Sample records for vivo fluorescence spectroscopy

  1. In Vivo Fluorescence Correlation and Cross-Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Mütze, Jörg; Ohrt, Thomas; Petrášek, Zden?k; Schwille, Petra

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

  2. Rapid multiexcitation fluorescence spectroscopy system for in vivo tissue diagnosis

    NASA Astrophysics Data System (ADS)

    Zângaro, Renato Amaro; Silveira, Landulfo, Jr.; Manoharan, Ramasamy; Zonios, George; Itzkan, Irving; Dasari, Ramachandra R.; van Dam, Jacques; Feld, Michael S.

    1996-09-01

    We have designed, fabricated, and tested a compact, transportable, excitation-emission spectrofluorimeter with optical-fiber light delivery and collection for use in rapid analysis of tissues in a clinical setting. This system provides up to eleven different excitation wavelengths, permitting collection of all the corresponding emission spectra in approximately 600 ms. It uses a N2 laser that pumps a sequence of dyes placed in cuvettes on a rotating wheel. A white-light excitation source permits acquisition of the tissue's diffuse reflectance spectrum on each cycle. Return fluorescence and reflected light are dispersed by a small spectrograph and detected by a photodiode-array detector. The system can collect a single-shot spectrum from biological tissue with a signal-to-noise ratio in excess of 50:1.

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

    NASA Astrophysics Data System (ADS)

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

    2010-05-01

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

  4. Laser-induced fluorescence spectroscopy at endoscopy: tissue optics, Monte Carlo modeling, and in vivo measurements

    Microsoft Academic Search

    Jianan Y. Qu; Calum E. MacAulay; Stephen Lam; Branko Palcic

    1995-01-01

    The optical properties (absorption coefficient, scattering coefficient and the anisotropic factor of scattering) and fluorescence characteristics of normal and abnormal bronchial tissue were measured in vitro. After adding additional blood optical properties to in vitro optical properties of tissue, the in vivo bronchial fluorescence was simulated and analyzed by Monte Carlo modeling. The Monte Carlo simulation results shows that with

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

    PubMed

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

    2013-05-01

    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

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

    PubMed Central

    Tóth, Katalin; Togashi, Denisio M.; Ryder, Alan G.; Langowski, Jörg; Nasheuer, Heinz Peter

    2012-01-01

    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. PMID:22536402

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

    NASA Astrophysics Data System (ADS)

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

    2012-09-01

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

  8. Use of acousto-optic tuneable filters for imaging fluorescence spectroscopy applications in vivo and in vitro

    NASA Astrophysics Data System (ADS)

    Bouhifd, Mounir; Whelan, Maurice P.; Aprahamian, Marc

    2005-04-01

    We describe the design and development two prototype spectroscopy imaging instruments based on custom-made acousto-optic tuneable filters (AOTF). These devices can be coupled to many standard imaging systems (e.g. an endoscope or a fluorescence microscope). The instruments developed offer significant advantages over typical fixed-filter imaging systems in terms of flexibility, performance and diagnostic potential. Any filtering wavelength in the visible range can be rapidly selected either by random access or continuous tuning. Since filtering is achieved through a diffractive process, an excellent signal-to-noise ratio is achieved that allows the detection of extremely low fluorescence signals such as autofluorescence. These adapters were designed to allow the simultaneous imaging of both the filtered and unfiltered signals. A first prototype instrument was developed and demonstrated for in-vivo applications. When attached to the eyepiece of a commercial endoscope, it allowed the simultaneous white light endoscopy and fluorescence imaging. Autofluorescence of protoporphyrin IX (PpIX), an endogenous chromophore that traces early-stage diseased tissue experiencing an inflammatory response, was mapped in vivo on a rat model. The system has also been approved for medical use and human clinical trials are underway. In addition, we are currently testing a second AOTF module for in vitro applications. This new AOTF adapter was designed to be coupled to the viewing port of a commercial fluorescence microscope to realise a fluorescence imaging spectrometer capable of detecting and mapping fluorescent biomolecules.

