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1

Following protein association in vivo with fluorescence fluctuation spectroscopy  

NASA Astrophysics Data System (ADS)

The combination of fluorescence correlation spectroscopy and two-photon excitation provides us with a powerful spectroscopic technique. Its submicron resolution and single molecule sensitivity make it an attractive technique for in vivo applications. Experiments have demonstrated that quantitative in vivo fluorescence fluctuation measurements are feasible, despite the presence of autofluorescence and the heterogeneity of the cellular environment. I will demonstrate that molecular brightness of proteins tagged with green fluorescent protein (GFP) is a useful and robust parameter for in vivo studies. Knowledge of photon statistics is crucial for the interpretation of fluorescence fluctuation experiments. I will describe photon counting histogram (PCH) analysis, which determines the molecular brightness and complements autocorrelation analysis. Non-ideal detector effects and their influence on the photon statistics will be discussed. The goal of in vivo fluorescence fluctuation experiments is to address functional properties of biomolecules. We will focus on retinoid X receptor (RXR), a nuclear receptor, which is crucial for the regulation of gene expression. The fluorescence brightness of RXR tagged with EGFP will be used to probe the oligomerization state of RXR.

Muller, Joachim D.

2003-07-01

2

Toward Quantitative "In Vivo Biochemistry" with Fluorescence Fluctuation Spectroscopy  

PubMed Central

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

2010-01-01

3

Fluorescence lifetime correlation spectroscopy for precise concentration detection in vivo by background subtraction  

NASA Astrophysics Data System (ADS)

In vivo studies of single molecule dynamics by means of Fluorescence correlation spectroscopy can suffer from high background. Fluorescence lifetime correlation spectroscopy provides a tool to distinguish between signal and unwanted contributions via lifetime separation. By studying the motion of the RNA-induced silencing complex (RISC) within two compartments of a human cell, the nucleus and the cytoplasm, we observed clear differences in concentration as well as mobility of the protein complex between those two locations. Especially in the nucleus, where the fluorescence signal is very weak, a correction for background is crucial to provide reliable results of the particle number. Utilizing the fluorescent lifetime of the different contributions, we show that it is possible to distinguish between the fluorescent signal and the autofluorescent background in vivo in a single measurement.

Gärtner, Maria; Mütze, Jörg; Ohrt, Thomas; Schwille, Petra

2009-07-01

4

Molecular Brightness Characterization of EGFP In Vivo by Fluorescence Fluctuation Spectroscopy  

Microsoft Academic Search

We characterize the molecular properties of autofluorescence and transiently expressed EGFP in the nucleus and in the cytoplasm of HeLa cells by fluorescence correlation spectroscopy (FCS) and by photon counting histogram (PCH) analysis. PCH has been characterized and applied in vitro, but its potential for in vivo studies needs to be explored. Thus, this study mainly focuses on the characterization

Yan Chen; Joachim D. Müller; QiaoQiao Ruan; Enrico Gratton

2002-01-01

5

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

6

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

NASA Astrophysics Data System (ADS)

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

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

2010-05-01

7

Dual-color fluorescence fluctuation spectroscopy in vitro and in vivo  

NASA Astrophysics Data System (ADS)

The combination of dual-color fluorescence correlation spectroscopy (FCS) and two-photon excitation is a powerful tool for probing protein-protein interactions. The submicron resolution and single molecule sensitivity of the technique make it attractive for in vivo applications. However, the strong spectral cross talk between the two emission channels of most fluorescent dye mixtures provides a challenge for the analysis of dual-color FCS experiments. We describe a new technique, dual-color photon counting histogram (PCH) analysis that overcomes some of the challenges associated with spectral cross talk. Dual-color PCH is an extension of regular PCH that simultaneously analyses the photon counts of two detection channels. We demonstrate that dual color PCH quantitatively resolves protein mixtures in vitro. We also apply dual-color PCH to study proteins in biological cells. The fluorescent proteins ECFP and EYFP, which are commonly used for dual-color studies in cells, have significant spectral cross talk. We will discuss the resolvability of these fluorescent proteins and present data that successfully resolve the protein mixtures in vitro and in vivo. Our results show that dual color PCH is a promising technique for the characterization of protein-protein interactions in intact cells.

Mueller, Joachim D.; Tekmen, Mohac; Hillesheim, Lindsey; Yang, Weidong; Chen, Yan

2004-06-01

8

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.

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

2012-01-01

9

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

PubMed

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

2012-06-05

10

Cell Cycle-Dependent Mobility of Cdc45 Determined in vivo by Fluorescence Correlation Spectroscopy  

PubMed Central

Eukaryotic DNA replication is a dynamic process requiring the co-operation of specific replication proteins. We measured the mobility of eGFP-Cdc45 by Fluorescence Correlation Spectroscopy (FCS) in vivo in asynchronous cells and in cells synchronized at the G1/S transition and during S phase. Our data show that eGFP-Cdc45 mobility is faster in G1/S transition compared to S phase suggesting that Cdc45 is part of larger protein complex formed in S phase. Furthermore, the size of complexes containing Cdc45 was estimated in asynchronous, G1/S and S phase-synchronized cells using gel filtration chromatography; these findings complemented the in vivo FCS data. Analysis of the mobility of eGFP-Cdc45 and the size of complexes containing Cdc45 and eGFP-Cdc45 after UVC-mediated DNA damage revealed no significant changes in diffusion rates and complex sizes using FCS and gel filtration chromatography analyses. This suggests that after UV-damage, Cdc45 is still present in a large multi-protein complex and that its mobility within living cells is consistently similar following UVC-mediated DNA damage.

Toth, Katalin; Togashi, Denisio M.; Ryder, Alan G.; Langowski, Jorg; Nasheuer, Heinz Peter

2012-01-01

11

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

12

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

13

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

PubMed Central

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

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

1997-01-01

14

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

NASA Astrophysics Data System (ADS)

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

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

2013-02-01

15

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.

2012-02-01

16

Quantitative Diffuse Reflectance and Fluorescence Spectroscopy: A Tool to Monitor Tumor Physiology In Vivo  

PubMed Central

This study demonstrates the use of optical spectroscopy for monitoring tumor oxygenation and metabolism in response to hyperoxic gas breathing. Hemoglobin saturation and redox ratio were quantified for a set of 14 and 9 mice, respectively, measured at baseline and during carbogen breathing (95% O2, 5% CO2). In particular, significant increases in hemoglobin saturation and fluorescence redox ratio were observed upon carbogen breathing. These data were compared to that obtained concurrently using an established invasive technique, the OxyLite pO2 system, which also showed a significant increase in pO2. It was found that the direction of changes were generally the same between all of the methods, but that the OxyLite system was much more variable in general, suggesting that optical techniques may provide a better assessment of global tumor physiology. Optical spectroscopy measurements are demonstrated to provide a reliable, reproducible indication of changes in tumor physiology in response to physiologic manipulation.

Palmer, Gregory M.; Viola, Ronald J.; Schroeder, Thies; Yarmolenko, Pavel S.; Dewhirst, Mark W.; Ramanujam, Nirmala

2009-01-01

17

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

NASA Astrophysics Data System (ADS)

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

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

2007-07-01

18

Fluorescence correlation spectroscopy.  

PubMed

Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure concentrations, mobilities, and interactions of fluorescent biomolecules. It can be applied to various biological systems such as simple homogeneous solutions, cells, artificial, or cellular membranes and whole organisms. Here, we introduce the basic principle of FCS, discuss its application to biological questions as well as its limitations and challenges, present an overview of novel technical developments to overcome those challenges, and conclude with speculations about the future applications of fluorescence fluctuation spectroscopy. PMID:22415816

Ries, Jonas; Schwille, Petra

2012-03-13

19

In-vivo optical detection of cancer using chlorin e6 – polyvinylpyrrolidone induced fluorescence imaging and spectroscopy  

Microsoft Academic Search

BACKGROUND: Photosensitizer based fluorescence imaging and spectroscopy is fast becoming a promising approach for cancer detection. The purpose of this study was to examine the use of the photosensitizer chlorin e6 (Ce6) formulated in polyvinylpyrrolidone (PVP) as a potential exogenous fluorophore for fluorescence imaging and spectroscopic detection of human cancer tissue xenografted in preclinical models as well as in a

William WL Chin; Patricia SP Thong; Ramaswamy Bhuvaneswari; Khee Chee Soo; Paul WS Heng; Malini Olivo

2009-01-01

20

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

21

In Vivo Hexamerization and Characterization of the Arabidopsis AAA ATPase CDC48A Complex Using F?rster Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy and Fluorescence Correlation Spectroscopy1[W][OA  

PubMed Central

The Arabidopsis (Arabidopsis thaliana) AAA ATPase CDC48A was fused to cerulean fluorescent protein and yellow fluorescent protein. AAA ATPases like CDC48 are only active in hexameric form. Förster resonance energy transfer-based fluorescence lifetime imaging microscopy using CDC48A-cerulean fluorescent protein and CDC48A-yellow fluorescent protein showed interaction between two adjacent protomers, demonstrating homo-oligomerization occurs in living plant cells. Interaction between CDC48A and the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) transmembrane receptor occurs in very restricted domains at the plasma membrane. In these domains the predominant form of the fluorescently tagged CDC48A protein is a hexamer, suggesting that SERK1 is associated with the active form of CDC48A in vivo. SERK1 trans-phosphorylates CDC48A on Ser-41. Förster resonance energy transfer-fluorescence lifetime imaging microscopy was used to show that in vivo the C-terminal domains of CDC48A stay in close proximity. Employing fluorescence correlation spectroscopy, it was shown that CDC48A hexamers are part of larger complexes.

Aker, Jose; Hesselink, Renske; Engel, Ruchira; Karlova, Rumyana; Borst, Jan Willem; Visser, Antonie J.W.G.; de Vries, Sacco C.

2007-01-01

22

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

23

Multimodal fluorescence imaging spectroscopy.  

PubMed

Multimodal fluorescence imaging is a versatile method that has a wide application range from biological studies to materials science. Typical observables in multimodal fluorescence imaging are intensity, lifetime, excitation, and emission spectra which are recorded at chosen locations at the sample. This chapter describes how to build instrumentation that allows for multimodal fluorescence imaging and explains data analysis procedures for the observables. PMID:24108642

Stopel, Martijn H W; Blum, Christian; Subramaniam, Vinod

2014-01-01

24

Fluorescence spectroscopy of peptides.  

PubMed

In peptides, steady-state fluorescence can be used to measure the intrinsic fluorescence of Trp, Tyr, and Phe residues, as well as associated dyes, which can change upon exposure to different environmental conditions. This technique can be thus used to detect changes in the conformational states of peptides. Time-resolved fluorescence can also be used to study fast motions of peptides and their interactions with fluorescence dyes. Data from several low-resolution spectroscopic techniques, including fluorescence, can be combined to generate an overall picture of peptide structure as a function of environmental conditions. PMID:24146408

Liyanage, Mangala R; Bakshi, Kunal; Volkin, David B; Middaugh, C Russell

2014-01-01

25

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

NASA Astrophysics Data System (ADS)

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.

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

2012-11-01

26

In vivo fluorescence lifetime tomography  

NASA Astrophysics Data System (ADS)

Local molecular and physiological processes can be imaged in vivo through perturbations in the fluorescence lifetime (FLT) of optical imaging agents. In addition to providing functional information, FLT methods can quantify specific molecular events and multiplex diagnostic and prognostic information. We have developed a fluorescence lifetime diffuse optical tomography (DOT) system for in vivo preclinical imaging. Data is captured using a time-resolved intensified charge coupled device (ICCD) system to measure fluorescence excitation and emission in the time domain. Data is then converted to the frequency domain, and we simultaneously reconstruct images of yield and lifetime using an extension to the normalized Born approach. By using differential phase measurements, we demonstrate DOT imaging of short lifetimes (from 350 ps) with high precision (+/-5 ps). Furthermore, this system retains the efficiency, speed, and flexibility of transmission geometry DOT. We demonstrate feasibility of FLT-DOT through a progressive series of experiments. Lifetime range and repeatability are first measured in phantoms. Imaging of subcutaneous implants then verifies the FLT-DOT approach in vivo in the presence of inhomogeneous optical properties. Use in a common research scenario is ultimately demonstrated by imaging accumulation of a targeted near-infrared (NIR) fluorescent-labeled peptide probe (cypate-RGD) in a mouse with a subcutaneous tumor.

Nothdurft, Ralph E.; Patwardhan, Sachin V.; Akers, Walter; Ye, Yunpeng; Achilefu, Samuel; Culver, Joseph P.

2009-03-01

27

Fluorescence Lifetime Selectivity in Multifrequency Phase-Resolved Fluorescence Spectroscopy,  

National Technical Information Service (NTIS)

Theoretical aspects of the implementation of fluorescence lifetime selectivity in multifrequency phase resolved fluorescence spectroscopy (PRFS) are discussed. The dependence of phase-resolved fluorescence intensities on the dynamic instrumental parameter...

L. McGown

1988-01-01

28

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

NASA Astrophysics Data System (ADS)

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

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

2010-01-01

29

Fluorescence Spectroscopy in a Shoebox  

Microsoft Academic Search

This article describes construction of a simple, inexpensive fluorometer. It utilizes a flashlight or sunlight source, highlighter marker ink, bowl of water with mirror as dispersing element, and colored cellophane sheets as filters. The human eye is used as a detector. This apparatus is used to demonstrate important concepts related to fluorescence spectroscopy. Using ink from a highlighter marker, one

M. Farooq Wahab

2007-01-01

30

Dispersion-Relation Fluorescence Spectroscopy  

NASA Astrophysics Data System (ADS)

Because of its ability to study specifically labeled structures, fluorescence microscopy is the most widely used technique for investigating live cell dynamics and function. Fluorescence correlation spectroscopy is an established method for studying molecular transport and diffusion coefficients at a fixed spatial scale. We propose a new approach, dispersion-relation fluorescence spectroscopy (DFS), to study the transport dynamics over a broad range of spatial and temporal scales. The molecules of interest are labeled with a fluorophore whose motion gives rise to spontaneous fluorescence intensity fluctuations that are analyzed to quantify the governing mass transport dynamics. These data are characterized by the effective dispersion relation. We report on experiments demonstrating that DFS can distinguish diffusive from advection motion in a model system, where we obtain quantitatively accurate values of both diffusivities and advection velocities. Because of its spatially resolved information, DFS can distinguish between directed and diffusive transport in living cells. Our data indicate that the fluorescently labeled actin cytoskeleton exhibits active transport motion along a direction parallel to the fibers and diffusive in the perpendicular direction.

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

2012-11-01

31

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  

Microsoft Academic Search

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 sub- nuclear 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 ho- modimers localize diffusely in the nucleus, whereas

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

2005-01-01

32

Recent advances in fluorescence correlation spectroscopy  

Microsoft Academic Search

Fluorescence correlation spectroscopy is a method in which fluctuations in the fluorescence arising from a very small sample volume are correlated to obtain information about the processes giving rise to the fluctuations. Recent progress has been made in methodologies such as two-photon excitation, photon counting histogram analysis, cross-correlation, image correlation and evanescent excitation. Fluorescence correlation spectroscopy techniques have been applied

Nancy L Thompson; Alena M Lieto; Noah W Allen

2002-01-01

33

Fluorescence spectroscopy in nucleic acid research  

Microsoft Academic Search

Fluorescence spectroscopy is a type of electromagnetic spectroscopy, which uses the UV light to excite certain molecules and cause them to emit light of lower energy, usually in the visible range of the spectrum. It has a wide area of applications such as in pharmaceutical sciences, chemistry, molecular biology, biotechnology etc. Fluorescence spectroscopy involves the electronic and vibrational states of

Mohammad Nasir Uddin

34

Calibrated fluorescence imaging of tissue in vivo  

SciTech Connect

A calibrated fluorescence imaging system utilizing a combination of fluorescence and cross-polarization imaging technology is described and applied to tissue examination in vivo. The results show that the inhomogeneity of fluorescence excitation and collection across the irregular surface of the examined tissue is calibrated to a great extent by taking the ratio of the raw fluorescence image to the cross-polarized reflection image. The effects of optical properties of tissue on the calibrated fluorescence signals are studies on simulated tissue phantoms systematically. Using the calibrated fluorescence imaging technology, we demonstrate that different tissue can be clearly separated endoscopically and in vivo based on the calibrated fluorescence signal. {copyright} 2001 American Institute of Physics.

Qu, Jianan Y.; Hua, Jianwen

2001-06-18

35

Photodynamics of Red Fluorescent Proteins Studied by Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

Red fluorescent proteins are important tools in fluorescence-based life science research. Recently, we have introduced eqFP611, a red fluorescent protein with advantageous properties from the sea anemone Entacmaea quadricolor. Here, we have studied the submillisecond light-driven intramolecular dynamics between bright and dark states of eqFP611 and, for comparison, drFP583 (DsRed) by using fluorescence correlation spectroscopy on protein solutions. A three-state

Andreas Schenk; Sergey Ivanchenko; Carlheinz Röcker; Jörg Wiedenmann; G. Ulrich Nienhaus

2004-01-01

36

Position-Sensitive Scanning Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) uses a stationary laser beam to illuminate a small sample volume and analyze the temporal behavior of the fluorescence fluctuations within the stationary observation volume. In contrast, scanning FCS (SFCS) collects the fluorescence signal from a moving observation volume by scanning the laser beam. The fluctuations now contain both temporal and spatial information about the sample.

Joseph P. Skinner; Yan Chen; Joachim D. Muller

2005-01-01

37

Microrheology with fluorescence correlation spectroscopy.  

PubMed

We show that fluorescence correlation spectroscopy (FCS) using a commercial spectrometer can be applied to passive microrheological (MR) experiments. The method probes the local rheological properties of materials on length scales of the focus dimension of the confocal microscope. For a feasibility study, we performed measurements on a high molecular weight poly(ethylene oxide)-water solution to allow direct comparison of the results to previous studies using diffusing wave spectroscopy, quasielastic light scattering, and particle tracking methods. We were able to detect mean-square center-of-mass displacements ranging from somewhat better than [SYMBOL: SEE TEXT]2(t) approximately 100 nm2 up to above Deltar2(t) approximately 10(6) nm2. Thus, we were able to derive the bulk rheological shear moduli covering more than five decades in frequency (from omegaspectroscopy a powerful tool in particular for biological and medical applications. PMID:19425563

Rathgeber, Silke; Beauvisage, Hans-Josef; Chevreau, Hubert; Willenbacher, Norbert; Oelschlaeger, Claude

2009-06-01

38

Glucose Recognition in Vitro Using Fluorescent Spectroscopy  

SciTech Connect

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

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

2001-04-25

39

In vivo fluorescence lifetime optical projection tomography  

PubMed Central

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

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

2011-01-01

40

Probing protein interactions in cells by fluorescence fluctuation spectroscopy  

NASA Astrophysics Data System (ADS)

The combination of fluorescence correlation spectroscopy and two-photon excitation provides us with a powerful spectroscopic technique. Its submicron resolution and single molecule sensitivity make it an attractive technique for in vivo applications. Experiments have demonstrated that quantitative in vivo fluorescence fluctuation measurements are feasible, despite the presence of autofluorescence and the heterogeneity of the cellular environment. I will demonstrate that molecular brightness of proteins tagged with green fluorescent protein (GFP) is a useful and robust parameter for in vivo studies. Knowledge of photon statistics is crucial for the interpretation of fluorescence fluctuation experiments. I will describe photon counting histogram (PCH) analysis, which determines the molecular brightness and complements autocorrelation analysis. Non-ideal detector effects and their influence on the photon statistics will be discussed. The goal of in vivo fluorescence fluctuation experiments is to address functional properties of biomolecules. We will focus on retinoid X receptor (RXR), a nuclear receptor, which is crucial for the regulation of gene expression. The fluorescence brightness of RXR tagged with EGFP will be used to probe the oligomerization state of RXR.

Mueller, Joachim

2003-03-01

41

Laser induced fluorescence spectroscopy for FTU.  

National Technical Information Service (NTIS)

Laser induced fluorescence spectroscopy (LIFS) is based on the absorption of a short pulse of tuned laser light by a group of atoms and the observation of the resulting fluorescence radiation from the excited state. Because the excitation is resonant it i...

T. P. Hughes

1995-01-01

42

Fluorescence Correlation Spectroscopy: Diagnostics for Sparse Molecules  

Microsoft Academic Search

The robust glow of molecular fluorescence renders even sparse molecules detectable and susceptible to analysis for concentration, mobility, chemistry, and photophysics. Correlation spectroscopy, a statistical-physics-based tool, gleans quantitative information from the spontaneously fluctuating fluorescence signals obtained from small molecular ensembles. This analytical power is available for studying molecules present at minuscule concentrations in liquid solutions (less than one nanomolar), or

Sudipta Maiti; Ulrich Haupts; Watt W. Webb

1997-01-01

43

Time-resolved fluorescence correlation spectroscopy  

Microsoft Academic Search

A new method of performing fluorescence correlation spectroscopy (FCS) measurements for mixtures of several fluorescent molecular species is introduced. It uses time-resolved fluorescence detection for separating the different FCS- contributions from the different species. This allows simultaneous and independent monitoring of the diffusion of several molecular species in one sample, or performing multi-label cross-correlation measurements. In this way, the proposed

Martin B; Michael Wahl; Rainer Erdmann

2002-01-01

44

Time-resolved fluorescence correlation spectroscopy  

Microsoft Academic Search

A new method of performing fluorescence correlation spectroscopy (FCS) measurements for mixtures of several fluorescent molecular species is introduced. It uses time-resolved fluorescence detection for separating the different FCS-contributions from the different species. This allows simultaneous and independent monitoring of the diffusion of several molecular species in one sample, or performing multi-label cross-correlation measurements. In this way, the proposed method

Martin Böhmer; Michael Wahl; Hans-Jürgen Rahn; Rainer Erdmann; Jörg Enderlein

2002-01-01

45

Fluorescence Correlation Spectroscopy in Living Cells  

Microsoft Academic Search

\\u000a In fluorescence correlation spectroscopy (FCS), information about molecular dynamics is extracted by recording the fluctuating\\u000a signal of a pico- to micromolar concentration of fluorescent molecules in solution. As primary parameters, FCS provides time\\u000a constants of the fluorescence emission, as well as numbers and dwell times of the observed particles diffusing through the\\u000a open volume. A biochemical reaction or macromolecular rearrangement

Thomas Weidemann; Petra Schwille

46

Global analysis in fluorescence correlation spectroscopy and fluorescence lifetime microscopy.  

PubMed

Fluorescence correlation spectroscopy (FCS) and related fluctuation spectroscopy and microscopy methods have become important research tools that enable detailed investigations of the chemical and physical properties of molecules and molecular systems in a variety of complex environments. Information recovery via curve fitting of fluctuation data can present complicating challenges due to limited resolution and/or problems with fitting model verification. We discuss a new approach to data analysis called ?FCS that couples multiple modes of signal acquisition, here specifically FCS and fluorescence lifetimes, with global analysis. We demonstrate enhanced resolution using ?FCS, including the capability to recover the concentration of both molecular species in a two-component mixture even when the species have identical diffusion coefficients and molecular brightness values, provided their fluorescent lifetimes are distinct. We also demonstrate how ?FCS provides useful tools for model discrimination in FCS curve fitting. PMID:23276539

Anthony, Neil; Berland, Keith

2013-01-01

47

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

2012-02-01

48

The Standard Deviation in Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

The standard deviation (SD) in fluorescence correlation spectroscopy (FCS) has been mostly neglected in applications. However, the knowledge of the correct SD is necessary for an accurate data evaluation, especially when fitting theoretical models to experimental data. In this work, an algorithm is presented that considers the essential features of FCS. It allows prediction of the performance of FCS measurements

Thorsten Wohland; Rudolf Rigler; Horst Vogel

2001-01-01

49

Dynamics of Fluorescence Fluctuations in Green Fluorescent Protein Observed by Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L\\/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence.

Ulrich Haupts; Sudipta Maiti; Petra Schwille; Watt W. Webb

1998-01-01

50

High Count Rates with Total Internal Reflection Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

We achieved photon count rates per molecule as high as with commonly used confocal fluorescence correlation spectroscopy instruments using a new total internal reflection fluorescence correlation spectroscopy system based on an epi-illumination configuration.

Kai Hassler; Tiemo Anhut; Rudolf Rigler; Michael Gösch; Theo Lasser

2005-01-01

51

Ultraviolet, Visible, and Fluorescence Spectroscopy  

NASA Astrophysics Data System (ADS)

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

Penner, Michael H.

52

Fluorescence Cross-Correlation Spectroscopy (FCCS) in Living Cells.  

PubMed

Fluorescence cross-correlation spectroscopy (FCCS) is a single-molecule sensitive technique to quantitatively study interactions among fluorescently tagged biomolecules. Besides the initial implementation as dual-color FCCS (DC-FCCS), FCCS has several powerful derivatives, including single-wavelength FCCS (SW-FCCS), two-photon FCCS (TP-FCCS), and pulsed interleaved excitation FCCS (PIE-FCCS). However, to apply FCCS successfully, one needs to be familiar with procedures ranging from fluorescent labeling, instrumentation setup and alignment, sample preparation, and data analysis. Here, we describe the procedures to apply FCCS in various biological samples ranging from live cells to in vivo measurements, with the focus on DC-FCCS and SW-FCCS. PMID:24108644

Ma, Xiaoxiao; Foo, Yong Hwee; Wohland, Thorsten

2014-01-01

53

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

54

Total internal reflection with fluorescence correlation spectroscopy  

Microsoft Academic Search

Total internal reflection–fluorescence correlation spectroscopy (TIR-FCS) is an emerging technique that is used to measure events at or near an interface, including local fluorophore concentrations, local translational mobilities and the kinetic rate constants that describe the association and dissociation of fluorophores at the interface. TIR-FCS is also an extremely promising method for studying dynamics at or near the basal membranes

Bridgett L Steele; Nancy L Thompson

2007-01-01

55

Dual-focus fluorescence correlation spectroscopy.  

PubMed

This chapter introduces into the technique of dual-focus fluorescence correlation spectroscopy or 2fFCS. In 2fFCS, the fluorescence signals generated in two laterally shifted but overlapping focal regions are auto- and crosscorrelated. The resulting correlation curves are then used to determine diffusion coefficients of fluorescent molecules or particles in solutions or membranes. Moreover, the technique can also be used for noninvasively measuring flow-velocity profiles in three dimensions. Because the distance between the focal regions is precisely known and not changed by most optical aberrations, this provides an accurate and immutable external length scale for determining diffusivities and velocities, making 2fFCS the method of choice for accurately measuring absolute values of these quantities at pico- to nanomolar concentration. PMID:23276540

Pieper, Christoph; Weiß, Kerstin; Gregor, Ingo; Enderlein, Jörg

2013-01-01

56

In vivo Porphyrin Fluorescence from Propionibacterium acnes. A Characterization of the Fluorescing Pigments  

Microsoft Academic Search

The natural fluorescence from Propionibacterium acnes has been investigated in vivo. The fluorescence spectrum can be decomposed into three distinct emission spectra due to the presence of metal-free coproporphyrin, protoporphyrin and uroporphyrin in the bacteria. The amounts of these substances can be estimated from the in vivo fluorescence measurements, which may serve as a nondestructive technique to study the porphyrin

T. B. Melø; M. Johnsson

1982-01-01

57

Intracellular applications of fluorescence correlation spectroscopy: prospects for neuroscience  

Microsoft Academic Search

Based on time-averaging fluctuation analysis of small fluorescent molecular ensembles in equilibrium, fluorescence correlation spectroscopy has recently been applied to investigate processes in the intracellular milieu. The exquisite sensitivity of fluorescence correlation spectroscopy provides access to a multitude of measurement parameters (rates of diffusion, local concentration, states of aggregation and molecular interactions) in real time with fast temporal and high

Sally A Kim; Petra Schwille

2003-01-01

58

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.

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

2009-01-01

59

Fluorescence spectroscopy characteristics of nasopharyngeal carcinoma cells  

NASA Astrophysics Data System (ADS)

The spectroscopic characteristics of autofluorescence for the nasopharyngeal carcinoma in vitro and nasopharyngeal carcinoma cells (CNE cells) were investigated, respectively. The characteristics of fluorescence agree with the results that deduced from the nasopharyngeal carcinoma in vivo, and the optimal excitation-emission wavelength was found at 350-500 nm. Secondly, the selectivity and optimal time for optical diagnosis of nasopharyngeal carcinoma by using the new photosensitizer of Hematoporphyrin Monomethyl Ether (HMME) has been demonstrated and determined by incubated CNE cells with HMME. The fluorescence emission peaks of 615 and 675 nm characterized the selective accumulation of HMME in CNE cells, and the optimal time for optical diagnostics with HMME was about 140 mins after clinic intravenous administration. Moreover, when the concentration of HMME in CNE cells below 32 ?g/mL, the fluorescence intensity versus HMME concentration reveals an obvious linearity. Finally, the fluorescence intensity of CNE cells increases linearly with concentration over the entire range up to 9.0E+05 cells/mL. These results can be used to helpfully improve the accuracy of optical diagnosis for nasopharyngeal carcinoma.

Li, Buhong; Zhang, Zhenxi; Xie, Shusen; Lin, Huiyun

2005-01-01

60

APD detectors for biological fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

2006-11-01

61

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.

Furukawa, Hiromitsu; Fukuda, Takashi

2012-01-01

62

The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) is generally used to obtain information about the number of fluorescent particles in a small volume and the diffusion coefficient from the autocorrelation function of the fluorescence signal. Here we demonstrate that photon counting histogram (PCH) analysis constitutes a novel tool for extracting quantities from fluorescence fluctuation data, i.e., the measured photon counts per molecule and

Yan Chen; Joachim D. Müller; Peter T. C. So; Enrico Gratton

1999-01-01

63

Observation of DOM Composition by Synchronous Fluorescence Spectroscopy.  

National Technical Information Service (NTIS)

Dilute solutions of model compounds of lignin degradation products and a freshwater pond sample were studied using synchronous fluorescence spectroscopy. Synchronous fluorescence spectra of the model compounds and of a caffeic acid and pond water during t...

A. Vodacek

1991-01-01

64

In vivo multiphoton fluorescence microscopy of epithelial precancer  

NASA Astrophysics Data System (ADS)

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

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

2011-02-01

65

Fluorescence Correlation Spectroscopy: Past, Present, Future  

PubMed Central

In recent years fluorescence correlation spectroscopy (FCS) has become a routine method for determining diffusion coefficients, chemical rate constants, molecular concentrations, fluorescence brightness, triplet state lifetimes, and other molecular parameters. FCS measures the spatial and temporal correlation of individual molecules with themselves and so provides a bridge between classical ensemble and contemporary single-molecule measurements. It also provides information on concentration and molecular number fluctuations for nonlinear reaction systems that complement single-molecule measurements. Typically implemented on a fluorescence microscope, FCS samples femtoliter volumes and so is especially useful for characterizing small dynamic systems such as biological cells. In addition to its practical utility, however, FCS provides a window on mesoscopic systems in which fluctuations from steady states not only provide the basis for the measurement but also can have important consequences for the behavior and evolution of the system. For example, a new and potentially interesting field for FCS studies could be the study of nonequilibrium steady states, especially in living cells.

Elson, Elliot L.

2011-01-01

66

Spectroscopy and fluorescence in esophageal diseases.  

