exist in zebrafish yolk sac. The superior properties of the probe make it of great potential use in other biosystems and in vivo studies. PMID:25064818

Li, Zhao; He, Xinyuan; Wang, Zhe; Yang, Ronghua; Shi, Wen; Ma, Huimin

2015-01-15

331

An in vivo comparison of photoactivatable fluorescent proteins in an avian embryo model.  

PubMed

Tracing the lineage or neighbor relationships of cells in a migratory population or deep within an embryo is difficult with current methods. The recent explosion of photoactivatable fluorescent proteins (PAFPs) offers a unique cell labeling tool kit, yet their in vivo performance in intact embryos and applicability have not been thoroughly explored. We report a comparison study of PAGFP, PSCFP2, KikGR, and Kaede analyzed in the avian embryo using confocal and 2-photon microscopy. PAFPs were introduced into the chick neural tube by electroporation and each photoconverted in the neural crest or cells in the neural tube with exposure to 405 nm light, but showed dramatic differences in photoefficiency and photostability when compared at the same 2% laser power. KikGR and Kaede photoconverted with ratios only slightly lower than in vitro results, but cells rapidly photobleached after reaching maximal photoefficiency. PSCFP2 had the lowest photoefficiency and photoconverted nearly 70 times slower than the other dual-color PAFPs tested, but was effective at single-cell marking, especially with 2-photon excitation at 760 nm. The dual-color PAFPs were more effective to monitor cell migratory behaviors, since non-photoconverted neighboring cells were fluorescently marked with a separate color. However, photoconverted cells were limited in all cases to be visually distinguishable for long periods, with PSCFP2 visible from background the longest (48 hr). Thus, photoactivation in embryos has the potential to selectively mark less accessible cells with laser accuracy and may provide an effective means to study cell-cell interactions and short-term cell lineage in developmental and stem cell biology. PMID:17486622

Stark, Danny A; Kulesa, Paul M

2007-06-01

332

Fluorescent probes concentration estimation in vitro and ex vivo as a model for early detection of Alzheimer's disease  

NASA Astrophysics Data System (ADS)

The pathogenic process of Alzheimer's disease (AD) begins years before clinical diagnosis. Here, we suggest a method that may detect AD several years earlier than current exams. The method is based on previous reports that relate the concentration ratio of biomarkers (amyloid-beta and tau) in the cerebrospinal fluid (CSF) to the development of AD. Our method replaces the lumbar puncture process required for CSF drawing by using fluorescence measurements. The system uses an optical fiber coupled to a laser source and a detector. The laser radiation excites two fluorescent probes which may bond to the CSF biomarkers. Their concentration ratio is extracted from the fluorescence intensities and can be used for future AD detection. First, we present a theoretical model for fluorescence concentration ratio estimation. The method's feasibility was validated using Monte Carlo simulations. Its accuracy was then tested using multilayered tissue phantoms simulating the epidural fat, CSF, and bone. These phantoms have various optical properties, thicknesses, and fluorescence concentrations in order to simulate human anatomy variations and different fiber locations. The method was further tested using ex vivo chicken tissue. The average errors of the estimated concentration ratios were low both in vitro (4.4%) and ex vivo (10.9%), demonstrating high accuracy.

Harbater, Osnat; Gannot, Israel

2014-12-01

333

Fluorescent probes concentration estimation in vitro and ex vivo as a model for early detection of Alzheimer's disease.  

PubMed

The pathogenic process of Alzheimer's disease (AD) begins years before clinical diagnosis. Here, we suggest a method that may detect AD several years earlier than current exams. The method is based on previous reports that relate the concentration ratio of biomarkers (amyloid-beta and tau) in the cerebrospinal fluid (CSF) to the development of AD. Our method replaces the lumbar puncture process required for CSF drawing by using fluorescence measurements. The system uses an optical fiber coupled to a laser source and a detector. The laser radiation excites two fluorescent probes which may bond to the CSF biomarkers. Their concentration ratio is extracted from the fluorescence intensities and can be used for future AD detection. First, we present a theoretical model for fluorescence concentration ratio estimation. The method's feasibility was validated using Monte Carlo simulations. Its accuracy was then tested using multilayered tissue phantoms simulating the epidural fat, CSF, and bone. These phantoms have various optical properties, thicknesses, and fluorescence concentrations in order to simulate human anatomy variations and different fiber locations. The method was further tested using ex vivo chicken tissue. The average errors of the estimated concentration ratios were low both in vitro (4.4%) and ex vivo (10.9%), demonstrating high accuracy. PMID:25545342

Harbater, Osnat; Gannot, Israel

2014-12-01

334

NEW MICROSCOPIC LASER-COUPLED SPECTROSCOPY INSTRUMENT COMBINING RAMAN, LIBS, AND FLUORESCENCE FOR PLANETARY SURFACE MINERALOGY. J. Blacksberg1  

E-print Network

-resolved laser spectroscopy in a Figure 1. Image of our pulsed 532 nm microchip laser focused to ~ 1 µm spotNEW MICROSCOPIC LASER-COUPLED SPECTROSCOPY INSTRUMENT COMBINING RAMAN, LIBS, AND FLUORESCENCE (LIBS, Raman) have been the subject of increasing attention and development [e.g., 1, 2, 3] because

Rossman. George R.

335

In vivo and in vitro 31P-NMR spectroscopy of rat liver treated with halocarbons.  

PubMed

Both in vivo and in vitro 31P-NMR spectroscopy were used to demonstrate metabolic changes in rat liver as a function of time after exposure to either carbon tetrachloride (CCl4) or bromotrichloromethane (BrCCl3). The inorganic phosphate resonance, measured in vivo, moves upfield, which is associated with a decrease in cytosolic pH over a 12 or 20 h period (for BrCCl3 or CCl4, respectively). Intoxication by CCl4 or BrCCl3 causes an intracellular acidosis to pH 7.05 or 6.82 (+/- 0.05), respectively. Also, it has been found that halocarbon exposure increases the amounts of phosphomonoesters (PME) detected. High resolution in vitro 31P-NMR spectroscopy studies of perchloric acid extracts of CCl4-treated rat livers indicated a significant increase in the height of the phosphocholine resonance in the PME region 4-5 h after CCl4 exposure. PMID:2804127

Towner, R A; Brauer, M; Janzen, E G; Ling, M F

1989-10-13

336

Multimodal fiber probe spectroscopy for tissue diagnostics applications: a combined Raman-fluorescence approach  

NASA Astrophysics Data System (ADS)

Two different optical fiber probes for combined Raman and fluorescence spectroscopic measurements were designed, developed and used for tissue diagnostics. Two visible laser diodes were used for fluorescence spectroscopy, whereas a laser diode emitting in the NIR was used for Raman spectroscopy. The two probes were based on fiber bundles with a central multimode optical fiber, used for delivering light to the tissue, and 24 surrounding optical fibers for signal collection. Both fluorescence and Raman spectra were acquired using the same detection unit, based on a cooled CCD camera, connected to a spectrograph. The two probes were successfully employed for diagnosing melanocytic lesions in a good agreement with common routine histology. The obtained results demonstrated that the multimodal approach is crucial for improving diagnostic capabilities. Further investigations were performed on colon and brain tissue samples in order to have a benchmark for diagnosing a broader range of tissue lesions and malignancies. The system presented here can improve diagnostic capabilities on a broad range of tissues and has the potential of being used for endoscopic inspections in the near future.

Cicchi, Riccardo; Anand, Suresh; Rossari, Susanna; Sturiale, Alessandro; Giordano, Flavio; De Giorgi, Vincenzo; Maio, Vincenza; Massi, Daniela; Nesi, Gabriella; Buccoliero, Anna Maria; Tonelli, Francesco; Guerrini, Renzo; Pimpinelli, Nicola; Pavone, Francesco S.

2014-03-01

337

Cure Monitoring of an Unsaturated Polyester Resin Using Near-Infrared and Fluorescence Spectroscopies  

NASA Astrophysics Data System (ADS)

The applicability of both near-infrared (NIR) and fluorescence spectroscopy for the purpose of cure monitoring an unsaturated polyester (UPE) resin was investigated. Based on standard reference mixtures, peak assignments in the NIR region of the spectrum were made. It was determined that the peak at 1629 nm was due to the first overtone band of RHC=CH2 stretching modes in styrene, while a combination of RHC=CHR and -C=C- stretching modes in diethyl fumarate were responsible for the peak observed at 2087 nm. NIR spectra of the UPE resin during isothermal cure at 75 C exhibited decreases in peak absorbance at 1629 and 2087 nm due to conversion of styrene and vinylene bonds, respectively. Conversion of styrene and vinylene with time calculated using NIR spectra showed similar trends found with FTIR analysis throughout the entire conversion range. Fluorescence spectroscopy was used to monitor the isothermal curing reaction of the UPE resin by exciting styrene at 250 nm. Emission intensity at ca. 306 nm remained unchanged for the initial 60-80 minutes then increased with cure time due to a reduced self-quenching effect as cure proceeded. The increase in fluorescence intensity was concurrent with an increase in styrene conversion up to 93% styrene conversion.

Sung, Chong S. P.; Grunden, Bradley L.

1998-03-01

338

Combining surface sensitive vibrational spectroscopy and fluorescence microscopy to study biological interfaces  

NASA Astrophysics Data System (ADS)

A multimodal system combining surface sensitive sum frequency generation (SFG) vibrational spectroscopy and total-internal reflection fluorescence (TIRF) microscopy for surface and interface study was developed. Interfacial molecular structural information can be detected using SFG spectroscopy while interfacial fluorescence signal can be visualized using TIRF microscopy from the same sample. As a proof of concept experiment, SFG spectra of fluorescent polystyrene (PS) beads with different surface coverage were correlated with TIRF signal observed. Results showed that SFG signals from the ordered surfactant methyl groups were detected from the substrate surface, while signals from PS phenyl groups on the beads were not seen. Additionally, a lipid monolayer labeled using lipid-associated dye was deposited on a silica substrate and studied in different environments. The contact with water of this lipid monolayer caused SFG signal to disappear, indicating a possible lipid molecular disorder and the formation of lipid bilayers or liposomes in water. TIRF was able to visualize the presence of lipid molecules on the substrate, showing that the lipids were not removed from the substrate surface by water. The integration of the two surface sensitive techniques can simultaneously visualize interfacial molecular dynamics and characterize interfacial molecular structures in situ, which is important and is expected to find extensive applications in biological interface related research.

Zhang, Chi; Jasensky, Joshua; Wu, Jing; Chen, Zhan

2014-03-01

339

Near-infrared emission Ba3(PO4)2:Mn5+ phosphor and potential application in vivo fluorescence imaging.  