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

    NASA Astrophysics Data System (ADS)

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

    2003-11-01

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

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

    PubMed

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

    2013-03-01

    Changes in the amounts of cellular eumelanin and pheomelanin have been associated with carcinogenesis. The goal of this work is to develop methods based on two-photon-excited-fluorescence (TPEF) for measuring relative concentrations of these compounds. We acquire TPEF emission spectra (?(ex)=1000? nm) of melanin in vitro from melanoma cells, hair specimens, and in vivo from healthy volunteers. We find that the pheomelanin emission peaks at approximately 615 to 625 nm and eumelanin exhibits a broad maximum at 640 to 680 nm. Based on these data we define an optical melanin index (OMI) as the ratio of fluorescence intensities at 645 and 615 nm. The measured OMI for the MNT-1 melanoma cell line is 1.6 ± 0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI=0.5 ± 0.05 and 0.17 ± 0.03, respectively). The measured values are in good agreement with chemistry-based melanin extraction methods. In order to better separate melanin fluorescence from other intrinsic fluorophores, we perform fluorescence lifetime imaging microscopy of in vitro specimens. The relative concentrations of keratin, eumelanin, and pheomelanin components are resolved using a phasor approach for analyzing lifetime data. Our results suggest that a noninvasive TPEF index based on spectra and lifetime could potentially be used for rapid melanin ratio characterization both in vitro and in vivo. PMID:23235925

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

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

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

  12. Fluorescence and Raman spectroscopy.

    PubMed

    Wong Kee Song, Louis-Michel; Marcon, Norman E

    2003-04-01

    Table 2 provides a summary of selected in vivo fluorescence and Raman studies performed in BE. Although the findings from these studies appear promising, these techniques are still under development, and it is anticipated that technological refinements will further enhance their diagnostic accuracy. Ultimately, however, large-scale prospective clinical trials are required to determine their true diagnostic potential in BE and other sites. Ideally, the instrumentation of choice would be a real-time endoscopic system that combines excellent diagnostic accuracy with wide-area sampling. In this regard, fluorescence imaging is most appealing, although a variety of issues remain to be resolved, including the choice between autofluorescence versus drug-induced fluorescence and the problematic distinction between dysplastic (true positive) and confounding background metaplastic fluorescence (false positive), among others. It is also not clear whether exogenous fluorophores are necessary to achieve clinically useful sensitivity and specificity for lesion detection in BE. Point spectroscopic techniques, either fluorescence or Raman scattering, are inherently limited by the small volume of tissue (biopsy specimen size) they sample, but more detailed information can be extracted from the spectra, which may increase diagnostic accuracy. Moreover, it may be that the optimal system will be a combination of multiple optical spectroscopic or imaging techniques (multimodality approach), as suggested by Georgakoudi et al. For instance, a lesion could be detected by fluorescence imaging and its dysplastic nature characterized (graded) by Raman spectroscopy. In this era of cost containment, however, the critical challenge is to demonstrate whether an increase in diagnostic accuracy merits investment in costly technology, regardless of the technique used. PMID:12916660

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

    NASA Astrophysics Data System (ADS)

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

    2005-04-01

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

  14. Quantification of in vivo fluorescence decoupled from the effects of tissue optical properties using fiber-optic spectroscopy measurements

    PubMed Central

    Kim, Anthony; Khurana, Mamta; Moriyama, Yumi; Wilson, Brian C.