PubMed

Malignant tumors of the esophagus continue to be a major health issue associated with high mortality primarily because most present with symptoms of dysphagia or anaemia. The disease at that stage is advanced and not likely curable. The big issue for squamous dysplasia and that associated with BE is that only a small proportion are discovered in surveillance programs when they are asymptomatic, either because the patient lives in a high-incidence geographical area, has a family history, previously diagnosed head and neck cancer or chronic reflux, as in Barrett's. Current endoscopic methods are hampered by the endoscopist's inability to recognize subtle topographic clues of dysplasia, sampling errors related to biopsy protocols, and confounding inflammation-induced artifacts both for the endoscopist and pathologist. What is desperately needed would be a biomarker (e.g. serological, fecal, urinary) that selects patients for endoscopy. However, such a test is not yet on the horizon. This article examines the current status in practice and research of novel optically based 'bioendoscopic' devices (i.e. fluorescence spectroscopy and imaging, confocal fluorescence microendoscopy (CFM), light scattering spectroscopy (LSS), Raman spectroscopy (RS), and immunophotodiagnostic endoscopy) which may enhance the diagnosis of dysplasia in all patients undergoing conventional white light endoscopy. Perhaps these new technologies will lead to more cost-effective diagnosis, mapping (e.g. surface), and staging (e.g. depth) of dysplasia, thereby allowing timely cure by endoscopic means (e.g. EMR and/or PDT), biological interventions (e.g. Cox-2 inhibitors) rather than esophajectomy. PMID:16473800

Dacosta, Ralph S; Wilson, Brian C; Marcon, Norman E

2006-02-01

67

Silicon nanoparticle characterization by fluorescence correlation spectroscopy  

NASA Astrophysics Data System (ADS)

This thesis aims to characterize the fluorescence brightness and size of silicon nanocrystals in solution by measuring equilibrium fluctuations through the techniques of Fluorescence Correlation Spectroscopy (FCS) as well as Photon Counting Histogram (PCH). It was found that Si nanocrystals are comparably bright to fluorescein, a standard organic fluorophore, as well as comparably small (˜1.1nm in diameter). Imaging results on single Si nanocrystals show that individual nanocrystals are photostable for over 150s of continuous illumination, orders of magnitude longer than possible with traditional organic fluorophores under similar conditions. Due to the poorly controlled sonication step in the preparation of the Si nanocrystal colloid from the porous Si precursor, it was desirable to quantify the heterogeneity of the Si nanocrystal colloid. This was achieved by extending the techniques of FCS and PCH by scanning the excitation energy. In this way each fraction excited at a given wavelength could be counted, and a spectrum of number density versus excitation wavelength could be built up. By directly measuring the molecular heterogeneity in this way it was found that there exists significant heterogeneity in the Si nanocrystal preparations (i.e. the number density changes as a function of excitation wavelength). This important new observable (number density spectrum) can now be used as a control variable in refining the production of Si nanocrystal colloids in the effort to produce homogenous samples, which would be a necessary condition for applications. Traditional ensemble techniques (fluorescence emission/excitation spectra, fluorescence lifetime) are also performed, corroborating the conclusion of heterogeneity, though such techniques are not able to quantify it at the molecular level.

Akcakir, Osman

2001-08-01

68

Anomalous Protein Diffusion in Living Cells as Seen by Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

We investigate the challenges and limitations that are encountered when studying membrane protein dynamics in vivo by means of fluorescence correlation spectroscopy (FCS). Based on theoretical arguments and computer simulations, we show that, in general, the fluctuating fluorescence has a fractal dimension D0?1.5, which is determined by the anomality ? of the diffusional motion of the labeled particles, i.e., by

Matthias Weiss; Hitoshi Hashimoto; Tommy Nilsson

2003-01-01

69

Counting Nucleosomes in Living Cells with a Combination of Fluorescence Correlation Spectroscopy and Confocal Imaging  

Microsoft Academic Search

Although methods for light microscopy of chromatin are well established, there are no quantitative data for nucleosome concentrations in vivo. To establish such a method we used a HeLa clone expressing the core histone H2B fused to the enhanced yellow fluorescent protein (H2B-EYFP). Quantitative gel electrophoresis and fluorescence correlation spectroscopy (FCS) of isolated oligonucleosomes show that 5% of the total

Thomas Weidemann; Malte Wachsmuth; Tobias A. Knoch; Gabriele Müller; Waldemar Waldeck; Jörg Langowski

2003-01-01

70

Fluorescence rejection by shifted excitation Raman difference spectroscopy  

Microsoft Academic Search

Raman spectroscopy is often hampered by strong fluorescence background that can easily bury the much weaker Raman signal. One of the most widely used techniques to reject the fluorescence disturbance is the shifted-excitation Raman difference spectroscopy (SERDS), which incorporates multiple wavelengths as excitation sources. This paper proposed a SERDS system with 532 nm and 526.5 nm DPSS lasers and a

Wenlong Zou; Zhijian Cai; Jianhong Wu

2010-01-01

71

The standard deviation in fluorescence correlation spectroscopy.  

PubMed Central

The standard deviation (SD) in fluorescence correlation spectroscopy (FCS) has been mostly neglected in applications. However, the knowledge of the correct SD is necessary for an accurate data evaluation, especially when fitting theoretical models to experimental data. In this work, an algorithm is presented that considers the essential features of FCS. It allows prediction of the performance of FCS measurements in various cases, which is important for finding optimal experimental conditions. The program calculates the SD of the experimental autocorrelation function online. This procedure leads to improved parameter estimation, compared to currently used theoretical approximations for the SD. Three methods for the calculation of the SD are presented and compared to earlier analytical solutions (D. E. Koppel. 1974. Phys. Rev. A. 10:1938-1945.), calculation directly from fluorescence intensity values, by averaging several FCS measurements, or by dividing one measurement into a set of shorter data packages. Although the averaging over several measurements yields accurate estimates for the SD, the other two methods are considerably less time consuming, can be run online, and yield comparable results.

Wohland, T; Rigler, R; Vogel, H

2001-01-01

72

Optical biopsy fiber-based fluorescence spectroscopy instrumentation  

NASA Astrophysics Data System (ADS)

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

Katz, Al; Ganesan, S.; Yang, Yuan-Long; Tang, Gui C.; Budansky, Y.; Celmer, Edward J.; Savage, Howard E.; Schantz, Stimson P.; Alfano, Robert R.

1996-04-01

73

Fluorescence lifetime spectroscopy for guided therapy of brain tumors.  

PubMed

This study evaluates the potential of time-resolved laser induced fluorescence spectroscopy (TR-LIFS) as intra-operative tool for the delineation of brain tumor from normal brain. Forty two patients undergoing glioma (WHO grade I-IV) surgery were enrolled in this study. A TR-LIFS prototype apparatus (gated detection, fast digitizer) was used to induce in-vivo fluorescence using a pulsed N2 laser (337 nm excitation, 0.7 ns pulse width) and to record the time-resolved spectrum (360-550 nm range, 10 nm interval). The sites of TR-LIFS measurement were validated by conventional histopathology (H&E staining). Parameters derived from the TR-LIFS data including intensity values and time-resolved intensity decay features (average fluorescence lifetime and Laguerre coefficients values) were used for tissue characterization and classification. 71 areas of tumor and normal brain were analyzed. Several parameters allowed for the differentiation of distinct tissue types. For example, normal cortex (N=35) and normal white matter (N=12) exhibit a longer-lasting fluorescence emission at 390 nm (?390=2.12±0.10 ns) when compared with 460 nm (?460=1.16±0.08 ns). High grade glioma (grades III and IV) samples (N=17) demonstrate emission peaks at 460 nm, with large variation at 390 nm while low grade glioma (I and II) samples (N=7) demonstrated a peak fluorescence emission at 460 nm. A linear discriminant algorithm allowed for the classification of low-grade gliomas with 100% sensitivity and 98% specificity. High-grade glioma demonstrated a high degree of heterogeneity thus reducing the discrimination accuracy of these tumors to 47% sensitivity and 94% specificity. Current findings demonstrate that TR-LIFS holds the potential to diagnose brain tumors intra-operatively and to provide a valuable tool for aiding the neurosurgeon-neuropathologist team in to rapidly distinguish between tumor and normal brain during surgery. PMID:21055475

Butte, Pramod V; Mamelak, Adam N; Nuno, Miriam; Bannykh, Serguei I; Black, Keith L; Marcu, Laura

2010-11-03

74

Tracking-FCS: Fluorescence correlation spectroscopy of individual particles  

Microsoft Academic Search

We exploit recent advances in single-particle tracking to perform fluorescence correlation spectroscopy on individual fluorescent particles, in contrast to traditional methods that build up statistics over a sequence of many measurements. By rapidly scanning the focus of an excitation laser in a circular pattern, demodulating the measured fluorescence, and feeding these results back to a piezoelectric translation stage, we track

Andrew J. Berglund; Hideo Mabuchi

2005-01-01

75

Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) extracts information about molecular dynamics from the tiny fluctuations that can be observed in the emission of small ensembles of fluorescent molecules in thermodynamic equilibrium. Employing a confocal setup in conjunction with highly dilute samples, the average number of fluorescent particles simultaneously within the measurement volume (?1fl) is minimized. Among the multitude of chemical and physical

Elke Haustein; Petra Schwille

2003-01-01

76

In vivo fluorescence hyperspectral imaging of oral neoplasia  

Microsoft Academic Search

A hyperspectral imaging system using a liquid-crystal tunable filter (LCTF) was constructed for the purpose of in vivo optical imaging of oral neoplasia. The system operates in fluorescence mode and has the dual capability of capturing high quality widefield images and detecting fluorescence emission spectra from arbitrary locations within the captured field of view (FOV). The system was calibrated and

Darren Roblyer; Cristina Kurachi; Ann M. Gillenwater; Rebecca Richards-Kortum

2009-01-01

77

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

PubMed

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; Hammond, Paula T; Swager, Timothy M

2013-06-24

78

Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy  

Microsoft Academic Search

Capillary flow experiments are described with fluorescent molecules, bacteria, and microspheres using fluorescence correlation spectroscopy as an analytical tool. The flow velocity in the microcapillary is determined by fitting autocorrelation traces with a model containing parameters related to diffusion and flow. The flow profile of pressure-driven flow inside a microcapillary is determined by using the fluorescence fluctuations of a small

Beno H. Kunst; Arjen Schots; Antonie J. W. G. Visser

2002-01-01

79

In vivo imaging with near-infrared fluorescence lifetime contrast  

NASA Astrophysics Data System (ADS)

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

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

2009-02-01

80

Fluorescence spectroscopy for noninvasive glucose measurement  

NASA Astrophysics Data System (ADS)

The interaction between gold nanoparticles and glucose and its effect on the fluorescence spectrum of nanoparticles were investigated experimentally. It was observed after this interaction the intensity of fluorescence peak becomes weaker and red shifted.

Bagheri, Z.; Massudi, R.; Ghanavi, J.; Latifi, H.

2013-06-01

81

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, M.; Jacobowitz, Larry; Alfano, Robert R.

1993-08-01

82

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

83

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

84

Fluorescence Correlation Spectroscopy: Inception, Biophysical Experimentations, and Prospectus  

Microsoft Academic Search

Fluorescence correlation spectroscopy examines the chemical and the photophysical dynamics of dilute molecular solutions by measurement of the dynamic optical fluctuations of the fluorescence of a few molecules, even averaging less than one molecule at a time, in open focal volumes that are usually less than a femtoliter ( <10-18 m3 ). It applies the same principles of statistical thermodynamics

Watt W. Webb

2001-01-01

85

Excitation saturation in two-photon fluorescence correlation spectroscopy  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) has become a powerful and sensitive research tool for the study of molecular dynamics at the single-molecule level. Because photophysical dynamics often dramatically influence FCS measurements, the role of various photophysical processes in FCS measurements must be understood to accurately interpret FCS data. We describe the role of excitation saturation in two-photon fluorescence correlation measurements. We

Keith Berland; Guoqing Shen

2003-01-01

86

Fluorescence correlation spectroscopy: the technique and its applications  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) is an experimental technique using statistical analysis of the fluctuations of fluorescence in a system in order to decipher dynamic molecular events, such as diffusion or conformational fluctuations of biomolecules. First introduced by Magde et al to measure the diffusion and binding of ethidium bromide onto double-stranded DNA, the technique has been undergoing a renaissance since

Oleg Krichevsky; Grégoire Bonnet

2002-01-01

87

Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy  

Microsoft Academic Search

A series of fluorophores with single-exponential fluorescence decays in liquid solution at 20 C were measured independently by nine laboratories using single-photon timing and multifrequency phase and modulation fluorometry instruments with lasers as excitation source. The dyes that can serve as fluorescence lifetime standards for time-domain and frequency-domain measurements are all commercially available, are photostable under the conditions of the

Noël Boens; Wenwu Qin; N. Basaric; Johan Hofkens; Marcel Ameloot; Jacques Pouget; J. P. Lefevre; Bernard Valeur; Enrico Gratton; Ven van de M; D. Silva jr; Yves Engelborghs; Katrien Willaert; Alain Sillen; Garry Rumbles; D. Philips; Antonie J. W. G. Visser; Arie van Hoek; Joseph R. Lakowicz; Henryk Malak; Ignacy Gryczynski; Arthur G. Szabo; Don T. Krajcarski; Naoto Tamai; Atsushi Miura

2007-01-01

88

Precise Measurement of Diffusion Coefficients using Scanning Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

We have implemented scanning fluorescence correlation spectroscopy (sFCS) for precise determination of diffusion coefficients of fluorescent molecules in solution. The measurement volume where the molecules are excited, and from which the fluorescence is detected, was scanned in a circle with radius comparable to its size at frequencies 0.5–2kHz. The scan radius R, determined with high accuracy by careful calibration, provides

Zden?k Petrášek; Petra Schwille

2008-01-01

89

The application of fluorescence correlation spectroscopy in detecting DNA condensation  

Microsoft Academic Search

We report the application of fluorescence correlation spectroscopy (FCS) in characterizing conformational changes (condensation) of chemically well-defined DNA plasmids. The plasmids: pH?APr-1-neo (10 kbp, contour length 3.4 ?m) and pBluescript SKt (2.96 kbp, contour length 1.02 ?m) were imaged by a confocal fluorescence microscope using two fluorescent probes: ethidium bromide (EtBr) and propidium iodide (PrIo). It became clear that the

Teresa Kral; Marek Langner; Martin Beneš; Dagmara Baczy?ska; Maciej Ugorski; Martin Hofb

2002-01-01

90

Recent Advances in Fluorescence Cross-correlation Spectroscopy  

Microsoft Academic Search

Fluorescence cross-correlation spectroscopy (FCCS) is a method that measures the temporal fluorescence fluctuations coming\\u000a from two differently labeled molecules diffusing through a small sample volume. Cross-correlation analysis of the fluorescence\\u000a signals from separate detection channels extracts information of the dynamics of the dual-labeled molecules. FCCS has become\\u000a an essential tool for the characterization of diffusion coefficients, binding constants, kinetic rates

Ling Chin Hwang; Thorsten Wohland

2007-01-01

91

Multiphoton cascade absorption in single molecule fluorescence saturation spectroscopy.  

PubMed

Saturation spectroscopy is a relevant method to investigate photophysical parameters of single fluorescent molecules. Nevertheless, the impact of a gradual increase, over a broad range, of the laser excitation on the intramolecular dynamics is not completely understood, particularly concerning their fluorescence emission (the so-called brightness). Thus, we propose a comprehensive theoretical and experimental study to interpret the unexpected evolution of the brightness with the laser power taking into account the cascade absorption of two and three photons. Furthermore, we highlight the key role played by the confocal observation volume in fluorescence saturation spectroscopy of single molecules in solution. PMID:23521543

Winckler, Pascale; Jaffiol, Rodolphe

2013-04-11

92

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

PubMed

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

Kapusta, Peter; Machá?, Radek; Benda, Aleš; Hof, Martin

2012-10-09

93

Fluorescence molecular tomography resolves protease activity in vivo  

Microsoft Academic Search

Systematic efforts are under way to develop novel technologies that would allow molecular sensing in intact organisms in vivo. Using near-infrared fluorescent molecular beacons and inversion techniques that take into account the diffuse nature of photon propagation in tissue, we were able to obtain three-dimensional in vivo images of a protease in orthopic gliomas. We demonstrate that enzyme-activatable fluorochromes can

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

2002-01-01

94

Pigment Organization Effects on Energy Transfer and Chl a Emission Imaged in the Diatoms C. meneghiniana and P. tricornutum In Vivo: A Confocal Laser Scanning Fluorescence (CLSF) Microscopy and Spectroscopy Study.  

PubMed

The (auto)fluorescence from three diatom strains, Cyclotella meneghiniana (Cm), Phaeodactylum tricornutum 1a (Pt1a), and Phaeodactylum UTex (PtUTex), has been imaged in vivo to submicrometer resolution using confocal laser scanning fluorescence (CLSF) microscopy. The diatoms are excited at 473 and 532 nm, energy primarily absorbed by the carotenoid fucoxanthin (Fx) found within the fucoxanthin chlorophyll a/c proteins (FCPs). On the basis of the fluorescence spectra measured in each image voxel, we obtain information about the spatial and energetic distribution of the terminal Chl a emitters, localized in the FCPs and the reaction centers of the PSII protein complexes, and the nature and location of the primary absorbers that are linked to these emitters; 532 nm excites the highly efficient Fxred light harvesters, and lesser amounts of Fxgreens, that are enriched in some FCPs and preferentially transfer energy to PSII, compared to 473 nm, which excites almost equal amounts of all three previously identified sets of Fx - Fxred, Fxgreen and Fxblue - as well as Chl c. The heterogeneous Chl a emission observed from the (C)LSF images indicates that the different Fx's serve different final emitters in P. tricornutum and suggest, at least in C. meneghiniana , a localization of FCPs with relatively greater Fxred content at the chloroplast edges, but with overall higher FCP concentration in the interior of the plastid. To better understand our results, the concentration-dependent ensemble-averaged diatom solution spectra are compared to the (auto)fluorescence spectra of individual diatoms, which indicate that pigment packing effects at an intracellular level do affect the diatoms' spectral properties, in particular, concerning a 710 nm emission band apparent under stress conditions. A species-specific response of the spectral signature to the incident light is also discussed in terms of the presence of a silica shell in Cm but not in Pt1a nor PtUTex. PMID:23844975

Premvardhan, Lavanya; Réfrégiers, Matthieu; Büchel, Claudia

2013-08-05

95

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

96

Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy.  

PubMed

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

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

2007-02-01

97

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

NASA Astrophysics Data System (ADS)

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

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

2012-12-01

98

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

NASA Astrophysics Data System (ADS)

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

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

1999-07-01

99

Fluorescence spectroscopy of gastrointestinal tumors using ?-ALA  

NASA Astrophysics Data System (ADS)

In the recent study delta-aminolevulinic acid/Protoporphyrin IX (?-ALA/PpIX) is used as fluorescent marker for dysplasia and tumor detection in esophagus and stomach. The ?-ALA is administered per os six hours before measurements at dose 20mg/kg weight. High-power light-emitting diode at 405 nm is used as an excitation source. Special opto-mechanical device is built to use the light guide of standard video-endoscopic system (Olimpus Corp.). Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer (USB4000, OceanOptics Inc.). The fluorescence detected from tumor sites has very complex spectral origins. It consists of autofluorescence, fluorescence from exogenous fluorophores and re-absorption from the chromophores accumulated in the tissue investigated. Mucosa autofluorescence lies at 450-600 nm region. The fluorescence of PpIX is clearly pronounced at the 630-710 nm region. Deep minima in the tumor fluorescence signals are observed in the region 540-575 nm, related to hemoglobin re-absorption. Such high hemoglobin content is an indication of the tumors neovascularisation and it is clearly pronounced in all dysplastic and tumor sites investigated. The lack of fluorescence peaks in the red spectral area for normal mucosa is an indication for selective accumulation of ?-ALA/PpIX only in abnormal sites and gives high contrast when lesion borders are determined from clinicians during video observation in the process of diagnostic procedure. Very good correlation between fluorescence signals and histology examination results of the lesions investigated is achieved.

Borisova, E. G.; Vladimirov, B. G.; Angelov, I. G.; Avramov, L. A.

2007-03-01

100

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.

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

2011-01-01

101

In vivo lipidomics using single-cell Raman spectroscopy.  

PubMed

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

2011-02-10

102

Approaches to localized NMR spectroscopy in vivo  

SciTech Connect

Nuclear magnetic resonance (NMR) techniques are developed which allow spatially localized spectra to be obtained from living tissue. The localization methods are noninvasive and exploit the enhanced sensitivity afforded by surface coil probes. Techniques are investigated by computer simulation and experimentally verified by the use of phantom samples. The feasibility and utility of the techniques developed in this research are demonstrated by /sup 31/P spatial localization experiments involving various in vivo organs. In the first part of the thesis, two feasible approaches to localized spectroscopy, which were developed by other laboratories are theoretically analyzed by computer simulation. An alternative approach is provided by the rotating frame zeugmatography experiment which affords chemical-shift spectra displayed as a function of penetration distance into the sample. The further modification of the rotating frame experiment is developed, the Fourier series window (FSW) approach, which utilizes various types of window functions to afford localization in one or a few tissue regions of interest with high sensitivity. Theoretical comparisons with depth pulse methods are also included, along with methods to refine adverse off-resonance behavior.

Garwood, M.G.

1985-01-01

103

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

PubMed

In the field of biomedical optics, Raman spectroscopy is a powerful tool for probing the chemical composition of biological samples. In particular, fiber Raman probes play a crucial role for in vivo and ex vivo tissue analysis. However, the high-fluorescence background typically contributed by the auto fluorescence from both a tissue sample and the fiber-probe interferes strongly with the relatively weak Raman signal. Here we demonstrate the implementation of wavelength-modulated Raman spectroscopy (WMRS) to suppress the fluorescence background while analyzing tissues using fiber Raman probes. We have observed a significant signal-to-noise ratio enhancement in the Raman bands of bone tissue, which have a relatively high fluorescence background. Implementation of WMRS in fiber-probe-based bone tissue study yielded usable Raman spectra in a relatively short acquisition time (?30??s), notably without any special sample preparation stage. Finally, we have validated its capability to suppress fluorescence on other tissue samples such as adipose tissue derived from four different species. PMID:22894519

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

2012-07-01

104

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

NASA Astrophysics Data System (ADS)

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

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

2012-07-01

105

Detection of experimental brain tumors using time-resolved laser-induced fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

Time-Resolved Laser-Induced Fluorescence Spectroscopy (TR-LIFS) has the potential to provide a non- invasive characterization and detection of tumors. We utilized TR-LIFS to detect gliomas in-vivo in the rat C6 glioma model. Time-resolved emission spectra of both normal brain and tumor were analyzed to determine if unique fluorescence signatures could be used to distinguish the two. Fluorescence parameters derived from both spectral and time domain were used for tissue characterization. Our results show that in the rat C6 glioma model, TR-LIFS can be used to differentiate brain tumors from normal tissue (gray and white mater) based upon time- resolved fluorescence signatures seen in brain tumors.

Thompson, Reid C.; Black, Keith L.; Kateb, Babak; Marcu, Laura

2002-05-01

106

X-ray Fluorescence Spectroscopy of Novel Materials  

Microsoft Academic Search

This review focuses on the capabilities and potential of soft x-ray fluorescence spectroscopy for the study of the electronic\\u000a structure and chemical bonding of novel materials. The basic principle of x-ray fluorescence measurements using synchrotron\\u000a radiation and the corresponding instrumentation issues are outlined. Particular attention is given to x-ray spectroscopic\\u000a studies of the electronic structure and characterization of nanostructures, thin

E. Z. Kurmaev

2005-01-01

107

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

108

Exonuclease Degradation of DNA Studied by Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

Here we developed an accurate method for kinetic analysis of enzymatic degradation processes of double and\\/or single-stranded DNA\\/oligonucleotides using fluorescent reporter dyes. 217-bp DNA fragments were produced by polymerase chain reaction and cleaved by the 3? to 5? exonuclease activity of T7-DNA polymerase. The analysis of the products was performed by Fluorescence Correlation Spectroscopy measuring autocorrelation amplitudes and diffusion times.

Zeno Földes-Papp; Per Thyberg; Sofie Björling; Arne Holmgen; Rudolf Rigler

1997-01-01

109

Method for X-ray fluorescence spectroscopy  

Microsoft Academic Search

A method and apparatus are described for performing X-ray fluorescence analysis where the physical relationship between the source\\/detector and the object being examined is not controlled. This technique and apparatus is particularly advantageous in performing an in situ analysis of the concentration of one or more elements present in a matrix of a material such as rock. In accordance with

W. C. Boyce; T. E. Hall; L. C. Howard; W. M. Lechelt; W. D. Wittekind

1985-01-01

110

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

111

Fluorescent Lifetime Spectroscopy in Random Media  

NASA Astrophysics Data System (ADS)

Recently, an abundance of near-infrared phosphorescent and fluorescent probes have been developed whose lifetime is sensitive to changes in the local environment. The lifetime of these probes can be readily determined in a dilute, non-scattering media using conventional time- and frequency-domain techniques. From the lifetime, the concentration of metabolites can be found using the Stern-Volmer relationship. However, in highly scattering media such as tissues and particulate process streams, measurement of lifetime is complicated by the time delay associated with light scatter. In this dissertation, frequency-domain measurements of photon migration are developed for measuring fluorescent lifetimes independent of absorption and scattering properties of tissues and other random media. The measurement consists of launching, onto the surface of the medium, excitation light whose intensity is sinusoidally modulated at megahertz frequencies. The fluorescent light generated within the medium is intensity modulated at the same frequency, but phase-shifted and amplitude demodulated relative to the incident excitation source. In addition, the excitation light is also phase-shifted and amplitude demodulated relative to the incident excitation source. From Green's function analysis, finite element computations, and experimental measurements of fluorescent phase-shift and amplitude demodulation, we show it is possible to determine fluorophore lifetime of common laser dyes in a tissue mimicking phantom. Furthermore, the finite element computations of excitation and fluorescent light fluence show that when the fluorophore is uniformly distributed within a medium, signals re-emitted at the surface do not originate from significant depths if its lifetime is greater than the photon migration time associated with scatter. Consequently, this research points to the development of short-lived fluorescent compounds for biodiagnostics using properly referenced frequency-domain measurements. Applications for lifetime-based sensing in tissues and other random media (such as monitoring changes in glucose, pH, pCO _2, pO_2, and other metabolites) depend upon the continued development of the referencing procedures developed herein, as well as engineering interactions with synthetic chemists for the future development of optical probes.

Hutchinson, Christina Laura

112

Fluorescence correlation spectroscopy at high concentrations using gold bowtie nanoantennas  

NASA Astrophysics Data System (ADS)

Fluorescence correlation spectroscopy (FCS) measures the fluorescence fluctuations of fluorophores in solution, but is restricted to extremely low concentrations. Plasmonic gold bowtie nanoantennas enhance a single molecule's fluorescence relative to a large background of unenhanced molecules, and here we show that bowties can extend FCS measurements to much higher concentrations. In this demonstration, the bowtie-FCS signal is dominated by molecules that transiently stick to the substrate near the bowtie gap, and photobleaching/photoblinking dynamics for two fluorophores are measured on the 10-100 ms time scale.

Kinkhabwala, Anika A.; Yu, Zongfu; Fan, Shanhui; Moerner, W. E.

2012-10-01

113

Nonlinear Laser Fluorescence Spectroscopy of Natural Organic Compounds  

NASA Astrophysics Data System (ADS)

Principles of nonlinear laser fluorescence spectroscopy of complicated organic compounds and of the method capable of determining photophysical parameters are considered in this chapter. Special attention is paid to the peculiarities of the method connected with specific photophysical processes in natural organic compounds, especially in proteins, and to the major role of intramolecular energy transfer and presence of localized donor-acceptor pairs (LDAP) of fluorophores within single macromolecules. These facts stimulated the development of models based on the collective states formalism describing fluorescent response of LDAP to pulsed laser excitation. Unique features of the method are illustrated by the example of proteins (proteins with intrinsic fluorescence (HSA, BSA) and fluorescent protein mRFP1) that can be used as fluorescent tags of intracellular processes while their photophysical parameters can be used as the information channel.

Fadeev, Victor V.; Shirshin, Evgeny A.

114

Fluorescence correlation spectroscopy study on room-temperature ionic liquids  

NASA Astrophysics Data System (ADS)

Unique fluorescence behavior was reported in room-temperature ionic liquids having imidazolium cations. Due to the molecular electronic energy level of the imidazolium cation, fluorescence in the visible range from pure ionic liquids is not expected, but was readily observed. Fluorescence correlation spectroscopy (FCS) was used to find the nature of this unique fluorescence by investigating the fluorescence fluctuation and the number density of fluorophores. FCS signal was observed in pure room-temperature ionic liquids having different cations and anions, which is considered to come from the molecular aggregate of room-temperature ionic liquid. The size and the number density of aggregates in pure ionic liquids were measured by using FCS.

Cha, Seoncheol; Kim, Doseok

2012-11-01

115

In vivo fluorescence hyperspectral imaging of oral neoplasia  

NASA Astrophysics Data System (ADS)

A hyperspectral imaging system using a liquid-crystal tunable filter (LCTF) was constructed for the purpose of in vivo optical imaging of oral neoplasia. The system operates in fluorescence mode and has the dual capability of capturing high quality widefield images and detecting fluorescence emission spectra from arbitrary locations within the captured field of view (FOV). The system was calibrated and evaluated for spectral resolution and accuracy. In vivo hyperspectral images were obtained from two normal volunteers and two patients with confirmed oral malignancy. Normal volunteer measurements revealed differences in intensity and lineshape of spectra between different anatomic locations, but intensity and lineshape were similar between different measurement sites from the same anatomic location. Measurements from normal and neoplastic areas of two patients with previously confirmed oral neoplasia showed differences in intensity, lineshape, and location of peak intensity. We have demonstrated that this system can provide both high quality widefield images, and spectral information at chosen locations within the field of view.

Roblyer, Darren; Kurachi, Cristina; Gillenwater, Ann M.; Richards-Kortum, Rebecca

2009-02-01

116

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

117

Triplet-state monitoring by fluorescence correlation spectroscopy  

Microsoft Academic Search

The effects of high excitation intensities in fluorescence correlation spectroscopy (FCS) in terms of saturation and triplet-state build-up have been studied for the case of Rh6G in aqueous solution. It was found that FCS provides a powerful means for the determination of intersystem crossing and triplet-state depopulation rates of fluorophores in solution.