PubMed

Fluorescence imaging in the second near-infrared window (NIR-II, 1000-1350 nm) is attracting attention due to negligible tissue scattering and lower tissue autofluorescence, etc. Here, Ba3(PO4)2:Mn(5+) phosphor is prepared via solid state reaction method in air, and NIR emission band peaking at ?1191 nm in the NIR-II region is observed. According to experiment results, Ba3(PO4)2:Mn(5+) phosphor has a great potential for the study of the NIR-II fluorescence imaging in vivo. PMID:24691378

Cao, Renping; Yu, Xiaoguang; Sun, Xinyuan; Cao, Chunyan; Qiu, Jianrong

2014-07-15

340

Detection of dimethyl sulfone in the human brain by in vivo proton magnetic resonance spectroscopy  

Microsoft Academic Search

We wish to report the detection of dimethyl sulfone (methylsulfonylmethane, C2H6O2S) in the brain of a normal 62-year-old male using in vivo proton magnetic resonance spectroscopy. The presence of this exogenous metabolite resulted from ingestion of a dietary supplement containing dimethyl sulfone. The concentration of this compound in the brain was measured to be 2.4 mmol, with a washout “half

Stephen E Rosea; Jonathan B Chalk; Graham J Galloway; David M Doddrell

2000-01-01

341

In vivo proton magnetic resonance spectroscopy of the normal aging human brain  

Microsoft Academic Search

The effect of age on brain metabolite concentrations was evaluated using localized proton magnetic resonance spectroscopy. This technique allows in vivo measurements of N-acetyl compounds (NA), total creatine (CR), choline-containing compounds (CHO), myo-inositol (MI), glutamate and glutamine (GLX), as well as the percentage of cerebrospinal fluid (CSF) and the brain water content within the brain region studied. Frontal gray matter

Linda Chang; Thomas Ernst; Russell E. Poland; Donald J. Jenden

1996-01-01

342

Intracellular localization of meso-tetraphenylporphine tetrasulphonate probed by time-resolved and microscopic fluorescence spectroscopy.  

PubMed

The effects of solvent pH on spectral properties and fluorescence decay kinetics were investigated in order to characterize the microenvironment of meso-tetraphenylporphine tetrasulphonate (TPPS4) taken up by cells. Steady-state absorption and fluorescence spectra of TPPS4 in buffer solutions of different pH were used to identify a ring protonated species at pH less than or equal to 4. This dictation could also be distinguished from the unprotonated form by its altered fluorescence decay time (3.5 vs. 11.4 ns). In addition, time-resolved spectroscopy gave some evidence of a monocationic species existing at pH 6-9. This was concluded from the occurrence of another component with a decay time of 5 ns. Measurements of the spectral and kinetic properties of the fluorescence emission of single epithelial cells (RR1022) incubated with TPPS4 indicated that the sensitizer was mainly localized in a microenvironment with a pH of 5, a value which occurs intracellularly only within lysosomes. Cells kept in the dark exhibited the characteristic spectra of both the dication and the neutral form. The fluorescence decay showed two components with decay times of 2.6 ns and 10.6 ns. Irradiation of the cells changed the decay times to 4.6 ns and 13.4 ns and the dication fluorescence emission peak vanished, which is in accordance with the results obtained from buffer solutions at pH greater than or equal to 6. Therefore, we deduce that the photodynamic action leads to a rupture of the lysosomes and that the sensitizer is released into the surrounding cytoplasm. PMID:1635012

Wessels, J M; Strauss, W; Seidlitz, H K; Rück, A; Schneckenburger, H

1992-02-28

343

Fluorescence lifetime imaging and intravascular ultrasound: co-registration study using ex vivo human coronaries.  

PubMed

Fluorescence lifetime imaging (FLIM) has demonstrated potential for robust assessment of atherosclerotic plaques biochemical composition and for complementing conventional intravascular ultrasound (IVUS), which provides information on plaque morphology. The success of such a bi-modal imaging modality depends on accurate segmentation of the IVUS images and proper angular registration between these two modalities. This paper reports a novel IVUS segmentation methodology addressing this issue. The image preprocessing consisted of denoising, using the Wiener filter, followed by image smoothing, implemented through the application of the alternating sequential filter on the edge separability metric images. Extraction of the lumen/intima and media/adventitia boundaries was achieved by tracing the gray-scale peaks over the A-lines of the IVUS preprocessed images. Cubic spline interpolation, in both cross-sectional and longitudinal directions, ensured boundary smoothness and continuity. The detection of the guide-wire artifact in both modalities is used for angular registration. Intraluminal studies were conducted in 13 ex vivo segments of human coronaries. The IVUS segmentation accuracy was assessed against independent manual tracings, providing 91.82% sensitivity and 97.55% specificity. The proposed methodology makes the bi-modal FLIM and IVUS approach feasible for comprehensive intravascular diagnosis by providing co-registered biochemical and morphological information of atherosclerotic plaques. PMID:25163056

Gorpas, Dimitris; Fatakdawala, Hussain; Bec, Julien; Ma, Dinglong; Yankelevich, Diego R; Qi, Jinyi; Marcu, Laura

2015-01-01

344

Endoscopic fluorescence lifetime imaging for in vivo intraoperative diagnosis of oral carcinoma.  

PubMed

A clinically compatible fluorescence lifetime imaging microscopy (FLIM) system was developed. The system was applied to intraoperative in vivo imaging of head and neck squamous cell carcinoma (HNSCC). The endoscopic FLIM prototype integrates a gated (down to 0.2 ns) intensifier imaging system and a fiber-bundle endoscope (0.5-mm-diameter, 10,000 fibers with a gradient index lens objective 0.5 NA, 4-mm field of view), which provides intraoperative access to the surgical field. Tissue autofluorescence was induced by a pulsed laser (337 nm, 700 ps pulse width) and collected in the 460 ± 25 nm spectral band. FLIM experiments were conducted at 26 anatomic sites in ten patients during head and neck cancer surgery. HNSCC exhibited a weaker florescence intensity (~50% less) when compared with healthy tissue and a shorter average lifetime (?(HNSCC) = 1.21 ± 0.04 ns) than the surrounding normal tissue (?N = 1.49 ± 0.06 ns). This work demonstrates the potential of FLIM for label-free head and neck tumor demarcation during intraoperative surgical procedures. PMID:23702007

Sun, Yinghua; Phipps, Jennifer E; Meier, Jeremy; Hatami, Nisa; Poirier, Brian; Elson, Daniel S; Farwell, D Gregory; Marcu, Laura

2013-08-01

345

In Vivo Fluorescence Resonance Energy Transfer Imaging for Targeted Anti-Cancer Drug Delivery Kinetics  

NASA Astrophysics Data System (ADS)

We describe an approach for the evaluation of targeted anti-cancer drug delivery in vivo. The method emulates the drug release and activation process through acceptor release from a targeted donor-acceptor pair that exhibits fluorescence resonance energy transfer (FRET). In this case, folate targeting of the cancer cells is used - 40 % of all human cancers, including ovarian, lung, breast, kidney, brain and colon cancer, over-express folate receptors. We demonstrate the reconstruction of the spatially-dependent FRET parameters in a mouse model and in tissue phantoms. The FRET parameterization is incorporated into a source for a diffusion equation model for photon transport in tissue, in a variant of optical diffusion tomography (ODT) called FRET-ODT. In addition to the spatially-dependent tissue parameters in the diffusion model (absorption and diffusion coefficients), the FRET parameters (donor-acceptor distance and yield) are imaged as a function of position. Modulated light measurements are made with various laser excitation positions and a gated camera. More generally, our method provides a new vehicle for studying disease at the molecular level by imaging FRET parameters in deep tissue, and allows the nanometer FRET ruler to be utilized in deep tissue.

Webb, Kevin; Gaind, Vaibhav; Tsai, Hsiaorho; Bentz, Brian; Chelvam, Venkatesh; Low, Philip

2012-02-01

346

Endoscopic Fluorescence Lifetime Imaging for In Vivo Intraoperative Diagnosis of Oral Carcinoma  

PubMed Central

A clinically compatible fluorescence lifetime imaging microscopy (FLIM) system was developed. The system was applied to intraoperative in vivo imaging of head and neck squamous cell carcinoma (HNSCC). The endoscopic FLIM prototype integrates a gated (down to 0.2 ns) intensifier imaging system and a fiber-bundle endoscope (0.5-mm-diameter, 10,000 fibers with a gradient index lens objective 0.5 NA, 4-mm field of view), which provides intraoperative access to the surgical field. Tissue autofluorescence was induced by a pulsed laser (337 nm, 700 ps pulse width) and collected in the 460 ± 25 nm spectral band. FLIM experiments were conducted at 26 anatomic sites in ten patients during head and neck cancer surgery. HNSCC exhibited a weaker florescence intensity (~50% less) when compared with healthy tissue and a shorter average lifetime (?HNSCC = 1.21 ± 0.04 ns) than the surrounding normal tissue (?N = 1.49 ± 0.06 ns). This work demonstrates the potential of FLIM for label-free head and neck tumor demarcation during intraoperative surgical procedures. PMID:23702007

Sun, Yinghua; Phipps, Jennifer E.; Meier, Jeremy; Hatami, Nisa; Poirier, Brian; Elson, Daniel S.; Farwell, D. Gregory; Marcu, Laura

2014-01-01

347

Photon correlation spectroscopy of light scattered by eye lenses in in vivo conditions.  

PubMed Central

The application of photon correlation spectroscopy on mammalian eye lenses in vivo is revisited. It is shown that the use of a short wavelength laser type and a logarithmic correlator improves the signal-to-noise ratio to such an extent that shorter measurement times are possible without impairing the information content of the correlation function. Experimental correlation functions obtained in vivo on a rabbit eye lens, are analyzed with several techniques. The histogram approach is most successful for the determination of the distribution function of relaxation processes in the correlation function and proposes four different populations of components in the lens. This result is comparable to that from in vitro measurements on highly concentrated solutions of alpha-crystallins and of fiber cell cytoplasm, the former proteins being the main scattering components both in vivo and in vitro in the eye lens system. Our results indicate that the application of photon correlation spectroscopy on eye lenses in vivo offers new perspectives to use this technique as a fast, noninvasive tool to study relaxation phenomena in normal and cataractous lenses. The sensitivity of the method allows it to be used as an important analytical technique in the study of prevention and treatment of cataract. PMID:2009358

Van Laethem, M; Babusiaux, B; Neetens, A; Clauwaert, J

1991-01-01

348

Applications of time-resolved laser fluorescence spectroscopy to the environmental biogeochemistry of actinides.  