    2010-01-01

    We present a method for tissue fluorescence quantification in situ using a handheld fiber optic probe that measures both the fluorescence and diffuse reflectance spectra. A simplified method to decouple the fluorescence spectrum from distorting effects of the tissue optical absorption and scattering is developed, with the objective of accurately quantifying the fluorescence in absolute units. The primary motivation is measurement of 5-aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX) concentration in tissue during fluorescence-guided resection of malignant brain tumors. This technique is validated in phantoms and ex vivo mouse tissues, and tested in vivo in a rabbit brain tumor model using ALA-PpIX fluorescence contrast. PMID:21198210

  15. Fluorescence Correlation Spectroscopy

    NSDL National Science Digital Library

    Haustein, Elke

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

  16. Nanosecond fluorescence spectroscopy

    SciTech Connect

    Leskovar, B.

    1985-03-01

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

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

    PubMed

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

    2014-02-01

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

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

    PubMed Central

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

    2011-01-01

    Biomarkers are indicators of biological processes and hold promise for the diagnosis and treatment of disease. Gliomas represent a heterogeneous group of brain tumors with marked intra- and inter-tumor variability. The extent of surgical resection is a significant factor influencing post-surgical recurrence and prognosis. Here, we used fluorescence and reflectance spectral signatures for in vivo quantification of multiple biomarkers during glioma surgery, with fluorescence contrast provided by exogenously-induced protoporphyrin IX (PpIX) following administration of 5-aminolevulinic acid. We performed light-transport modeling to quantify multiple biomarkers indicative of tumor biological processes, including the local concentration of PpIX and associated photoproducts, total hemoglobin concentration, oxygen saturation, and optical scattering parameters. We developed a diagnostic algorithm for intra-operative tissue delineation that accounts for the combined tumor-specific predictive capabilities of these quantitative biomarkers. Tumor tissue delineation achieved accuracies of up to 94% (specificity = 94%, sensitivity = 94%) across a range of glioma histologies beyond current state-of-the-art optical approaches, including state-of-the-art fluorescence image guidance. This multiple biomarker strategy opens the door to optical methods for surgical guidance that use quantification of well-established neoplastic processes. Future work would seek to validate the predictive power of this proof-of-concept study in a separate larger cohort of patients. PMID:22112112

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

    NASA Astrophysics Data System (ADS)

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

    2011-11-01

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

  20. In vivo fluorescence lifetime tomography

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

    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.

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

    Microsoft Academic Search

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

    2010-01-01

    Native fluorescence spectroscopy has shown potential to characterize and diagnose oral malignancy. We aim at extending the native fluorescence spectroscopy technique to characterize normal and oral submucous fibrosis (OSF) patients under pre- and post-treated conditions, and verify whether this method could also be considered in the monitoring of therapeutic prognosis noninvasively. In this study, 28 normal subjects and 28 clinically

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

    NASA Astrophysics Data System (ADS)

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

    2010-01-01

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

  3. Reflectance and fluorescence spectroscopies in photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Finlay, Jarod C.

    In vivo fluorescence spectroscopy during photodynamic therapy (PDT) has the potential to provide information on the distribution and degradation of sensitizers, the formation of fluorescent photoproducts and changes in tissue autofluorescence induced by photodynamic treatment. Reflectance spectroscopy allows quantification of light absorption and scattering in tissue. We present the results of several related studies of fluorescence and reflectance spectroscopy and their applications to photodynamic dosimetry. First, we develop and test an empirical method for the correction of the distortions imposed on fluorescence spectra by absorption and scattering in turbid media. We characterize the irradiance dependence of the in vivo photobleaching of three sensitizers, protoporphyrin IX (PpIX), Photofrin and mTHPC, in a rat skin model. The photobleaching and photoproduct formation of PpIX exhibit irradiance dependence consistent with singlet oxygen (1O2)-mediated bleaching. The bleaching of mTHPC occurs in two phases, only one of which is consistent with a 1O 2-mediated mechanism. Photofrin's bleaching is independent of irradiance, although its photoproduct formation is not. This can be explained by a mixed-mechanism bleaching model. Second, we develop an algorithm for the determination of tissue optical properties using diffuse reflectance spectra measured at a single source-detector separation and demonstrate the recovery of the hemoglobin oxygen dissociation curve from tissue-simulating phantoms containing human erythrocytes. This method is then used to investigate the heterogeneity of oxygenation response in murine tumors induced by carbogen inhalation. We find that while the response varies among animals and within each tumor, the majority of tumors exhibit an increase in blood oxygenation during carbogen breathing. We present a forward-adjoint model of fluorescence propagation that uses the optical property information acquired from reflectance spectroscopy to obtain the undistorted fluorescence spectrum over a wide range of optical properties. Finally, we investigate the ability of the forward-adjoint theory to extract undistorted fluorescence and optical property information simultaneously from a single measured fluorescence spectrum. This method can recover the hemoglobin oxygen dissociation curve in tissue-simulating phantoms with an accuracy comparable to that of reflectance-based methods while correcting distortions in the fluorescence over a wide range of absorption and scattering coefficients.