Jerker Widengren; Rudolf Rigler; Ülo Mets

1994-01-01

118

Fluorescence correlation spectroscopy: lead discovery by miniaturized HTS  

Microsoft Academic Search

Miniaturized high-throughput screening offers great promise for increasing the productivity of the pharmaceutical drug discovery process. By monitoring interactions of single molecules in femtoliter volumes, fluorescence correlation spectroscopy (FCS) offers the highest potential as the detection technique in the nanoscale. The authors (Appendix A) summarize the current status of practical experiences with FCS assays for HTS and explore the scope

Manfred Auer; Keith J Moore; Franz J Meyer-Almes; Rolf Guenther; Andrew J Pope; Kurt A Stoeckli

1998-01-01

119

Measuring diffusion in cell membranes by fluorescence correlation spectroscopy  

Microsoft Academic Search

Fluorescence Correlation Spectroscopy (FCS) can measure diffusion on the cell surface with unparalleled sensitivity. In appropriate situations, this can be the most sensitive and accurate method for measuring receptor interaction and oligomerization. Here we attempt to describe FCS in sufficient detail so that the reader is able to judge when there is a compelling reason to choose this technique, understand

Parijat Sengupta; J Balaji; Sudipta Maiti

2002-01-01

120

Dynamics of Large Semiflexible Chains Probed by Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

Fluorescence correlation spectroscopy was used to probe the dynamics of lambda-phage DNA in aqueous solution labeled with the randomly intercalating dye TOTO. The linear macromolecules (i) carry more than one chromophore and (ii) are larger than the waist of the focal volume. The correlation function decays significantly faster than expected for a stiff globule of corresponding size but is in

D. Lumma; S. Keller; T. Vilgis; J. O. Rädler

2003-01-01

121

Soft x-ray fluorescence spectroscopy using tunable synchrotron radiation  

Microsoft Academic Search

The introduction of high brightness synchrotron radiation sources has opened up new possibilities for using soft x-ray fluorescence spectroscopy in the study of electronic structure. Selective excitation allows site specific emission to be studied, satellite structure due to multiple excitation to be separated, resonant phenomena to be investigated, etc. In particular, it has been found that inelastic scattering becomes dominating

Joseph E. Nordgren; Nial Wassdahl

1995-01-01

122

Applications of Fluorescence Correlation Spectroscopy— Particle Size Effect  

Microsoft Academic Search

Both computer simulations and experiments have been employed to study the fluorescence correlation spectroscopy autocorrelation function of particles with size comparable to the size of the light beam. When such particles are labeled by several fluorophores the characteristic time of diffusion and the particle number in the light beam are larger than those expected from the existing theory developed for

Konstantin Starchev; Jingwu Zhang; Jacques Buffle

1998-01-01

123

Fluorescence cross-correlation spectroscopy in living cells  

Microsoft Academic Search

Cell biologists strive to characterize molecular interactions directly in the intracellular environment. The intrinsic resolution of optical microscopy, however, allows visualization of only coarse subcellular localization. By extracting information from molecular dynamics, fluorescence cross-correlation spectroscopy (FCCS) grants access to processes on a molecular scale, such as diffusion, binding, enzymatic reactions and codiffusion, and has become a valuable tool for studies

Kirsten Bacia; Sally A Kim; Petra Schwille

2006-01-01

124

Dual fluorescent protein reporters for studying cell behaviors in vivo  

PubMed Central

Fluorescent proteins (FPs) are useful tools for visualizing live cells and their behaviors. Protein domains that mediate subcellular localization have been fused to FPs to highlight cellular structures. FPs fused with histone H2B incorporate into chromatin allowing visualization of nuclear events. FPs fused to a glycosylphosphatidylinositol anchor signal sequence (GPI) label the plasma membrane, highlighting cellular shape. Thus, a reporter gene containing both types of FP fusions would allow for effective monitoring of cell shape, movement, mitotic stage, apoptosis and other cellular activities. Here we report a binary color-coding system using four different colored fluorescent protein reporters that generates 16 distinct color codes to label the nuclei and plasma membranes of live cells in culture and in transgenic mice. As an initial test of this system in vivo, the promoter of the human Ubiquitin C (UBC) gene was used to widely express one of the color code reporters. Widespread expression of the reporter was attained in embryos; however, both male and female transgenic mice were infertile. In contrast, the promoter of the mouse Oct4/Pou5f1 gene linked to two different color code reporters specifically labeled blastocysts, primordial germ cells, and postnatal germ cells and these mice were fertile. Time-lapse movies of fluorescently-labeled primordial germs cells demonstrate the utility of the color code system to visualize cell behaviors. This set of new fluorescent protein reporters should be a useful tool for labeling distinct cell populations and studying their behaviors in complex tissues in vivo.

Stewart, M. David; Jang, Chuan-Wei; Hong, Nicholas W.; Austin, Anthony P.; Behringer, Richard R.

2011-01-01

125

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

Microsoft Academic Search

Fluorescence anisotropy and fluorescence correlation spectroscopy measurements of rhodamine-labeled DNA oligonucleotide duplexes have been used to determine equilibrium binding constants for DNA binding of the prokaryotic transcription activator protein NtrC. Measurements were made with wild-type NtrC from Escherichia coli and the constitutively active mutant NtrC S160F from Salmonella using DNA duplexes with one or two binding sites. The following results

Franz W. Sevenich; J. Langowski; Karsten Rippe; V. Weiss

1998-01-01

126

Photonic Methods to Enhance Fluorescence Correlation Spectroscopy and Single Molecule Fluorescence Detection  

PubMed Central

Recent advances in nanophotonics open the way for promising applications towards efficient single molecule fluorescence analysis. In this review, we discuss how photonic methods bring innovative solutions for two essential questions: how to detect a single molecule in a highly concentrated solution, and how to enhance the faint optical signal emitted per molecule? The focus is set primarily on the widely used technique of fluorescence correlation spectroscopy (FCS), yet the discussion can be extended to other single molecule detection methods.

Wenger, Jerome; Rigneault, Herve

2010-01-01

127

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

NASA Astrophysics Data System (ADS)

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

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

2008-03-01

128

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

129

Quantifying Lipid-Protein Interaction by Fluorescence Correlation Spectroscopy (FCS).  

PubMed

Fluorescence correlation spectroscopy (FCS) is a powerful method to investigate molecular interactions based on the variation of diffusion properties at the single-molecule level. This technique allows studying quantitatively the interaction of fluorescently labeled proteins/peptides with lipid vesicles. Here, we describe how to acquire and analyze FCS partition data in order to accurately determine the protein/peptide partition coefficients between the aqueous and lipid phases. It is shown that the recovery of unbiased partition coefficients from FCS partition curves (fractional amplitude of the bound species versus lipid concentration) requires considering explicitly the Poissonian loading of the lipid vesicles with the fluorescently labeled protein in order to account for the variable liposome brightness in each sample. Additionally, the impact of a trace amount of a fluorescent non-binding component on the partition curves determined by FCS is also discussed. PMID:24108645

Melo, Ana M; Prieto, Manuel; Coutinho, Ana

2014-01-01

130

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

PubMed

In an effort to elucidate the mechanism of movement of nanovehicles on nonconducting surfaces, the synthesis and optical properties of five fluorescently tagged nanocars are reported. The nanocars were specifically designed for studies by single-molecule fluorescence spectroscopy and bear a tetramethylrhodamine isothiocyanate fluorescent tag for excitation at 532 nm. The molecules were designed such that the arrangement of their molecular axles and p-carborane wheels relative to the chassis would be conducive to the control of directionality in the motion of these nanovehicles. PMID:20828172

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

2010-10-01

131

Extracting decay curves of the correlated fluorescence photons measured in fluorescence correlation spectroscopy  

NASA Astrophysics Data System (ADS)

We have developed a new method to extract the decay curves of the correlated fluorescence photons from the data of fluorescence correlation spectroscopy using time-correlated single photon counting. In this method, a two-dimensional correlation map of photon pairs is generated at an absolute delay time with reference to the excitation-emission delay of each photon. Using a dye-labeled DNA as an example, we have demonstrated that the decay curve of the correlated fluorescence photons is separated from the uncorrelated background signals simply by subtracting a two-dimensional correlation map at sufficiently long delay time without additional prior information.

Ishii, Kunihiko; Tahara, Tahei

2012-01-01

132

EPR Spectroscopy of Function In Vivo  

Microsoft Academic Search

EPR can be used to study free radicals in vivo, environmental and biophysical parameters in cells and tissues, and to report metabolism, physiology, and biochemistry. The authors have attempted to judge which of these types of measurements will be productive for studies in animals and in humans. It is envisioned that a large number of in vivo applications of EPR

Harold M. Swartz; Nadeem Khan

133

In vivo diagnosis of cervical intraepithelial neoplasia using 337-nm-excited laser-induced fluorescence.  

PubMed Central

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

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

1994-01-01

134

Detection of H2O2 by fluorescence correlation spectroscopy.  

PubMed

Fluorescence correlation spectroscopy (FCS) is a technique in which measurement of fluorescence intensity fluctuations is used to clarify dynamic molecular interactions within a very small space in a solution containing a small number of fluorescent molecules. The FCS-based analysis gives the average number and average diffusion time of the fluorescent molecules during their passage through a very small space. One advantage of FCS is that physical separation between free and bound fluorescent probes is not required because the properties of fluorescence fluctuations are accounted for. Therefore, when fluorescent probes are bound with proteins by peroxidase and hydrogen peroxide (H2O2), FCS enables us to detect H2O2 with high sensitivity. In addition, because H2O2 is generated by oxidase-catalyzed reactions, a highly sensitive method for detecting H2O2 is applicable to the measurement of low levels of various oxidases and their substrates, such as glucose. We here describe the protocol of a de novo, highly sensitive method for the measurement of H2O2 and glucose using FCS. PMID:23791098

Ito, Etsuro; Watabe, Satoshi; Morikawa, Mika; Kodama, Hiromi; Okada, Ryuichi; Miura, Toshiaki

2013-01-01

135

Diagnosis of corneal pathology by laser fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

2012-09-01

136

Diffusivity of asphaltene molecules by fluorescence correlation spectroscopy.  

PubMed

Using fluorescence correlation spectroscopy (FCS) we measure the translational diffusion coefficient of asphaltene molecules in toluene at extremely low concentrations (0.03-3.0 mg/L): where aggregation does not occur. We find that the translational diffusion coefficient of asphaltene molecules in toluene is about 0.35 x 10(-5) cm(2)/s at room temperature. This diffusion coefficient corresponds to a hydrodynamic radius of approximately 1 nm. These data confirm previously estimated size from rotational diffusion studied using fluorescence depolarization. The implication of this concurrence is that asphaltene molecular structures are monomeric, not polymeric. PMID:16805495

Andrews, A Ballard; Guerra, Rodrigo E; Mullins, Oliver C; Sen, Pabitra N

2006-07-01

137

Femtosecond broadband fluorescence upconversion spectroscopy: Improved setup and photometric correction  

SciTech Connect

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

Zhang, X.-X. [Photonics Center, College of Physical Science, Nankai University, Tianjin (China); Department of Chemistry, Humboldt Universitaet zu Berlin (Germany); Wuerth, C.; Resch-Genger, U. [Federal Institute for Materials Research and Testing, Berlin (Germany); Zhao, L. [Photonics Center, College of Physical Science, Nankai University, Tianjin (China); Ernsting, N. P.; Sajadi, M. [Department of Chemistry, Humboldt Universitaet zu Berlin (Germany)

2011-06-15

138

Fluorescence correlation spectroscopy in the nanosecond time range: Photon antibunching in dye fluorescence  

Microsoft Academic Search

Possibilities for the use of fluorescence correlation spectroscopy in the nanosecond time range are demonstrated. The experiment is based on a cw argon ion laser, a microfluorimeter, two photon detectors, and a time-to-analog converting system. Experiments using solutions of rhodamine 6G and pyronine G in water at concentrations of about 20 molecules per sample volume are reported. The photon anticorrelation

P. Kask; P. Piksarv; Ü. Mets

1985-01-01

139

Determination of the size of quantum dots by fluorescence spectroscopy.  

PubMed

There has been a lack of quick, simple and reliable methods for determination of nanoparticle size. An investigation of the size of hydrophobic (CdSe) and hydrophilic (CdSe/ZnS) quantum dots was performed by using the maximum position of the corresponding fluorescence spectrum. It has been found that fluorescence spectroscopy is a simple and reliable methodology to estimate the size of both quantum dot types. For a given solution, the homogeneity of the size of quantum dots is correlated to the relationship between the fluorescence maximum position (FMP) and the quantum dot size. This methodology can be extended to the other fluorescent nanoparticles. The employment of evolving factor analysis and multivariate curve resolution-alternating least squares for decomposition of the series of quantum dots fluorescence spectra recorded by a specific measuring procedure reveals the number of quantum dot fractions having different diameters. The size of the quantum dots in a particular group is defined by the FMP of the corresponding component in the decomposed spectrum. These results show that a combination of the fluorescence and appropriate statistical method for decomposition of the emission spectra of nanoparticles may be a quick and trusted method for the screening of the inhomogeneity of their solution. PMID:21491050

Mutavdži?, Dragosav; Xu, Jianmin; Thakur, Garima; Triulzi, Robert; Kasas, Sandor; Jeremi?, Milorad; Leblanc, Roger; Radoti?, Ksenija

2011-04-14

140

Fluorescence spectroscopy of excitation transfer in Photosystem 1  

SciTech Connect

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

Mukerji, I.

1990-12-01

141

Fluorescence lifetime imaging microscopy of nanodiamonds in vivo  

NASA Astrophysics Data System (ADS)

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

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

2013-03-01

142

Emerging In Vivo Analyses of Cell Function Using Fluorescence Imaging?  

PubMed Central

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

Lippincott-Schwartz, Jennifer

2013-01-01

143

The power and prospects of fluorescence microscopies and spectroscopies.  

PubMed

Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory's interest in single-molecule microscopy and spectroscopy. PMID:12598370

Michalet, Xavier; Kapanidis, Achillefs N; Laurence, Ted; Pinaud, Fabien; Doose, Soeren; Pflughoefft, Malte; Weiss, Shimon

2003-02-11

144

Surface-plasmon field-enhanced fluorescence spectroscopy  

Microsoft Academic Search

We describe the combination of surface plasmon- and fluorescence spectroscopy for sensor applications. The resonant excitation of PSP modes at a metal\\/buffer-interface in a flow cell results in optical field intensities largely enhanced compared to the incoming laser light: a factor of 16, calculated for a Au\\/water interface by Fresnel formulas was experimentally confirmed. This field enhancement can be used

Thorsten Liebermann; Wolfgang Knoll

2000-01-01

145

Fluorescence correlation spectroscopy of molecular motions and kinetics  

Microsoft Academic Search

The foundations for fluorescence correlation spectroscopy (FCS) were already laid in the early 1970s, but this technique did not become widely used until single-molecule detection was established almost 20 years later with the use of diffraction-limited confocal volume element. The analysis of molecular noise from the GHz- to the Hz-region facilitates measurements over a large dynamic range covering photophysics, conformational

Michael Gösch; Rudolf Rigler

2005-01-01

146

Near-infrared fluorescent proteins for multicolor in vivo imaging.  

PubMed

Near-infrared fluorescent proteins (FPs) are in high demand for in vivo imaging. We developed four spectrally distinct near-infrared FPs--iRFP670, iRFP682, iRFP702 and iRFP720--from bacterial phytochromes. iRFPs exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies. iRFP670 and iRFP720 enable two-color imaging with standard approaches in living cells and mice. The four new iRFPs and the previously engineered iRFP713 allow multicolor imaging with spectral unmixing in living mice. PMID:23770755

Shcherbakova, Daria M; Verkhusha, Vladislav V

2013-06-16

147

A new approach for fluorescence correlation spectroscopy (FCS) based immunoassays.  

PubMed

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring physicochemical properties, such as concentration and diffusion constant, of bio-molecules in complex mixtures. Although, as such, FCS is well suited for development of homogeneous immunoassays, a major obstacle lies in the relatively high molecular weight of antibodies. This is because in FCS discrimination between unbound fluorescently-labelled antibodies and the same antibodies bound to immune complexes is based on the difference of their respective diffusion coefficients. To overcome this limitation we here propose to use a fluorescently-labelled tag which has two crucial properties: (a) its molecular weight is significantly lower than that of an antibody and (b) it is capable to discriminate between free antibodies and immune complexes. We have evaluated the feasibility of this approach in a model system consisting of mouse monoclonal IgG directed against the Lewis X antigen, and Protein A as a low molecular weight tag. PMID:14711501

Mayboroda, Oleg A; van Remoortere, Alexandra; Tanke, Hans J; Hokke, Cornelis H; Deelder, André M

2004-01-22

148

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

PubMed Central

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

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

2010-01-01

149

Interaction between carbamazepine and humic substances: a fluorescence spectroscopy study.  

PubMed

Carbamazepine is a popular drug that has been detected in natural environments, but little is known about its biogeochemical cycling, influencing factors, and eco-environmental effects in aquatic ecosystems. Interaction between carbamazepine and humic substances, including fulvic and humic acids, was studied using three-dimensional excitation-emission matrix fluorescence spectroscopy and synchronous-scan fluorescence spectroscopy. The intrinsic fluorescence of humic substances was quenched on the addition of carbamazepine, and static quenching was the primary mechanism. The binding parameters on their interaction, including the conditional binding constants (log K) and binding capacities (C(L)), were estimated by the Ryan-Weber nonlinear theory equation. Log K ranged from 3.41 to 5.04 L/mol at 25 degrees C and pH 7.0. The influence of pH on the complexation and the competition between carbamazepine and Cu(II) for fluorescence-binding sites also were discussed. The present results would be helpful in understanding the fate and biogeochemical cycling of other pharmaceuticals and personal care products in aquatic ecosystems. PMID:18092851

Bai, Yingchen; Wu, Fengchang; Liu, Congqiang; Guo, Jianyang; Fu, Pingqing; Li, Wen; Xing, Baoshan

2008-01-01

150

In vivo magnetic resonance spectroscopy: basic methodology and clinical applications  

Microsoft Academic Search

The clinical use of in vivo magnetic resonance spectroscopy (MRS) has been limited for a long time, mainly due to its low\\u000a sensitivity. However, with the advent of clinical MR systems with higher magnetic field strengths such as 3 Tesla, the development\\u000a of better coils, and the design of optimized radio-frequency pulses, sensitivity has been considerably improved. Therefore,\\u000a in vivo

Marinette van der Graaf

2010-01-01

151

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.

2006-03-01

152

Measurement of diffusion in articular cartilage using fluorescence correlation spectroscopy  

PubMed Central

Background Fluorescence correlation spectroscopy (FCS) provides information about translational diffusion of fluorescent molecules in tiny detection volumes at the single-molecule level. In normal states, cartilage tissue lacks vascularity, so chondrocyte metabolism depends on diffusion for molecular exchanges. The abundant extracellular matrix (ECM) of cartilage is maintained by a limited number of chondrocytes. ECM plays an important role in the regulation of chondrocyte functions. In this study, FCS was used to measure diffusion behaviors of albumin, the major protein of the intra-articular space, using normal and degenerated cartilage. Preliminary investigation of fluorescence dyes including Alexa 488, Rhodamine 6G and Rhodamine 123 was conducted to evaluate their properties in cartilage. Results The results indicate that the diffusion behaviors of fluorescently lableded albumin can be observed using FCS in both normal and chemically degenerated cartilage. Conclusions This work demonstrates the capability of FCS for direct measurement of diffusion in cartilaginous ECM. When the diffusion characteristics of fluorescent probes in ECM are clarified using FCS evaluation, FCS will be applicable as a method for early diagnosis of osteoarthritis, which is accompanied by increased abnormalities of ECM and also as tool for evaluating bio-engineered artificial cartilage for autologous chondrocyte implantation.

2011-01-01

153

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

154

Modeling in vivo fluorescence of small animals using TracePro software  

Microsoft Academic Search

The theoretical modeling of fluorescence excitation, emission, and propagation within living tissue has been a limiting factor in the development and calibration of in vivo small animal fluorescence imagers. To date, no definitive calibration standard, or phantom, has been developed for use with small animal fluorescence imagers. Our work in the theoretical modeling of fluorescence in small animals using solid

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

2007-01-01

155

What does it take to improve existing fluorescent proteins for in vivo imaging applications?  

PubMed

Although fluorescent proteins are ubiquitously used as genetic tracers and imaging agents, there is significant room for improvement. This chapter discusses how new improved fluorescent proteins can be designed. It focuses on the design of far-red and infrared fluorescent proteins, since the currently-available red fluorescent proteins are not optimal for in vivo applications. PMID:22700415

Zimmer, Marc

2012-01-01

156

Application of Fluorescence Correlation Spectroscopy (FCS) to Measure the Dynamics of Fluorescent Proteins in Living Cells.  

PubMed

Fluorescence correlation spectroscopy (FCS) can add dynamic molecular information to images of live cells. For example, a confocal laser scanning microscope (CLSM) equipped with an accessory FCS unit provides the possibility to first image the spatial distribution of a fluorescent protein before probing its mobility within defined regions of interest. Whereas specific protein-protein interactions are preferably assayed with a dual-color approach, single-color FCS can still provide valuable information about the size of the diffusing entities and potential interactions with other, nonfluorescent, proteins or subcellular structures. Because number fluctuations are measured, the concentrations of freely diffusing complexes and their state of oligomerization are accessible. PMID:24108643

Weidemann, Thomas

2014-01-01

157

Multiphoton spectroscopy of human skin in vivo  

NASA Astrophysics Data System (ADS)

In vivo multiphoton-intensity images and emission spectra of human skin are reported. Optical sections from different depths of the epidermis and dermis have been measured with near-infrared laser-pulse excitation. While the intensity images reveal information on the morphology, the spectra show emission characteristics of main endogenous skin fluorophores like keratin, NAD(P)H, melanin, elastin and collagen as well as of second harmonic generation induced by the excitation-light interaction with the dermal collagen network.

Breunig, Hans G.; Weinigel, Martin; König, Karsten

2012-02-01

158

Forcing a Connection: Impacts of Single-Molecule Force Spectroscopy on In Vivo Tension Sensing  

PubMed Central

Mechanical tension plays a large role in cell development ranging from morphology to gene expression. On the molecular level, the effects of tension can be seen in the dynamic arrangement of membrane proteins as well as the recruitment and activation of intracellular proteins. Forces applied to biopolymers during in vitro force measurements offer greater understanding of the effects of tension on molecules in live cells, and experimental techniques in test tubes and live cells can often overlap. Indeed, when forces exerted on cellular components can be calibrated ex vivo with force spectroscopy, a powerful tool is available for researchers in probing cellular mechanotransduction on the molecular scale. This review will discuss the techniques used in measuring both cellular traction forces and single-molecule force spectroscopy. Emphasis will be placed on the use of fluorescence reporter systems for the development of in vivo tension sensors that can be used for calibration with single molecule force methods.

Brenner, Michael D.; Zhou, Ruobo; Ha, Taekjip

2011-01-01

159

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy.  

PubMed

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

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

2013-09-11

160

Spectroscopy analysis of tissues in vivo  

NASA Astrophysics Data System (ADS)

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

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

1995-01-01

161

Coded spectroscopy for ethanol detection in diffuse, fluorescent media  

NASA Astrophysics Data System (ADS)

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

McCain, Scott Thomas

162

Fluorescent nanocrystals as colloidal probes in complex fluids measured by fluorescence correlation spectroscopy.  

PubMed

The diffusion properties of fluorescent colloidal CdSe and CdSe/ZnS nanocrystals (QDs) with different hydrophilic coatings were characterized in complex fluids such as actin solutions using fluorescence correlation spectroscopy (FCS). The hydrodynamic radii of the QDs were determined both in organic solvents and water. Attention was given to the potential artifacts arising from the fluorescence properties of the QDs. With increasing excitation intensities, the apparent particle concentration and diffusion times are overestimated if using a simple diffusion model. This can be explained by a numerical simulation. The diffusion behavior of QDs in actin networks of different concentrations was determined to demonstrate the potential use of nanocrystals as probes in soft biological matter. The decreasing diffusion coefficient of the nanocrystals with increasing actin concentration results in an intrinsic polymer viscosity of 0.12+/-0.02 ml mg(-1), in accordance with literature values. PMID:17193385

Liedl, Tim; Keller, Simon; Simmel, Friedrich C; Rädler, Joachim O; Parak, Wolfgang J

2005-10-01

163

Single-molecule fluorescence spectroscopy in (bio)catalysis.  

PubMed

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

2007-07-30

164

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

165

Light emitting diode excitation emission matrix fluorescence spectroscopy.  

PubMed

An excitation emission matrix (EEM) fluorescence instrument has been developed using a linear array of light emitting diodes (LED). The wavelengths covered extend from the upper UV through the visible spectrum: 370-640 nm. Using an LED array to excite fluorescence emission at multiple excitation wavelengths is a low-cost alternative to an expensive high power lamp and imaging spectrograph. The LED-EEM system is a departure from other EEM spectroscopy systems in that LEDs often have broad excitation ranges which may overlap with neighboring channels. The LED array can be considered a hybrid between a spectroscopic and sensor system, as the broad LED excitation range produces a partially selective optical measurement. The instrument has been tested and characterized using fluorescent dyes: limits of detection (LOD) for 9,10-bis(phenylethynyl)-anthracene and rhodamine B were in the mid parts-per-trillion range; detection limits for the other compounds were in the low parts-per-billion range (< 5 ppb). The LED-EEMs were analyzed using parallel factor analysis (PARAFAC), which allowed the mathematical resolution of the individual contributions of the mono- and dianion fluorescein tautomers a priori. Correct identification and quantitation of six fluorescent dyes in two to six component mixtures (concentrations between 12.5 and 500 ppb) has been achieved with root mean squared errors of prediction (RMSEP) of less than 4.0 ppb for all components. PMID:12537381

Hart, Sean J; JiJi, Renée D

2002-12-01

166

Single molecule fluorescence spectroscopy: Toward observation of single molecule reaction  

NASA Astrophysics Data System (ADS)

Advances in room temperature single molecule spectroscopy by laser induced fluorescence provide new tools for the study of individual biological macromolecules under physiological conditions. Two properties of a single fluorescent probe attached to a macromolecule can be exploited to provide local, dynamic structural information. The first, is the very high sensitivity of the fluorophore to its immediate local environment, including the sensitivity to the presence of other fluorophores and quenchers near-by. The second is its unique absorption and emission transition dipoles, which can be interrogated by polarized excitation light and/or by analyzing the emission polarization. Conformational changes can be detected by measuring distance and orientation changes on the macromolecule. Distance changes between two sites on the macromolecule can be measured via single-pair fluorescence resonance energy transfer (spFRET). Orientation changes can be detected by measuring the changes in the dipole orientation of a rigidly attached probe or changes in the rotational freedom of motion of a tethered probe via single molecule fluorescence polarization anisotropy (smFPA). We report spFRET and smFPA measurements done on individual biological macromolecules (DNA, proteins) under physiological condition. Strong inhomogeneity among molecules and time dependent behavior, which would otherwise be hidden in conventional ensemble studies, are clearly revealed. spFRET and smFPA are ideal tools for observing single molecule reactions such as folding/unfolding and enzymatic activities of proteins. Recent results toward establishing structure-function relationship using these tools will be discussed.

Ha, Taekjip

1998-03-01

167

Cytoskeleton dynamics studied by dispersion-relation fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

2013-03-01

168

Hybrid phosphorescence and fluorescence native spectroscopy for breast cancer detection.  

PubMed

Fluorescence and phosphorescence measurements are performed on normal and malignant ex vivo human breast tissues using UV LED and xenon lamp excitation. Tryptophan (trp) phosphorescence intensity is higher in both normal glandular and adipose tissue when compared to malignant tissue. An algorithm based on the ratio of trp fluorescence intensity at 345 nm to phosphorescence intensity at 500 nm is successfully used to separate normal from malignant tissue types. Normal specimens consistently exhibited a low I(345)I(500) ratio (<10), while for malignant specimens, the I(345)I(500) ratio is consistently high (>15). The ratio analysis correlates well with histopathology. Intensity ratio maps with a spatial resolution of 0.5 mm are generated in which local regions of malignancy could be identified. PMID:17343479

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

169

Continuous Fluorescence Microphotolysis and Correlation Spectroscopy Using 4Pi Microscopy  

PubMed Central

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

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

2007-01-01

170

Optical fiber fluorescence spectroscopy for detecting AFM1 in milk  

NASA Astrophysics Data System (ADS)

Fluorescence spectroscopy carried out by means of optical fibers was used for the rapid screening of M1 aflatoxin in milk, enabling the detection of concentrations up to the legal limit, which is 50 ppt. A compact fluorometric device equipped with a LED source, a miniaturized spectrometer, and optical fibers for illumination/detection of the measuring micro-cell was tested for measuring threshold values of AFM1 in pre-treated milk samples. Multivariate processing of the spectral data made it possible to obtain a preliminary screening at the earlier stages of the industrial process, as well as to discard contaminated milk stocks before their inclusion in the production chain.

Mignani, A. G.; Cucci, C.; Ciaccheri, L.; Dall'Asta, C.; Galaverna, G.; Dossena, A.; Marchelli, R.

2008-06-01

171

Pulsed interleaved excitation fluorescence spectroscopy with a supercontinuum source.  

PubMed

Pulsed Interleaved Excitation (PIE) improves fluorescence cross-correlation spectroscopy (FCCS) and single pair Förster Resonance Energy Transfer (spFRET) measurements, by correlating each detected photon to the excitation source that generated it. It relies on the interleaving of two picosecond laser sources and time correlated single photon counting (TCSPC) detection. Here, we present an optical configuration based on a commercial supercontinuum laser, which generates multicoulour interleaved picosecond pulses with arbitrary spacing and wavelengths within the visible spectrum. This simple, yet robust configuration can be used as a versatile source for PIE experiments, as an alternative to an array of picosecond lasers and drivers. PMID:23481797

Olofsson, Linnea; Margeat, Emmanuel

2013-02-11

172

Dynamics of Large Semiflexible Chains Probed by Fluorescence Correlation Spectroscopy  

NASA Astrophysics Data System (ADS)

Fluorescence correlation spectroscopy was used to probe the dynamics of ?-phage DNA in aqueous solution labeled with the randomly intercalating dye TOTO. The linear macromolecules (i) carry more than one chromophore and (ii) are larger than the waist of the focal volume. The correlation function decays significantly faster than expected for a stiff globule of corresponding size but is in good agreement with the dynamic model of semiflexible chains including hydrodynamic interactions. As the chromophore density is lowered the correlation time decreases in accordance with this model.

Lumma, D.; Keller, S.; Vilgis, T.; Rädler, J. O.

2003-05-01

173

Detectors for single-molecule fluorescence imaging and spectroscopy  

PubMed Central

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

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

2010-01-01

174

Identification of active fluorescence stained bacteria by Raman spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

2008-05-01

175

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

176

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

NASA Astrophysics Data System (ADS)

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

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

1995-03-01

177

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

NASA Astrophysics Data System (ADS)

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

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

1994-10-01

178

Total Internal Reflection with Fluorescence Correlation Spectroscopy: Combined Surface Reaction and Solution Diffusion  

Microsoft Academic Search

Total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) is a method for measuring the surface association\\/dissociation rates and absolute densities of fluorescent molecules at the interface of solution and a planar substrate. This method can also report the apparent diffusion coefficient and absolute concentration of fluorescent molecules very close to the surface. An expression for the fluorescence fluctuation autocorrelation function

Tammy E. Starr; Nancy L. Thompson

2001-01-01

179

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

180

Studying Slow Membrane Dynamics with Continuous Wave Scanning Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

Here we discuss the application of scanning fluorescence correlation spectroscopy (SFCS) using continuous wave excitation to analyze membrane dynamics. The high count rate per molecule enables the study of very slow diffusion in model and cell membranes, as well as the application of two-foci fluorescence cross-correlation spectroscopy for parameter-free determination of diffusion constants. The combination with dual-color fluorescence cross-correlation spectroscopy

Jonas Ries; Petra Schwille

2006-01-01

181

Fluorescence correlation spectroscopy of repulsive systems: theory, simulation, and experiment.  

PubMed

The theoretical basis of fluorescence correlation spectroscopy (FCS) for repulsive systems, such as charged colloids or macromolecules, has been further expanded and developed. It is established that the collective correlation function can no longer be fitted using the theoretical model of non-interacting systems. Also, it is discovered that the collective correlation function can be divided into two parts: a self-part and a distinct-part, named as the self-correlation and cross-correlation function, respectively. The former indicates the self-diffusion of objects, while the latter describes mutual interactions. Dual-color fluorescence cross-correlation spectroscopy provides the direct measurements of the two parts. The particle concentration and mean squared displacement of single particles can be deduced from the self-correlation function, while the correlation volume between particles can be approximated from the cross-correlation function. In the case of charged colloids, the Debye length of the solution and particle surface charge number can be fitted from the cross-correlation function. These theoretical results are successfully proven using Brownian dynamics simulations and preliminary FCS experiments for model charged colloidal systems. PMID:23758396

Feng, Ligang; Yang, Jingfa; Zhao, Jiang; Wang, Dapeng; Koynov, Kaloian; Butt, Hans-Jürgen

2013-06-01

182

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.