PubMed

Time-resolved laser fluorescence spectroscopy (TRLFS) is a useful means of identifying certain actinide species resulting from various biogeochemical processes. In general, TRLFS differentiates chemical species of a fluorescent metal ion through analysis of different excitation and emission spectra and decay lifetimes. Although this spectroscopic technique has largely been applied to the analysis of actinide and lanthanide ions having fluorescence decay lifetimes on the order of microseconds, such as UO , Cm, and Eu, continuing development of ultra-fast and cryogenic TRLFS systems offers the possibility to obtain speciation information on metal ions having room-temperature fluorescence decay lifetimes on the order of nanoseconds to picoseconds. The main advantage of TRLFS over other advanced spectroscopic techniques is the ability to determine in situ metal speciation at environmentally relevant micromolar to picomolar concentrations. In the context of environmental biogeochemistry, TRLFS has principally been applied to studies of (i) metal speciation in aqueous and solid phases and (ii) the coordination environment of metal ions sorbed to mineral and bacterial surfaces. In this review, the principles of TRLFS are described, and the literature reporting the application of this methodology to the speciation of actinides in systems of biogeochemical interest is assessed. Significant developments in TRLFS methodology and advanced data analysis are highlighted, and we outline how these developments have the potential to further our mechanistic understanding of actinide biogeochemistry. PMID:21546659

Collins, Richard N; Saito, Takumi; Aoyagi, Noboru; Payne, Timothy E; Kimura, Takaumi; Waite, T David

2011-01-01

349

Charge transfer to solvent identified using dark channel fluorescence-yield L-edge spectroscopy  

NASA Astrophysics Data System (ADS)

Aqueous ions are central to catalysis and biological function and play an important role in radiation biology as sources of damage-inducing electrons. Detailed knowledge of solute-solvent interactions is therefore crucial. For transition-metal ions, soft X-ray L-edge spectroscopy allows access to d orbitals, which are involved in chemical bonding. Using this technique, we show that the fluorescence-yield spectra of aqueous ionic species exhibit additional features compared with those of non-aqueous solvents. Some features dip below the fluorescence background of the solvent and this is rationalized by the competition between the fluorescence yields of the solute and solvent species, and between the solute radiative (fluorescence) and non-radiative channels; in particular, electron transfer to the water molecules. This method allows us to determine the nature, directionality and timescale of the electron transfer. Remarkably, we observe such features even for fully ligated metal atoms, which indicates a direct interaction with the water molecules.

Aziz, Emad F.; Rittmann-Frank, M. Hannelore; Lange, Kathrin M.; Bonhommeau, Sébastien; Chergui, Majed

2010-10-01

350

Charge transfer to solvent identified using dark channel fluorescence-yield L-edge spectroscopy.  

PubMed

Aqueous ions are central to catalysis and biological function and play an important role in radiation biology as sources of damage-inducing electrons. Detailed knowledge of solute-solvent interactions is therefore crucial. For transition-metal ions, soft X-ray L-edge spectroscopy allows access to d orbitals, which are involved in chemical bonding. Using this technique, we show that the fluorescence-yield spectra of aqueous ionic species exhibit additional features compared with those of non-aqueous solvents. Some features dip below the fluorescence background of the solvent and this is rationalized by the competition between the fluorescence yields of the solute and solvent species, and between the solute radiative (fluorescence) and non-radiative channels; in particular, electron transfer to the water molecules. This method allows us to determine the nature, directionality and timescale of the electron transfer. Remarkably, we observe such features even for fully ligated metal atoms, which indicates a direct interaction with the water molecules. PMID:20861901

Aziz, Emad F; Rittmann-Frank, M Hannelore; Lange, Kathrin M; Bonhommeau, Sébastien; Chergui, Majed

2010-10-01

351

Metal-Enhanced Fluorescence Lifetime Imaging and Spectroscopy on a Modified SERS Substrate  

PubMed Central

In this paper, we developed a metal-enhanced fluorescence (MEF) substrate by modification of the commercially available surface enhanced Raman spectroscopy (SERS) substrate that may meet the reproducibility and sensitivity challenge of MEF. In spite of many studies and interest on MEF from a number of research groups, application to real-world situations and its commercial use remain challenging mainly due to the difficulties in fabricating reproducible MEF substrates. Specifically, one of the challenges is achieving a standardized MEF substrate for reproducible fluorescence intensity enhancement and/or changes in lifetime. The gold standard klarite substrates for SERS were coated with a thin layer of silver nanoparticles for MEF studies. To test the newly developed MEF substrates, a monolayer of streptavidin conjugated Alexa-647 was assembled on biotinylated-glass or MEF substrates. We observed over 50-fold increase in the fluorescence intensity from a monolayer of streptavidin conjugated Alexa-647 on the biotinylated MEF substrate compared to the same on glass substrate. A significant reduction in the lifetime and increased photostability of Alexa-647 on MEF substrate was observed. Fluorescence lifetime imaging was performed on the monolayer of dye assembled on the modified SERS substrates. We expect this study will serve as a platform to encourage the future use of a standardized MEF substrate for a plethora of sensing applications. PMID:24416457

Ray, Krishanu; Lakowicz, Joseph R.

2013-01-01

352

In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves  

PubMed Central

Accurate localization of phytoalexins is a key for better understanding their role. This work aims to localize stilbenes, the main phytoalexins of grapevine. The cellular localization of stilbene fluorescence induced by Plasmopara viticola, the agent of downy mildew, was determined in grapevine leaves of very susceptible, susceptible, and partially resistant genotypes during infection. Laser scanning confocal microscopy and microspectrofluorimetry were used to acquire UV-excited autofluorescence three-dimensional images and spectra of grapevine leaves 5–6 days after inoculation. This noninvasive technique of investigation in vivo was completed with in vitro spectrofluorimetric studies on pure stilbenes as their fluorescence is largely affected by the physicochemical environment in various leaf compartments. Viscosity was the major physicochemical factor influencing stilbene fluorescence intensity, modifying fluorescence yield by more than two orders of magnitude. Striking differences in the localization of stilbene fluorescence induced by P. viticola were observed between the different genotypes. All inoculated genotypes displayed stilbene fluorescence in cell walls of guard cells and periclinal cell walls of epidermal cells. Higher fluorescence intensity was observed in guard-cell walls than in any other compartment due to increased local viscosity. In addition stilbene fluorescence was found in epidermal cell vacuoles of the susceptible genotype and in the infected spongy parenchyma of the partially resistant genotype. The very susceptible genotype was devoid of fluorescence both in the epidermal vacuoles and the mesophyll. This strongly suggests that the resistance of grapevine leaves to P. viticola is correlated with the pattern of localization of induced stilbenes in host tissues. PMID:22412183

Bellow, Sébastien; Latouche, Gwendal; Brown, Spencer C.; Poutaraud, Anne; Cerovic, Zoran G.

2012-01-01

353

X-ray fluorescence/Auger-electron coincidence spectroscopy of vacancy cascades in atomic argon  

SciTech Connect

Argon L{sub 2.3}-M{sub 2.3}M{sub 2.3} Auger-electron spectra were measured in coincidence with K{alpha} fluorescent x-rays in studies of Ar K-shell vacancy decays at several photon energies above the K-threshold and on the 1s-4p resonance in atomic argon. The complex spectra recorded by conventional electron spectroscopy are greatly simplified when recorded in coincidence with fluorescent x-rays, allowing a more detailed analysis of the vacancy cascade process. The resulting coincidence spectra are compared with Hartree-Fock calculations which include shake-up transitions in the resonant case. Small energy shifts of the coincidence electron spectra are attributed to post-collision interaction with 1s photoelectrons.

Arp, U. [National Inst. of Standards and Technology, Gaithersburg, MD (United States). Electron and Optical Physics Div.; LeBrun, T.; Southworth, S.H.; Jung, M. [Argonne National Lab., IL (United States). Physics Div.; MacDonald, M.A. [E.P.S.R.C. Daresbury Lab., Warrington (United Kingdom)

1996-12-01

354

Investigation of polymer electrolyte membrane chemical degradation and degradation mitigation using in situ fluorescence spectroscopy  

PubMed Central

A fluorescent molecular probe, 6-carboxy fluorescein, was used in conjunction with in situ fluorescence spectroscopy to facilitate real-time monitoring of degradation inducing reactive oxygen species within the polymer electrolyte membrane (PEM) of an operating PEM fuel cell. The key requirements of suitable molecular probes for in situ monitoring of ROS are presented. The utility of using free radical scavengers such as CeO2 nanoparticles to mitigate reactive oxygen species induced PEM degradation was demonstrated. The addition of CeO2 to uncatalyzed membranes resulted in close to 100% capture of ROS generated in situ within the PEM for a period of about 7 h and the incorporation of CeO2 into the catalyzed membrane provided an eightfold reduction in ROS generation rate. PMID:22219367

Prabhakaran, Venkateshkumar; Arges, Christopher G.; Ramani, Vijay

2012-01-01

355

Combining total internal reflection sum frequency spectroscopy spectral imaging and confocal fluorescence microscopy.  

PubMed

Understanding surface and interfacial lateral organization in material and biological systems is critical in nearly every field of science. The continued development of tools and techniques viable for elucidation of interfacial and surface information is therefore necessary to address new questions and further current investigations. Sum frequency spectroscopy (SFS) is a label-free, nonlinear optical technique with inherent surface specificity that can yield critical organizational information on interfacial species. Unfortunately, SFS provides no spatial information on a surface; small scale heterogeneities that may exist are averaged over the large areas typically probed. Over the past decade, this has begun to be addressed with the advent of SFS microscopy. Here we detail the construction and function of a total internal reflection (TIR) SFS spectral and confocal fluorescence imaging microscope directly amenable to surface investigations. This instrument combines, for the first time, sample scanning TIR-SFS imaging with confocal fluorescence microscopy. PMID:25506739

Allgeyer, Edward S; Sterling, Sarah M; Gunewardene, Mudalige S; Hess, Samuel T; Neivandt, David J; Mason, Michael D

2015-01-27

356

Application of fluorescence spectroscopy for on-line bioprocess monitoring and control  

NASA Astrophysics Data System (ADS)

12 Modern bioprocess control requires fast data acquisition and in-time evaluation of bioprocess variables. On-line fluorescence spectroscopy for data acquisition and the use of chemometric methods accomplish these requirements. The presented investigations were performed with fluorescence spectrometers with wide ranges of excitation and emission wavelength. By detection of several biogenic fluorophors (amino acids, coenzymes and vitamins) a large amount of information about the state of the bioprocess are obtained. For the evaluation of the process variables partial least squares regression is used. This technique was applied to several bioprocesses: the production of ergotamine by Claviceps purpurea, the production of t-PA (tissue plasminogen activator) by animal cells and brewing processes. The main point of monitoring the brewing processes was to determine the process variables cell count and extract concentration.

Boehl, Daniela; Solle, D.; Toussaint, Hans J.; Menge, M.; Renemann, G.; Lindemann, Carsten; Hitzmann, Bernd; Scheper, Thomas-Helmut

2001-02-01

357

Synthesis of Ag clusters in microemulsions: A time-resolved UV vis and fluorescence spectroscopy study  

NASA Astrophysics Data System (ADS)

The combined use of the microemulsion technique and the kinetic control allows the preparation of small silver clusters. By using UV-vis and fluorescence spectroscopy the main stages by which the clusters grow, before the formation of nanoparticles, were elucidated. Transmission electron microscopy (TEM) and scanning tunnelling microscopy (STM) were used to further characterize the samples. Two main stages were clearly identified, which are associated with: (1) the formation of Ag n clusters with n<10, which self-aggregate into one atom high 2D nanodiscs of 3.2 nm size and (2) Ag n clusters, which self-aggregate into 3D nanostructures of 1.5 nm in size. The fluorescence properties observed with both stages show that the formed clusters are small enough to display a molecule-like behaviour.