  4. Fluorescence of Photoreceptor Cells Observed in vivo

    Microsoft Academic Search

    N. Franceschini; K. Kirschfeld; B. Minke

    1981-01-01

    Most rhabdomeres in the eye of the fly (Musca domestica) are fluorescent. One kind of fluorescent emission emanates from a photoproduct of the visual pigment, other kinds may be ascribed to photostable pigments. These phenomena provide not only a means of spectrally mapping the retina but also a new spectroscopic tool for analyzing the primary visual processes in vivo.

  5. Fluorescence Spectroscopy in a Shoebox

    NASA Astrophysics Data System (ADS)

    Farooq Wahab, M.

    2007-08-01

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

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

    Microsoft Academic Search

    Scott T. McCain; Michael E. Gehm; Yanqia Wang

    2005-01-01

    Optical diagnostics in biological materials are hindered by fluorescence and scattering. We have developed a multimodal, multiplex, coded-aperture Raman spectrometer to detect alcohol in a lipid tissue phantom solution. Raman spectroscopy is a powerful diagnostic tool due to its high specificity and possibility for in vivo applications. At the same time, its very weak signal strength and incoherent scattering properties

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  8. Fluorescence spectroscopy applied to orange trees

    NASA Astrophysics Data System (ADS)

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

    2006-05-01

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

  9. Online fluorescence suppression in modulated Raman spectroscopy.

    PubMed

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

    2010-01-15

    Label-free chemical characterization of single cells is an important aim for biomedical research. Standard Raman spectroscopy provides intrinsic biochemical markers for noninvasive analysis of biological samples but is often hindered by the presence of fluorescence background. In this paper, we present an innovative modulated Raman spectroscopy technique to filter out the Raman spectra from the fluorescence background. The method is based on the principle that the fluorescence background does not change whereas the Raman scattering is shifted by the periodical modulation of the laser wavelength. Exploiting this physical property and importantly the multichannel lock-in detection of the Raman signal, the modulation technique fulfills the requirements of an effective fluorescence subtraction method. Indeed, once the synchronization and calibration procedure is performed, minimal user intervention is required, making the method online and less time-consuming than the other fluorescent suppression methods. We analyze the modulated Raman signal and shifted excitation Raman difference spectroscopy (SERDS) signal of 2 mum-sized polystyrene beads suspended in a solution of fluorescent dye as a function of modulation rate. We show that the signal-to-noise ratio of the modulated Raman spectra at the highest modulation rate is 3 times higher than the SERDS one. To finally evaluate the real benefits of the modulated Raman spectroscopy, we apply our technique to Chinese hamster ovary cells (CHO). Specifically, by analyzing separate spectra from the membrane, cytoplasm, and nucleus of CHO cells, we demonstrate the ability of this method to obtain localized sensitive chemical information from cells, away from the interfering fluorescence background. In particular, statistical analysis of the Raman data and classification using PCA (principal component analysis) indicate that our method allows us to distinguish between different cell locations with higher sensitivity and specificity, avoiding potential misinterpretation of the data obtained using standard background procedures. PMID:20017474