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

2011-01-01

183

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

184

Study plasma disintegration based on the fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

It is known that plasma is very important in the diagnosis and therapy of disease so that more and more scientific workers attach importance to the plasma storage life or storage environment. We research plasma disintegration with the increases of storage time based on the fluorescence spectroscopy. Based on the experimental researches and theoretical analysis, we find that their plasma fluorescence intensity is increasing within about 10 hours, and but is decreasing gradually and is nearly a straight line after this. It is indicated that plasma proteins have begun to disintegrate so as to make the fluorescence quenching after storage time beyond 10 hours. And the disintegration speed of plasma in the case of different concentration is different, the concentration is higher and the speed is lower in them, and but they are almost same after about 35 hours. Therefore, we think that plasma under higher concentration is deposited easier. These research consequences may order a theoretical and experimental reference to know the changes of plasma in structure in different disintegration time. It may make sense for understand the plasma disintegrative mechanism and distinguishing the fine plasma with faulty.

Gao, Shumei; Li, Rongqing; Chen, Guoqing; Lu, Jun

2007-10-01

185

Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy  

PubMed Central

Fluorescence fluctuation spectroscopy provides information about protein interactions in the intercellular environment from naturally occurring equilibrium fluctuations. We determine the molecular brightness of fluorescent proteins from the fluctuations by analyzing the photon counting histogram (PCH) or its moments and demonstrate the use of molecular brightness in probing the oligomerization state of proteins. We report fluorescence fluctuation measurements of enhanced GFP (EGFP) in cells up to concentrations of 10 ?M by using an improved PCH theory. The molecular brightness of EGFP is constant in the concentration range studied. The brightness of a tandem EGFP construct, which carries two fluorophores, increases by a factor of two compared with EGFP alone, demonstrating the sensitivity of molecular brightness as a probe for protein complex formation. Oligomerization of nuclear receptors plays a crucial role in the regulation of gene expression. We probe the oligomerization state of the testicular receptor 4 and the ligand-binding domains of retinoid X receptor and retinoic acid receptor by observing molecular brightness changes as a function of protein concentration. The large concentration range accessible by experiment allows us to perform titration experiments on EGFP fusion proteins. An increase in the molecular brightness with protein concentration indicates the formation of homocomplexes. We observe the formation of homodimers of retinoid X receptor ligand binding domain upon addition of ligand. Resolving protein interactions in a cell is an important step in understanding cellular function on a molecular level. Brightness analysis promises to develop into an important tool for determining protein complex formation in cells.

Chen, Yan; Wei, Li-Na; Muller, Joachim D.

2003-01-01

186

Scanning fluorescence correlation spectroscopy in model membrane systems.  

PubMed

Fluorescence correlation spectroscopy (FCS) is an emerging technique employed in biophysical studies that exploits the temporal autocorrelation of fluorescence intensity fluctuations measured in a tiny volume (in the order of fL). The autocorrelation curve derived from the fluctuations can then be fitted with diffusion models to obtain parameters such as diffusion time and number of particles in the diffusion volume/area. Application of FCS to membranes allows studying membrane component dynamics, which includes mobility and interactions between the components. However, FCS encounters several difficulties like accurate positioning and stability of the setup when applied to membranes.Here, we describe the theoretical basis of point FCS as well as the scanning FCS (SFCS) approach, which is a practical way to address the challenges of FCS with membranes. We also list materials necessary for FCS experiments on two model membrane systems: (1) supported lipid bilayers and (2) giant unilamellar vesicles. Finally, we present simple protocols for the preparation of these model membrane systems, calibration of the microscope setup for FCS, and acquisition and analysis of point FCS and SFCS data so that diffusion coefficients and concentrations of fluorescent probes within lipid membranes can be calculated. PMID:23996179

Unsay, Joseph D; García-Sáez, Ana J

2013-01-01

187

In-vivo fluorescence imaging with a multivariate curve resolution spectral unmixing technique  

Microsoft Academic Search

Spectral unmixing is a useful technique in fluorescence imaging for reducing the effects of native tissue autofluorescence and separating multiple fluorescence probes. While spectral unmixing methods are well established in fluorescence microscopy, they typically rely on precharacterized in-vitro spectra for each fluorophore. However, there are unique challenges for in-vivo applications, since the tissue absorption and scattering can have a significant

Heng Xu; Brad W. Rice

2009-01-01

188

Probing the interaction between fluorophores and DNA nucleotides by fluorescence correlation spectroscopy and fluorescence quenching.  

PubMed

We have investigated the association interactions between the fluorescent dyes TAMRA, Cy3B and Alexa-546 and the DNA deoxynucleoside monophosphates by means of fluorescence quenching and fluorescence correlation spectroscopy (FCS). The interactions of Cy3B and TAMRA with the nucleotides produce a decrease in the apparent diffusion coefficient of the dyes, which result in a shift toward longer times in the FCS autocorrelation decays. Our results with Cy3B demonstrate the existence of Cy3B-nucleotide interactions that do not affect the fluorescence intensity or lifetime of the dye significantly. The same is true for TAMRA in the presence of dAMP, dCMP and dTMP. In contrast, the diffusion coefficient of Alexa 546 remains practically unchanged even at high concentrations of nucleotide. These results demonstrate that interactions between this dye and the four dNMPs are not significant. The presence of the negatively charged sulfonates and the bulky chlorine atoms in the phenyl group of Alexa 546 possibly prevent strong interactions that are otherwise possible for TAMRA. The characterization of dye-DNA interactions is important in biophysical research because they play an important role in the interpretation of energy transfer experiments, and because they can potentially affect the structure and dynamics of the DNA. PMID:22364288

Ranjit, Suman; Levitus, Marcia

2012-03-28

189

Relaxation Kinetics by Fluorescence Correlation Spectroscopy: Determination of Kinetic Parameters in the Presence of Fluorescent Impurities  

PubMed Central

The use of Fluorescence Correlation Spectroscopy (FCS) in combination with Förster Resonance Energy Transfer (FRET) is gaining popularity as a tool to investigate kinetics in equilibrium conditions. The technique is based on the study of fluorescence fluctuations in small numbers of molecules, and is particularly well-suited to investigate conformational dynamics in biopolymers. In practice, its applicability is often hindered by the presence of certain impurities such as partially labeled biomolecules, excess of free fluorophore, or partially dissociated multi-subunit complexes. Here, we show that the simultaneous measurement of the fluctuations in the donor and acceptor intensities allows the determination of the kinetic relaxation time of the reaction in the presence of donor-only particles when cross-talk is negligible, or in cases where all species have the same diffusion coefficient. Theoretical predictions are supported with the results of Monte Carlo simulations, and demonstrate that the applicability of the technique is more general than previously thought.

Levitus, Marcia

2010-01-01

190

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

NASA Astrophysics Data System (ADS)

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

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

2010-03-01

191

Fluorescence correlation spectroscopy: molecular complexing in solution and in living cells.  

PubMed

This chapter describes how the microscope can be used to measure a fluorescence signal from a small, confined volume of the sample-the confocal volume-and how these measurements are used to quantitate the dynamics and complexing of molecules, the technique of fluorescence correlation spectroscopy (FCS). FCS represents a significant example of how the microscope can be used to extract information beyond the resolution limit of classical optics. FCS enables studying events at the level of single molecules. With FCS, one can measure the diffusion times and the interaction of macromolecules, the absolute concentration of fluorescently labeled particles, and the kinetics of chemical reactions. Practical applications of FCS include studies on ligand-receptor binding, protein-protein and protein-DNA interactions, and the aggregation of fluorescently labeled particles. The chapter focuses on the principles of FCS, demonstrates how FCS is used to study macromolecular interactions in solution and in living cells, and examines critical experimental parameters that must be considered. The chapter also discusses the minimum requirements for building a microscope-based FCS instrument and illustrates the key criteria for both instrument sensitivity and analysis of FCS data. It can be used to study single molecules both in solution and in living cells and can be used to monitor a variety of macromolecular interactions. When used as an in vitro technique, FCS measurements are easy to conduct and can be made on simplified instrumentation. When used in vivo on living cells, many additional factors must be considered when evaluating experimental data. Despite these concerns, FCS represents a new approach that has broad applicability for the determination of molecular stoichiometry both in vivo and in vitro for a variety of membrane and soluble receptor systems. PMID:23931520

Bulseco, Dylan A; Wolf, David E

2013-01-01

192

In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue  

Microsoft Academic Search

DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal

Pernille Hojman; Jens Eriksen; Julie Gehl

2009-01-01

193

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.

Wachsmann-Hogiu, Sebastian; Weeks, Tyler

2009-01-01

194

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

195

Laser optogalvanic and fluorescence spectroscopy in glow discharge plasmas  

NASA Astrophysics Data System (ADS)

Diagnostic techniques based on laser optogalvanic spectroscopy and laser induced fluorescence are providing spatial and temporal maps of key quantities in glow discharge plasma. Such maps are particularly useful in studying the cathode region. This region is of fundamental interest because of the failure of the local field approximation, and of practical interest because of widespread use of glow discharges in plasma processing and other areas. Laser techniques are used to map space charge electric fields, charged particle densities, average energies of charged particles, flux densities of charged particles, excited atom densities, and other key quantities. These maps provide valuable insights into the dominant physical processes in the cathode region and provide a stringent test of numerical models. A more quantitative understanding of the cathode region is emerging from laser studies and from advanced modeling efforts.

Lawler, J. E.; den Hartog, E. A.

1990-05-01

196

Fluorescence and UV-vis Spectroscopy of Synovial Fluids  

NASA Astrophysics Data System (ADS)

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

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

2009-10-01

197

Fluorescence correlation spectroscopy to study diffusion through diatom nanopores.  

PubMed

The intricate pore architecture of diatom frustules has been extensively studied mainly as the basis of diatom classification. There have, however, been very few reports on understanding the effect of the pore architecture on the movement of molecules through the pores. Information on molecular transport though diatom membrane pores has the potential to help develop more efficient membrane filtration systems. In this paper the transport of molecules through individual diatom nanopores is investigated. Fluorescence correlation spectroscopy (FCS) is used to determine diffusion coefficients. Thus, for the very first time, we measure the effect of the three dimensional pore structure of the diatom Coscinodiscus wailesii, on the mean diffusion coefficient through the pores. The results show almost a 50% decrease in the diffusion coefficient relative to that in the free solution. PMID:19908596

Bhatta, Hemant; Enderlein, Jörg; Rosengarten, Gary

2009-11-01

198

In vivo fluorescence lifetime tomography of a FRET probe expressed in mouse  

PubMed Central

Förster resonance energy transfer (FRET) is a powerful biological tool for reading out cell signaling processes. In vivo use of FRET is challenging because of the scattering properties of bulk tissue. By combining diffuse fluorescence tomography with fluorescence lifetime imaging (FLIM), implemented using wide-field time-gated detection of fluorescence excited by ultrashort laser pulses in a tomographic imaging system and applying inverse scattering algorithms, we can reconstruct the three dimensional spatial localization of fluorescence quantum efficiency and lifetime. We demonstrate in vivo spatial mapping of FRET between genetically expressed fluorescent proteins in live mice read out using FLIM. Following transfection by electroporation, mouse hind leg muscles were imaged in vivo and the emission of free donor (eGFP) in the presence of free acceptor (mCherry) could be clearly distinguished from the fluorescence of the donor when directly linked to the acceptor in a tandem (eGFP-mCherry) FRET construct.

McGinty, James; Stuckey, Daniel W.; Soloviev, Vadim Y.; Laine, Romain; Wylezinska-Arridge, Marzena; Wells, Dominic J.; Arridge, Simon R.; French, Paul M. W.; Hajnal, Joseph V.; Sardini, Alessandro

2011-01-01

199

Study on the interaction between theasinesin and human serum albumin by fluorescence spectroscopy  

Microsoft Academic Search

The binding properties on theasinesin to human serum albumin (HSA) have been studied for the first time using fluorescence spectroscopy in combination with UV–vis absorbance spectroscopy. The results showed that theasinesin strongly quenched the intrinsic fluorescence of HSA through a static quenching procedure, and non-radiation energy transfer happened within molecules. The number of binding site was 1, and the efficiency

Feng Ge; Chaoyin Chen; Diqiu Liu; Benyong Han; Xiangfeng Xiong; Shenglan Zhao

2010-01-01

200

Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

2007-10-01

201

Quantifying lipid diffusion by fluorescence correlation spectroscopy: a critical treatise.  

PubMed

Fluorescence correlation spectroscopy (FCS) measurements are widely used for determination of diffusion coefficients of lipids and proteins in biological membranes. In recent years, several variants of FCS have been introduced. However, a comprehensive comparison of these methods on identical systems has so far been lacking. In addition, there exist no consistent values of already determined diffusion coefficients for well-known or widely used membrane systems. This study aims to contribute to a better comparability of FCS experiments on membranes by determining the absolute diffusion coefficient of the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) in giant unilamellar vesicles (GUVs) made of dioleoylphosphatidylcholine (DOPC), which can in future studies be used as a reference value. For this purpose, five FCS variants, employing different calibration methods, were compared. Potential error sources for each particular FCS method and strategies to avoid them are discussed. The obtained absolute diffusion coefficients for DiD in DOPC were in good agreement for all investigated FCS variants. An average diffusion coefficient of D = 10.0 ± 0.4 ?m(2) s(-1) at 23.5 ± 1.5 °C was obtained. The independent confirmation with different methods indicates that this value can be safely used for calibration purposes. Moreover, the comparability of the methods also in the case of slow diffusion was verified by measuring diffusion coefficients of DiD in GUVs consisting of DOPC and cholesterol. PMID:22891610

Heinemann, Fabian; Betaneli, Viktoria; Thomas, Franziska A; Schwille, Petra

2012-08-31

202

Continuous fluorescence microphotolysis and correlation spectroscopy using 4Pi microscopy.  

PubMed

Continuous fluorescence microphotolysis (CFM) and fluorescence correlation spectroscopy (FCS) permit measurement of molecular mobility and association reactions in single living cells. CFM and FCS complement each other ideally and can be realized using identical equipment. So far, the spatial resolution of CFM and FCS was restricted by the resolution of the light microscope to the micrometer scale. However, cellular functions generally occur on the nanometer scale. Here, we develop the theoretical and computational framework for CFM and FCS experiments using 4Pi microscopy, which features an axial resolution of approximately 100 nm. The framework, taking the actual 4Pi point spread function of the instrument into account, was validated by measurements on model systems, employing 4Pi conditions or normal confocal conditions together with either single- or two-photon excitation. In all cases experimental data could be well fitted by computed curves for expected diffusion coefficients, even when the signal/noise ratio was small due to the small number of fluorophores involved. PMID:17704168

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

2007-08-17

203

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

204

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

PubMed

Fluorescence Correlation Spectroscopy (FCS) is widely used to quantitate reaction rates and concentrations of molecules in vitro and in vivo. We recently reported Fluorescence Triple Correlation Spectroscopy (F3CS), which correlates three signals together instead of two. F3CS can analyze the stoichiometries of complex mixtures and detect irreversible processes by identifying time-reversal asymmetries. Here we report the computational developments that were required for the realization of F3CS and present the results as the Triple Correlation Toolbox suite of programs. Triple Correlation Toolbox is a complete data analysis pipeline capable of acquiring, correlating and fitting large data sets. Each segment of the pipeline handles error estimates for accurate error-weighted global fitting. Data acquisition was accelerated with a combination of off-the-shelf counter-timer chips and vectorized operations on 128-bit registers. This allows desktop computers with inexpensive data acquisition cards to acquire hours of multiple-channel data with sub-microsecond time resolution. Off-line correlation integrals were implemented as a two delay time multiple-tau scheme that scales efficiently with multiple processors and provides an unprecedented view of linked dynamics. Global fitting routines are provided to fit FCS and F3CS data to models containing up to ten species. Triple Correlation Toolbox is a complete package that enables F3CS to be performed on existing microscopes. PMID:23525193

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

2012-12-31

205

Probing transcription factor diffusion dynamics in the living mammalian embryo with photoactivatable fluorescence correlation spectroscopy.  

PubMed

Transcription factors use diffusion to search the DNA, yet the mechanisms controlling transcription factor diffusion during mammalian development remain poorly understood. Here we combine photoactivation and fluorescence correlation spectroscopy to study transcription factor diffusion in developing mouse embryos. We show that the pluripotency-associated transcription factor Oct4 displays both fast and Brownian and slower subdiffusive behaviours that are controlled by DNA interactions. Following cell lineage specification, the slower DNA-interacting diffusion fraction distinguishes pluripotent from extraembryonic cell nuclei. Similar to Oct4, Sox2 shows slower diffusion in pluripotent cells while Cdx2 displays opposite dynamics, suggesting that slow diffusion may represent a general feature of transcription factors in lineages where they are essential. Slow Oct4 subdiffusive behaviours are conserved in embryonic stem cells and induced pluripotent stem cells (iPS cells), and lost during differentiation. We also show that Oct4 diffusion depends on its interaction with ERG-associated protein with SET domain. Photoactivation and fluorescence correlation spectroscopy provides a new intravital approach to study transcription factor diffusion in complex in vivo systems. PMID:23535658

Kaur, Gurpreet; Costa, Mauro W; Nefzger, Christian M; Silva, Juan; Fierro-González, Juan Carlos; Polo, Jose M; Bell, Toby D M; Plachta, Nicolas

2013-01-01

206

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.

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

2013-04-01

207

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

208

Localized in vivo13C NMR spectroscopy of the brain  

PubMed Central

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

Gruetter, Rolf; Adriany, Gregor; Choi, In-Young; Henry, Pierre-Gilles; Lei, Hongxia; Oz, Gulin

2006-01-01

209

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

PubMed Central

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

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

2004-01-01

210

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

211

Ligand-Receptor Kinetics Measured by Total Internal Reflection with Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

Total internal reflection excitation used in combination with fluorescence correlation spectroscopy (TIR-FCS) is a method for characterizing the dynamic behavior and absolute concentrations of fluorescent molecules near or at the interface of a planar substrate and a solution. In this work, we demonstrate for the first time the use of TIR-FCS for examining the interaction kinetics of fluorescent ligands in

Alena M. Lieto; Randall C. Cush; Nancy L. Thompson

2003-01-01

212

Fluorescence Correlation Spectroscopy in Small Cytosolic Compartments Depends Critically on the Diffusion Model Used  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion constants. In the standard case, fluorescence fluctuations are measured in an open detection volume defined by the confocal optics. However, if FCS measurements are carried out in cellular processes that confine the detection volume, the standard FCS model leads to erroneous results.

Arne Gennerich; Detlev Schild

2000-01-01

213

Optical Saturation in Fluorescence Correlation Spectroscopy under Continuous-Wave and Pulsed Excitation  

Microsoft Academic Search

A detailed theoretical and experimental study of the dependence of fluorescence correlation measurements on optical excitation power due to optical saturation effects is presented. It is shown that the sensitivity of a fluorescence correlation measurement on excitation power becomes increasingly stronger for decreasing excitation power. This makes exact measurements of diffusion co- efficients with fluorescence correlation spectroscopy rather diffi- cult.

Ingo Gregor; Digambara Patra

2005-01-01

214

Assessing topographic cutaneous autofluorescence variation using fluorescence UV and visible excitation emission matrix (EEM) spectroscopy  

Microsoft Academic Search

Cutaneous autofluorescence properties were systematically studied using fluorescence excitation emission matrix spectroscopy. Twenty-six healthy subjects with a mean age of 34 (range 21-74) participated in this study. The fluorescence of major skin fluorophores such as tryptophan, collagen, elastin and NADH could be readily identified. On average, facial skin shows strong tryptophan and measurable porphyrin fluorescence; the palm and nail show

Jianhua Zhao; Soodabeh Zandi; Florina Feng; Haishan Zeng; David I. McLean; Harvey Lui

2011-01-01

215

Tracing dissolved organic matter in aquatic environments using a new approach to fluorescence spectroscopy  

Microsoft Academic Search

Dissolved organic matter (DOM) is a complex and poorly understood mixture of organic polymers that plays an influential role in aquatic ecosystems. In this study we have successfully characterised the fluorescent fraction of DOM in the catchment of a Danish estuary using fluorescence excitation–emission spectroscopy and parallel factor analysis (PARAFAC). PARAFAC aids the characterisation of fluorescent DOM by decomposing the

Colin A Stedmon; Stiig Markager; Rasmus Bro

2003-01-01

216

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

PubMed Central

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

Palmer, A G; Thompson, N L

1987-01-01

217

X-ray microprobe for micro x-ray fluorescence and absorption spectroscopies at GSECARS  

Microsoft Academic Search

The hard x-ray microprobe for x-ray fluorescence and absorption spectroscopy at GeoSoilEnviroCARS is presented. Using focused synchrotron radiation from an undulator beamline at the Advanced Photon Source at Argonne National Lab, the x-ray microprobe provides bright, monochromatic x-rays with typical spot sizes down to 1x1 mum for x-ray fluorescence and absorption spectroscopies. Quantitative x-ray fluorescence (XRF) analysis gives precise elemental

M. Newville; S. Sutton; M. Rivers

2002-01-01

218

Combined Photoacoustic and Molecular Fluorescence Imaging In Vivo  

Microsoft Academic Search

Because of the overwhelming scattering of light in biological tissues, the spatial resolution and imaging depth of conventional fluorescent imaging is unsatisfactory. Therefore, we present a dual modality imaging technique by combining fluorescence imaging with high-resolution noninvasive photoacoustic tomography (PAT) for the study of an animal tumor model. PAT provides high-resolution structural images of tumor angiogenesis, and fluorescence imaging offers

Lihong V. Wang; Xueyi Xie; Jung-Taek Oh; Meng-Lin Li; Geng Ku; Shi Ke; Sergiu Similache; Chun Li; George Stoica

2005-01-01

219

The Fluorescent Materials produced in vivo by certain Dermatophytes  

Microsoft Academic Search

SUMMARY: The fluorescent materials present in human and cat hair as a result of infection with Microsporon canis, M. audouini and Trichophyton schoenleini were extracted with dilute ammonia. Paper chromatography and electrophoresis showed that the principal fluorescent material was common to all these infections and that further fluorescent substances were present in the extract from 2'. schoenleini infected hair. Chemical

F. W. Chattaway; A. J. E. Barlow

1954-01-01

220

In vivo infrared and Raman spectroscopy of human stratum corneum  

NASA Astrophysics Data System (ADS)

ATR-FTIR spectroscopy and Raman spectroscopy were employed to obtain information about the molecular composition and hydration of skin in vivo. Both techniques enable the in vivo acquisition of high quality spectra within 10-30s at a spectral resolution of 8cm-1. The penetration depth of ATR-FTIR is about 1.5 (Mu) m. Raman spectra could be obtained with a resolution of about 5 micrometers . ATR-FTIR spectra of hydrated stratum corneum were analyzed using a band fitting algorithm. By means of this algorithm the signal contributions of water relative to protein signal contributions could be determined. The results of Raman microspectroscopic experiments on frozen sections and isolated skin components were used for the interpretation of Raman spectra obtained in vivo. Information was obtained about lipid components present in the stratum corneum. These were shown to vary widely between individuals and between different locations on the body. The combination of these spectroscopic techniques may prove to be valuable for applications in dermatology and skin care.

Lucassen, Gerald W.; Caspers, Peter J.; Puppels, Gerwin J.

1998-04-01

221

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

2012-02-08

222

Fluorescence Spectroscopy of Human Nonmalignant and Malignant Cells and Tissues.  

NASA Astrophysics Data System (ADS)

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

Glassman, Wenling Sha

223

Evaluation of fiber optic probes for in-vivo Raman spectroscopy  

Microsoft Academic Search

Raman spectroscopy has been sued for the analysis of biological tissue. Preliminary studies, which have been performed ex vivo, indicate that potentially useful diagnostic information may be obtained from the spectra. A new fiber optic-based in vivo Raman system has been constructed which can obtain spectra in vivo from tissue in less than 30 s. Unfortunately, tissue spectroscopy is hindered

Martin G. Shim; Brian C. Wilson; Eric Marple; Michael L. Wach

1998-01-01

224

Interactions between 1-benzoyl-4- p-chlorophenyl thiosemicarbazide and serum albumin: investigation by fluorescence spectroscopy  

Microsoft Academic Search

The interactions between 1-benzoyl-4-p-chlorphenyl thiosemicarbazide (BCPT) and bovine serum albumin (BSA) or human serum albumin (HSA) have been studied by fluorescence spectroscopy. By the analysis of fluorescence spectrum and fluorescence intensity, it was showed that BCPT has a strong ability to quench the intrinsic fluorescence of both bovine serum albumin and human serum albumin through a static quenching procedure. The

Feng-Ling Cui; Jing Fan; Jian-Ping Li; Zhi-De Hu

2004-01-01

225

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

226

Microbiochemical analysis of carious dentine using Raman and fluorescence spectroscopy.  

PubMed

The aim of this study was to evaluate and correlate objectively the microspectroscopically derived biochemical components of sound, infected and affected carious dentine with their microhardness and autofluorescence (AF) characteristics. Over 3 million high-resolution Raman spectra from 8 extracted human carious teeth were recorded using Raman spectrometer with parallel spectrum acquisition. Green AF signals across each carious lesion from all samples were acquired with a similar spatial resolution using confocal fluorescence microscopy. The Knoop microhardness (KHN) from a total of 233 co-localized areas was recorded from the same samples and allocated subjectively into the three zones. Cluster analysis of the Raman data, performed using in-house software, produced five independent spectral components representing mineral content, protein content, porphyrin fluorescence (PF), putative infected dentine signal (IDS) and affected dentine signal (ADS). The distributions of the 5 Raman components and the AF signal were matched across all samples and their average values were calculated for each corresponding KHN area. The infected dentine was defined significantly by the KHN, AF and by the relative contribution of the mineral, PF and IDS clusters. Protein cluster was not statistically related to the KHN or AF. A delineation between affected and sound dentine was observed using the KHN, AF, PF and ADS parameters. This study concludes that micro-Raman spectroscopy can provide a non-invasive and objective evaluation of different carious dentine zones. Being able to detect and assess clinically the caries-affected dentine during minimally invasive operative caries management is important to control the risk of unnecessary tissue removal. PMID:22739587

Almahdy, A; Downey, F C; Sauro, S; Cook, R J; Sherriff, M; Richards, D; Watson, T F; Banerjee, A; Festy, F

2012-06-27

227

Fluorescence spectroscopy of atmospherically relevant bacterial and fungal spores and potential interferences  

NASA Astrophysics Data System (ADS)

Single-particle fluorescence spectroscopy was used to study fluorescence properties of fungal spores and bacteria, selected for their possible atmospheric relevance. In addition, aromatic organic acid aerosols, potentially interfering with laser induced fluorescence measurement, were studied. The results indicate that fungal spores and bacteria have dissimilar fluorescence spectra. The tested aromatic organic acids had fluorescence properties rather similar to common biological molecules. It may be possible to classify atmospheric bacterial and fungal spores through the dissimilar fluorescence properties, but the influence of the potential interferences must be taken into account.

Saari, S. E.; Putkiranta, M. J.; Keskinen, J.

2013-06-01

228

Fluorescence spectroscopy as a diagnostic of the radiation environment in high energy density experiments (invited)  

SciTech Connect

A fluorescence spectroscopy technique has been developed to measure conditions in high energy density (HED) experiments. The experimental technique and modeling of the spectra are described and results of fluorescence measurements are presented. Fluorescence spectra were measured from an aluminium microdot over a small hole in the wall of an experimental package or a hohlraum. The aluminium was photopumped from a broadband radiation source, without perturbing the temperature. To date, fluorescence spectroscopy has been used to diagnose the radiative heating of plasmas in the temperature range 20-80 eV. Fluorescence spectroscopy has several advantages over x-ray absorption and self-emission spectroscopy in the diagnosis of HED experiments and these are discussed in the article. Extension of the technique to higher temperature plasma is discussed.

Hoarty, D.J.; Smith, C.C.; Clark, E.L.; Foster, J.M.; Gales, S.G.; Magelssen, G.; Workman, J.; Wood, W.M.; Caldwell, S.; Chrien, R.; Sandoval, J.; Sedillo, T.; Walsh, P.; Carpenter, B.; Compton, S.; Perry, T. [AWE Plasma Physics Department, Reading Berkshire, RG7 4PR (United Kingdom); Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (United States); Lawrence Livermore National Laboratory, Livermore, California 94550-9234 (United States)

2004-10-01

229

A New Method for in vivo Quantification of Changes in Initial Enamel Caries with Laser Fluorescence  

Microsoft Academic Search

A new method for the in vivo assessment of changes in initial enamel caries lesions was developed and tested. A CCD camera equipped with a high-pass filter (? > 520 nm) collects the fluorescence image of carious teeth, illuminated intraorally with diffuse laser light (?=488 nm). Incipient lesions show a loss in fluorescence to be expressed as a percentage of

E. de Josselin de Jong; F. Sundström; H. Westerling; S. Tranaeus; J. J. Ten Bosch; B. Angmar-Månsson

1995-01-01

230

Monte Carlo methods for the in vivo analysis of Cisplatin using x-ray fluorescence  

Microsoft Academic Search

A Monte Carlo method has been used to model the measument of cisplatin uptake with in vivo X-ray fluorescence. A user-code has been written for the EGS4 Monte Carlo system that incorporates linear polarisation and multiple element fluorescence extensoions. The yield of fluorescent photons to the mainly Compton scattered background is computed for our detector arrangement. The detector consists of

R. P. Hugtenburg; J. R. Turner; D. M. Mannering; B. A. Robinson

1998-01-01

231

In vivo hyperspectral confocal fluorescence imaging to determine pigment localization and distribution in cyanobacterial cells  

Microsoft Academic Search

Hyperspectral confocal fluorescence imaging provides the opportunity to obtain individual fluorescence emission spectra in small (≈0.03-mum3) volumes. Using multivariate curve resolution, individual fluorescence components can be resolved, and their intensities can be calculated. Here we localize, in vivo, photosynthesis-related pigments (chlorophylls, phycobilins, and carotenoids) in wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Cells were excited at

Wim F. J. Vermaas; Jerilyn A. Timlin; Howland D. T. Jones; Michael B. Sinclair; Linda T. Nieman; Sawsan W. Hamad; David K. Melgaard; David M. Haaland

2008-01-01

232

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

233

Fluorescence correlation spectroscopy on nano-fakir surfaces  

NASA Astrophysics Data System (ADS)

Single biomolecule behaviour can reveal crucial information about processes not accessible by ensemble measurements. It thus represents a real biotechnological challenge. Common optical microscopy approaches require pico- to nano-molar concentrations in order to isolate an individual molecule in the observation volume. However, biologically relevant conditions often involve micromolar concentrations, which impose a drastic reduction of the conventional observation volume by at least three orders of magnitude. This confinement is also crucial for mapping sub-wavelength heterogeneities in cells, which play an important role in many biological processes. We propose an original approach, which couples Fluorescence Correlation Spectroscopy (FCS), a powerful tool to retrieve essential information on single molecular behaviour, and nano-fakir substrates with strong field enhancements and confinements at their surface. These electromagnetic singularities at nanometer scale, called "hotspots", are the result of the unique optical properties of surface plasmons. They provide an elegant means for studying single-molecule dynamics at high concentrations by reducing dramatically the excitation volume and enhancing the fluorophore signal by several orders of magnitude. The nano-fakir substrates used are obtained from etching optical fiber bundles followed by sputtering of a gold thin-film. It allows one to design reproducible arrays of nanotips.