Ledo, Ana; Martínez, F.; López-Quintela, M. A.; Rivas, J.

2007-09-01

358

High-resolution X-ray fluorescence and excitation spectroscopy of metalloproteins.  

PubMed

A spectrograph has been developed with sufficient efficiency to make high-resolution fluorescence experiments on metalloproteins possible. The resolution of this spectrometer can reach 0.45 eV at 7.1 keV emission energy. The focus images of this multiple curved-crystal array spectrometer are presented. The chemical sensitivity of Kbeta emission spectra can be used to identify chemical states, and the spin-polarized near-edge structure provides a new measure of the spin density. The high-resolution fluorescence metalloprotein studies should become routine with third-generation synchrotron facilities, and the strength of both site and spin selectivity should complement the structural information from other spectroscopies. PMID:16699236

Wang, X; Grush, M M; Froeschner, A G; Cramer, S P

1997-07-01

359

Fluorescence line narrowing spectroscopy of polycyclic aromatic hydrocarbons on solid-liquid extraction membranes.  

PubMed

The potential of solid-liquid extraction fluorescence line narrowing spectroscopy is evaluated for screening polycyclic aromatic hydrocarbons in aqueous samples. Octadecyl silica membranes are used with the dual purpose of sample preconcentration and solid substrate for spectroscopic measurements. 4.2 K fluorescence line narrowed spectra are directly recorded from the membrane with the aid of a fiber-optic probe. The experimental procedure is free from organic solvents and takes less than 5 min per sample. With 10 mL of water sample, the limits of detection are at the parts-per-billion level. Qualitative analysis is based on wavelength time matrices, which provide a unique format for compound identification based on spectral and lifetime data. The selectivity of this approach is demonstrated with the unambiguous determination of naphthalene in a heavily contaminated water sample. PMID:14658704

Bystol, Adam J; Campiglia, Andres D

2003-06-01

360

Studies on the structure of actin gels using time correlation spectroscopy of fluorescent beads.  

PubMed

Fluorescence correlation spectroscopy (FCS) has been used to measure the diffusion of fluorescently labeled beads in solutions of polymerized actin or buffer. The results, obtained at actin concentrations of 1 mg/ml, show that small beads (0.09 micron in diameter) diffuse nearly as rapidly in the actin gel as in buffer, whereas the largest beads tested (0.5 micron in diameter) are immobilized. Measured autocorrelation times for motions of beads with intermediate sizes show that the diffusion is retarded (relative to buffer) and that the time behavior cannot be represented as a single diffusive process. In addition to the retarded diffusion observed over distances > 1 micron, 0.23-micron beads also show a faster motion over smaller distances. Based on the measured rate of this faster motion, we estimate that the beads may be constrained within a cage approximately 0.67 micron on a side, equal to a filament length of approximately 250 subunits. Fluorescence correlation spectroscopy measurements made in the same small spot (radius of 1.4 microns) of the gel vary over time. From the variations of both the autocorrelation functions and the mean fluorescence, we conclude that, corresponding to a spatial scale of 1.4 microns, the actin gel is a dynamic structure with slow rearrangement of the gel occurring over periods of 20-50 s at 21-22 degrees C. This rearrangement may result from local reorganization of the actin matrix. Data for the retardation of beads by the actin gel are consistent with a detailed theory of the diffusion of particles through solutions of rigid rods that have longitudinal diffusion coefficients much less than that of the particles (Ogston, A. G., B. N. Preston, and J. D. Wells. 1973. Proc. R. Soc. Lond. A. 333:297-316). PMID:1420920

Qian, H; Elson, E L; Frieden, C

1992-10-01

361

Intermolecular disulfide bond formation promotes immunoglobulin aggregation: Investigation by fluorescence correlation spectroscopy.  

PubMed

Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (?D ) of ?200 µsec for Rhodamine-labeled IgG, corresponding to a hydrodynamic radius (RH ) of 56 Å for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of ?D under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation-dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation. Proteins 2015; 83:169-177. © 2014 Wiley Periodicals, Inc. PMID:25371040

Nag, Moupriya; Bera, Kallol; Basak, Soumen

2015-01-01

362

Plant abiotic stress diagnostic by laser induced chlorophyll fluorescence spectral analysis of in vivo leaf tissue of biofuel species  

NASA Astrophysics Data System (ADS)

Laser induced fluorescence is exploited to evaluate the effect of abiotic stresses upon the evolution and characteristics of in vivo chlorophyll emission spectra of leaves tissues of brazilian biofuel plants species(Saccharum officinarum and Jatropha curcas). The chlorophyll fluorescence spectra of 20 min predarkened intact leaves were studied employing several excitation wavelengths in the UV-VIS spectral region. Red(Fr) and far-red (FFr) chlorophyll fluorescence emission signals around 685 nm and 735 nm, respectively, were analyzed as a function of the stress intensity and the time of illumination(Kautsky effect). The Chl fluorescence ratio Fr/FFr which is a valuable nondestructive indicator of the chlorophyll content of leaves was investigated during a period of time of 30 days. The dependence of the Chl fluorescence ratio Fr/FFr upon the intensity of the abiotic stress(salinity) was examined. The results indicated that the salinity plays a major hole in the chlorophyll concentration of leaves in both plants spieces, with a significant reduction in the chlorophyll content for NaCl concentrations in the 25 - 200 mM range. The laser induced chlorophyll fluorescence analysis allowed detection of damage caused by salinity in the early stages of the plants growing process, and can be used as an early-warning indicator of salinity stress

Gouveia-Neto, Artur S.; Silva, Elias A., Jr.; Costa, Ernande B.; Bueno, Luciano A.; Silva, Luciana M. H.; Granja, Manuela M. C.; Medeiros, Maria J. L.; Câmara, Terezinha J. R.; Willadino, Lilia G.

2010-02-01

363

In Vitro and In Vivo Infection of Neural Cells by a Recombinant Measles Virus Expressing Enhanced Green Fluorescent Protein  

Microsoft Academic Search

This study focused on the in vitro infection of mouse and human neuroblastoma cells and the in vivo infection of the murine central nervous system with a recombinant measles virus. An undifferentiated mouse neuroblastoma cell line (TMN) was infected with the vaccine strain of measles virus (MVeGFP), which expresses enhanced green fluorescent protein (EGFP). MVeGFP infected the cells, and cell-to-cell

W. PAUL DUPREX; STEPHEN MCQUAID; BRANKA ROSCIC-MRKIC; ROBERTO CATTANEO; CECILIA MCCALLISTER; BERT K. RIMA

2000-01-01

364

Ultrasensitive detection of waste products in water using fluorescence emission cavity-enhanced spectroscopy  

PubMed Central

Clean water is paramount to human health. In this article, we present a technique for detection of trace amounts of human or animal waste products in water using fluorescence emission cavity-enhanced spectroscopy. The detection of femtomolar concentrations of urobilin, a metabolic byproduct of heme metabolism that is excreted in both human and animal waste in water, was achieved through the use of an integrating cavity. This technique could allow for real-time assessment of water quality without the need for expensive laboratory equipment. PMID:24799690

Bixler, Joel N.; Cone, Michael T.; Hokr, Brett H.; Mason, John D.; Figueroa, Eleonora; Fry, Edward S.; Yakovlev, Vladislav V.; Scully, Marlan O.

2014-01-01

365

Detection of orange juice frauds using front-face fluorescence spectroscopy and Independent Components Analysis.  

PubMed

The aim of this study was to find simple objective analytical methods to assess the adulteration of orange juice by grapefruit juice. The adulterations by addition of grapefruit juice were studied by 3D-front-face fluorescence spectroscopy followed by Independent Components Analysis (ICA) and by classical methods such as free radical scavenging activity and total flavonoid content. The results of this study clearly indicate that frauds by adding grapefruit juice to orange juice can be detected at percentages as low as 1%. PMID:25172702

Ammari, Faten; Redjdal, Lamia; Rutledge, Douglas N

2015-02-01

366

Layer with reduced viscosity at water-oil interfaces probed by fluorescence correlation spectroscopy.  

PubMed

The two-dimensional diffusion of isolated molecular tracers at the water-n-alkane interface was studied with fluorescence correlation spectroscopy. The interfacial diffusion coefficients of larger tracers with a hydrodynamic radius of 4.0 nm agreed well with the values calculated from the macroscopic viscosities of the two bulk phases. However, for small molecule tracers with hydrodynamic radii of only 1.0 and 0.6 nm, notable deviations were observed, indicating the existence of an interfacial region with reduced effective viscosity and increased mobility. PMID:23410340

Wang, Dapeng; Pevzner, Leonid; Li, Chen; Peneva, Kalina; Li, Christopher Y; Chan, Derek Y C; Müllen, Klaus; Mezger, Markus; Koynov, Kaloian; Butt, Hans-Jürgen

2013-01-01

367

Detection of Non-Cavitated Occlusal Caries with Impedance Spectroscopy and Laser Fluorescence: an In Vitro Study  

PubMed Central

Objective: To evaluate the performance of an impedance spectroscopy technology for detecting non-cavitated occlusal caries lesions in permanent teeth in vitro. The method was compared with a commonly used laser fluorescence device and validated against histology. Material and Methodology: A non-cavitated sample of 100 extracted posterior teeth was randomly selected and assessed for caries on enamel and dentin level with aid of CarioScan PRO (ACIS) and DIAGNOdent pen (LF pen) by three examiners. After the measurements, the extension of the lesion was histologically determined as gold standard. Sensitivity, specificity, accuracy and receiver-operating curves were calculated. Intra- and inter-examiner reproducibility was expressed by intra class correlation coefficients. Results: The histological caries prevalence was 99% and 41% exhibited dentin caries. The ACIS technique displayed high specificities but almost negligible sensitivities at readings >50. A similar pattern was noted for the LF pen at readings >30. The intra- and inter-examiner reproducibility varied between 0.47 and 0.98 and the values were generally lower for the ACIS technique than for the LF pen. The inter-examiner agreement reached excellent levels with both methods. Conclusions: In vitro,the ACIS technique showed a low ability to disclose occlusal caries lesions in the enamel and/or dentin of non-cavitated permanent molars. However, further in vivo studies of permanent occlusal surfaces are needed to mirror the clinical situation. PMID:24799965

Mortensen, Diana; Dannemand, Katrine; Twetman, Svante; Keller, Mette Kirstine

2014-01-01

368

In Vivo Fluorescence-Based Endoscopic Detection of Colon Dysplasia in the Mouse Using a Novel Peptide Probe  

PubMed Central

Colorectal cancer (CRC) is a major cause of cancer-related deaths in much of the world. Most CRCs arise from pre-malignant (dysplastic) lesions, such as adenomatous polyps, and current endoscopic screening approaches with white light do not detect all dysplastic lesions. Thus, new strategies to identify such lesions, including non-polypoid lesions, are needed. We aim to identify and validate novel peptides that specifically target dysplastic colonic epithelium in vivo. We used phage display to identify a novel peptide that binds to dysplastic colonic mucosa in vivo in a genetically engineered mouse model of colo-rectal tumorigenesis, based on somatic Apc (adenomatous polyposis coli) gene inactivation. Binding was confirmed using confocal microscopy on biopsied adenomas and excised adenomas incubated with peptide ex vivo. Studies of mice where a mutant Kras allele was somatically activated in the colon to generate hyperplastic epithelium were also performed for comparison. Several rounds of in vivo T7 library biopanning isolated a peptide, QPIHPNNM. The fluorescent-labeled peptide bound to dysplastic lesions on endoscopic analysis. Quantitative assessment revealed the fluorescent-labeled peptide (target/background: 2.17±0.61) binds ?2-fold greater to the colonic adenomas when compared to the control peptide (target/background: 1.14±0.15), p<0.01. The peptide did not bind to the non-dysplastic (hyperplastic) epithelium of the Kras mice. This work is first to image fluorescence-labeled peptide binding in vivo that is specific towards colonic dysplasia on wide-area surveillance. This finding highlights an innovative strategy for targeted detection to localize pre-malignant lesions that can be generalized to the epithelium of hollow organs. PMID:21408169

Miller, Sharon J.; Joshi, Bishnu P.; Feng, Ying; Gaustad, Adam; Fearon, Eric R.; Wang, Thomas D.