  10. Biomolecular shape and interactions determined by fluorescence correlation spectroscopy

    E-print Network

    Rippe, Karsten

    Biomolecular shape and interactions determined by fluorescence correlation spectroscopy J monitor an optical parameter such as absorbance, fluorescence intensity or depolarization, or circular 10 8 M -1 can be measured only with great difficulty because of the limited sensitivity. Fluorescence

  11. In vivo multiphoton fluorescence microscopy of epithelial precancer

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

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

    PubMed Central

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

    2012-01-01

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

  13. Ultraviolet, Visible, and Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Penner, Michael H.

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

  14. Long Range Surface Plasmon Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Kasry, Amal; Knoll, Wolfgang

    2007-03-01

    Surface plasmon modes, excited at the two sides of a thin metal layer surrounded by two (nearly) identical dielectric media interact via the overlap of their electromagnetic fields. This overlap results in two new-coupled modes, a short and a long-range surface plasmon (LRSP). We demonstrate that combining the LRSP optics with fluorescence spectroscopy can result in a huge enhancement of the fluorescence signal due to the enhanced optical field of the LRSP at the metal dielectric interface, and to its increased evanescent depth into the analyte. This was demonstrated for the detection of the fluorescence intensity of chromophore labeled protein bound to the surface sensor. Beside that, some fundamentals were studied leading to some interesting difference between SPFS and LRSPFS.

  15. Thermosensitive porphyrin-incorporated hydrogel with four-arm PEG-PCL copolymer: preparation, characterization and fluorescence imaging in vivo.

    PubMed

    Lv, Feng; Mao, Lina; Liu, Tianjun

    2014-10-01

    A biodegradable thermosensitive hydrogel based on four-arm PEG-PCL copolymer was prepared with porphyrin as a fluorescence tag. Its structure and composition were characterized by FTIR, (1)H NMR and GPC. Sol-gel-sol transition was evaluated by the test tube-inverting method and rheological analysis. The optical properties of hydrogel were investigated by UV-vis and fluorescence spectroscopy in vitro and by fluorescence imaging system in vivo. The results show that the thermosensitive hydrogel possesses dual function of fluorescence and injectability in vivo with good biocompatibility. Consequently it can be potentially applied in biomedical field as a visible implant for in situ monitoring. PMID:25175208

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

    PubMed Central

    Ahmed, Eilaf; Morton, Stephen W.

    2014-01-01

    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

  17. Theory and applications of fluorescence spectroscopy in food research

    Microsoft Academic Search

    Gale M. Strasburg; Richard D. Ludescher

    1995-01-01

    Fluorescence spectroscopy is a rapid, sensitive method for characterizing molecular environments and events. In spite of its utility, food researchers have been slow to adopt fluorescence methodology, partly because its value has gone unrecognized. This article presents a brief overview of the theory of fluorescence spectroscopy, together with some examples of applications of this technique to illustrate its potential for

  18. Fluorescence spectroscopy characteristics of nasopharyngeal carcinoma cells

    NASA Astrophysics Data System (ADS)

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

    2005-01-01

    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.

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

    PubMed Central

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

    2013-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

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

  1. Trp aporepressor engineered for fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    1992-04-01

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

  2. Two-Photon Fluorescence Correlation Spectroscopy

    NASA Technical Reports Server (NTRS)

    Zimmerli, Gregory A.; Fischer, David G.