Delahaye, Julie; Gresillon, Samuel; Lévêque-Fort, Sandrine; Sojic, Neso; Fort, Emmanuel

2010-02-01

234

Clinical Measurement of von Willebrand Factor by Fluorescence Correlation Spectroscopy  

PubMed Central

BACKGROUND Identification of von Willebrand factor (vWF) abnormalities in a variety of conditions is hampered by the limitations of currently available diagnostic tests. Although direct multimer visualization by immunoelectrophoresis is a commonly used method, it is impractical as a routine clinical test. In this study, we used a biophysical analysis tool, fluorescence correlation spectroscopy (FCS), to measure vWF distributions. The goals were to develop a method that is quicker and simpler than vWF gel electrophoresis and to evaluate the potential of FCS as a clinical diagnostic technique. METHODS We analyzed plasma from 12 patients with type 1 von Willebrand disease (vWD), 14 patients with type 2 vWD, and 10 healthy controls using a fluctuation-based immunoassay approach. RESULTS FCS enabled identification and proper classification of type 1 and type 2 vWD, producing quantitative results that correspond to qualitative gel multimer patterns. FCS required minimal sample preparation and only a 5-min analysis time. CONCLUSIONS This study represents the first implementation of FCS for clinical diagnostics directly on human plasma. The technique shows potential for further vWF studies and as a generally applicable laboratory test method.

Torres, Richard; Genzen, Jonathan R.; Levene, Michael J.

2013-01-01

235

DOM transformations in stream biofilms shown by fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

2012-04-01

236

Resonance fluorescence spectroscopy in laser-induced cavitation bubbles.  

PubMed

Laser-induced breakdown spectroscopy (LIBS) in liquids using a double-pulse Q-switched Nd:YAG laser system has provided reliable results that give trace detection limits in water. Resonant laser excitation has been added to enhance detection sensitivity. A primary laser pulse (at 532 nm), transmitted via an optical fiber, induces a cavitation bubble and shockwave at a target immersed in a 10 mg l(-1)-100 mg l(-1) indium (In) water suspension. The low-pressure rear of the shockwave induces bubble expansion and a resulting reduction in cavity pressure as it extends away from the target. Shortly before the maximum diameter is expected, a secondary laser pulse (also at 532 nm) is fed into the bubble in order to reduce quenching processes. The plasma field generated is then resonantly excited by a fiber-guided dye laser beam to increase detection selectivity. The resulting resonance fluorescence emission is optically detected and processed by an intensified optical multichannel analyzer system. PMID:16520936

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

2006-03-07

237

Two fundamentally different types of variable chlorophyll fluorescence in vivo  

Microsoft Academic Search

Upon onset of saturating continuous light only the first part of the observed polyphasic fluorescence rise follows QA reduction (photochemical phase), whereas the remaining part (thermal phases) is kinetically limited by relatively slow reactions with light saturated half-times in the order of 10–50 ms. A simple hypothesis is presented for the interpretation of these fundamentally different types of variable fluorescence.

Ulrich Schreiber; Anja Krieger

1996-01-01

238

Polymer Dynamics, Fluorescence Correlation Spectroscopy, and the Limits of Optical Resolution  

NASA Astrophysics Data System (ADS)

In recent years, fluorescence correlation spectroscopy has been increasingly applied for the study of polymer dynamics on the nanometer scale. The core idea is to extract, from a measured autocorrelation curve, an effective mean-square displacement function that contains information about the underlying conformational dynamics. This Letter presents a fundamental study of the applicability of fluorescence correlation spectroscopy for the investigation of nanoscale conformational and diffusional dynamics. We find that fluorescence correlation spectroscopy cannot reliably elucidate processes on length scales much smaller than the resolution limit of the optics used and that its improper use can yield spurious results for the observed dynamics.

Enderlein, Jörg

2012-03-01

239

Fluorescence spectroscopy and imaging for noninvasive diagnostics: applications to early cancer detection in the lung  

NASA Astrophysics Data System (ADS)

Tissue fluorescence spectroscopy and imaging are being investigated as potential methods for non-invasive detection of pre-neoplastic change in the lung and other organ systems. A substantial contribution to tissue fluorescence is known to arise from endogenous cellular fluorophores. Using steady-state and time-resolved fluorescence spectroscopy and imaging, we characterized the endogenous fluorescence properties of immortalized and carcinogen-transformed human bronchial epithelial cells. Non-invasive sensing of endogenous molecular biomarkers associated with human bronchial pre-neoplasia will be discussed.

Mycek, Mary-Ann; Urayama, Paul; Zhong, Wei; Sloboda, Roger D.; Dragnev, Konstantin H.; Dmitrovsky, Ethan

2003-10-01

240

In vivo two-dimensional NMR correlation spectroscopy  

NASA Astrophysics Data System (ADS)

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

Kraft, Robert A.

1999-10-01

241

Lactate Turnover in Rat Glioma Measured by in Vivo Nuclear Magnetic Resonance Spectroscopy1  

Microsoft Academic Search

Elevated tissue lactate concentrations typically found in tumors can be measured by in vivo nuclear magnetic resonance (NMR) spectroscopy. In this study, lactate turnover in rat C6 glioma was determined from in vivo 'II NMR measurements of (3-\\

Melissa Terpstra; Rolf Gruetter; Wanda B. High; Marlene Mescher; Lance DelaBarre; Mollumi Merkle; Michael Garwood

242

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.

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

2009-01-01

243

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.

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

2013-01-01

244

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

NASA Astrophysics Data System (ADS)

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

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

2009-05-01

245

Identification of oral carcinogenesis using autofluorescence spectroscopy: an in-vivo study  

NASA Astrophysics Data System (ADS)

A study on in vivo measurement of autofluorescence spectra for hamster buccal pouch and development of oral carcinogenesis identification algorithm is presented. The measurement was preceded with a fiber-optics based fluorescence spectroscopy system. In total 75 samples, including 14 hyperkratosis, 23 normal, 28 dysplasia, and 10 SCC, were separated into 4 categories. All the spectra were normalized to have the same area below the spectrum curve. The results show that the autofluorescence spectra start to change as soon as the tissues have morphological alternation (eg hyperkratosis). The differences of ratios between the areas under 380+/- 15 nm and 460+/- 15 nm (denoted as A380+/- 15/ A460+/- 15) among categories are statistically significant. To develop a diagnostic algorithm for early neoplasia detection and evaluate its performance, a PLS discriminant analysis with cross-validation technique was proceeded. Sample points on the PLS score plot were grouped as four categories. By selecting suitable threshold, the accuracy rates for classifying 4 categories of samples are 86% (hyperkratosis), 87% (normal), 90% (dysplasia), and 100% (SCC), respectively. The results reveal that the autofluorescence spectroscopy technique is potential for in vivo detection of early neoplasia of oral tissues.

Tsai, Tsui-min; Chen, Chin-Tin; Wang, Chih-Yu; Chiang, Chun-Pin; Kuo, Yu-Luan

2001-10-01

246

Long-term in vivo glucose monitoring using fluorescent hydrogel fibers  

PubMed Central

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.

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

2011-01-01

247

Injectable hydrogel microbeads for fluorescence-based in vivo continuous glucose monitoring.  

PubMed

Fluorescent microbeads hold great promise for in vivo continuous glucose monitoring with wireless transdermal transmission and long-lasting activity. The full potential of fluorescent microbeads has yet to be realized due to insufficient intensity for transdermal transmission and material toxicity. This paper illustrates the highly-sensitive, biostable, long-lasting, and injectable fluorescent microbeads for in vivo continuous glucose monitoring. We synthesized a fluorescent monomer composed of glucose-recognition sites, a fluorogenic site, spacers, and polymerization sites. The spacers are designed to be long and hydrophilic for increasing opportunities to bind glucose molecules; consequently, the fluorescent monomers enable high-intensive responsiveness to glucose. We then fabricated injectable-sized fluorescent polyacrylamide hydrogel beads with high uniformity and high throughput. We found that our fluorescent beads provide sufficient intensity to transdermally monitor glucose concentrations in vivo. The fluorescence intensity successfully traced the blood glucose concentration fluctuation, indicating our method has potential uses in highly-sensitive and minimally invasive continuous blood glucose monitoring. PMID:20921374

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

2010-10-04

248

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

PubMed

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

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

2014-01-01

249

Single Molecule Spectral Diffusion in a Solid Detected via Fluorescence Spectroscopy.  

National Technical Information Service (NTIS)

Recent advances in fluorescence excitation spectroscopy have produced significant increases in detection sensitivity for single performance molecules in crystals of p-terphenyl at low temperatures. With the increased signal to noise ratio, we have observe...

T. Basche W. E. Moerner W. P. Ambrose

1991-01-01

250

Quantification of Element Abundances of Stardust Interstellar Candidates by Synchrotron Radiation X-Ray Fluorescence Spectroscopy  

NASA Astrophysics Data System (ADS)

Orion and Sirius, two Interstellar Dust Candidates from the NASA Stardust mission were analyzed using hyperspectral fluorescence/diffraction nano-X-ray imaging. Correlation spectroscopy of associated elements helped propose an associated mineralogy.

Simionovici, A. S.; Lemelle, L.; Cloetens, P.; Solé, V. A.; Sans Tresseras, J.-A.; Butterworth, A. L.; Westphal, A. J.; Gainsforth, Z.; Stodolna, J.; Allen, C.; Anderson, D.; Ansari, A.; Bajt, S.; Bassim, N.; Bastien, R. S.; Bechtel, H. A.; Borg, J.; Brenker, F. E.; Bridges, J.; Brownlee, D. E.; Burchell, M.; Burghamme, M.; Changela, H.; Davis, A. M.; Doll, R.; Floss, Ch.; Flynn, G. J.; Frank, D. R.; Grün, E.; Heck, Ph. R.; Hillier, J. K.; Hoppe, P.; Hudson, B.; Huth, J.; Hvide, B.; Kearsley, A.; King, A. J.; Lai, B.; Leitner, J.; Leonard, A.; Leroux, H.; Lettieri, R.; Marchant, W.; Nittler, L. R.; Ogliore, R.; Ja Ong, W. J.; Postberg, F.; Price, M. C.; Sandford, S. A.; Schmitz, S.; Schoonjans, T.; Schreiber, K.; Silversmit, G.; Srama, R.; Stephan, Th.; Sterken, V. J.; Stroud, R. M.; Sutton, S.; Trieloff, M.; Tsou, P.; Tsuchiyama, A.; Tyliszczak, T.; Vekemans, B.; Vincze, L.; Von Korff, J.; Wordsworth, N.; Zevin, D.; Zolensky, M. E.

2013-09-01

251

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

252

Assessing topographic cutaneous autofluorescence variation using fluorescence UV and visible excitation emission matrix (EEM) spectroscopy  

NASA Astrophysics Data System (ADS)

Cutaneous autofluorescence properties were systematically studied using fluorescence excitation emission matrix spectroscopy. Twenty-six healthy subjects with a mean age of 34 (range 21-74) participated in this study. The fluorescence of major skin fluorophores such as tryptophan, collagen, elastin and NADH could be readily identified. On average, facial skin shows strong tryptophan and measurable porphyrin fluorescence; the palm and nail show strong tryptophan and keratin fluorescence. These results demonstrate that regional topographic variations exist not only in the amount of fluorescence but also in the relative distribution of fluorophores in normal skin. Moreover this provides a basis for future interpretation of autofluorescence in diseased skin.

Zhao, Jianhua; Zandi, Soodabeh; Feng, Florina; Zeng, Haishan; McLean, David I.; Lui, Harvey

2011-02-01

253

Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy 1 1 Edited by W. Baumeister  

Microsoft Academic Search

We have investigated spatial variations of the diffusion behavior of the green fluorescent protein mutant EGFP (F64L\\/S65T) and of the EGFP-?-galactosidase fusion protein in living cells with fluorescence correlation spectroscopy. Our fluorescence correlation spectroscopy device, in connection with a precision x-y translation stage, provides submicron spatial resolution and a detection volume smaller than a femtoliter. The fluorescence fluctuations in cell

Malte Wachsmuth; Waldemar Waldeck; Jörg Langowski

2000-01-01

254

Novel detection of aerosols: combined cavity ring-down and fluorescence spectroscopy.  

PubMed

High fluences inside cavity ring-down spectroscopy optical resonators lend themselves to fluorescence or Raman spectroscopy. An instrument at 488 nm was developed to measure extinction, and fluorescence of aerosols. A detection limit of 6 x 10;-9 cm;-1Hz;-1/2 (0.6 Mm;-1Hz;-1/2) was achieved. The fluorescence spectral power collected from a single fluorescent microsphere was 10 to 20 pW/nm. This power is sufficient to obtain the spectrum of a single microsphere with a resolution of 10 nm and signal-to-noise ratio of ~10. The relative concentrations of two types of fluorescent microspheres were determined from a time-integrated fluorescence measurement of a mixture of both. PMID:19495240

Richman, Bruce; Kachanov, Alexander; Paldus, Barbara; Strawa, Anthony

2005-05-01

255

Sensitivity Enhancement in Fluorescence Correlation Spectroscopy of Multiple Species Using Time-Gated Detection  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure chemical reaction rates and diffusion coefficients of molecules in thermal equilibrium. The capabilities of FCS can be enhanced by measuring the energy, polarization, or delay time between absorption and emission of the collected fluorescence photons in addition to their arrival times. This information can be used to change the relative

Don C. Lamb; Andreas Schenk; Carlheinz Röcker; C. Scalfi-Happ; G. Ulrich Nienhaus

2000-01-01

256

Microenvironment of Endosomal Aqueous Phase Investigated by the Mobility of Microparticles Using Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

Temporal observation of the dynamic behavior of molecules in cells gives information about the physiological environment at the region of interest. Here we report the direct measurement of the mobility of rhodamine-labeled microparticles (14 and 35 nm in diameter) ingested in endosomes of cultured bovine aortic endothelial cells using fluorescence correlation spectroscopy (FCS). The fluctuation of fluorescent signals from microparticles

Naoto Yoshida; Masataka Kinjo; Mamoru Tamura

2001-01-01

257

Determination of concentration of living immobilized yeast cells by fluorescence spectroscopy  

Microsoft Academic Search

Fluorescence spectroscopy is one of suitable methods for the direct monitoring of immobilized cells because it does not require (in certain arrangement) transparent environment like absorbance measurements. The aim of this work was to develop a method allowing measurements of concentrations of living immobilized cells using only intrinsic fluorescence of biogenic fluorophores in cells. Saccharomyces cerevisiae yeast cells were immobilized

O. Podrazky; G. Kuncova

2005-01-01

258

Application of Fluorescence Spectroscopy and Chemometrics in the Evaluation of Processed Cheese During Storage  

Microsoft Academic Search

Front face fluorescence spectroscopy is applied for an evaluation of the stability of processed cheese during storage. Fluorescence landscapes with excitation from 240 to 360 nm and emission in the range of 275 to 475 nm were obtained from cheese samples stored in darkness and light in up to 259 d, at 5, 20 and 37°C, respectively. Parallel factor (PARAFAC)

J. Christensen; V. T. Povlsen; J. Sørensen

2003-01-01

259

Fluorescence Cross-correlation Spectroscopy (FCCS) to Observe Dimerization of Transcription Factors in Living Cells.  

PubMed

Fluorescence cross-correlation spectroscopy (FCCS) is an established spectroscopic method to observe the interaction between the different fluorescent molecules. Using FCCS, researchers can assess the interaction of target molecules in the aqueous condition, and can apply the technique in cultured cells. Here, we describe the method of FCCS to demonstrate direct observation of dimerization between transcription factors in a living cell. PMID:23436366

Sadamoto, Hisayo; Muto, Hideki

2013-01-01

260

The effects of daunomycin antibiotic on histone H 1: thermal denaturation and fluorescence spectroscopy studies  

Microsoft Academic Search

Using thermal denaturation and fluorescence spectroscopy, we have investigated the interaction of antitumor antibiotic, daunomycin, with calf thymus histone H1 under several ionic strengths. The results show that daunomycin binds to histone H1 and increases its melting temperature. Increasing ionic strength elevates this effect. Fluorescence emission data show that the interaction of daunomycin with histone H1 decreases the emission intensity

Seyed Jalal Zargar; Azra Rabbani

2000-01-01

261

Dissociation and unfolding of jack bean urease studied by fluorescence emission spectroscopy  

Microsoft Academic Search

Thermal and chemical denaturation of urease has been studied by fluorescence spectroscopy. For thermal denaturation, a change in structure was indicated by a change in the wavelength of maximal intensity, ?max. The fluorescence peak position shifted from 330 nm to 335 nm upon heating urease from room temperature to 75° C, with a Tm corresponding to 72.4° C. The thermal

S. Omar; M. Beauregard

1995-01-01

262

Direct measurement of Gag-Gag interaction during retrovirus assembly with FRET and fluorescence correlation spectroscopy  

Microsoft Academic Search

uring retrovirus assembly, the polyprotein Gag di- rects protein multimerization, membrane binding, and RNA packaging. It is unknown whether assembly initiates through Gag-Gag interactions in the cytosol or at the plasma membrane. We used two fluorescence techniques— two-photon fluorescence resonance energy transfer and fluorescence correlation spectroscopy—to examine Rous sarcoma virus Gag-Gag and -membrane interactions in living cells. Both techniques provide

Daniel R. Larson; Yu May Ma; Volker M. Vogt; Watt W. Webb

2003-01-01

263

Fluorescence Correlation Spectroscopy Studies of Peptide and Protein Binding to Phospholipid Vesicles  

Microsoft Academic Search

We used fluorescence correlation spectroscopy (FCS) to analyze the binding of fluorescently labeled peptides to lipid vesicles and compared the deduced binding constants to those obtained using other techniques. We used a well-characterized peptide corresponding to the basic effector domain of myristoylated alanine-rich C kinase substrate, MARCKS(151–175), that was fluorescently labeled with Alexa488, and measured its binding to large unilamellar

Laura Rusu; Alok Gambhir; Stuart McLaughlin; Joachim Rädler

2004-01-01

264

Single-Molecule Analysis of Restriction DNA Fragments Using Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

The cleavage of fluorescence-labeled M13DNA (7250 bp) usingHaeIII,HgaI,BsmAI, andBspMI was analyzed by fluorescence correlation spectroscopy (FCS) in a small volume (1.5 × 10?15liters). The digestion process can be monitored by the decrease in amplitude of the fluorescence correlation function while the original DNA molecule is divided into several fragments by the enzymes. To analyze this reaction by FCS, we derived

Masataka Kinjo; Goro Nishimura; Tomiyasu Koyama; Ülo Mets; Rudolf Rigler

1998-01-01

265

Laser-induced fluorescence-cued, laser-induced breakdown spectroscopy biological-agent detection  

Microsoft Academic Search

Methods for accurately characterizing aerosols are required for detecting biological warfare agents. Currently, fluorescence-based biological agent sensors provide adequate detection sensitivity but suffer from high false-alarm rates. Combining single-particle fluorescence analysis with laser-induced breakdown spectroscopy (LIBS) provides additional discrimination and potentially reduces false-alarm rates. A transportable UV laser-induced fluorescence-cued LIBS test bed has been developed and used to evaluate the

John D. Hybl; Shane M. Tysk; Shaun R. Berry; Michael P. Jordan

2006-01-01

266

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.

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

267

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

2012-02-01

268

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.

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

2011-01-01

269

In-vivo optical imaging and spectroscopy of cerebral hemodynamics  

NASA Astrophysics Data System (ADS)

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

Zhou, Chao

270

Cystic intracranial mass lesions: Possible role of in vivo MR spectroscopy in its differential diagnosis  

Microsoft Academic Search

Thirty-four patients showing cystic intracranial mass lesions on MR imaging were evaluated by in vivo proton MR spectroscopy (MRS) with the aim of detecting lesion-specific spectral patterns that may assist imaging in better tissue characterization. In vivo spectroscopy was performed using stimulated echo acquisition mode with echo times 20 m and 270 m in all, and spin echo with echo

Harish Poptani; Rakesh K. Gupta; Vijendera K. Jain; Raja Roy; Rakesh Pandey

1995-01-01

271

Near-infrared fluorescent sensor for in vivo copper imaging in a murine Wilson disease model  

PubMed Central

Copper is an essential metal nutrient that is tightly regulated in the body because loss of its homeostasis is connected to severe diseases such as Menkes and Wilson diseases, Alzheimer’s disease, prion disorders, and amyotrophic lateral sclerosis. The complex relationships between copper status and various stages of health and disease remain challenging to elucidate, in part due to a lack of methods for monitoring dynamic changes in copper pools in whole living organisms. Here we present the synthesis, spectroscopy, and in vivo imaging applications of Coppersensor 790, a first-generation fluorescent sensor for visualizing labile copper pools in living animals. Coppersensor 790 combines a near-infrared emitting cyanine dye with a sulfur-rich receptor to provide a selective and sensitive turn-on response to copper. This probe is capable of monitoring fluctuations in exchangeable copper stores in living cells and mice under basal conditions, as well as in situations of copper overload or deficiency. Moreover, we demonstrate the utility of this unique chemical tool to detect aberrant increases in labile copper levels in a murine model of Wilson disease, a genetic disorder that is characterized by accumulation of excess copper. The ability to monitor real-time copper fluxes in living animals offers potentially rich opportunities to examine copper physiology in health and disease.

Hirayama, Tasuku; Van de Bittner, Genevieve C.; Gray, Lawrence W.; Lutsenko, Svetlana; Chang, Christopher J.

2012-01-01

272

Near-infrared fluorescent sensor for in vivo copper imaging in a murine Wilson disease model.  

PubMed

Copper is an essential metal nutrient that is tightly regulated in the body because loss of its homeostasis is connected to severe diseases such as Menkes and Wilson diseases, Alzheimer's disease, prion disorders, and amyotrophic lateral sclerosis. The complex relationships between copper status and various stages of health and disease remain challenging to elucidate, in part due to a lack of methods for monitoring dynamic changes in copper pools in whole living organisms. Here we present the synthesis, spectroscopy, and in vivo imaging applications of Coppersensor 790, a first-generation fluorescent sensor for visualizing labile copper pools in living animals. Coppersensor 790 combines a near-infrared emitting cyanine dye with a sulfur-rich receptor to provide a selective and sensitive turn-on response to copper. This probe is capable of monitoring fluctuations in exchangeable copper stores in living cells and mice under basal conditions, as well as in situations of copper overload or deficiency. Moreover, we demonstrate the utility of this unique chemical tool to detect aberrant increases in labile copper levels in a murine model of Wilson disease, a genetic disorder that is characterized by accumulation of excess copper. The ability to monitor real-time copper fluxes in living animals offers potentially rich opportunities to examine copper physiology in health and disease. PMID:22308360

Hirayama, Tasuku; Van de Bittner, Genevieve C; Gray, Lawrence W; Lutsenko, Svetlana; Chang, Christopher J

2012-01-30

273

Correct Diffusion Coefficients of Proteins in Fluorescence Correlation Spectroscopy. Application to Tubulin Oligomers Induced by Mg 2+ and Paclitaxel  

Microsoft Academic Search

In view of recent warnings for artifacts in fluorescence correlation spectroscopy, the diffusion coefficient of a series of labeled proteins in a wide range of molecular mass (43–670 kD) was determined and shown to be correct with respect to published values and the theory. Fluorescence correlation spectroscopy was then applied to the study of fluorescently labeled tubulin and its oligomerization

Tatiana Krouglova; J. Vercammen; Yves Engelborghs

2004-01-01

274

Modeling in vivo fluorescence of small animals using TracePro software  

NASA Astrophysics Data System (ADS)

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

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

2007-03-01

275

Advanced in vivo applications of blue light photoreceptors as alternative fluorescent proteins.  

PubMed

The ultimate ambition in cell biology, microbiology and biomedicine is to unravel complex physiological and pathophysiological processes within living organisms. To conquer this challenge, fluorescent proteins (FPs) are used as versatile in vivo reporters and biosensors to study gene regulation as well as the synthesis, localization and function of proteins in living cells. The most widely used FPs are the green fluorescent protein (GFP) and its derivatives and relatives. Their use as in vivo reporter proteins, however, is sometimes restricted by different environmental and cellular factors. Consequently, a whole range of alternative, cofactor-dependent reporter proteins have been developed recently. In this perspective, we summarize the advantages and limitations of the novel class of cyan-green fluorescent flavoproteins in comparison to members of the GFP family and discuss some correlated consequences for the use of FPs as in vivo reporters. PMID:23660639

Drepper, Thomas; Gensch, Thomas; Pohl, Martina

2013-07-01

276

Mini review of ultrafast fluorescence polarization spectroscopy [invited].  

PubMed

A mini review is presented on the theory, experiment, and application of the ultrafast fluorescence polarization dynamics and anisotropy with examples of two important medical dyes, namely Indocyanine Green and fluorescein. The time-resolved fluorescence polarization spectra of fluorescent dyes were measured with the excitation of a linearly polarized femtosecond laser pulse, and detected using a streak camera. The fluorescence emitted from the dyes is found to be partially oriented (polarized), and the degree of polarization of emission decreases with time. The decay of the fluorescence component polarized parallel to the excitation beam was found to be faster than that of the perpendicular one. Based on the physical model on the time-resolved polarized emission spectra in nanosecond range first described by Weber [J. Chem. Phys.52, 1654 (1970)], a set of first-order linear differential equations was used to model fluorescence polarization dynamics and anistropy of dye in picoseconds range. Using this model, two important decay parameters were identified separately: the decay rate of total emission intensity and the decay rate of the emission polarization affected by the rotation of fluorescent molecules causing the transfer of emission polarization from one orthogonal component to another. These two decay rates were separated and extracted from the measured time-resolved fluorescence polarization spectra. The emission polarization difference among dyes arising from different molecular volumes was used to enhance the image contrast. PMID:23400053

Pu, Yang; Wang, Wubao; Dorshow, Richard B; Das, Bidyut B; Alfano, Robert R

2013-02-10

277

Imaging a photodynamic therapy photosensitizer in vivo with a time-gated fluorescence tomography system  

NASA Astrophysics Data System (ADS)

We report the tomographic imaging of a photodynamic therapy (PDT) photosensitizer, 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH) in vivo with time-domain fluorescence diffuse optical tomography (TD-FDOT). Simultaneous reconstruction of fluorescence yield and lifetime of HPPH was performed before and after PDT. The methodology was validated in phantom experiments, and depth-resolved in vivo imaging was achieved through simultaneous three-dimensional (3-D) mappings of fluorescence yield and lifetime contrasts. The tomographic images of a human head-and-neck xenograft in a mouse confirmed the preferential uptake and retention of HPPH by the tumor 24-h post-injection. HPPH-mediated PDT induced significant changes in fluorescence yield and lifetime. This pilot study demonstrates that TD-FDOT may be a good imaging modality for assessing photosensitizer distributions in deep tissue during PDT monitoring.

Mo, Weirong; Rohrbach, Daniel; Sunar, Ulas

2012-07-01

278

Imaging a photodynamic therapy photosensitizer in vivo with a time-gated fluorescence tomography system  

PubMed Central

Abstract. We report the tomographic imaging of a photodynamic therapy (PDT) photosensitizer, 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH) in vivo with time-domain fluorescence diffuse optical tomography (TD-FDOT). Simultaneous reconstruction of fluorescence yield and lifetime of HPPH was performed before and after PDT. The methodology was validated in phantom experiments, and depth-resolved in vivo imaging was achieved through simultaneous three-dimensional (3-D) mappings of fluorescence yield and lifetime contrasts. The tomographic images of a human head-and-neck xenograft in a mouse confirmed the preferential uptake and retention of HPPH by the tumor 24-h post-injection. HPPH-mediated PDT induced significant changes in fluorescence yield and lifetime. This pilot study demonstrates that TD-FDOT may be a good imaging modality for assessing photosensitizer distributions in deep tissue during PDT monitoring.

Mo, Weirong; Rohrbach, Daniel; Sunar, Ulas

2012-01-01

279

The fluorescence characteristics of furfurylated wood studied by fluorescence spectroscopy and confocal laser scanning microscopy  

Microsoft Academic Search

The fluorescence of furfurylated wood was studied. Furfurylation of wood introduced fluorescent curing products into the cell\\u000a walls. Fluorescent products were also found in the lumina if high loadings were used and\\/or if the wood contained cells with\\u000a small radii. More intense fluorescence was obtained from lignin-rich parts of the compound cell wall (i.e., the middle lamella\\u000a and the cell

L. G. Thygesen; S. Barsberg; T. M. Venås

2010-01-01

280

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

281

Imaging metastatic cell trafficking at the cellular level in vivo with fluorescent proteins.  

PubMed

Fluorescent proteins have revolutionized biology, allowing what was formerly invisible to be clearly seen. The Nobel Prize in Chemistry was awarded in 2008 for the discovery and early use of green fluorescent protein (GFP) as a genetic reporter. Our laboratory pioneered the use of GFP for in vivo imaging. In this chapter we review the developments within our research on subcellular imaging of metastatic trafficking of cancer cells carried out in real time in mice. Dual-color fluorescent cells, with one color fluorescent protein in the nucleus and another color fluorescent protein in the cytoplasm, enable real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. In the dual-color cells, red fluorescent protein (RFP) is expressed in the cytoplasm of cancer cells, and GFP is linked to histone H2B and is expressed in the nucleus. Nuclear GFP expression enables visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression allows visualization of nuclear cytoplasmic ratios in addition to simultaneous cell and nuclear shape changes. With the use of dual-color fluorescent cells, it is possible to achieve subcellular real-time imaging of cancer cell trafficking in live mice. Extravasation can also be imaged in real time. Dual-color imaging has shown that cytoplasmic processes of cancer cells exit the vessels first, with nuclei following along the cytoplasmic projections [Yamauchi et al., Cancer Res 66:4208-4214, 2006]. Dual-color in vivo cellular imaging was used to visualize cancer cell trafficking blood vessels, as well as in the lymphatic systems of the mice. The real-time imaging of cancer cell seeding on the lung has now been achieved with dual-color cells. Subcellular in vivo imaging confers great promise for understanding metastasis at the cellular level in vivo. PMID:24092439

Hoffman, Robert M

2014-01-01

282

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

NASA Astrophysics Data System (ADS)

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

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

2008-04-01

283

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

PubMed

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

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

2007-03-15

284

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

PubMed

The analysis of various varnishes from different origins, which are commonly found on historical musical instruments was carried out for the first time with both fluorescence excitation emission spectroscopy and laser-induced time-resolved fluorescence spectroscopy. Samples studied include varnishes prepared using shellac, and selected diterpenoid and triterpenoid resins from plants, and mixtures of these materials. Fluorescence excitation emission spectra have been collected from films of naturally aged varnishes. In parallel, time-resolved fluorescence spectroscopy of varnishes provides means for discriminating between short- (less than 2.0 ns) and long-lived (greater than 7.5 ns) fluorescence emissions in each of these complex materials. Results suggest that complementary use of the two non destructive techniques allows a better understanding of the main fluorophores responsible for the emission in shellac, and further provides means for distinguishing the main classes of other varnishes based on differences in fluorescence lifetime behaviour. Spectrofluorimetric data and time resolved spectra presented here may form the basis for the interpretation of results from future in situ fluorescence examination and time resolved fluorescence imaging of varnished musical instruments. PMID:19782228

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

2009-07-04

285

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.

Aouani, Heykel; Schon, Peter; Brasselet, Sophie; Rigneault, Herve; Wenger, Jerome

2010-01-01

286

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

287

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

288

Near-infrared fluorescent probe traces bisphosphonate delivery and retention in vivo.  