2011-01-01

369

X-Ray Fluorescence and Laser-Induced Breakdown Spectroscopy analysis of Roman silver denarii  

NASA Astrophysics Data System (ADS)

In this paper we present the results of a study performed on a large collection of silver Roman republican denarii, encompassing about two centuries of history. The joint use of Laser-Induced Breakdown Spectroscopy (LIBS) and X-Ray Fluorescence (XRF) spectroscopy allowed for an accurate determination of the coins' elemental composition; the measurements, performed mostly in situ at the 'Monetiere' in Florence, revealed a striking connection between the 'quality' of the silver alloy and some crucial contemporary events. This finding was used to classify a group of denarii whose dating was otherwise impossible. The comparison with other contemporary denarii disproves a recent theory on the origin of the so called 'serrated' denarii (denarii showing notched chisel marks on the edge of the coin).

Pardini, L.; El Hassan, A.; Ferretti, M.; Foresta, A.; Legnaioli, S.; Lorenzetti, G.; Nebbia, E.; Catalli, F.; Harith, M. A.; Diaz Pace, D.; Anabitarte Garcia, F.; Scuotto, M.; Palleschi, V.

2012-08-01

370

Tracking Biological Organic Compounds In Atmospheric Deposition In Alpine Environments With Fluorescence Spectroscopy  

NASA Astrophysics Data System (ADS)

Alpine environments, such as those of the Colorado Rocky Mountains, USA and the Sierra Nevada Mountains, Spain, contain undeveloped, barren soils that are carbon-limited. Atmospheric wet and dry deposition of organic carbon (OC) represents a substantial fraction of the OC load available to alpine soils, and includes contributions from atmospheric pollutants, dust, and biological aerosols, such as bacteria, algae, fungi, and plant debris. To evaluate the seasonal variability and sources of atmospheric deposition at these alpine sites, we measured the chemical characteristics of weekly wet and dry deposition and snowpack samples, including characterization of dissolved organic matter (DOM) and water soluble organic matter (WSOM) with fluorescence spectroscopy. The excitation-emission matrix (EEM) spectra we acquired show the presence of recurring peaks at low excitation and emission wavelengths typically associated with highly biodegradable organic carbon, presumably derived from the aromatic amino acids, tyrosine and tryptophan. Solar simulation experiments demonstrated that amino acid-like fluorescent components were more resistant to photo-degradation than humic- and fulvic-like fluorescent components. Our results also reveal the presence of a unique fluorophore, not previously described, that is found in both Rocky Mountains and the Sierra Nevada snowpack, wet deposition, and dry deposition and may be attributed to fluorescent pigments in bacteria. Biological aerosols may represent a labile source of carbon for alpine soil microbes, and consequently their deposition has important consequences for biogeochemical processes occurring in barren, alpine soils. Excitation emission matrix image of 24 Aug 2010 wet deposition sample from the Soddie site at Niwot Ridge, Colorado showing a unique fluorescent component with dual excitation peaks (285 nm and 340 nm) at 410 nm emission.

Mladenov, N.; Oldani, K. M.; Williams, M. W.; Schmidt, S. K.; Darcy, J.; Lemons, S.; Reche, I.

2013-12-01

371

Unraveling transcription factor interactions with heterochromatin protein 1 using fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy  

NASA Astrophysics Data System (ADS)

The epigenetic control of heterochromatin deposition is achieved through a network of protein interactions mediated by the heterochromatin protein 1 (HP1). In earlier studies, we showed that the CCAAT/enhancer-binding protein alpha (C/EBP?), a transcription factor that controls cell differentiation, localizes to heterochromatin, and interacts with HP1?. Here, deletion and mutagenesis are combined with live-cell imaging approaches to characterize these protein interactions. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBP? is sufficient for the interaction with HP1? in regions of heterochromatin. Fluorescence correlation spectroscopy and cross-correlation (FCS and FCCS) revealed very different diffusion profiles for HP1? and the BZip protein, and co-expression studies indicated that the mobile fractions of these nuclear proteins diffuse independently of one another. The steady-state interactions of these proteins in regions of heterochromatin were monitored using Förster resonance energy transfer (FRET). A point mutation in HP1?, W174A, which disrupts the interactions with proteins containing the common PxVxL motif did not affect the interaction with the BZip protein. In contrast, the HP1? W41A mutation, which prevents binding to methylated histones, exhibited greatly reduced FRET efficiency when compared to the wild type HP1? or HP1?W174A. The functional significance of these interactions is discussed.

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

2013-02-01

372

Application of NIR fluorescent markers to quantify expression level of HER2 receptors in carcinomas in vivo  

NASA Astrophysics Data System (ADS)

HER2 overexpression has been associated with a poor prognosis and resistance to therapy in breast cancer patients. However, quantitative estimates of this important characteristic have been limited to ex vivo ELISA essays of tissue biopsies and/or PET. We develop a novel approach in optical imaging, involving specific probes, not interfering with the binding of the therapeutic agents, thus, excluding competition between therapy and imaging. Affibody-based molecular probes seem to be ideal for in vivo analysis of HER2 receptors using near-infrared optical imaging. Fluorescence intensity distributions, originating from specific markers in the tumor area, can reveal the corresponding fluorophore concentration. We use temporal changes of the signal from a contrast agent, conjugated with HER2-specific Affibody as a signature to monitor in vivo the receptors status in mice with different HER2 over-expressed tumor models. Kinetic model, incorporating saturation of the bound ligands in the tumor area due to HER2 receptor concentration, is suggested to analyze relationship between tumor cell characteristics, i.e., HER2 overexpression, obtained by traditional ("golden standard") ex vivo methods (ELISA), and parameters, estimated from the series of images in vivo. Observed correlation between these parameters and HER2 overexpression substantiates application of our approach to quantify HER2 concentration in vivo.

Chernomordik, Victor; Hassan, Moinuddin; Lee, Sang Bong; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir

2010-02-01

373

[Discussion on diagenesis of Xilingang pluton-constrained by X-ray Fluorescence spectroscopy, plasma mass spectrometry and Raman spectroscopy].  

PubMed

The results on Xilingang pluton, mainly consisting of red beds, granites containing numerous debris of red beds and granites, obtained by X-ray fluorescence spectroscopy, plasma mass spectrometry and Raman spectroscopy show: (1) Xilingang pluton from red beds, granites containing numerous debris of red beds to granites has obvious characteristics of decreasing silicon and alkali content, and rising ignition loss, dark mineral content and oxidation index; (2) Chondrite-normalized REE distribution curves and primitive mantle-normalized spider diagram for trace elements of redbed, granites containing numerous debris of red beds and granites have a good consistency, the distribution characteristics of elements are similar to Nanling transformation-type granite; (3) The value of Raman spectrogram characteristic peak of quartz crystal in Xilingang granite decreased from the center of quartz crystal, and FWHM is steady. According to the above, the authors believe that Xilingang granite formed was related to in-situ melting of red beds and underlying strata and magma consolidation. Volatile components were discharged continuously, and oxidation index decreased gradually in the melting process. In the process of diagenesis, the top of pluton tend to be an ongoing silicon and alkali increase, while TFeO and MgO continue to migrate to bottom, and crystallization environment is a relatively closed and steady system. PMID:23905354

Tang, Yu-Kun; Chen, Guo-Neng; Zhang, Ke; Huang, Hai-Hua

2013-05-01

374

Healing and evaluating guinea pig skin incision after surgical suture and laser tissue by welding using in vivo Raman spectroscopy  

Microsoft Academic Search

Changes in collagen in the wound during the healing process of guinea pig skin following surgical incisions and LTW was evaluated using in vivo, using Raman spectroscopy. Raman spectroscopy provided information regarding the internal structure of the proteins. After the incisions were closed either by suturing or by LTW the ratio of the Raman peaks of the amide III (1247

A. Alimova; V. Sriramoju; R. Chakraverty; R. Muthukattil; R. R. Alfano

2010-01-01

375

Application of time-resolved polarization fluorescence spectroscopy in the femtosecond range to photosynthetic systems.  

PubMed

Time-resolved polarization fluorescence spectroscopy in the femtosecond range was applied to a photosynthetic antenna system. Specific signals of excited states were obtained by simultaneous measurements of fluorescence rise and decay curves and polarized spectroscopy. Relaxation processes of carotenoids, energy transfer from carotenoids to chlorophyll (Chl) a, and energy migration among pigment pools of Chl a and Chl b were clearly resolved. Two new characteristics of carotenoid molecules were revealed only by anisotropy measurements. A new singlet excited state between the well known S2 (1Bu(+)) and S1 (2Ag(-)) states was resolved by an intermediary anisotropy (r(t) = 0.30) for siphonaxanthin in chloroplasts of Codium fragile. Time-dependent changes in anisotropy with an initial value of 0.52 (r(0) = 0.52) were recorded during the relaxation of lutein molecules in the light-harvesting complexes II of Arabidopsis thaliana, and this was interpreted as a strong interaction between two lutein molecules in the pigment-protein complexes. Other examples of the application of this method were also discussed. PMID:16643087

Akimoto, Seiji; Mimuro, Mamoru

2007-01-01

376

A rapid and noninvasive method to detect dried saliva stains from human skin using fluorescent spectroscopy  

PubMed Central

Objective: Saliva is one of the vital fluids secreted in human beings. Significant amount of saliva is deposited on the skin during biting, sucking or licking, and can act as an important source in forensic evidence. An enzyme, ? amylase, gives a characteristic emission spectrum at 345–355 nm when excited at 282 nm and this can be identified by using fluorescent spectroscopy and can help in forensic identification. This study describes a rapid method to detect dried saliva on the human skin by fluorescent spectroscopy. Materials and Methods: This study included 10 volunteers, who deposited their own saliva on skin of their ventral forearm by licking and water on the contralateral arm as control. This study was carried out at Central Leather Research Institute, Chennai. Study design: Ten volunteers deposited their own saliva on skin of their ventral forearm by licking. A control sample of water was deposited at the contralateral arm. Each sample was excited at 282 nm and emission spectrum was recorded. Results: The emission spectra of 10 swab samples taken from dried saliva were characterized at the primary peak of 345 to 355 nm whereas the emission spectrum of water as a control was recorded at 362 nm. Conclusion: The presence of emission spectrum at 345–355 nm with excitation at 282 nm proves to be a strong indicator of saliva deposited on human skin. PMID:21731273

Nanda, Kanwar Deep Singh; Ranganathan, K; Umadevi, KM; Joshua, Elizabeth

2011-01-01

377

The use of solid-phase fluorescence spectroscopy in the characterisation of organic matter transformations.  

PubMed

Given its high sensitivity and non-destructive nature, fluorescence excitation-emission matrix (EEM) spectroscopy is widely used to differentiate changes and transformations of dissolved or water-extracted organic matter (OM) in natural environments. The same technique applied directly on solid samples (solid-phase fluorescence spectroscopy, SPF-EEM) provides accurate results when used with pharmaceutical products or food samples, but only a few studies have considered natural OM. This study reports on the use of SPF-EEM on solid compost samples and emphasises the way the different maturation phases can be distinguished with fluorophores closely resembling those found in dissolved samples. A very good correlation has been found with data from Rock-Eval pyrolysis, nuclear magnetic resonance ((13)C CPMAS NMR), and humic-fulvic acid ratios determined by conventional NaOH-extraction. SPF-EEM appears as a much simpler method than the conventional ones to detect transformations in natural OM samples with low mineral contents. However, direct application to soil samples requires some additional studies. PMID:25618693

Albrecht, R; Verrecchia, E; Pfeifer, H-R

2015-03-01

378

Silicon photon-counting avalanche diodes for single-molecule fluorescence spectroscopy.  

PubMed

Solution-based single-molecule fluorescence spectroscopy is a powerful experimental tool with applications in cell biology, biochemistry and biophysics. The basic feature of this technique is to excite and collect light from a very small volume and work in a low concentration regime resulting in rare burst-like events corresponding to the transit of a single molecule. Detecting photon bursts is a challenging task: the small number of emitted photons in each burst calls for high detector sensitivity. Bursts are very brief, requiring detectors with fast response time and capable of sustaining high count rates. Finally, many bursts need to be accumulated to achieve proper statistical accuracy, resulting in long measurement time unless parallelization strategies are implemented to speed up data acquisition. In this paper we will show that silicon single-photon avalanche diodes (SPADs) best meet the needs of single-molecule detection. We will review the key SPAD parameters and highlight the issues to be addressed in their design, fabrication and operation. After surveying the state-of-the-art SPAD technologies, we will describe our recent progress towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. The potential of this approach is illustrated with single-molecule Förster resonance energy transfer measurements. PMID:25309114

Michalet, Xavier; Ingargiola, Antonino; Colyer, Ryan A; Scalia, Giuseppe; Weiss, Shimon; Maccagnani, Piera; Gulinatti, Angelo; Rech, Ivan; Ghioni, Massimo

2014-11-01

379

Application of second-derivative fluorescence spectroscopy to monitor subtle changes in a monoclonal antibody structure.  

PubMed

In this study, the tertiary structure of a monoclonal antibody was analyzed under thermal and chemical stresses using second-derivative fluorescence spectroscopy. The effect of polyols, sucrose, and ethylene glycol on the tertiary structure of monoclonal antibody-U (mAb-U) (pH 7.0) was studied under thermal stress (25°C-75°C). The tertiary structure of mAb-U was also analyzed upon chemical denaturation using urea (2.0-8.0 M). The second derivative of mAb-U showed three bands corresponding to the three spectral classes of tryptophan, class I (330 nm), class II (340 nm), and class III (350 nm). Class II was higher in intensity in the presence of polyols compared with the solution without any polyol. Thermally denatured structure of mAb-U in sucrose and ethylene glycol was distinctly different than that in buffer. Addition of urea resulted in a decrease in intensity of class I and II, and an increase in intensity of class III implying unfolding. This study showed that second-derivative fluorescence spectroscopy is an effective tool to monitor subtle alterations in the tertiary structure of proteins. The unfolding of a protein is reflected as an increase in the intensity of the polar class III accompanied with a decrease in the intensity of class I. PMID:23132555

Abbas, Shermeen A; Gaspar, Greta; Sharma, Vikas K; Patapoff, Thomas W; Kalonia, Devendra S

2013-01-01

380

Bayesian approach to the analysis of fluorescence correlation spectroscopy data I: theory.  

PubMed

Fluorescence correlation spectroscopy (FCS) is a powerful tool to infer the physical process of macromolecules including local concentration, binding, and transport from fluorescence intensity measurements. Interpretation of FCS data relies critically on objective multiple hypothesis testing of competing models for complex physical processes that are typically unknown a priori. Here, we propose an objective Bayesian inference procedure for testing multiple competing models to describe FCS data based on temporal autocorrelation functions. We illustrate its performance on simulated temporal autocorrelation functions for which the physical process, noise, and sampling properties can be controlled completely. The procedure enables the systematic and objective evaluation of an arbitrary number of competing, non-nested physical models for FCS data, appropriately penalizing model complexity according to the Principle of Parsimony to prefer simpler models as the signal-to-noise ratio decreases. In addition to eliminating overfitting of FCS data, the procedure dictates when the interpretation of model parameters are not justified by the signal-to-noise ratio of the underlying sampled data. The proposed approach is completely general in its applicability to transport, binding, or other physical processes, as well as spatially resolved FCS from image correlation spectroscopy, providing an important theoretical foundation for the automated application of FCS to the analysis of biological and other complex samples. PMID:22423978

He, Jun; Guo, Syuan-Ming; Bathe, Mark

2012-05-01

381

In vivo interstitial glucose characterization and monitoring in the skin by ATR-FTIR spectroscopy  

NASA Astrophysics Data System (ADS)

Successful development of real-time non-invasive glucose monitoring would represent a major advancement not only in the treatment and management of patients with diabetes mellitus and carbohydrate metabolism disorders, but also for understanding in those biochemical, metabolic and (patho-)physiological processes of glucose at the molecular level in vivo. Here, ATR-FTIR spectroscopy technique has been challenged not only for in vivo measurement of interstitial glucose levels, but also for their non-invasive molecular qualitative and quantitative comparative characterization in the skin tissue. The results, based on calculated mean values of determined 5 glucose-specific peaks in the glucose-related 1000-1160 cm-1 region, showed intra- and inter-subject differences in interstitial glucose activity levels with their changes at different times and doses of OGTT, while raising questions about the relationships between interstitial and blood glucose levels. In conclusion, the introduction of ATR-FTIR spectroscopy technique has opened up an access to the interstitial fluid space in the skin tissue for interstitial glucose characterization and monitoring in vivo. Though interstitial versus blood glucose monitoring has different characteristics, it can be argued that accurate and precise measurements of interstitial glucose levels may be more important clinically.

Skrebova Eikje, Natalja

2011-03-01

382

In vivo optical analysis of pancreatic cancer tissue in living model mice using fluorescence and Raman spectroscopic techniques  

NASA Astrophysics Data System (ADS)

Living pancreatic cancer tissues grown subcutaneously in nude mice are studied by in vivo Raman spectroscopy and autofluorescence imaging. Comparing the same point spectra of alive pancreatic cancer tissue to that of the dead tissue, it is found that they are different each other. The results suggest that the spectral changes reflect the protein conformational changes in the tumor tissue with death of the host animal. From the result of autofluorescence study, in vivo autofluorescence imaging has potential as a method to assign the histological elements of the pancreatic cancer tissue without any staining. These results strongly suggest that combination of these techniques is very important to study biological tissue.

Suzuki, Toshiaki; Hattori, Yusuke; Katagiri, Takashi; Mitsuoka, Hiroki; Sato, Ken-ichi; Asakura, Toru; Shimosegawa, Toru; Sato, Hidetoshi

2009-02-01

383

Characterizing chlorine oxidation of dissolved organic matter and disinfection by-product formation with fluorescence spectroscopy and parallel factor analysis  

NASA Astrophysics Data System (ADS)

Relationships between chlorine demand and disinfection by-product (DBP) formation during chlorination and fluorescence of dissolved organic matter (DOM) were developed. Fluorescence excitation and emission (EEM) spectroscopy was employed, and parameters including fluorescence index, redox index, and overall fluorescence intensity (OFI) were correlated to chlorine demand and DBP formation. The EEMs were also analyzed using a well established global parallel factor analysis (PARAFAC) model which resolves the fluorescence signal into 13 components, including quinone-like and protein-like components. Over an 8-day chlorination period the OFI and sum of the 13 PARAFAC loadings decreased by more than 70%. The remaining identified quinone-like compounds within the DOM were shifted to a more oxidized state. Quinone fluorescence was strongly correlated to both reduced fluorescence intensity and to chlorine demand which indicates that fluorescence may be used to track the chlorine oxidation of DOM. Quinone fluorescence was also correlated strongly with both classes of regulated DBPs: total trihalomethanes and haloacetic acids. Quinone-like components were found to be strongly correlated to overall, short-term, and long-term specific DBP formation. The results of this study show that fluorescence is a useful tool in tracking both DOM oxidation and DBP formation during chlorination.

Beggs, Katherine M. H.; Summers, R. Scott; McKnight, Diane M.

2009-12-01

384

On-the-flow differentiation between cells based on native fluorescence spectroscopy on a chip  

NASA Astrophysics Data System (ADS)

Native fluorescence spectroscopy is a promising approach for the detection of pathogens without specific binding or tagging of the analyte. The distinction between different species is possible with (multi-color) UV excitation together with the detection of several spectral bands. We have developed a compact platform that combines a microfluidic quartz channel with chip-size wavelength-selective detection of the fluorescence from particles traversing the channel. The interaction between the UV excitation light and the analyte is enhanced by anti-resonantly guiding the light within fluid. We have recorded the intrinsic fluorescence of single cells (e.g. yeast, e-coli, and BT) passing the detection area. Knowing the particle speed and the physical dimensions of the observation window, we are able to determine particle positions with microscopic (˜10 microns) resolution. A special modulation technique allows us to achieve a high signal to noise ratio even for high particle speeds. Combining our technique with a cell sorting mechanism would allow for on-the-chip characterization and sorting of untagged cells.