PubMed

Bisphosphonate use has expanded beyond traditional applications to include treatment of a variety of low-bone-mass conditions. Complications associated with long-term bisphosphonate treatment have been noted, generating a critical need for information describing the local bisphosphonate-cell interactions responsible for these observations. This study demonstrates that a fluorescent bisphosphonate analogue, far-red fluorescent pamidronate (FRFP), is an accurate biomarker of bisphosphonate deposition and retention in vivo and can be used to monitor site-specific local drug concentration. In vitro, FRFP is competitively inhibited from the surface of homogenized rat cortical bone by traditional bisphosphonates. In vivo, FRFP delivery to the skeleton is rapid, with fluorescence linearly correlated with bone surface area. Limb fluorescence increases linearly with injected dose of FRFP; injected FRFP does not interfere with binding of standard bisphosphonates at the doses used in this study. Long-term FRFP retention studies demonstrated that FRFP fluorescence decreases in conditions of normal bone turnover, whereas fluorescence was retained in conditions of reduced bone turnover, demonstrating preservation of local FRFP concentration. In the mandible, FRFP localized to the alveolar bone and bone surrounding the periodontal ligament and molar roots, consistent with findings of osteonecrosis of the jaw. These findings support a role for FRFP as an effective in vivo marker for bisphosphonate site-specific deposition, turnover, and long-term retention in the skeleton. PMID:20200982

Kozloff, Kenneth M; Volakis, Leo I; Marini, Joan C; Caird, Michelle S

2010-08-01

289

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

290

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

2013-05-20

291

Biocompatible fluorescent nanoparticles for in vivo stem cell tracking  

NASA Astrophysics Data System (ADS)

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.

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

2013-06-01

292

Fluorescence spectroscopy: A promising tool for carbonate petrology  

SciTech Connect

Responses of depositional and diagenetic components in samples of the Mission Canyon Limestone to blue-light excitation vary most noticeably with mineralogy and crystal size. The finely crystalline micrites, dolomicrites and argillaceous carbonates fluoresce more intensely than the more coarsely crystalline sparry calcite cements, dolospar cements and coarsely crystalline dolomites. Low intensity spectral analysis of cherts, anhydrites, and the carbonate phases provides an objective manner for quantifying fluorescence responses and for comparing them statistically. Nineteen of the optical parameters used in organic petrology are evaluated for their utility in carbonate petrology. Results of the discriminant function analysis suggest that red-weighted fluorescence chromaticity indices and yellow-weighted ones are more useful for mineral identification than the blue-weighted or equal-energy chromaticity indices. Statistical analysis of the optical data, mineralogy, and minor element compositions suggests correlations between the fluorescence responses and major minerals, carbonate diagenetic components, and the minor element geochemistry of carbonate components. Although no single element is identified as an activator of fluorescence in this study, the complex correlations of optical indices with Fe suggest that it does act to quench fluorescence. The four fluorescence cy chromaticity indices correlate significantly and positively with mineralogy and negatively with MgCo[sub 3]. In organic petrology, these indices are related to maceral content. The positive correlations of the four fluorescence cx chromaticity indices with Fe and Mn likely reflect fluorescence response to changes in compositions of pore fluids during diagenesis. This trend parallels the increase in cx indices with increasing maturation of organic materials.

Vice, M.A.; Bensley, D.F.; Utgaard, J.E. (Southern Illinois Univ., Carbondale, IL (United States). Dept. of Geology)

1992-01-01

293

Fluorescent DNA base analogs: preparation, incorporation into oligonucleotides, and time-resolved fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

The synthesis of fluorescent DNA base analogs that can replace the natural bases adenine, thymine and cytosine and their incorporation into synthetic oligodeoxynucleotides is described. The effect on the stability of such modified nucleotides like 2-aminopurine and some pteridine derivatives, is studied by thermal melting studies and comparison with the corresponding unaltered oligonucleotides. The fluorescence spectroscopic properties and several applications of these new fluorescent DNA probes are described in greater detail. Structural information on the conformation of special oligonucleotides like hairpins, junctions and bulged duplexes can be obtained from fluorescence lifetime and fluorescence depolarization data. For example the fluorescence lifetime pattern of 2-aminopurine is a sensitive indicator of DNA base pairing. As examples the structure of the oligonucleotide-linker junction in a synthetically linked oligonucleotide hairpin and the base-pairing of the first 'deoxyribozyme' are discussed. A third application uses doubly, i.e., donor and acceptor, labeled oligonucleotides to measure distances by fluorescence resonance energy transfer.

Hochstrasser, Remo A.

1996-04-01

294

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

NASA Astrophysics Data System (ADS)

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

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

2007-07-01

295

Development of a dual-modal tissue diagnostic system combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy  

PubMed Central

We report a tissue diagnostic system which combines two complementary techniques of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonic backscatter microscopy (UBM). TR-LIFS evaluates the biochemical composition of tissue, while UBM provides tissue microanatomy and enables localization of the region of diagnostic interest. The TR-LIFS component consists of an optical fiber-based time-domain apparatus including a spectrometer, gated multichannel plate photomultiplier, and fast digitizer. It records the fluorescence with high sensitivity (nM concentration range) and time resolution as low as 300 ps. The UBM system consists of a transducer, pulser, receiving circuit, and positioning stage. The transducer used here is 45 MHz, unfocused, with axial and lateral resolutions 38 and 200 ?m. Validation of the hybrid system and ultrasonic and spectroscopic data coregistration were conducted both in vitro (tissue phantom) and ex vivo (atherosclerotic tissue specimens of human aorta). Standard histopathological analysis of tissue samples was used to validate the UBM-TRLIFS data. Current results have demonstrated that spatially correlated UBM and TR-LIFS data provide complementary characterization of both morphology (necrotic core and calcium deposits) and biochemistry (collagen, elastin, and lipid features) of the atherosclerotic plaques at the same location. Thus, a combination of fluorescence spectroscopy with ultrasound imaging would allow for better identification of features associated with tissue pathologies. Current design and performance of the hybrid system suggests potential applications in clinical diagnosis of atherosclerotic plaque.

Sun, Yang; Park, Jesung; Stephens, Douglas N.; Jo, Javier A.; Sun, Lei; Cannata, Jonathan M.; Saroufeem, Ramez M. G.; Shung, K. Kirk; Marcu, Laura

2009-01-01

296

Time-Resolved Single Vibronic Level Fluorescence Spectroscopy: Glyoxal.  

National Technical Information Service (NTIS)

Fluorescence originating from a single vibronic level of glyoxal 5 superscript 1 has been simultaneously resolved with respect to wavelength and time. The resultant time-resolved single vibronic level (TRSVL) spectra provide data concerning excited-state ...

E. Photos G. H. Atkinson

1975-01-01

297

Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid–base specificity  

Microsoft Academic Search

Green fluorescent protein (GFP), isolated from Aequorea victoria jellyfish, has been used extensively as a noninvasive intracellular pH indicator and site-specific fluorescent marker in biochemistry, cell biology, and molecular genetics. Numerous mutations, aimed at optimizing spectroscopic and thermodynamic properties of GFP, have been created for different applications. Fluorescence correlation spectroscopy (FCS) reveals that the enhanced green fluorescent protein mutant (EGFP;

Ahmed A Heikal; Samuel T Hess; Watt W Webb

2001-01-01

298

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

2007-06-01

299

In vivo fluorescence imaging with high-resolution microlenses  

PubMed Central

Micro-optics are increasingly used for minimally invasive in vivo imaging, in miniaturized microscopes and in lab-on-a-chip devices. Owing to optical aberrations and lower numerical apertures, a main class of microlens, gradient refractive index lenses, has not achieved resolution comparable to conventional microscopy. Here we describe high-resolution microlenses, and illustrate two-photon imaging of dendritic spines on hippocampal neurons and dual-color nonlinear optical imaging of neuromuscular junctions in live mice.

Barretto, Robert P J; Messerschmidt, Bernhard; Schnitzer, Mark J

2010-01-01

300

[In vivo single molecular fluorescence imaging for analysis of pharmacokinetics].  

PubMed

Nano-materials are expected for research on molecular imaging of pharmacokinetics. We measured in vivo migration of CdSe nano-particles(Quantum Dots(QDs))conjugated with monoclonal anti-HER2 antibody(trastuzumab)in tumor vessel to breast cancer cells. We established a high resolution in vivo 3D microscopic system for a novel imaging method at single molecular level. The HER2 protein expressed in cancer cells and its dynamics were visualized by QDs in vivo at the spatial resolution of 30 nm. It suggests future utilization of the system in medical applications to improve the drug delivery system to target primary and metastatic tumors for made-to-order treatment. Future innovation in cancer imaging by nano-technology and novel measurement technology will provide great improvement, not only in the clinical field, but also in basic medical science. Advances in nano-biotechnology have great potential to improve prevention, diagnosis and treatment of human disease. PMID:18701837

Takeda, Motohiro; Gonda, Kohsuke; Higuchi, Hideo; Ohuchi, Noriaki

2008-08-01

301

Improved in vivo whole-animal detection limits of green fluorescent protein-expressing tumor lines by spectral fluorescence imaging.  

PubMed

Green fluorescent protein (GFP) has been used for cell tracking and imaging gene expression in superficial or surgically exposed structures. However, in vivo murine imaging is often limited by several factors, including scatter and attenuation with depth and overlapping autofluorescence. The autofluorescence signals have spectral profiles that are markedly different from the GFP emission spectral profile. The use of spectral imaging allows separation and quantitation of these contributions to the total fluorescence signal seen in vivo by weighting known pure component profiles. Separation of relative GFP and autofluorescence signals is not readily possible using epifluorescent continuous-wave single excitation and emission bandpass imaging (EFI). To evaluate detection thresholds using these two methods, nude mice were subcutaneously injected with a series of GFP-expressing cells. For EFI, optimized excitation and emission bandpass filters were used. Owing to the ability to separate autofluorescence contributions from the emission signal using spectral imaging compared with the mixed contributions of GFP and autofluorescence in the emission signal recorded by the EFI system, we achieved a 300-fold improvement in the cellular detection limit. The detection limit was 3 x 10(3) cells for spectral imaging versus 1 x 10(6) cells for EFI. Despite contributions to image stacks from autofluorescence, a 100-fold dynamic range of cell number in the same image was readily visualized. Finally, spectral imaging was able to separate signal interference of red fluorescent protein from GFP images and vice versa. These findings demonstrate the utility of the approach in detecting low levels of multiple fluorescent markers for whole-animal in vivo applications. PMID:17711782

Tam, Jenny M; Upadhyay, Rabi; Pittet, Mikael J; Weissleder, Ralph; Mahmood, Umar

302

In vivo measurement of the carotenoid level using portable resonance Raman spectroscopy  

NASA Astrophysics Data System (ADS)

Carotenoid is an important antioxidant in human body, which can eliminate the free radicals and other harmful reactive oxygen species. The standard technique for measuring carotenoid is high-pressure liquid chromatography which involves using chemicals and is invasive. In this paper, we present a portable resonance Raman spectroscopy system for measuring carotenoid in vivo, which is noninvasive, highly sensitive and compact. A small diode-pumped all solid-state 473nm laser instead of a 488nm Argon ion laser is used to excite in vivo the carotenoid in the thumb, and the resonance Raman scattering light intensity is measured to assess the carotenoid level. Basically, it is difficult to detect the very weak resonance Raman scattering light because it is overlapping with the strong fluorescence. Our investigation shows that matching glycerol can help to reduce tissue scattering and increase light collecting efficiency. We demonstrate that the employment of optical matching technology for measuring carotenoid resonance Raman spectra in tissue can improve the signal-to-noise ratio by 3.9dB.

Shao, Yonghong; Qu, Junle; He, Yonghong

2007-07-01

303

In vivo impedance spectroscopy of deep brain stimulation electrodes  

NASA Astrophysics Data System (ADS)

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

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

2009-08-01

304

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.

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

2010-01-01

305

In vivo blood glucose quantification using Raman spectroscopy.  

PubMed

We here propose a novel Raman spectroscopy method that permits the noninvasive measurement of blood glucose concentration. To reduce the effects of the strong background signals produced by surrounding tissue and to obtain the fingerprint Raman lines formed by blood analytes, a laser was focused on the blood in vessels in the skin. The Raman spectra were collected transcutaneously. Characteristic peaks of glucose (1125 cm(-1)) and hemoglobin (1549 cm(-1)) were observed. Hemoglobin concentration served as an internal standard, and the ratio of the peaks that appeared at 1125 cm(-1) and 1549 cm(-1) peaks was used to calculate the concentration of blood glucose. We studied three mouse subjects whose blood glucose levels became elevated over a period of 2 hours using a glucose test assay. During the test, 25 Raman spectra were collected transcutaneously and glucose reference values were provided by a blood glucose meter. Results clearly showed the relationship between Raman intensity and concentration. The release curves were approximately linear with a correlation coefficient of 0.91. This noninvasive methodology may be useful for the study of blood glucose in vivo. PMID:23133555

Shao, Jingwei; Lin, Manman; Li, Yongqing; Li, Xue; Liu, Junxian; Liang, Jianpin; Yao, Huilu

2012-10-25

306

Intradermal Indocyanine Green for In Vivo Fluorescence Laser Scanning Microscopy of Human Skin: A Pilot Study  

PubMed Central

Background In clinical diagnostics, as well as in routine dermatology, the increased need for non-invasive diagnosis is currently satisfied by reflectance laser scanning microscopy. However, this technique has some limitations as it relies solely on differences in the reflection properties of epidermal and dermal structures. To date, the superior method of fluorescence laser scanning microscopy is not generally applied in dermatology and predominantly restricted to fluorescein as fluorescent tracer, which has a number of limitations. Therefore, we searched for an alternative fluorophore matching a novel skin imaging device to advance this promising diagnostic approach. Methodology/Principal Findings Using a Vivascope®-1500 Multilaser microscope, we found that the fluorophore Indocyanine-Green (ICG) is well suited as a fluorescent marker for skin imaging in vivo after intradermal injection. ICG is one of few fluorescent dyes approved for use in humans. Its fluorescence properties are compatible with the application of a near-infrared laser, which penetrates deeper into the tissue than the standard 488 nm laser for fluorescein. ICG-fluorescence turned out to be much more stable than fluorescein in vivo, persisting for more than 48 hours without significant photobleaching whereas fluorescein fades within 2 hours. The well-defined intercellular staining pattern of ICG allows automated cell-recognition algorithms, which we accomplished with the free software CellProfiler, providing the possibility of quantitative high-content imaging. Furthermore, we demonstrate the superiority of ICG-based fluorescence microscopy for selected skin pathologies, including dermal nevi, irritant contact dermatitis and necrotic skin. Conclusions/Significance Our results introduce a novel in vivo skin imaging technique using ICG, which delivers a stable intercellular fluorescence signal ideal for morphological assessment down to sub-cellular detail. The application of ICG in combination with the near infrared laser opens new ways for minimal-invasive diagnosis and monitoring of skin disorders.

Jonak, Constanze; Skvara, Hans; Kunstfeld, Rainer; Trautinger, Franz; Schmid, Johannes A.

2011-01-01

307

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

PubMed

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

2012-08-06

308

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.

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

2012-01-01

309

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.

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

2011-01-01

310

Probing the plasma membrane organization in living cells by spot variation fluorescence correlation spectroscopy.  

PubMed

While intrinsic Brownian agitation within a lipid bilayer does homogenize the molecular distribution, the extremely diverse composition of the plasma membrane, in contrast, favors the development of inhomogeneity due to the propensity of such a system to minimize its total free energy. Precisely, deciphering such inhomogeneous organization with appropriate spatiotemporal resolution remains, however, a challenge. In accordance with its ability to accurately measure diffusion parameters, fluorescence correlation spectroscopy (FCS) has been developed in association with innovative experimental strategies to monitor modes of molecular lateral confinement within the plasma membrane of living cells. Here, we describe a method, namely spot variation FCS (svFCS), to decipher the dynamics of the plasma membrane organization. The method is based on questioning the relationship between the diffusion time ?(d) and the squared waist of observation w(2). Theoretical models have been developed to predict how geometrical constraints such as the presence of adjacent or isolated domains affect the svFCS observations. These investigations have allowed significant progress in the characterization of cell membrane lateral organization at the suboptical level, and have provided, for instance, compelling evidence for the in vivo existence of raft nanodomains. PMID:23280115

Billaudeau, Cyrille; Mailfert, Sébastien; Trombik, Tomasz; Bertaux, Nicolas; Rouger, Vincent; Hamon, Yannick; He, Hai-Tao; Marguet, Didier

2013-01-01

311

Integrated liquid jet waveguide for fluorescence spectroscopy on chip  

NASA Astrophysics Data System (ADS)

An optofluidic jet waveguide for on chip fluorescence analysis is presented. The waveguide consists of an high speed water jet produced by means of a micro-channel coupled with a multimode optical fiber collecting the fluorescence opportunely excited. The liquid jet acts, at the same time, as the solution to analyse and as an optical waveguide. This configuration allows a strong reduction of the scattering and fluorescence of non analyte substances enabling a very low limit of detection (LOD). The integrated device is fabricated by PMMA micro-machining allowing a self-alignment between the liquid jet waveguide and the optical fiber used to deliver the fluorescence to the detector. The performance of the system has been tested on Cy5 water solutions and LOD of 2.56 nM has been obtained. A proof-of-concept of filter-free measurements has been performed demonstrating that fluorescence measurements can be performed also by using a photodiode with an LOD of 6.11 nM.

Persichetti, Gianluca; Testa, Genni; Bernini, Romeo

2013-03-01

312

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

NASA Astrophysics Data System (ADS)

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

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

2012-03-01

313

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

PubMed

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

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

2012-03-01

314

The multiple uses of fluorescent proteins to visualize cancer in vivo  

Microsoft Academic Search

Naturally fluorescent proteins have revolutionized biology by enabling what was formerly invisible to be seen clearly. These proteins have allowed us to visualize, in real time, important aspects of cancer in living animals, including tumour cell mobility, invasion, metastasis and angiogenesis. These multicoloured proteins have allowed the colour-coding of cancer cells growing in vivo and enabled the distinction of host

Robert M. Hoffman

2005-01-01

315

Current developments in animal in vivo optical imaging technologies with bioluminescence and fluorescence  

Microsoft Academic Search

Bioluminescence and fluorescence have been proved to be a versatile tool for in vivo imaging. Reporters that confer optical signals on a given biological process have been used widely in cell biology and more recently to interrogate biological process in animal models of human biology and disease. The theories, technologies, and the applications of these technologies will be compared and

ZHANG Yi; HAN Yu; ZHAO Chun-Lin

316

In-vivo fluorescence and reflectance imaging of human cervical tissue  

Microsoft Academic Search

A hyperspectral imaging spectrograph has been used to measure the fluorescence and reflectance of cervical tissue in vivo. The instrument was employed in a clinical trial in Vilnius, Lithuania, where 111 patients were examined. The patients were initially screened by Pap smear, examined by colposcopy and a tissue sampling procedure was performed. Detailed histopathological assessments were performed on the biopsies,

Ulf P. Gustafsson; Elisabeth McLaughlin; Ellen Jacobsen; Johan Hakansson; Paul Troy; Michael J. DeWeert; Katarina Svanberg; Sara Palsson; Marcelo Soto Thompson; Sune Svanberg; Aurelija Vaitkuviene

2003-01-01

317

Immune Response Induced by Fluorescent Nanocrystal Quantum Dots In Vitro and In Vivo  

Microsoft Academic Search

Fluorescent nanocrystal quantum dots (QDs) are widely used as novel tools in various biological fields including cellular biology, molecular biology, and even in basic and clinical medical fields, due to their far brighter photoemission and photostability. Although many amounts of biological studies, including in vivo experiments, were circumstantially investigated, there is no informative report that investigates whether the QDs affect

Akiyoshi Hoshino; Sanshiro Hanada; Noriyoshi Manabe; Toshinori Nakayama; Kenji Yamamoto

2009-01-01

318

Fiber optic based fluorescence detection system for in vivo studies of exogenous chromophore pharmacokinetics  

Microsoft Academic Search

The detection and quantification of the concentration of exogenous chromophores in-vivo by their fluorescence is complicated by many physical and geometrical parameters. Measurement of such signals is advantageous in determining the pharmacokinetics of photosensitizers such as those used in photodynamic therapy (PDT) or to assist in the diagnosis of tissue histological state. To overcome these difficulties a ratio based fiber

Daniel R. Doiron; J. B. Dunn; W. L. Mitchell; Brian K. Dalton; Greta M. Garbo; Jon A. Warner

1995-01-01

319

In vivo diagnosis of skin cancer using polarized and multiple scattered light spectroscopy  

NASA Astrophysics Data System (ADS)

This thesis research presents the development of a non-invasive diagnostic technique for distinguishing between skin cancer, moles, and normal skin using polarized and multiple scattered light spectroscopy. Polarized light incident on the skin is single scattered by the epidermal layer and multiple scattered by the dermal layer. The epidermal light maintains its initial polarization while the light from the dermal layer becomes randomized and multiple scattered. Mie theory was used to model the epidermal light as the scattering from the intercellular organelles. The dermal signal was modeled as the diffusion of light through a localized semi-homogeneous volume. These models were confirmed using skin phantom experiments, studied with in vitro cell cultures, and applied to human skin for in vivo testing. A CCD-based spectroscopy system was developed to perform all these experiments. The probe and the theory were tested on skin phantoms of latex spheres on top of a solid phantom. We next extended our phantom study to include in vitro cells on top of the solid phantom. Optical fluorescent microscope images revealed at least four distinct scatterers including mitochondria, nucleoli, nuclei, and cell membranes. Single scattering measurements on the mammalian cells consistently produced PSD's in the size range of the mitochondria. The clinical portion of the study consisted of in vivo measurements on cancer, mole, and normal skin spots. The clinical study combined the single scattering model from the phantom and in vitro cell studies with the diffusion model for multiple scattered light. When parameters from both layers were combined, we found that a sensitivity of 100% and 77% can be obtained for detecting cancers and moles, respectively, given the number of lesions examined.

Bartlett, Matthew Allen

320

Investigation of Laser Induced Fluorescence Spectroscopy for Making In-situ Species Concentration Measurements in Turbulent Combustion Flows.  

National Technical Information Service (NTIS)

The application is made for Laser Induced Fluorescence Spectroscopy (LIFS) to measurement of species concentrations and temperatures in flame adds. Abstracts: Pulsed Resonance Spectroscopy Applied to Turbulent Combustion Flows; Saturation Effects in Laser...

J. W. Daily

1981-01-01

321

In Vivo Fluorescent Labeling of Tumor Cells with the HaloTag(R) Technology  

PubMed Central

Many fluorescent sensors are currently available for in vitro bio-physiological microscopic imaging. The ability to label cells in living animals with these fluorescent sensors would help translate some of these assays into in vivo applications. To achieve this goal, the first step is to establish a method for selectively labeling target cells with exogenous fluorophores. Here we tested whether the HaloTag® protein tagging system provides specific labeling of xenograft tumors in living animals. After systemic delivery of fluorophore-conjugated ligands, we performed whole animal planar fluorescent imaging to determine uptake in tag-expressing HCT116 xenografts. Our results demonstrate that HaloTag ligands containing red or near-infrared fluorophores have enhanced tumor uptake and are suitable for non-invasive in vivo imaging. Our proof-of-concept results establish feasibility for using HaloTag technology for bio-physiological imaging in living animals.

Tseng, Jen-Chieh; Benink, Helene A; McDougall, Mark G; Chico-Calero, Isabel; Kung, Andrew L

2012-01-01

322

Cyanine-loaded lipid nanoparticles for improved in vivo fluorescence imaging.  

PubMed

Fluorescence is a very promising radioactive-free technique for functional imaging in small animals and, in the future, in humans. However, most commercial near-infrared dyes display poor optical properties, such as low fluorescence quantum yields and short fluorescence lifetimes. In this paper, we explore whether the encapsulation of infrared cyanine dyes within the core of lipid nanoparticles (LNPs) could improve their optical properties. Lipophilic dialkylcarbocyanines DiD and DiR are loaded very efficiently in 30-35-nm-diam lipid droplets stabilized in water by surfactants. No significant fluorescence autoquenching is observed up to 53 dyes per particle. Encapsulated in LNP, which are stable for more than one year at room temperature in HBS buffer (HEPES 0.02 M, EDTA 0.01 M, pH 5.5), DiD and DiR display far improved fluorescence quantum yields Phi (respectively, 0.38 and 0.25) and longer fluorescence lifetimes tau (respectively, 1.8 and 1.1 ns) in comparison to their hydrophilic counterparts Cy5 (Phi=0.28, tau=1.0 ns) and Cy7 (Phi=0.13, tau=0.57 ns). Moreover, dye-loaded LNPs are able to accumulate passively in various subcutaneous tumors in mice, thanks to the enhanced permeability and retention effect. These new fluorescent nanoparticles therefore appear as very promising labels for in vivo fluorescence imaging. PMID:19895107

Texier, Isabelle; Goutayer, Mathieu; Da Silva, Anabela; Guyon, Laurent; Djaker, Nadia; Josserand, Véronique; Neumann, Emmanuelle; Bibette, Jéro; Vinet, Françoise

323

Real-time Raman spectroscopy for in vivo, online gastric cancer diagnosis during clinical endoscopic examination.  

PubMed

Optical spectroscopic techniques including reflectance, fluorescence and Raman spectroscopy have shown promising potential for in vivo precancer and cancer diagnostics in a variety of organs. However, data-analysis has mostly been limited to post-processing and off-line algorithm development. In this work, we develop a fully automated on-line Raman spectral diagnostics framework integrated with a multimodal image-guided Raman technique for real-time in vivo cancer detection at endoscopy. A total of 2748 in vivo gastric tissue spectra (2465 normal and 283 cancer) were acquired from 305 patients recruited to construct a spectral database for diagnostic algorithms development. The novel diagnostic scheme developed implements on-line preprocessing, outlier detection based on principal component analysis statistics (i.e., Hotelling's T2 and Q-residuals) for tissue Raman spectra verification as well as for organ specific probabilistic diagnostics using different diagnostic algorithms. Free-running optical diagnosis and processing time of < 0.5 s can be achieved, which is critical to realizing real-time in vivo tissue diagnostics during clinical endoscopic examination. The optimized partial least squares-discriminant analysis (PLS-DA) models based on the randomly resampled training database (80% for learning and 20% for testing) provide the diagnostic accuracy of 85.6% [95% confidence interval (CI): 82.9% to 88.2%] [sensitivity of 80.5% (95% CI: 71.4% to 89.6%) and specificity of 86.2% (95% CI: 83.6% to 88.7%)] for the detection of gastric cancer. The PLS-DA algorithms are further applied prospectively on 10 gastric patients at gastroscopy, achieving the predictive accuracy of 80.0% (60/75) [sensitivity of 90.0% (27/30) and specificity of 73.3% (33/45)] for in vivo diagnosis of gastric cancer. The receiver operating characteristics curves further confirmed the efficacy of Raman endoscopy together with PLS-DA algorithms for in vivo prospective diagnosis of gastric cancer. This work successfully moves biomedical Raman spectroscopic technique into real-time, on-line clinical cancer diagnosis, especially in routine endoscopic diagnostic applications. PMID:23224179

Duraipandian, Shiyamala; Sylvest Bergholt, Mads; Zheng, Wei; Yu Ho, Khek; Teh, Ming; Guan Yeoh, Khay; Bok Yan So, Jimmy; Shabbir, Asim; Huang, Zhiwei

2012-08-01

324

Real-time Raman spectroscopy for in vivo, online gastric cancer diagnosis during clinical endoscopic examination  

NASA Astrophysics Data System (ADS)

Optical spectroscopic techniques including reflectance, fluorescence and Raman spectroscopy have shown promising potential for in vivo precancer and cancer diagnostics in a variety of organs. However, data-analysis has mostly been limited to post-processing and off-line algorithm development. In this work, we develop a fully automated on-line Raman spectral diagnostics framework integrated with a multimodal image-guided Raman technique for real-time in vivo cancer detection at endoscopy. A total of 2748 in vivo gastric tissue spectra (2465 normal and 283 cancer) were acquired from 305 patients recruited to construct a spectral database for diagnostic algorithms development. The novel diagnostic scheme developed implements on-line preprocessing, outlier detection based on principal component analysis statistics (i.e., Hotelling's T2 and Q-residuals) for tissue Raman spectra verification as well as for organ specific probabilistic diagnostics using different diagnostic algorithms. Free-running optical diagnosis and processing time of < 0.5 s can be achieved, which is critical to realizing real-time in vivo tissue diagnostics during clinical endoscopic examination. The optimized partial least squares-discriminant analysis (PLS-DA) models based on the randomly resampled training database (80% for learning and 20% for testing) provide the diagnostic accuracy of 85.6% [95% confidence interval (CI): 82.9% to 88.2%] [sensitivity of 80.5% (95% CI: 71.4% to 89.6%) and specificity of 86.2% (95% CI: 83.6% to 88.7%)] for the detection of gastric cancer. The PLS-DA algorithms are further applied prospectively on 10 gastric patients at gastroscopy, achieving the predictive accuracy of 80.0% (60/75) [sensitivity of 90.0% (27/30) and specificity of 73.3% (33/45)] for in vivo diagnosis of gastric cancer. The receiver operating characteristics curves further confirmed the efficacy of Raman endoscopy together with PLS-DA algorithms for in vivo prospective diagnosis of gastric cancer. This work successfully moves biomedical Raman spectroscopic technique into real-time, on-line clinical cancer diagnosis, especially in routine endoscopic diagnostic applications.

Duraipandian, Shiyamala; Sylvest Bergholt, Mads; Zheng, Wei; Yu Ho, Khek; Teh, Ming; Guan Yeoh, Khay; Bok Yan So, Jimmy; Shabbir, Asim; Huang, Zhiwei

2012-08-01

325

Time-resolved Hyperspectral Fluorescence Spectroscopy using Frequency Modulated Excitation  

SciTech Connect

An intensity-modulated excitation light source is used together with a micro channel plate intensified CCD (ICCD) detector gated at a slightly different frequency to generate a beat frequency from a fluorescent sample. The addition of a spectrograph produces a hyperspectral time-resolved data product where the resulting beat frequency is detected with a low frame rate camera. Measuring the beat frequency of the spectrum as a function of time allows separation of the excited fluorescence from ambient constant light sources. The excitation and detector repetition rates are varied over a range of discrete frequencies, and the phase shift of the beat wave maps out the emission decay rate(s).

,; Neill, M

2012-07-01

326

Ultrasmall near-infrared gold nanoclusters for tumor fluorescence imaging in vivo  

NASA Astrophysics Data System (ADS)

In this paper, we explore the possibility of using ultrasmall near-infrared (NIR) gold nanoclusters (AuNCs) as novel contrast imaging agents for tumor fluorescence imaging in vivo. The fluorescence imaging signal of the tail vein administrated AuNCs in living organisms can spectrally be well distinguished from the background with maximum emission wavelength at about 710 nm, and the high photostability of AuNCs promises continuous imaging in vivo. The uptake of AuNCs by the reticuloendothelial system is relatively low in comparison with other nanoparticle-based contrast imaging agents due to their ultrasmall hydrodynamic size (~2.7 nm). Through the body weight change analysis, the results show that the body weight of the mice administrated with AuNCs has not been changed obviously in comparison with that of the control mice injected with PBS. Furthermore, using MDA-MB-45 and Hela tumor xenograft models, in vivo and ex vivo imaging studies show that the ultrasmall NIR AuNCs are able to be highly accumulated in the tumor areas, thanks to the enhanced permeability and retention (EPR) effects. And the tumor-to-background ratio is about 15 for 6 h postinjection. The results indicate that the ultrasmall NIR AuNCs appear as very promising contrast imaging agents for in vivo fluorescence tumor imaging.

Wu, Xu; He, Xiaoxiao; Wang, Kemin; Xie, Can; Zhou, Bing; Qing, Zhihe

2010-10-01

327

Ultrasmall near-infrared gold nanoclusters for tumor fluorescence imaging in vivo.  