Beck, Markus; Bassler, Michael; Kiesel, Peter; Johnson, Noble M.; Schmidt, Oliver

2008-03-01

385

Plasmon-Enhanced Raman and Fluorescence Spectroscopy with Gold and Silver Nanoparticles  

NASA Astrophysics Data System (ADS)

This thesis contains five major contributions to the field of plasmon-enhanced spectroscopy. We start with the report of a unique SERS study of the amino acid hydroxyproline and a deuterated analogue. Later, we move on to the exploration of a major new research path known as shell-isolated nanoparticle-enhanced fluorescence (SHINEF), consisting in the application of silica-shelled noble metal nanoparticles to achieve surface-enhanced fluorescence. The proof of concept of this technique is explained in one chapter. The two following chapters are devoted to the exploration of the plasmonic properties of SHINEF: spectral profile modification showing the close relationship between the observed enhanced fluorescence and the nanoparticle scattering. The SHIN particles are employed to experimentally prove the relationship between the SEF and SERS enhancement factors, theoretically predicted before, but never verified experimentally until now. The thesis ends with an investigation, in aqueous solutions, of several different factors that play a role in the origin of SEF, showing greater enhancement for SHINEF after inducing nanoparticle aggregation.

Guerrero Hernandez, Ariel Rodrigo

386

In Vivo Imaging Of Bone Using a Deep-Red Fluorescent Molecular Probe Bearing Multiple Iminodiacetate Groups  

PubMed Central

Deep-red fluorescent molecular probes are described that have dendritic molecular architecture with a squaraine rotaxane core scaffold and multiple peripheral iminodiacetate groups as the bone targeting units. Iminodiacetates have inherently lower bone affinity than bisphosphonates and a major goal of the study was to determine how many appended iminodiacetate groups are required for effective deep-red fluorescence imaging of bone in living rodents. A series of in vitro and in vivo imaging studies showed that a tetra(iminodiacetate) probe stains bones much more strongly than an analogous bis(iminodiacetate) probe. In addition, a control tetra(iminodiapropionate) probe exhibited no bone targeting ability. The tetra(iminodiacetate) probe targeted the same regions of high bone turnover as the near-infrared bisphosphonate probe OsteoSense®750. Longitudinal studies showed that the fluorescence image signal from living mice treated with the tetra(iminodiacetate) probe was much more stable over 19 days than the signal from OsteoSense®750. The narrow emission band of the tetra(iminodiacetate) probe makes it very attractive for inclusion in multiplex imaging protocols that employ a mixture of multiple fluorescent probes in preclinical studies of bone growth or in fluorescence guided surgery. The results also suggest that molecules or nanoparticles bearing multivalent iminodiacetate groups have promise as bone targeting agents with tunable properties for various pharmaceutical applications. PMID:24099089

Harmatys, Kara M.; Cole, Erin L.; Smith, Bradley D.

2013-01-01

387

Activatable cell penetrating peptides linked to nanoparticles as dual probes for in vivo fluorescence and MR imaging of proteases  

PubMed Central

High-resolution imaging of molecules intrinsically involved in malignancy and metastasis would be of great value for clinical detection and staging of tumors. We now report in vivo visualization of matrix metalloproteinase activities by MRI and fluorescence of dendrimeric nanoparticles coated with activatable cell penetrating peptides (ACPPs), labeled with Cy5, gadolinium, or both. Uptake of such nanoparticles in tumors is 4- to 15-fold higher than for unconjugated ACPPs. With fluorescent molecules, we are able to detect residual tumor and metastases as small as 200 ?m, which can be resected under fluorescence guidance and analyzed histopathologically with fluorescence microscopy. We show that uptake via this mechanism is comparable to that of other near infrared protease sensors, with the added advantage that the approach is translatable to MRI. Once activated, the Gd-labeled nanoparticles deposit high levels (30–50 ?M) of Gd in tumor parenchyma with even higher amounts deposited in regions of infiltrative tumor, resulting in useful T1 contrast lasting several days after injection. These results should improve MRI-guided clinical staging, presurgical planning, and intraoperative fluorescence-guided surgery. The approach may be generalizable to deliver radiation-sensitizing and chemotherapeutic agents. PMID:20160077

Olson, Emilia S.; Jiang, Tao; Aguilera, Todd A.; Nguyen, Quyen T.; Ellies, Lesley G.; Scadeng, Miriam; Tsien, Roger Y.

2010-01-01

388

Local conformations and excited state dynamics of porphyrins and nucleic acids by 2-dimensional fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

Biological systems present many challenges to researchers attempting to study them using spectroscopy. Low specificity, low sensitivity, and broad and overlapping lineshapes limit the amount of information that can be obtained in experiments. Two-dimensional fluorescence spectroscopy (2D FS) is a highly sensitive and information-rich spectroscopic technique that was developed to study the conformations and excited state dynamics of systems exhibiting exciton coupling. In this dissertation, I describe a variety of extensions of 2D FS that further increase its utility for the study of biological systems. I describe experiments on a dimer of zinc tetraphenylporphyrin embedded in a membrane, in which the signals from two conformational subpopulations were separated in order to study the thermodynamics of their interconversion. I present proof-of-principle experiments on nucleic acids that utilize fluorescence resonance energy transfer to separate signals from different subpopulations. I also describe experiments in which 2D FS was performed using ultraviolet excitation to determine the conformation of a dinucleotide of a fluorescent analogue of the nucleic acid base adenine. I discuss experiments on porphyrin dimers in which 2D FS was used as a probe of excited state dynamics. Finally, I present model calculations for a proposed variation of 2D FS in which entangled photons would be used as the excitation source. These calculations suggest that this approach has the potential to yield significantly narrower spectral lineshapes than conventional 2D FS. These experiments and calculations yield new insight into the systems investigated and establish a `toolbox' of variations of 2D FS that can be used to gain as much information as possible from experiments on challenging systems such as protein-DNA complexes.

Widom, Julia R.

389

Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in the rat.  

PubMed

Abstract The mononuclear phagocyte system (MPS) which provides protection against infection is made up of phagocytic cells that engulf and digest bacteria or other foreign substances. Suppression of the MPS may lead to decreased clearance of pathogenic microbes. Drug delivery systems and immunomodulatory therapeutics that target phagocytes have a potential to inhibit MPS function. Available methods to measure inhibition of MPS function use uptake of radioactively-labeled cells or labor-intensive semi-quantitative histologic techniques. The objective of this work was to develop a non-radioactive quantitative method to measure MPS function in vivo by administering heat-killed E. coli conjugated to a pH-sensitive fluorescent dye (Bioparticles®). Fluorescence of the Bioparticles® is increased at low pH when they are in phagocytic lysosomes. The amount of Bioparticles® phagocytosed by MPS organs in rats was determined by measuring fluorescence intensity in livers and spleens ex vivo using an IVIS® Spectrum Pre-clinical In Vivo Imaging System. Phagocytosis of the particles by peripheral blood neutrophils was measured by flow cytometry. To assess method sensitivity, compounds likely to suppress the MPS [clodronate-containing liposomes, carboxylate-modified latex particles, maleic vinyl ether (MVE) polymer] were administered to rats prior to injection of the Bioparticles®. The E. coli particles consistently co-localized with macrophage markers in the liver but not in the spleen. All of the compounds tested decreased phagocytosis in the liver, but had no consistent effects on phagocytic activity in the spleen. In addition, administration of clodronate liposomes and MVE polymer increased the percentage of peripheral blood neutrophils that phagocytosed the Bioparticles®. In conclusion, an in vivo rat model was developed that measures phagocytosis of E. coli particles in the liver and may be used to assess the impact of test compounds on MPS function. Still, the detection of inhibition of splenic macrophage function will require further assay development. PMID:25027674

Tartaro, Karrie; VanVolkenburg, Maria; Wilkie, Dean; Coskran, Timothy M; Kreeger, John M; Kawabata, Thomas T; Casinghino, Sandra

2014-07-16

390

Hyperspectral fluorescence lifetime fibre probe spectroscopy for use in the study and diagnosis of osteoarthritis and skin cancer  

NASA Astrophysics Data System (ADS)

We present the application of two fibre-optic-coupled time-resolved spectrofluorometers and a compact steady-state diffuse reflected light/fluorescence spectrometer to in vivo and ex vivo studies of skin cancer and osteoarthritis. In a clinical study of skin cancer, 27 lesions on 25 patients were investigated in vivo before surgical excision of the region measured. Preliminary analysis reveals a statistically significant decrease in the autofluorescence lifetime of basal cell carcinomas compared to neighbouring healthy tissue. A study of autofluorescence signals associated with the onset of osteoarthritis indicates autofluorescence lifetime changes associated with collagen degradation.

Thompson, Alex; Manning, Hugh; Brydegaard, Mikkel; Coda, Sergio; Kennedy, Gordon; Patalay, Rakesh; Waitong-Braemming, Ulrika; De Beule, Pieter; Neil, Mark; Andersson-Engels, Stefan; Itoh, Yoshifumi; Bendsøe, Niels; Dunsby, Christopher; Svanberg, Katarina; French, Paul M.

2011-03-01

391

Characterization of dissolved organic matter in urban sewage using excitation emission matrix fluorescence spectroscopy and parallel factor analysis  

Microsoft Academic Search

Wastewater dissolved organic matter (DOM) from different processing stages of a sewage treatment plant in Xiamen was characterized using fluorescence and absorption spectroscopy. Parallel factor analysis modeling of excitation-emission matrix spectra revealed five fluorescent components occurring in sewage DOM: one protein-like (C1), three humic-like (C2, C4 and C5) and one xenobiotic-like (C3) components. During the aerated grit chamber and primary

Weidong Guo; Jing Xu; Jiangping Wang; Yingrou Wen; Jianfu Zhuo; Yuchao Yan

2010-01-01

392

A system for endoscopic mechanically scanned localized proton MR and light-induced fluorescence emission spectroscopies  

NASA Astrophysics Data System (ADS)

Molecular and near-cellular modalities offer new opportunities in assessing living tissue in situ, and multimodality approaches, which offer complementary information, may lead to improved characterization of tissue pathophysiology benefiting diagnosis and focal therapy. However, many such modalities are limited by their low penetration through tissue, which has led to minimally invasive trans-cannula approaches to place the corresponding sensors locally at the area of interest. This work presents a system for performing localized fluorescence emission and proton magnetic resonance (MR) spectroscopies via endoscopic access. The in-house developed side-firing 1.9-mm wide dual-sensor integrates a three-fiber optical sensor for fluorescence emission optical spectroscopy and a 1-mm circular radiofrequency (RF) coil for localized MR proton spectroscopy. An MR-compatible manipulator was developed for carrying and mechanically translating the dual-sensor along a linear access channel. The hardware and software control of the system allows reconfigurable synchronization of the manipulator-assisted translation of the sensor, and MR and optical data collection. The manipulator serves as the mechanical link for the three modalities and MR images, MR spectra and optical spectra are inherently co-registered to the MR scanner coordinate system. These spectra were then used to generate spatio-spectral maps of the fluorophores and proton MR-signal sources in three-compartment phantoms with optically- and MR-visible, and distinguishable, materials. These data demonstrate a good spatial match between MR images, MR spectra and optical spectra along the scanned path. In addition to basic research, such a system may have clinical applications for assessing and characterizing cancer in situ, as well as guiding focal therapies.

Sonmez, Ahmet E.; Webb, Andrew G.; Spees, William M.; Ozcan, Alpay; Tsekos, Nikolaos V.

2012-09-01

393

A system for endoscopic mechanically scanned localized proton MR and light-induced fluorescence emission spectroscopies.  

PubMed

Molecular and near-cellular modalities offer new opportunities in assessing living tissue in situ, and multimodality approaches, which offer complementary information, may lead to improved characterization of tissue pathophysiology benefiting diagnosis and focal therapy. However, many such modalities are limited by their low penetration through tissue, which has led to minimally invasive trans-cannula approaches to place the corresponding sensors locally at the area of interest. This work presents a system for performing localized fluorescence emission and proton magnetic resonance (MR) spectroscopies via endoscopic access. The in-house developed side-firing 1.9-mm wide dual-sensor integrates a three-fiber optical sensor for fluorescence emission optical spectroscopy and a 1-mm circular radiofrequency (RF) coil for localized MR proton spectroscopy. An MR-compatible manipulator was developed for carrying and mechanically translating the dual-sensor along a linear access channel. The hardware and software control of the system allows reconfigurable synchronization of the manipulator-assisted translation of the sensor, and MR and optical data collection. The manipulator serves as the mechanical link for the three modalities and MR images, MR spectra and optical spectra are inherently co-registered to the MR scanner coordinate system. These spectra were then used to generate spatio-spectral maps of the fluorophores and proton MR-signal sources in three-compartment phantoms with optically- and MR-visible, and distinguishable, materials. These data demonstrate a good spatial match between MR images, MR spectra and optical spectra along the scanned path. In addition to basic research, such a system may have clinical applications for assessing and characterizing cancer in situ, as well as guiding focal therapies. PMID:22820260

Sonmez, Ahmet E; Webb, Andrew G; Spees, William M; Ozcan, Alpay; Tsekos, Nikolaos V

2012-09-01

394

Fluorescence spectroscopy: a promising tool for gear-oil condition monitoring  

NASA Astrophysics Data System (ADS)

Wind power is one of the most promising green energy sources, especially when produced in offshore power plants. Corrective operations in wind turbines cause a considerable part of the maintenance costs of such plants. One preventive action for reducing such operations is the periodic off-line control of oil samples from the wind turbines. The time delay between sampling and availability of the results is a major disadvantage of this kind of controlling. In-situ condition monitoring is a solution to this problem. In-situ monitoring allows real time detection of random, time discrete events, thus enabling a better scheduling of preventive actions and reducing costs and downtime. Fluorescence spectroscopy is a complementary technique to absorption spectroscopy. Due to absorption of UV or visible light, the electrons of specific molecules are excited from a ground electronic state to a vibrational state of higher energy. By collision with other molecules, the excited electron looses a part of the acquired energy and relaxes to a lower vibrational state. The remaining acquired energy is emitted during the electron's transition to the ground state. The resulting frequency shift between excitation and emission energy, known as Stokes shift, is unique and characteristic for each active molecule. In this paper gear-oil condition monitoring based on fluorescence spectroscopy is proposed. Three typical commercial gear-oils for wind turbines were studied. The spectra gained by UV excitation of the samples were analyzed by means of partial least square (PLS) regression. Good prediction results were obtained for the total acid number (TAN). The latter is a measure for the oil acidity and is considered to be a proxy variable for oil age. Other parameters delivering information about gear-oil additive depletion and the related oil aging condition, like phosphor, sulfur and molybdenum concentration, were also analyzed.

Dorigo, Daniel D.; Wiesent, Benjamin R.; Simsek, Özlem; Pérez Grassi, A.; Koch, Alexander W.

2012-04-01

395

In vivo spectroscopic photoacoustic tomography imaging of a far red fluorescent protein expressed in the exocrine pancreas of adult zebrafish  

NASA Astrophysics Data System (ADS)

Fluorescent proteins brought a revolution in life sciences and biological research in that they make a powerful tool for researchers to study not only the structural and morphological information, but also dynamic and functional information in living cells and organisms. While green fluorescent proteins (GFP) have become a common labeling tool, red-shifted or even near infrared fluorescent proteins are becoming the research focus due to the fact that longer excitation wavelengths are more suitable for deep tissue imaging. In this study, E2-Crimson, a far red fluorescent protein whose excitation wavelength is 611 nm, was genetically expressed in the exocrine pancreas of adult zebrafish. Using spectroscopic all optical detection photoacoustic tomography, we mapped the distribution of E2-Crimson in 3D after imaging the transgenic zebrafish in vivo using two different wavelengths. With complementary morphological information provided by imaging the same fish using a spectral domain optical coherence tomography system, the E2-Crimson distribution acquired from spectroscopic photoacoustic tomography was confirmed in 2D by epifluorescence microscopy and in 3D by histology. To the authors' knowledge, this is the first time a far red fluorescent protein is imaged in vivo by spectroscopic photoacoustic tomography. Due to the regeneration feature of zebrafish pancreas, this work preludes the longitudinal studies of animal models of diseases such as pancreatitis by spectroscopic photoacoustic tomography. Since the effective penetration depth of photoacoustic tomography is beyond the transport mean free path length, other E2-Crimson labeled inner organs will also be able to be studied dynamically using spectroscopic photoacoustic tomography.

Liu, Mengyang; Schmitner, Nicole; Sandrian, Michelle G.; Zabihian, Behrooz; Hermann, Boris; Salvenmoser, Willi; Meyer, Dirk; Drexler, Wolfgang

2014-03-01

396

Leaf water dynamics of Arabidopsis thaliana monitored in-vivo using terahertz time-domain spectroscopy  

PubMed Central

The declining water availability for agriculture is becoming problematic for many countries. Therefore the study of plants under water restriction is acquiring extraordinary importance. Botanists currently follow the dehydration of plants comparing the fresh and dry weight of excised organs, or measuring their osmotic or water potentials; these are destructive methods inappropriate for in-vivo determination of plants' hydration dynamics. Water is opaque in the terahertz band, while dehydrated biological tissues are partially transparent. We used terahertz spectroscopy to study the water dynamics of Arabidopsis thaliana by comparing the dehydration kinetics of leaves from plants under well-irrigated and water deficit conditions. We also present measurements of the effect of dark-light cycles and abscisic acid on its water dynamics. The measurements we present provide a new perspective on the water dynamics of plants under different external stimuli and confirm that terahertz can be an excellent non-contact probe of in-vivo tissue hydration. PMID:24105302

Castro-Camus, E.; Palomar, M.; Covarrubias, A. A.

2013-01-01

397

Leaf water dynamics of Arabidopsis thaliana monitored in-vivo using terahertz time-domain spectroscopy  

NASA Astrophysics Data System (ADS)

The declining water availability for agriculture is becoming problematic for many countries. Therefore the study of plants under water restriction is acquiring extraordinary importance. Botanists currently follow the dehydration of plants comparing the fresh and dry weight of excised organs, or measuring their osmotic or water potentials; these are destructive methods inappropriate for in-vivo determination of plants' hydration dynamics. Water is opaque in the terahertz band, while dehydrated biological tissues are partially transparent. We used terahertz spectroscopy to study the water dynamics of Arabidopsis thaliana by comparing the dehydration kinetics of leaves from plants under well-irrigated and water deficit conditions. We also present measurements of the effect of dark-light cycles and abscisic acid on its water dynamics. The measurements we present provide a new perspective on the water dynamics of plants under different external stimuli and confirm that terahertz can be an excellent non-contact probe of in-vivo tissue hydration.

Castro-Camus, E.; Palomar, M.; Covarrubias, A. A.

2013-10-01

398

Immunoglobulin surface-binding kinetics studied by total internal reflection with fluorescence correlation spectroscopy.  

PubMed Central

An experimental application of total internal reflection with fluorescence correlation spectroscopy (TIR/FCS) is presented. TIR/FCS is a new technique for measuring the binding and unbinding rates and surface diffusion coefficient of fluorescent-labeled solute molecules in equilibrium at a surface. A laser beam totally internally reflects at the solid-liquid interface, selectively exciting surface-adsorbed molecules. Fluorescence collected by a microscope from a small, well-defined surface area approximately 5 micron2 spontaneously fluctuates as solute molecules randomly bind to, unbind from, and/or diffuse along the surface in chemical equilibrium. The fluorescence is detected by a photomultiplier and autocorrelated on-line by a minicomputer. The shape of the autocorrelation function depends on the bulk and surface diffusion coefficients, the binding rate constants, and the shape of the illuminated and observed region. The normalized amplitude of the autocorrelation function depends on the average number of molecules bound within the observed area. TIR/FCS requires no spectroscopic or thermodynamic change between dissociated and complexed states and no extrinsic perturbation from equilibrium. Using TIR/FCS, we determine that rhodamine-labeled immunoglobulin and insulin each nonspecifically adsorb to serum albumin-coated fused silica with both reversible and irreversible components. The characteristic time of the most rapidly reversible component measured is approximately 5 ms and is limited by the rate of bulk diffusion. Rhodamine-labeled bivalent antibodies to dinitrophenyl (DNP) bind to DNP-coated fused silica virtually irreversibly. Univalent Fab fragments of these same antibodies appear to specifically bind to DNP-coated fused silica, accompanied by a large amount of nonspecific binding. TIR/FCS is shown to be a feasible technique for measuring absorption/desorption kinetic rates at equilibrium. In suitable systems where nonspecific binding is low, TIR/FCS should prove useful for measuring specific solute-surface kinetic rates. Images FIGURE 3 PMID:6882857

Thompson, N L; Axelrod, D

1983-01-01

399

Linking fluorescence spectroscopy to diffuse soil source for dissolved humic substances in the Daning River, China.  

PubMed

Dissolved organic matter collected in Daning River (China) in July 2009 was investigated with parallel factor analysis (PARAFAC) and fluorescence spectroscopy with the aim of identifying the origin of dissolved humic substance (HS) components. Two HS-like fluorescence components (peak M and C) with excitation/emission (ex/em) maxima at 305/406 nm and 360/464 nm showed relatively uniform distribution in the vertical direction for each sampling site but a trend of accumulation down the river, independent of the highly heterogeneous water environment as implicated by water quality parameters (i.e., water temperature, algae density, chlorophyll a, dissolved oxygen, dissolved organic carbon, pH, conductivity and turbidity), while an amino acid/protein-like component (peak T; ex/em = 280/334 nm) was quite variable in its spatial distribution, implying strong influence from point sources (e.g. sewage discharge) and local microbial activities. The fluorescence intensity (F max in Raman units) at these ex/em wavelength pairs fell in the range of 0.031-0.358, 0.051-0.224 and 0.026-0.115 for peak T, M and C, respectively. In addition, the F max values of peak C covaried with M (i.e. C = 0.503 ×M, p < 0.01, R (2) = 0.973). Taken together, these results indicate that peak M and C originated primarily and directly from the same soil sources that were diffusive in the catchment, but peak T was more influenced by local point sources (e.g. wastewater discharge) and in situ microbial activities. This study presents new insights into the currently controversial origin of some HS components (e.g."peak M", as commonly referred to in the literature). This study highlights that