PubMed

In this paper, we explore the possibility of using ultrasmall near-infrared (NIR) gold nanoclusters (AuNCs) as novel contrast imaging agents for tumor fluorescence imaging in vivo. The fluorescence imaging signal of the tail vein administrated AuNCs in living organisms can spectrally be well distinguished from the background with maximum emission wavelength at about 710 nm, and the high photostability of AuNCs promises continuous imaging in vivo. The uptake of AuNCs by the reticuloendothelial system is relatively low in comparison with other nanoparticle-based contrast imaging agents due to their ultrasmall hydrodynamic size (?2.7 nm). Through the body weight change analysis, the results show that the body weight of the mice administrated with AuNCs has not been changed obviously in comparison with that of the control mice injected with PBS. Furthermore, using MDA-MB-45 and Hela tumor xenograft models, in vivo and ex vivo imaging studies show that the ultrasmall NIR AuNCs are able to be highly accumulated in the tumor areas, thanks to the enhanced permeability and retention (EPR) effects. And the tumor-to-background ratio is about 15 for 6 h postinjection. The results indicate that the ultrasmall NIR AuNCs appear as very promising contrast imaging agents for in vivo fluorescence tumor imaging. PMID:20835443

Wu, Xu; He, Xiaoxiao; Wang, Kemin; Xie, Can; Zhou, Bing; Qing, Zhihe

2010-09-08

328

Determination of the PSI/PSII ratio in living plant cells at room temperature by spectrally resolved fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

Leaf cells of living plants exhibit strong fluorescence from chloroplasts, the reaction centers of photosynthesis. Mutations in the photosystems change their structure and can, thus, be monitored by recording the fluorescence spectra of the emitted chlorophyll light. These measurements have, up to now, mostly been carried out at low temperatures (77 K), as these conditions enable the differentiation between the fluorescence of Photosystem I (PSI) and Photosystem II (PSII). In contrast, at room temperature, energy transfer processes between the various photosynthetic complexes result in very similar fluorescence emissions, which mainly consist of fluorescence photons emitted by PSII hindering a discrimination based on spectral ROIs (regions of interest). However, by statistical analysis of high resolution fluorescence spectra recorded at room temperature, it is possible to draw conclusions about the relative PSI/PSII ratio. Here, the possibility of determining the relative PSI/PSII ratio by fluorescence spectroscopy is demonstrated in living maize plants. Bundle-sheath chloroplasts of mature maize plants have a special morphologic characteristic; they are agranal, or exhibit only rudimentary grana, respectively. These chloroplasts are depleted in PSII activity and it could be shown that PSII is progressively reduced during leaf differentiation. A direct comparison of PSII activity in isolated chloroplasts is nearly impossible, since the activity of PSII in both mesophyll- and bundle-sheath chloroplasts decays with time after isolation and it takes significantly longer to isolate bundle-sheath chloroplasts. Considering this fact the measurement of PSI/PSII ratios with the 77K method, which includes taking fluorescence spectra from a diluted suspension of isolated chloroplasts at 77K, is questionable. These spectra are then used to analyze the distribution of energy between PSI and PSII. After rapid cooling to 77K secondary biochemical influences, which attenuate the fluorescence emanated from PSI, are frozen out. Due to their characteristic morphology, maize chloroplasts of mesophyll and bundle-sheath cells are an appropriate system for demonstrating the applicability of our in vivo method which, unlike the common 77K method, does not require the isolation of chloroplasts. In mesophyll chloroplasts of higher land plants, the thylakoids have a heterogenic morphology of appressed and non-appressed membrane domains, called the grana and the stroma lamellae. PSII is enriched in the grana, whereas PSI is enriched in the stroma lamellae. Changes in chloroplast membrane structure and composition, according to changes in the PSI/ PSII ratio, can be triggered by light quality and carbon source deficiency. Here, we demonstrate the applicability of statistical analysis of fluorescence spectra to detect changes in the PSI/PSII ratio resulting from structure changes in the thylakoid membrane.

Elgass, Kirstin; Zell, Martina; Maurino, Veronica G.; Schleifenbaum, Frank

2011-02-01

329

Amplitude and Phase Fluorescence-Spectroscopy Methods for Dissolved Oxygen Concentration Evaluation: Comparative Practical Results  

Microsoft Academic Search

This paper shows the practical results from a detailed comparative study of amplitude and phase fluorescence-spectroscopy methods for dissolved oxygen concentration evaluation. These results were obtained with an implemented optoelectronic measurement system that guarantees near-optimal operation conditions for both methods and a commercial fluorescence optical-fiber sensor, which is excited by a continuous-regulated sinusoidal-amplitude modulated light beam. The comparison was made

Gustavo J. Grillo; Miguel A. Pérez; Marta Valledor; Rubén Ramos

2005-01-01

330

The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy  

Microsoft Academic Search

Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluores- cence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spec- troscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by invest- igating bimolecular quenching

Jiho Kim; Soren Doose; Hannes Neuweiler; Markus Sauer

2006-01-01

331

Probing Lipid Mobility of Raft-exhibiting Model Membranes by Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

Confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS) have been employed to investigate the lipid spatial and dynamic organization in giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine\\/sphingomyelin\\/cholesterol. For a certain range of cholesterol concentration, formation of domains with raft-like properties was observed. Strikingly, the lipophilic probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C18) was excluded from sphingomyelin-enriched regions, where the raft

Kirsten Bacia; Dag Scherfeld; Nicoletta Kahya; Bert Poolman; Petra Schwille

2003-01-01

332

On-the-flow differentiation between cells based on native fluorescence spectroscopy on a chip  

Microsoft Academic Search

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

Markus Beck; Michael Bassler; Peter Kiesel; Noble M. Johnson; Oliver Schmidt

2008-01-01

333

Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy  

Microsoft Academic Search

We report the detection of heterogeneities in the diffusion of lipid molecules for the three-component mixture dipalmitoyl-PC\\/dilauroyl-PC\\/cholesterol, a chemically simple lipid model for the mammalian plasma membrane outer leaflet. Two-color fluorescence correlation spectroscopy (FCS) was performed on giant unilamellar vesicles (GUVs) using fluorescent probes that have differential lipid phase partition behavior—DiO-C18:2 favors disordered fluid lipid phases, whereas DiI-C20:0 prefers spatially

Jonas Korlach; Tobias Baumgart; Watt W. Webb; Gerald W. Feigenson

2005-01-01

334

Raman spectroscopy of a coal liquid shows that fluorescence interference is minimized with ultraviolet excitation  

SciTech Connect

The first ultraviolet resonance Raman measurements of a coal liquid are reported. The spectra detail the presence of numerous polycyclic aromatic hydrocarbons with ring systems similar to those of naphthalene, fluorene, phenanthrene, pyrene, and triphenylene. The ultraviolet resonance Raman measurements of this highly complex sample show no significant interference from fluorescence. The lack of fluorescence interference and the high selectivity indicate that ultraviolet resonance Raman spectroscopy is a powerful new technique for characterizing highly complex samples and mixtures. 11 references, 2 figures.

Asher, S.A.; Johnson, C.R.

1984-07-20

335

Raman spectroscopy of a coal liquid shows that fluorescence interference is minimized with ultraviolet excitation  

SciTech Connect

The first ultraviolet resonance Raman measurements of a coal liquid are reported. The spectra detail the presence of numerous polycyclic aromatic hydrocarbons with ring systems similar to those of naphthalene, fluorene, phenanthrene, pyrene, and triphenylene . The ultraviolet resonance Raman measurements of this highly complex sample show no significant interference from fluorescence. The lack of fluorescence interference and the high selectivity indicate that ultraviolet resonance Raman spectroscopy is a powerful new technique for characterizing highly complex samples and mixtures.

Asher, S.A.; Johnson, C.R.

1984-07-20

336

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; Marine, Vladimir I.; Sentis, Marc L.

2000-04-01

337

Two-photon fluorescence excitation spectroscopy of biological molecules  

NASA Astrophysics Data System (ADS)

The UV fluorescence spectra of aromatic amino-acids and some proteins at two photon excitation by second harmonic of Nd:YAG laser are received. Two-photon absorption cross sections of tryptophan, tyrosine, phenylalanine and proteins: bovine serum albumin, lysozyme, trypsin, (alpha) - chymotrypsinogen and pepsin at wavelength 532 nm were measured by means of the two-quantum standard method.

Meshalkin, Yu. P.; Alfimov, E. E.; Groshev, D. E.; Makukha, V. K.

1996-06-01

338

Short communication: rapid detection of milk fat adulteration with vegetable oil by fluorescence spectroscopy.  

PubMed

This study assessed the potential application of fluorescence spectroscopy in detecting adulteration of milk fat with vegetable oil and characterizing the samples according to the source of the fat. Pure butterfat was adulterated with different vegetable oils at various concentrations (0, 5, 10, 15, 20, 30, and 40%). Nonfat and reduced-fat milk were also adulterated with vegetable oils to simulate full-fat milk (3.2%). The 2- and 3-dimensional front-face fluorescence spectroscopy and gas chromatography were used to obtain the fluorescence spectra and fatty acid profile, respectively. Principal component analysis and 3-way partial least squares regression analysis were applied to analyze the data. The pure and adulterated samples were discriminated based on the total concentration of saturated fatty acids and unsaturated fatty acids, and also on the 3 major fluorophores: tryptophan, tocopherols, and riboflavin. Fluorescence spectroscopy was able to detect up to 5% of adulteration of vegetable oil into the butterfat. The saturated fatty acids showed higher predictability than the unsaturated fatty acids (R(2) = 0.73-0.92 vs. 0.20-0.65, respectively). The study demonstrated the high potential of fluorescence spectroscopy to rapidly detect adulteration of milk fat with vegetable oil, and discriminate commercial butter and milk according to the source of the fat. PMID:23415535

Ntakatsane, M P; Liu, X M; Zhou, P

2013-02-15

339

Distinguished photons: a review of in vivo spectral fluorescence imaging in small animals.  

PubMed

Fluorescence-based molecular imaging in small animals is having a major impact on drug development and disease research and the ability to detect multiple molecular species at once is becoming increasingly important. Unlike bioluminescence, in fluorescence, ubiquitous autofluorescent signals from the skin need to be separated from that of labeled fluorophores to ensure proper quantitative data. Since its introduction in 2004, spectral imaging methods have become an important part of in vivo fluorescence imaging of small animals by enabling easy multiplexed imaging methods and through the quantitative removal of interfering skin autofluorescence signals. This article is a review of the literature on spectral imaging methods and applications in fluorescence imaging of small animals. PMID:20497114

Mansfield, James R

2010-09-01

340

Simultaneous fluorescence and positron emission tomography for in vivo imaging of small animals  

NASA Astrophysics Data System (ADS)

Simultaneous positron emission tomography (PET) and fluorescence tomography (FT) for in vivo imaging of small animals is proposed by a dual-modality system. This system combines a charge-coupled device-based near-infrared fluorescence imaging with a planar detector pair-based PET. With [18F]-2-fluoro-2-deoxy-d-glucose radioactive tracer and the protease activated fluorescence probe, on the one hand, the simultaneous metabolic activity and protease activity in tumor region are revealed by the PET and FT, respectively. On the other hand, the protease activity both on the surface layer and the deep tissue of the tumor is provided by the fluorescence reflection imaging and FT, respectively.

Zhang, Bin; Liu, Shuangquan; Liu, Fei; Zhang, Xiaochun; Xu, Yanyan; Luo, Jianwen; Shan, Baoci; Bai, Jing

2011-12-01

341

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

342

Multi-Photon Fluorescence Spectroscopy of Fluorescent Bio-Probes and Bio-Molecules.  

National Technical Information Service (NTIS)

Multi-photon fluorescence spectra of a number of commonly used biological probes were measured in this study. Significant spectral variation has been detected between single and multi-photon excitation. The result is important for the proper selection of ...

B. Lin C. Sun F. Kao P. Cheng Y. Wang

2000-01-01

343

Characterization of Lipid Bilayer Phases by Confocal Microscopy and Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

We report the application of confocal imaging and fluorescence correlation spectroscopy (FCS) to characterize chemically well-defined lipid bilayer models for biomembranes. Giant unilamellar vesicles of dilauroyl phosphatidylcholine\\/dipalmitoyl phosphatidylcholine (DLPC\\/DPPC)\\/cholesterol were imaged by confocal fluorescence microscopy with two fluorescent probes, 1,1'-dieicosanyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiI-C20) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)- 1-hexadecanoyl-sn-glycero-3-phosphocholine (Bodipy-PC). Phase separation was visualized by differential probe partition into the coexisting phases. Three-dimensional

Jonas Korlach; Petra Schwille; Watt W. Webb; Gerald W. Feigenson

1999-01-01

344

Characterization of the main transition of dinervonoylphosphocholine liposomes by fluorescence spectroscopy  

Microsoft Academic Search

The structural dynamics of the main phase transition of large unilamellar dinervonoylphosphocholine (DNPC) vesicles was investigated by steady state and time-resolved fluorescence spectroscopy of the membrane incorporated fluorescent lipid analog, 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC). These data were supplemented by differential scanning calorimetry (DSC) and fluorescence anisotropy measured for 1-palmitoyl-2-(3-(diphenylhexatrienyl) propanoyl)-sn-glycero-3-phosphocholine (DPHPC). The collected data displayed several discontinuities in the course of the

Antti J Metso; Juha-Pekka Mattila; Paavo K. J Kinnunen

2004-01-01

345

O2(1?) Yield Measurement by Raman Spectroscopy With Elimination of Chlorine Fluorescence Interference  

NASA Astrophysics Data System (ADS)

Deleterious chlorine fluorescence was found to occur at the same frequency as the Raman scattering of O2(1?) and O2(3?), seriously affecting the O2(1?) yield measurement in the reaction of chlorine with basic hydrogen peroxide by use of the Raman spectroscopy technique. To solve this problem we have taken advantage of the fact that Raman radiation is always strongly polarized while fluorescence is essentially non-polarized in a gaseous medium. When chlorine utilization of a singlet oxygen generator is 88%, O2(1?) yield reaches (42.4±7.4)% with the effect of chlorine fluorescence completely eliminated.

Cui, Rong-rong; Shi, Wen-bo; Deng, Lie-zheng; Yang, He-ping; Sha, Guo-he; Zhang, Cun-hao

2012-04-01

346

Native fluorescence spectroscopy reveals spectral differences among prostate cancer cell lines with different risk levels  

NASA Astrophysics Data System (ADS)

The spectral changes of native fluorophores among normal fibroblasts and cancer cell lines of different metastatic ability are investigated by fluorescence spectroscopy. The normal (fibroblast), moderately metastatic (DU-145), and advanced metastatic (PC-3) cell lines were each selectively excited at 300 nm, and their fluorescence emission spectra are analyzed using principal component analysis to explore the differences of the relative contents of tryptophan and reduced nicotinamide adenine dinucleotide in these cell lines. The results show that the tryptophan emission featured predominantly in the fluorescence spectra of the advanced metastatic cancer cells in comparison with the moderately metastatic cancer and normal cells.

Pu, Yang; Xue, Jianpeng; Wang, Wubao; Xu, Baogang; Gu, Yueqing; Tang, Rui; Ackerstaff, Ellen; Koutcher, Jason A.; Achilefu, Samuel; Alfano, Robert R.

2013-08-01

347

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

PubMed

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

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

2011-06-16

348

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

PubMed Central

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

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

2011-01-01

349

Fluorescence spectroscopy: a diagnostic tool for cervical intraepithelial neoplasia (CIN).  

PubMed

A spectroscopic system incorporating a pulsed nitrogen laser, an optical fiber probe, and an optical multichannel analyzer was utilized to record fluorescence spectra of the intact cervix at colposcopy. Spectra were obtained from 66 colposcopically normal areas and 49 histologically abnormal areas (5 pathologic inflammation, 21 HPV infection, 9 CIN I, 10 CIN II, and 4 CIN III) in 28 patients. The resulting spectra could be used to differentiate histologically abnormal tissues from colposcopically normal tissues with a sensitivity, specificity, and positive predictive value of 92, 90, and 88%. Furthermore, CIN could be differentiated from nonneoplastic abnormal tissues with a sensitivity, specificity, and positive predictive value of 87, 73, and 74%. These results suggest that laser-induced fluorescence can be used in the recognition and differential diagnosis of CIN at colposcopy. PMID:8307499

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

1994-01-01

350

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).

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

2011-01-01

351

Intestine pH measurements using fluorescence imaging: an in-vivo preliminary study  

NASA Astrophysics Data System (ADS)

Measurement of gastrointestinal intramucosal pH has been recognized as an important factor in the detection of hypoxia-induced dysfunctions. However, current pH measurement techniques are limited in terms of time and spatial resolution. A major advance in accurate pH measurement was the development of the ratiometric fluorescent indicator dye, 2',7'-bis(carboxyethyl)-4,5- carboxyfluorescein (BCECF). This study aimed to demonstrate the feasibility of fluorescence imaging technique to measure in vivo the pH of intestine. The intestine was inserted in an optical chamber placed under a microscope. Animals were injected i.v. with the pH-sensitive fluorescent dye BCECF. Fluorescence was visualized by illuminating the intestine alternately at 490 and 470 nm. The emitted fluorescence was directed to an intensified camera. The ratio of emitted fluorescence at excitation wavelengths of 490 and 470 nm was measured, corrected and converted to pH by constructing a calibration curve. The pH controls were performed with a pH microelectrode correlated with venous blood gas sampling. We concluded that accurate pH measurements of rat intestine can be obtained by fluorescence imaging using BCECF. This technology could be easily adapted for endoscopic pH measurement.

Marechal, Xavier-Marie; Mordon, Serge R.; Devoisselle, Jean-Marie; Begu, Sylvie; Mathieu, D.; Buys, Bruno; Dhelin, Guy; Lesage, Jean C.; Neviere, Remi; Chopin, Claude

1999-02-01

352

Laser Induced Fluorescence Spectroscopy of a Langmuir Monolayer of C-16 Fluorescent Dipyrrinone Liquid Crystal  

NASA Astrophysics Data System (ADS)

A C-16 Fluorescent Dipyrrinone Liquid Crystal synthesized by the Chemistry department, University of West Florida, has been prepared in a Langmuir monolayer using a Nima Langmuir-Blodgett Trough. DeLuca et al. [1] studied how the length of the hydrocarbon tail influences the behavior of the pressure-area isotherm of the Langmuir film. The C-16 Fluorescent Dipyrrinone Liquid Crystal film produced a stable film at 20 mN/m and a stable, optical quality film at 40 mN/m. We present a study of the fluorescence properties of the C-16 fluorescent dipyrrinone liquid crystal film. Once the monolayer is compressed the sample is excited using a 410 nm wavelength laser and the fluorescence is measured using an Oriel MS260i 1/4 m Spectrograph. [4pt] [1] Deluca, Giovanni; Carroll, Alexander; Prayaga, Chandra; Wade, Aaron; Heath, Christopher; Renaud, Amy; Huggins, Michael. ``Preparation and Characterization of C-16 and C-10 Fluorescent Dipyrrinone Liquid Crystal Langmuir-Blodgett Films.'' American Physical Society, APS March Meeting 2012, 02/2012.

Struebing, Christian; Deluca, Giovanni; Prayaga, Chandra; Wade, Aaron; Huggins, Michael; Renaud, Amy; Chandler, Rebecca

2013-03-01

353

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

354

In-vivo fluorescence imaging with a multivariate curve resolution spectral unmixing technique.  

PubMed

Spectral unmixing is a useful technique in fluorescence imaging for reducing the effects of native tissue autofluorescence and separating multiple fluorescence probes. While spectral unmixing methods are well established in fluorescence microscopy, they typically rely on precharacterized in-vitro spectra for each fluorophore. However, there are unique challenges for in-vivo applications, since the tissue absorption and scattering can have a significant impact on the measured spectrum of the fluorophore, and therefore make the in-vivo spectra substantially different to that of in vitro. In this work, we introduce a spectral unmixing algorithm tailored for in-vivo optical imaging that does not rely on precharacterized spectral libraries. It is derived from a multivariate curve resolution (MCR) method, which has been widely used in studies of chemometrics and gene expression. Given multispectral images and a few straightforward constraints such as non-negativity, the algorithm automatically finds the signal distribution and the pure spectrum of each component. Signal distribution maps help separate autofluorescence from other probes in the raw images and hence provide better quantification and localization for each probe. The algorithm is demonstrated with an extensive set of in-vivo experiments using near-infrared dyes and quantum dots in both epi-illumination and transillumination geometries. PMID:20059249

Xu, Heng; Rice, Brad W

355

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

356

Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC).  

PubMed

Fluorescence correlation spectroscopy (FCS) is a frequently applied technique that allows for the precise and sensitive analysis of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time-correlated single-photon counting methods. Here, we demonstrate the suppression of autofluorescence in FCS using ADOTA-labeled hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the overexpression of which has been observed in many types of cancer. PMID:23564284

Rich, Ryan M; Mummert, Mark; Gryczynski, Zygmunt; Borejdo, Julian; Sørensen, Thomas Just; Laursen, Bo W; Foldes-Papp, Zeno; Gryczynski, Ignacy; Fudala, Rafal

2013-04-06

357

Application of in vivo ESR spectroscopy to measurement of cerebrovascular ROS generation in stroke  

Microsoft Academic Search

This study used an in vivo ESR spectroscopy\\/spin probe technique to measure directly the generation of reactive oxygen species (ROS) in the brain after cerebral ischemia-reperfusion. Transient middle cerebral artery occlusion (MCAO) was induced in rats by inserting a nylon thread into the internal carotid artery for 1 h. The in vivo generation of ROS and its location in the

Mayumi Yamato; Toru Egashira; Hideo Utsumi

2003-01-01

358

Combined In Vivo Confocal Raman Spectroscopy and Confocal Microscopy of Human Skin  

Microsoft Academic Search

In vivo confocal Raman spectroscopy is a noninvasive optical method to obtain detailed information about the molecular composition of the skin with high spatial resolution. In vivo confocal scanning laser microscopy is an imaging modality that provides optical sections of the skin without physically dissecting the tissue. A combination of both techniques in a single instrument is described. This combination

P. J. Caspers; G. W. Lucassen; G. J. Puppels

2003-01-01

359

In vivo hyperspectral confocal fluorescence imaging to determine pigment localization and distribution in cyanobacterial cells  

PubMed Central

Hyperspectral confocal fluorescence imaging provides the opportunity to obtain individual fluorescence emission spectra in small (?0.03-?m3) volumes. Using multivariate curve resolution, individual fluorescence components can be resolved, and their intensities can be calculated. Here we localize, in vivo, photosynthesis-related pigments (chlorophylls, phycobilins, and carotenoids) in wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Cells were excited at 488 nm, exciting primarily phycobilins and carotenoids. Fluorescence from phycocyanin, allophycocyanin, allophycocyanin-B/terminal emitter, and chlorophyll a was resolved. Moreover, resonance-enhanced Raman signals and very weak fluorescence from carotenoids were observed. Phycobilin emission was most intense along the periphery of the cell whereas chlorophyll fluorescence was distributed more evenly throughout the cell, suggesting that fluorescing phycobilisomes are more prevalent along the outer thylakoids. Carotenoids were prevalent in the cell wall and also were present in thylakoids. Two chlorophyll fluorescence components were resolved: the short-wavelength component originates primarily from photosystem II and is most intense near the periphery of the cell; and the long-wavelength component that is attributed to photosystem I because it disappears in mutants lacking this photosystem is of higher relative intensity toward the inner rings of the thylakoids. Together, the results suggest compositional heterogeneity between thylakoid rings, with the inner thylakoids enriched in photosystem I. In cells depleted in chlorophyll, the amount of both chlorophyll emission components was decreased, confirming the accuracy of the spectral assignments. These results show that hyperspectral fluorescence imaging can provide unique information regarding pigment organization and localization in the cell.

Vermaas, Wim F. J.; Timlin, Jerilyn A.; Jones, Howland D. T.; Sinclair, Michael B.; Nieman, Linda T.; Hamad, Sawsan W.; Melgaard, David K.; Haaland, David M.

2008-01-01

360

Excitation spectroscopy in multispectral optical fluorescence tomography: methodology, feasibility and computer simulation studies  

Microsoft Academic Search

Molecular probes used for in vivo optical fluorescence tomography (OFT) studies in small animals are typically chosen such that their emission spectra lie in the 680–850 nm wavelength range. This is because tissue attenuation in this spectral band is relatively low, allowing optical photons even from deep sites in tissue to reach the animal surface and consequently be detected by

Abhijit J Chaudhari; Sangtae Ahn; Richard Levenson; Ramsey D Badawi; Simon R Cherry; Richard M Leahy

2009-01-01

361

Statistical Analysis of Fluorescence Correlation Spectroscopy: The Standard Deviation and Bias  

Microsoft Academic Search

We present a detailed statistical analysis of fluorescence correlation spectroscopy for a wide range of timescales. The derivation is completely analytical and can provide an excellent tool for planning and analysis of FCS experiments. The dependence of the signal-to-noise ratio on different measurement conditions is extensively studied. We find that in addition to the shot noise and the noise associated

Saveez Saffarian; Elliot L. Elson

2003-01-01

362

Kinetic investigations by fluorescence correlation spectroscopy: The analytical and diagnostic potential of diffusion studies  

Microsoft Academic Search

This review demonstrates the large analytical and diagnostic potential of fluorescence correlation spectroscopy applied to freely diffusing biomolecules in solution. All applications discussed here in detail are based on changes in the diffusion characteristics of fluorescenctly labeled complementary strands of nucleic acids when they associate. However, the principle of the measurement can be extended to many different reactions with characteristic

Petra Schwille; Jan Bieschke; Frank Oehlenschläger

1997-01-01

363

Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) is carried out with an electron multiplying CCD (EMCCD). This new strategy is compared to standard detection by an avalanche photo diode showing good agreement with respect to the resulting autocorrelation curves. Applying different readout modes, a time resolution of 20 µs can be achieved, which is sufficient to resolve the diffusion of free dye in

Markus Burkhardt; Petra Schwille

2006-01-01

364

Measuring Size Distribution in Highly Heterogeneous Systems with Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) is a sensitive and widely used technique for measuring diffusion. FCS data are conventionally modeled with a finite number of diffusing components and fit with a least-square fitting algorithm. This approach is inadequate for analyzing data obtained from highly heterogeneous systems. We introduce a Maximum Entropy Method based fitting routine (MEMFCS) that analyzes FCS data in

Parijat Sengupta; K. Garai; J. Balaji; N. Periasamy; S. Maiti

2003-01-01

365

Fluorescence Correlation Spectroscopy Diffusion Laws to Probe the Submicron Cell Membrane Organization  

Microsoft Academic Search

To probe the complexity of the cell membrane organization and dynamics, it is important to obtain simple physical observables from experiments on live cells. Here we show that fluorescence correlation spectroscopy (FCS) measurements at different spatial scales enable distinguishing between different submicron confinement models. By plotting the diffusion time versus the transverse area of the confocal volume, we introduce the

Laure Wawrezinieck; Hervé Rigneault; Didier Marguet; Pierre-François Lenne

2005-01-01

366

Two-Focus Fluorescence Correlation Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements  

Microsoft Academic Search

We present a new method to measure absolute diffusion coeffi- cients at nanomolar concentrations with high precision. Based on a modified fluorescence correlation spectroscopy (FCS)-setup, this method is improved by introducing an external ruler for measuring the diffusion time by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by a

Thomas Dertinger; Victor Pacheco; Iris von der Hocht; Rudolf Hartmann; Ingo Gregor; Jörg Enderlein

2007-01-01

367

Probing Diffusion Laws within Cellular Membranes by Z-Scan Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

The plasma membrane of various mammalian cell types is heterogeneous in structure and may contain microdomains, which can impose constraints on the lateral diffusion of its constituents. Fluorescence correlation spectroscopy (FCS) can be used to investigate the dynamic properties of the plasma membrane of living cells. Very recently, Wawrezinieck et al. (Wawrezinieck, L., H. Rigneault, D. Marguet, and P. F.

Jana Humpolickova ´; Ellen Gielen; Aleš Benda; Veronika Fagulova; J. Vercammen; Martin vandeVen; Martin Hof; Marcel Ameloot; Yves Engelborghs

2006-01-01

368

Focal Volume Optics and Experimental Artifacts in Confocal Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) can provide a wealth of information about biological and chemical systems on a broad range of time scales (<1?s to >1s). Numerical modeling of the FCS observation volume combined with measurements has revealed, however, that the standard assumption of a three-dimensional Gaussian FCS observation volume is not a valid approximation under many common measurement conditions. As

Samuel T. Hess; Watt W. Webb

2002-01-01

369

?-Repressor Oligomerization Kinetics at High Concentrations Using Fluorescence Correlation Spectroscopy in Zero-Mode Waveguides  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) has demonstrated its utility for measuring transport properties and kinetics at low fluorophore concentrations. In this article, we demonstrate that simple optical nanostructures, known as zero-mode waveguides, can be used to significantly reduce the FCS observation volume. This, in turn, allows FCS to be applied to solutions with significantly higher fluorophore concentrations. We derive an empirical

K. T. Samiee; M. Foquet; L. Guo; E. C. Cox; H. G. Craighead

2005-01-01

370

Dynamics of ANS Binding to Tuna Apomyoglobin Measured with Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

The dynamics of the binding reaction of ANS to native and partly folded (molten globule) tuna and horse apomyoglobins has been investigated by fluorescence correlation spectroscopy and frequency domain fluorometry. The reaction rate has been measured as a function of apomyoglobin and ANS concentrations, pH, and temperature. Examination of the autocorrelation functions shows that the reaction rate is fast enough

Ettore Bismuto; Enrico Gratton; Don C. Lamb

2001-01-01

371

Spatial-Temporal Studies of Membrane Dynamics: Scanning Fluorescence Correlation Spectroscopy (SFCS)  

Microsoft Academic Search

Giant unilamellar vesicles (GUVs) have been widely used as a model membrane system to study membrane organization, dynamics, and protein-membrane interactions. Most recent studies have relied on imaging methods, which require good contrast for image resolution. Multiple sequential image processing only detects slow components of membrane dynamics. We have developed a new fluorescence correlation spectroscopy (FCS) technique, termed scanning FCS

Qiaoqiao Ruan; Melanie A. Cheng; Moshe Levi; Enrico Gratton; William W. Mantulin

2004-01-01

372

Photochemistry of Bisphenol-A Based Polycarbonate: Early Detection of Photoproducts by Fluorescence Spectroscopy.  

National Technical Information Service (NTIS)

The utility of fluorescence spectroscopy in detection of salicylic acid and 2,2'biphenol type products in the very early stages of photolysis of polycarbonate and its model compounds has been demonstrated. The broad structureless peak around 41 0 nm produ...

C. E. Hoyle H. Shah I. B. Rufus

1993-01-01

373

Microwave digestion and analysis of foliage for total mercury by cold vapor atomic fluorescence spectroscopy  

Microsoft Academic Search

A microwave technique for digesting foliage samples was developed and evaluated for quantifying low levels of Hg by cold vapor atomic fluorescence spectroscopy, CVAFS. The method meets three criteria: (1) to digest all sample material completely and consistently, (2) to reduce sample digestion time to less than one hour, and (3) to maintain a low analytical blank. Mean recovery of

Anne W. Rea; Gerald J. Keeler

1998-01-01

374

Characterization of natural carious lesions by fluorescence spectroscopy at 405-nm excitation wavelength  

Microsoft Academic Search

We aim to characterize natural caries enamel lesions by fluorescence spectroscopy. Sixty human samples with natural non- cavitated caries lesions on smooth surfaces were selected and classi- fied into three groups: dull, shiny, and brown lesions. All the samples were analyzed externally at the natural surface and after hemisec- tionig internally at the center of the lesion. The lesions were

Denise Maria Zezell; Adriana Costa Ribeiro; Luciano Bachmann; Anderson Stevens Leonidas Gomes; Christel Rousseau; John Girkin

2007-01-01

375

Water and surface contamination monitoring using deep UV laser induced native fluorescence and Raman spectroscopy  

Microsoft Academic Search

Reagentless water and surface sensors employing laser induced native fluorescence (LINF) and resonance Raman spectroscopy (RRS) in the deep UV are making significant progress in detecting chemical and biological targets and differentiating them against a wide range of background materials. Methods for optimizing sensor performance for specific target and backgrounds materials will be discussed in relationship to closed industrial environments

William F. Hug; Rohit Bhartia; Alexandre Tsapin; Pamela Conrad; Kripa Sijapati; Ray D. Reid

2006-01-01

376

Ground state C 2 density measurement in carbon plume using laser-induced fluorescence spectroscopy  

Microsoft Academic Search

The temporal evolution and spatial distribution of C2 molecules produced by laser ablation of a graphite target is studied using optical emission spectroscopy, dynamic imaging and laser-induced fluorescence (LIF) investigations. We observe peculiar bifurcation of carbon plume into two parts; stationary component close to the target surface and a component moving away from the target surface which splits further in

Dheerendra Yadav; Varun Gupta; Raj K. Thareja

2009-01-01

377

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

378

Fluorescence correlation spectroscopy on dielectric surfaces in total internal reflection geometries  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) has evolved to a valuable tool for biomolecular analysis on the single molecule level. Measurements on a single molecule level can only be performed if the measurement volume is small enough to contain on average only very few molecules. Common FCS-systems are therefore based on a confocal geometry in which a laser spot is focused into

Tiemo Anhut; Kai Hassler; Theo Lasser; Karsten Koenig; Rudolf Rigler

2005-01-01

379

Time resolved fluorescence spectroscopy of quercetin and morin complexes with Al 3+  

Microsoft Academic Search

The association process of Al3+ with quercetin and morin in methanol was studied by electronic absorption and emission spectroscopies. The number of species in solution with different absorption spectra were determined by the method of Rank analysis of the absorbance matrix, and the stoichiometries of the complexes were evaluated using the Job method. The number of fluorescent species in solution

Amanda C. Gutierrez; Marcelo H. Gehlen

2002-01-01

380

In-vivo Fluorescent X-ray CT Imaging of Mouse Brain  

SciTech Connect

Using a non-radioactive iodine-127 labeled cerebral perfusion agent (I-127 IMP), fluorescent X-ray computed tomography (FXCT) clearly revealed the cross-sectional distribution of I-127 IMP in normal mouse brain in-vivo. Cerebral perfusion of cortex and basal ganglion was depicted with 1 mm in-plane spatial resolution and 0.1 mm slice thickness. Degree of cerebral perfusion in basal ganglion was about 2-fold higher than that in cortical regions. This result suggests that in-vivo cerebral perfusion imaging is realized quantitatively by FXCT at high volumetric resolution.

Takeda, T.; Wu, J.; Lwin, Thet-Thet; Huo, Q.; Minami, M. [Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575 (Japan); Sunaguchi, N.; Murakami, T.; Mouri, S.; Nasukawa, S.; Yuasa, T.; Akatsuka, T. [Faculty of Engineering, Yamagata University, Yonezawa, Yamagata 992-8510 (Japan); Hyodo, K. [Institute of Material Science, High Energy Accelerator Research Organization, Tsukuba, Ibaraki 305-0801 (Japan); Hontani, H. [Department of Computer Science and Engineering, Nagoya Institute of Technology, Nagoya, Aichi 466-8555 (Japan)

2007-01-19

381

Lead nephropathy: In vivo x ray fluorescence (XRF) for assessing body lead stores  

SciTech Connect

The EDTA lead mobilization test has proven of value in the diagnosis of renal disease due to lead (lead nephropathy) but is unsuitable for large scale studies in patients with end-stage renal disease. A rapid, safe, non-invasive technique for determining body lead stores by in vivo tibial x ray fluorescence (XRF) is described. These studies show that the chelation test can be replaced by in vivo XRF in patients with end-stage renal disease. XRF, for the first time, will permit epidemiologic studies of large populations which may be at risk for lead nephropathy from excessive exposure to environmental lead. 15 refs., 2 figs.

Wedeen, R.P.; Batuman, V.; Quinless, F.; Williams, F.H. Jr.; Bogden, J.; Schidlovsky, G.; Jones, K.W.

1986-01-01

382

Synchronous fluorescence spectroscopy: tool for monitoring thermally stressed edible oils.  

PubMed

Total synchronous fluorescence spectra are proposed for monitoring edible oils during thermal stress. Synchronous fluorescence spectra obtained at an 80 nm wavelength interval combined with principal component analysis are suitable for classification of vegetable oil deterioration. Spectroscopic features in the range of 300-500 nm have been used for extra virgin olive, olive pomace, and sesame oil and the range of 320-520 nm has been used for corn, soybean, sunflower, and a commercial blend of oils. The score in the first two principal components explains 91.1% of the data matrix variance for extra virgin olive, sesame, and olive pomace oil and 89.3% for corn, soybean, sunflower, and the commercial blend of oils. The objective of this study is to develop a rapid method for the prediction of edible oil quality during thermal stress. Spectroscopic changes are indicative of oxidative deterioration as measured through wet chemistry methods: peroxide value, p-anisidine value, totox value, and radical-scavenging capacity. PMID:19722493

Poulli, Konstantina I; Chantzos, Nickolaos V; Mousdis, George A; Georgiou, Constantinos A

2009-09-23

383

Internal Structure and Dynamics of Isolated Escherichia coli Nucleoids Assessed by Fluorescence Correlation Spectroscopy  

PubMed Central

The morphology and dynamics of DNA in a bacterial nucleoid affects the kinetics of such major processes as DNA replication, gene expression. and chromosome segregation. In this work, we have applied fluorescence correlation spectroscopy to assess the structure and internal dynamics of isolated Escherichia coli nucleoids. We show that structural information can be extracted from the amplitude of fluorescence correlation spectroscopy correlation functions of randomly labeled nucleoids. Based on the developed formalism we estimate the characteristic size of nucleoid structural units for native, relaxed, and positively supercoiled nucleoids. The degree of supercoiling was varied using the intercalating agent chloroquine and evaluated from fluorescence microscopy images. The relaxation of superhelicity was accompanied by 15-fold decrease in the length of nucleoid units (from ?50 kbp to ?3 kbp).

Romantsov, Tatyana; Fishov, Itzhak; Krichevsky, Oleg

2007-01-01

384

Fluorescence correlation spectroscopy and photon-counting histogram analysis of receptor-receptor interactions.  

PubMed

Fluorescence correlation spectroscopy (FCS) performed using a laser scanning confocal microscope is a technique with single-molecule sensitivity that is becoming more accessible to cell biologists. In this chapter, we describe the use of FCS for the analysis of diffusion coefficients and receptor-receptor interactions in live cells in culture. In particular, we describe a protocol to collect fluorescence fluctuation data from fluorescence-tagged receptors as they diffuse into an out of a small laser-illuminated observation volume using a commercially available system such as the Zeiss ConfoCor 3 or LSM-780 microscope. Autocorrelation analysis of the fluctuations in fluorescence intensity provides information about the diffusion time and number of fluorescent molecules in the observation volume. A photon-counting histogram can be used to examine the relationship between fluorescence intensity and the number of fluorescent molecules to estimate the average molecular brightness of the sample. Since molecular brightness is directly proportional to the number of fluorescent molecules, it can be used to monitor receptor-receptor interactions and to decode the number of receptor monomers present in an oligomeric complex. PMID:24143978

Herrick-Davis, Katharine; Mazurkiewicz, Joseph E

2013-01-01

385

Interactions between 1-benzoyl-4-p-chlorophenyl thiosemicarbazide and serum albumin: investigation by fluorescence spectroscopy.  

PubMed

The interactions between 1-benzoyl-4-p-chlorphenyl thiosemicarbazide (BCPT) and bovine serum albumin (BSA) or human serum albumin (HSA) have been studied by fluorescence spectroscopy. By the analysis of fluorescence spectrum and fluorescence intensity, it was showed that BCPT has a strong ability to quench the intrinsic fluorescence of both bovine serum albumin and human serum albumin through a static quenching procedure. The binding constants of BCPT with BSA or HSA were determined at different temperatures based on the fluorescence quenching results. The binding sites were obtained and the binding force were suggested to be mainly hydrophobic. The effect of common ions on the binding constants was also investigated. A new fluorescence spectroscopy assay of the proteins is presented. The linear range is 5.36-67.0 microg mL(-1) with recovery of 101.1% for BSA, and the linear range is 8.28-144.9 microg mL(-1) with recovery of 102.6% for HSA. Determination of the proteins in bovine serum or in human serum by this method gives results which are very close to those obtained by using Coomassie Brilliant Blue G-250 colorimetry. A practical method was proposed for the determination of BCPT in human serum samples. PMID:14697780

Cui, Feng-Ling; Fan, Jing; Li, Jian-Ping; Hu, Zhi-De

2004-01-01

386

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

NASA Astrophysics Data System (ADS)

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

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

2013-06-01

387

A dark-field scanning spectroscopy platform for localized scatter and fluorescence imaging of tissue  

NASA Astrophysics Data System (ADS)

Tissue ultra-structure and molecular composition provide native contrast mechanisms for discriminating across pathologically distinct tissue-types. Multi-modality optical probe designs combined with spatially confined sampling techniques have been shown to be sensitive to this type of contrast but their extension to imaging has only been realized recently. A modular scanning spectroscopy platform has been developed to allow imaging localized morphology and molecular contrast measures in breast cancer surgical specimens. A custom designed dark-field telecentric scanning spectroscopy system forms the core of this imaging platform. The system allows imaging localized elastic scatter and fluorescence measures over fields of up to 15 mm x 15 mm at 100 microns resolution in tissue. Results from intralipid and blood phantom measurements demonstrate the ability of the system to quantify localized scatter parameters despite significant changes in local absorption. A co-registered fluorescence spectroscopy mode is also demonstrated in a protophorphyrin-IX phantom.

Krishnaswamy, Venkataramanan; Laughney, Ashley M.; Paulsen, Keith D.; Pogue, Brian W.

2011-02-01

388

The Monte Carlo modelling of in vivo X-ray fluorescence measurement of lead in tissue  

Microsoft Academic Search

A Monte Carlo model has been developed, using the EGS4 code, to model the in vivo X-ray fluorescence (XRF) measurement of Pb in non-superficial bone\\/tissue. Unlike previous work in this field the current model incorporates a correction for Doppler broadening of the Compton scatter peak due to the electron momentum distribution of the medium (tissue\\/water) in which the photons are

J. D. Wallace

1994-01-01

389

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

Microsoft Academic Search

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

Jeffrey C. Lodwick

2003-01-01

390

Fluorescence diagnosis of the status of the human lens in vivo  

NASA Astrophysics Data System (ADS)

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

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

2012-03-01

391

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

392

Fluorescent core-shell star polymers based bioassays for ultrasensitive DNA detection by surface plasmon fluorescence spectroscopy.  

PubMed

Multilayers containing a perylene diimide labelled star polymers (FSP) donor adjacent to phosphorus dendrimer layer on a silver substrate were constructed by layer by layer (LBL) approach. Using Surface Plasmon Enhanced Fluorescence Spectroscopy (SPFS) technique, a time-resolved ultrasensitive and selective detection of DNA targets relying on enhanced optical fields associated with energy transfer (ET) were achieved under the excitation at 543 nm. The detection limit is about 8 orders of magnitude better than the achieved one under the excitation at 632 nm, which is ascribed to no energy transfer from the donor to the acceptor under the excitation at 632 nm, resulting in much weak detection signal in turn. PMID:21480427

Feng, Chuan Liang; Yin, Meizhen; Zhang, Di; Zhu, Shenmin; Caminade, Anne Marie; Majoral, Jean Pierre; Müllen, Klaus

2011-03-07

393

Doppler-free Yb spectroscopy with the fluorescence spot technique  

NASA Astrophysics Data System (ADS)

We demonstrate a simple technique to measure the resonant frequency of the 398.9-nm 1S0?1P1 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.

2010-10-01

394

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

395

In vivo chlorophyll fluorescence study of hazardous waste site vegetation under field and controlled conditions  

SciTech Connect

Cattail (Typha sp.) and Arrow Arum (Peltandra virginica) were studied to determine the effects of cadmium and nickel contamination in a freshwater tidal marsh. An in vivo chlorophyll fluorescence instrument was used in the field to estimate photosynthetic capacity. No definitive effects on photosynthesis were observed. A laboratory study was then designed to determine whether fluorescence could detect sublethal impacts of cadmium and whether tolerant plants had developed in the contaminated area. Arrow Arum seeds collected from a reference wetland and from the contaminated wetland were grown in horticultural vermiculite with cadmium concentrations of 0, 1, 2, 5 and 10 mg/L. Results indicate that, regardless of seed origin, fluorescence can detect an effect at cadmium levels at which there are no visual signs of stress. However, the plants from the contaminated wetland exhibited reduced growth, and deformities in several individuals.

Mayasich, S.A.; Zygmont, N.J. (Roy F. Weston, Inc., Edison, NJ (United States) CDM Federal Programs Corp., South Plainfield, NJ (United States))

1993-06-01

396

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

397

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

NASA Astrophysics Data System (ADS)

In vivo fluorescence of Chlorophyll-a (Chl-a) is a potentially useful property to study the vertical distribution of phytoplankton biomass. However the technique is presently not fully exploited as it should be, essentially because of the difficulties in converting the fluorescence signal into an accurate Chl-a concentration. These difficulties arise noticeably from natural variations in the Chl-a fluorescence relationship, which is under the control of community composition as well as of their nutrient and light status. As a consequence, although vertical profiles of fluorescence are likely the most recorded biological property in the open ocean, the corresponding large databases are underexploited. Here with the aim to convert a fluorescence profile into a Chl-a concentration profile, we test the hypothesis that the Chl-a concentration can be gathered from the sole knowledge of the shape of the fluorescence profile. We analyze a large dataset from 18 oceanographic cruises conducted in case-1 waters from the highly stratified hyperoligotrophic waters (surface Chl-a = 0.02 mg m-3) of the South Pacific Gyre to the eutrophic waters of the Benguela upwelling (surface Chl-a = 32 mg m-3) and including the very deep mixed waters in the North Atlantic (Mixed Layer Depth = 690 m). This dataset encompasses more than 700 vertical profiles of Chl-a fluorescence as well as accurate estimations of Chl-a by High Performance Liquid Chromatography (HPLC). Two typical fluorescence profiles are identified, the uniform profile, characterized by a homogeneous layer roughly corresponding to the mixed layer, and the non-uniform profile, characterized by the presence of a Deep Chlorophyll Maximum. Using appropriate mathematical parameterizations, a fluorescence profile is subsequently represented by 3 or 5 shape parameters for uniform or non-uniform profiles, respectively. For both situations, an empirical model is developed to predict the "true" Chl-a concentration from these shape parameters. This model is then used to calibrate a fluorescence profile in Chl-a units. The validation of the approach provides satisfactory results with a median absolute percent deviation of 33 % when comparing the HPLC Chl-a profiles to the Chl-a-calibrated fluorescence. The proposed approach thus opens the possibility to produce Chl-a climatologies from uncalibrated fluorescence profile databases that have been acquired in the past and to which numerous new profiles will be added, thanks to the recent availability of autonomous platforms (profiling floats, gliders and animals) instrumented with miniature fluorometers.

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

2011-08-01

398

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

NASA Astrophysics Data System (ADS)

In vivo fluorescence of Chlorophyll-a (Chl-a) is a potentially useful property to study the vertical distribution of phytoplankton biomass. However the technique is presently not fully exploited as it should be, essentially because of the difficulties in converting the fluorescence signal into an accurate Chl-a concentration. These difficulties arise noticeably from natural variations in the Chl-a fluorescence relationship, which is under the control of community composition as well as of their nutrient and light status. As a consequence although vertical profiles of fluorescence are likely the most recorded biological property in the open ocean, the corresponding large databases are underexploited. Here with the aim to convert a fluorescence profile into a Chl-a concentration profile, we test the hypothesis that the Chl-a concentration can be gathered from the sole knowledge of the shape of the fluorescence profile. We analyze a large dataset from 18 oceanographic cruises conducted in case-1 waters from the highly stratified hyperoligotrophic waters (surface Chl-a = 0.02 mg m-3) of the South Pacific Gyre to the eutrophic waters of the Benguela upwelling (surface Chl-a = 32 mg m-3) and including the very deep mixed waters in the North Atlantic (Mixed Layer Depth = 690 m). This dataset encompasses more than 700 vertical profiles of Chl-a fluorescence as well as accurate estimations of Chl-a by High Performance Liquid Chromatography (HPLC). Two typical fluorescence profiles are identified, the uniform profile, characterized by a homogeneous layer roughly corresponding to the mixed layer, and the non-uniform profile, characterized by the presence of a Deep Chlorophyll Maximum. Using appropriate mathematical parameterizations, a fluorescence profile is subsequently represented by 3 or 5 shape parameters for uniform or non-uniform profiles, respectively. For both situations, an empirical model is developed to predict the "true" Chl-a concentration from these shape parameters. This model is then used to calibrate a fluorescence profile in Chl-a units. The validation of the approach provides satisfactory results with a median absolute percent deviation of 33% when comparing the HPLC Chl-a profiles to the Chl-a-calibrated fluorescence. The proposed approach thus opens the possibility to produce Chl-a climatologies from uncalibrated fluorescence profile databases that have been acquired in the past and to which numerous new profiles will be added, thanks to the recent availability of autonomous platforms (profiling floats, gliders and animals) instrumented with miniature fluorometers.

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

2011-04-01

399

Determination of lipid raft partitioning of fluorescently-tagged probes in living cells by Fluorescence Correlation Spectroscopy (FCS).  

PubMed

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases. Yet their existence is still a matter of controversy. Indeed, lipid raft size has been estimated to be around 20 nm, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells. Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models. FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change. PMID:22508446

Marquer, Catherine; Lévêque-Fort, Sandrine; Potier, Marie-Claude

2012-04-06

400

Global analysis of autocorrelation functions and photon counting distributions in fluorescence fluctuation spectroscopy.  

PubMed

In fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis, the same experimental fluorescence intensity fluctuations are used, but each analytical method focuses on a different property of the signal. The time-dependent decay of the correlation of fluorescence fluctuations is measured in FCS yielding molecular diffusion coefficients and triplet-state parameters such as fraction and decay time. The amplitude distribution of these fluctuations is calculated by PCH analysis yielding the molecular brightness. Both FCS and PCH give information about the molecular concentration. Here we describe a global analysis protocol that simultaneously recovers relevant and common parameters in model functions of FCS and PCH from a single fluorescence fluctuation trace. Application of a global analysis approach allows increasing the information content available from a single measurement that results in more accurate values of molecular diffusion coefficients and triplet-state parameters and also in robust, time-independent estimates of molecular brightness and number of molecules. PMID:24108652

Skakun, Victor V; Digris, Anatoli V; Apanasovich, Vladimir V

2014-01-01

401

Laser-induced fluorescence-cued, laser-induced breakdown spectroscopy biological-agent detection  

SciTech Connect

Methods for accurately characterizing aerosols are required for detecting biological warfare agents. Currently, fluorescence-based biological agent sensors provide adequate detection sensitivity but suffer from high false-alarm rates. Combining single-particle fluorescence analysis with laser-induced breakdown spectroscopy (LIBS) provides additional discrimination and potentially reduces false-alarm rates. A transportable UV laser-induced fluorescence-cued LIBS test bed has been developed and used to evaluate the utility of LIBS for biological-agent detection. Analysis of these data indicates that LIBS adds discrimination capability to fluorescence-based biological-agent detectors.However, the data also show that LIBS signatures of biological agent simulants are affected by washing. This may limit the specificity of LIBS and narrow the scope of its applicability in biological-agent detection.

Hybl, John D.; Tysk, Shane M.; Berry, Shaun R.; Jordan, Michael P

2006-12-01

402

Determination of the in vivo redox potential using roGFP and fluorescence spectra obtained from one-wavelength excitation  

NASA Astrophysics Data System (ADS)

The analysis of molecular processes in living (plant) cells such as signal transduction, DNA replication, carbon metabolism and senescence has been revolutionized by the use of green fluorescent protein (GFP) and its variants as specific cellular markers. Many cell biological processes are accompanied by changes in the intracellular redox potential. To monitor the redox potential, a redox-sensitive mutant of GFP (roGFP) was created, which shows changes in its optical properties in response to changes in the redox state of its surrounding medium. For a quantitative analysis in living systems, it is essential to know the optical properties of roGFP in vitro. Therefore, we applied spectrally resolved fluorescence spectroscopy on purified roGFP exposed to different redox potentials to determine shifts in both the absorption and the emission spectra of roGFP. Based on these in vitro findings, we introduce a new approach using one-wavelength excitation to use roGFP for the in vivo analysis of cell biological processes. We demonstrate the ability this technique by investigating chloroplast-located Grx1-roGFP2 expressing Arabidopsis thaliana cells as example for dynamically moving intracellular compartments. This is not possible with the two-wavelength excitation technique established so far, which hampers a quantitative analysis of highly mobile samples due to the time delay between the two measurements and the consequential displacement of the investigated area.

Wierer, S.; Elgass, K.; Bieker, S.; Zentgraf, U.; Meixner, A. J.; Schleifenbaum, F.

2011-02-01

403

Ultrafast fluorescence quenching by electron transfer and fluorescence from the second excited state of a charge transfer complex as studied by femtosecond up-conversion spectroscopy  

Microsoft Academic Search

Photoinduced electron transfer dynamics between fluorescer (acceptor, A) and quencher (donor, D) was investigated by measuring the fluorescence quenching using femtosecond up-conversion spectroscopy. The measurements were made in a quencher concentration range of 0.15 M-1 M and also in a neat quencher solvent. Fluorescence decay at times longer than 5 ps can be explained by combining the diffusion equation with

Shinichiro Iwai; Shigeo Murata; M. Tachiya

1998-01-01

404

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

405

Photochemistry and fluorescence spectroscopy of polymeric materials containing triphenylsulfonium salts  

NASA Astrophysics Data System (ADS)

Triphenylsulfonium salts (TPS) have been formulated with polymers to make photosensitive systems for optical and optoelectronic applications. Photolysis of these salts generates strong acid which has been used in cross-linking reactions, deprotection reactions, and depolymerization reactions for photosensitive polymers, photodeformable polymers, and photo-doped conducting polymers. In addition, materials best described as polymeric sulfonium salts have been found to become conducting after photolysis. We have studied the photochemistry of TPS in polymer films and in solution. TPS photodecomposes by a mechanism that gives both in-cage recombination reactions and cage-escape products, and by reaction with solvent or polymer matrix. These products give cage/escape (C/E) ratios which are sensitive to the viscosity, rigidity, and polarity of the environment, and also the excited state from which the photochemistry occurs. Details of the reactivity and C/E ratios from photolysis of TPS salts in solution, have made it possible to determine their reactivity in polymers. In some cases the polymer behaves as a viscous solvent leading to high C/E ratios and in other systems the polymer excited state can sensitize the decomposition of TPS salts to give lower C/E ratios. Fluorescence studies on these polymers and quenching studies with TPS salts have helped to determine which excited states of the polymer and TPS salts are involved, and whether there is static or dynamic quenching in these systems. The photochemistry of model compounds for the polymeric sulfonium salts is also described.

Hacker, Nigel P.; Welsh, Kevin M.

1991-12-01

406

In vivo Fluorescence Imaging of Muscle Cell Regeneration by Transplanted EGFP-labeled Myoblasts  

PubMed Central

In vivo fluorescence imaging (FLI) enables monitoring fluorescent protein (FP)-labeled cells and proteins in living organisms noninvasively. Here, we examined whether this modality could reach a sufficient sensitivity to allow evaluation of the regeneration process of enhanced green fluorescent protein (eGFP)-labeled muscle precursors (myoblasts). Using a basic FLI station, we were able to detect clear fluorescence signals generated by 40,000 labeled cells injected into a tibialis anterior (TA) muscle of mouse. We observed that the signal declined to ~25% on the 48 hours of cell injection followed by a recovery starting at the second day and reached a peak of ~45% of the original signal by the 7th day, suggesting that the survived population underwent a limited run of proliferation before differentiation. To assess whether transplanted myoblasts could form satellite cells, we injured the transplanted muscles repeatedly with cardiotoxin. We observed a recovery of fluorescence signal following a disappearance of the signal after each cardiotoxin injection. Histology results showed donor-derived cells located underneath basal membrane and expressing Pax7, confirming that the regeneration observed by imaging was indeed mediated by donor-derived satellite cells. Our results show that FLI is a powerful tool that can extend our ability to unveil complicated biological processes such as stem cell-mediated regeneration.

Xu, Xiaoyin; Yang, Zhong; Liu, Qiang; Wang, Yaming

2010-01-01

407

In vivo 31P spectroscopy by fully adiabatic extended image selected in vivo spectroscopy: a comparison between 3 T and 7 T.  

PubMed

An improved image selected in vivo spectroscopy (ISIS) sequence for localized (31)P magnetic resonance spectroscopy at 7 T was developed. To reduce errors in localization accuracy, adiabatic excitation, gradient offset independent adiabatic inversion pulses, and a special extended ISIS ordering scheme were used. The localization accuracy of extended ISIS was investigated in phantoms. The possible spectral quality and reproducibility in vivo was explored in a volunteer (brain, muscle, and liver). A comparison between 3 T and 7 T was performed in five volunteers. Adiabatic extended ISIS provided high spectral quality and accurate localization. The contamination in phantom experiments was only ?5%, even if a pulse repetition time ? 1.2·T(1) was chosen to maximize the signal-to-noise ratio per unit time. High reproducibility was found in the calf muscle for 2.5 cm isotropic voxels at 7 T. When compared with 3 T, localized (31)P magnetic resonance spectroscopy in the human calf muscle at 7 T provided ?3.2 times higher signal-to-noise ratio (as judged from phosphocreatine peak amplitude in frequency domain after matched filtering). At 7 T, extended ISIS allowed the performance of high-quality localized (31)P magnetic resonance spectroscopy in a short measurement time (?3 to 4 min) and isotropic voxel sizes of ?2.5 to 3 cm. With such short measurement times, localized (31)P magnetic resonance spectroscopy has the potential to be applied not only for clinical research but also for routine clinical practice. PMID:21446033

Bogner, W; Chmelik, M; Andronesi, O C; Sorensen, A G; Trattnig, S; Gruber, S

2011-03-28

408

High-wavenumber FT-Raman spectroscopy for in vivo and ex vivo measurements of breast cancer  

Microsoft Academic Search

The identification of normal and cancer breast tissue of rats was investigated using high-frequency (HF) FT-Raman spectroscopy\\u000a with a near-infrared excitation source on in vivo and ex vivo measurements. Significant differences in the Raman intensities\\u000a of prominent Raman bands of lipids and proteins structures (2,800–3,100 cm?1) as well as in the broad band of water (3,100–3,550 cm?1) were observed in mean normal

A. F. García-Flores; L. Raniero; R. A. Canevari; K. J. Jalkanen; R. A. Bitar; H. S. Martinho; A. A. Martin

409

Laser-induced fluorescence spectroscopy of human normal and cancerous tissues  

NASA Astrophysics Data System (ADS)

Fluorescence properties of stomach tissues have been investigated to determine whether malignant specimens can be discriminated in vitro. Differences between normal and tumor tissues are found that concern both the intensity distribution and spectral shape of the autofluorescence emission. According to the distinctions tumor tissues would be differed from the normal tissues. The resulting spectra could be differentiate histologically stomach abnormal tissues from normal tissues with a sensitivity and specificity value of 90 percent and 92 percent. Furthermore, fluorescence spectra from human stomach normal and malignant tissues have been measured in real time in vivo.

Chen, Wei; He, Bin; Wei, Guanghui; Gao, Ge; Li, Shirong

1996-09-01

410

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

PubMed

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

2013-06-11

411

Fluorescence Correlation Spectroscopy of Tryptophan-containing Proteins in Sugar Solutions using Two Photon Excitation  

NASA Astrophysics Data System (ADS)

Sugars are common ingredients for many commercial cryopreserving agents yet their function in this role is poorly understood. Some believe that sugars preferentially bind with a protein surface thereby replacing hazardous, ice-forming water. In an attempt to test idea, we have undertaken studies of the diffusion of proteins and protein-coated nanospheres using fluorescence correlation spectroscopy in an effort to determine if the hydrodynamic size is influenced by the addition of sugars. Some novelty of our approach lies in exploiting the native fluorescence of tryptophan (a common flurophore found in many proteins) by use of two-photon excitation.

Wang, Yuli; Holman, Nathan; Sidebottom, David; Nichols, Micheal; Haas, Eric

2012-02-01

412

Strengths and weaknesses of recently engineered red fluorescent proteins evaluated in live cells using fluorescence correlation spectroscopy.  

PubMed

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

2013-10-14

413

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

414

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

Todd, A C; Chettle, D R

1994-01-01

415

An (125)I-labeled octavalent peptide fluorescent nanoprobe for tumor-homing imaging in vivo.  

PubMed

Targeting radiopeptides are promising agents for radio-theranostics. However, in vivo evaluation of their targeting specificity is often obscured by their short biologic half-lives and low binding affinities. Here, we report an approach to efficiently examine targeting radiopeptides with a new class of octavalent peptide fluorescent nanoprobe (Octa-FNP) platform, which is composed of candidate targeting peptides and a tetrameric far-red fluorescent protein (tfRFP) scaffold. To shed light on this process, (125)I-Octa-FNP, (125)I-tfRFP and (125)I-peptide were synthesized, and their targeting functionalities were compared. Both fluorescence imaging and radioactive quantification results confirmed that (125)I-Octa-FNP had a significantly higher cellular binding capability than (125)I-tfRFP. In vivo biodistribution studies show that at 6 h post-injection, (125)I-Octa-FNP had 2-fold and 30-fold higher tumor uptake than that of (125)I-tfRFP and (125)I-peptide, respectively. Moreover, ?-imaging at 24 h post-injection revealed a remarkable accumulation of (125)I-Octa-FNP in the tumor while maintaining an extremely low background contrast, which was further confirmed by immunofluorescence analysis. These data suggested that, as an engineered and multivalent platform, Octa-FNP could enhance the tumor targeting of a designed peptide and provide excellent contrast radioimaging, making it a valuable tool for the evaluation of the targeting ability of specifically designed radiopeptides for cancer theranostics. PMID:22494886

Luo, Haiming; Shi, Jiyun; Jin, Honglin; Fan, Di; Lu, Lisen; Wang, Fan; Zhang, Zhihong

2012-04-10

416

Chromosome orientation fluorescence in situ hybridization (CO-FISH) to study sister chromatid segregation in vivo  

PubMed Central

Previously, assays for sister chromatid segregation patterns relied on incorporation of BrdU and indirect methods to infer segregation patterns after two cell divisions. Here we describe a method to differentially label sister chromatids of murine cells and directly assay sister chromatid segregation patterns following one cell division in vitro and in vivo by adaptation of the well-established CO-FISH (chromosome orientation fluorescent in situ hybridization) technique. 5-bromo-2?-deoxyuridine (BrdU) is incorporated into newly-formed DNA strands, followed by photolysis and exonuclease digestion to create single-stranded sister chromatids containing parental template DNA only. Such single-stranded sister chromatids are differentially labeled using unidirectional probes to major satellite sequences coupled to fluorescent markers. Differentially-labeled sister chromatids in post-mitotic cells are visualized using fluorescence microscopy and sister chromatid segregation patterns can be directly assayed after one cell division. This procedure requires four days for in vivo mouse tissues, and two days for in vitro cultured cells.

Falconer, Ester; Chavez, Elizabeth; Henderson, Alexander; Lansdorp, Peter M.

2013-01-01

417

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

418

Longitudinal in vivo imaging of bone formation and resorption using fluorescence molecular tomography.  

PubMed

Bone research often focuses on anatomical imaging of the bone microstructure, but in order to gain better understanding in how bone remodeling is modulated through interventions also bone formation and resorption processes should be investigated. With this in mind, the purpose of this study was to establish a longitudinal in vivo imaging approach of bone formation a