    2002-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2004-07-01

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

  4. Time-resolved two-photon excited fluorescence spectroscopy based on a streak camera

    NASA Astrophysics Data System (ADS)

    Liu, Lixin; Qu, Junle; Chen, Danni; Lin, Ziyang; Xu, Gaixia; Guo, Baoping; Niu, Hanben

    2006-09-01

    Combination of fluorescence spectral and temporal resolutions can improve the sensitivity and specificity of biomedical diagnostics. In this paper, we present the development of a time resolved two-photon excited fluorescence spectroscopy system that consists of a Ti: Sapphire femtosecond laser, a fluorescence microscope objective, a prism spectrophotometer and a high repetition rate picosecond streak camera. The streak camera and the time-resolved fluorescence spectroscopy system. have been calibrated with an F-P etalon and a spectral line lamp respectively. Validation experiment of the system is also performed on two standard fluorescent dyes (Rhodamine 6G and Coumarin 314), and the results agree well with those reported in the literatures. Preliminary experimental results on autofluorescence spectra and lifetimes of freshly picked leaves and in vivo human skin are also presented, which demonstrates the potential applications of this system in tissue discrimination and clinical diagnostics.

  5. Fluorescence Correlation Spectroscopy: Past, Present, Future

    PubMed Central

    Elson, Elliot L.

    2011-01-01

    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. PMID:22208184

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

    PubMed

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

    2014-03-01

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

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

    PubMed Central

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

    2014-01-01

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

  8. Imaging cellular dynamics in vivo with multicolor fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Hoffman, Robert M.

    2005-04-01

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

  9. Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy

    Microsoft Academic Search

    Yan Chen; Li-Na Wei; Joachim D. Müller

    2003-01-01

    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

  10. Optimizing Disinfection Pretreatment using Excitation-emission Matrix Fluorescence Spectroscopy

    Microsoft Academic Search

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

    2012-01-01

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

  11. In vivo fluorescence imaging with high-

    E-print Network

    Schnitzer, Mark

    Messerschmidt2 & Mark J Schnitzer1,3 Micro-optics are increasingly used for minimally invasive in vivo imaging-photon imaging of dendritic spines on hippocampal neurons and dual-color nonlinear optical imaging with 920 nm laser excitation. The loss of resolution impairs image quality and hinders examination

  12. Fluorescence Spectroscopy Investigations of Cutaneous Tissues

    NASA Astrophysics Data System (ADS)

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

    2007-04-01

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

  13. In-vivo tissue characterization by Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Puppels, Gerwin J.; van Aken, Matthijs; Wolthuis, Rolf; Caspers, Peter J.; Bakker Schut, Tom C.; Bruining, Hajo A.; Roemer, Tjeerd J.; Buschman, Hendrik P. J.; Wach, Michael L.; Robinson, J. S., Jr.

    1998-04-01

    Vibrational spectroscopies hold great promise for applications in medical diagnosis, especially if they can be applied in vivo. Recent advances in flexible fiber probe design, enable good quality Raman spectra of tissue to be obtained in vivo. Here we illustrate this with Raman spectra of rat tissues, obtained ex vivo and in vivo.

  14. Imaging of the fluorescence spectrum of a single fluorescent molecule by prism-based spectroscopy

    Microsoft Academic Search

    Yoshikazu Suzuki; Tomomi Tani; Kazuo Sutoh; Shinji Kamimura

    2002-01-01

    We have devised a novel method to visualize the fluorescence spectrum of a single fluorescent molecule using prism-based spectroscopy. Equiping a total internal reflection microscope with a newly designed wedge prism, we obtained a spectral image of a single rhodamine red molecule attached to an essential light chain of myosin. We also obtained a spectral image of single-pair fluorescence resonance

  15. [Synthesis of byrazoline fluorescent compounds and studies by infrared spectroscopy].

    PubMed

    Xian, Yuan-fang; Li, Dong-feng; Li, Hai-dong; Yu, Bao-hui

    2005-03-01

    Benzothiazole compounds with byrazoline group or benximidazole group are new fluorescent compounds. The fluorescent compounds have been used in many fields, but their development has beeo slow. According to Schellhammer theory on the relation between chemical structure and fluorescent quality, the authors designed now fluorescent compounds with benximidazole group or the byrazoline group in the 1-benzothiazole and with biphenyl in the 3,5-benzothiazole, and their possess fluorescent nature. Two new benximidazole and benzothiazole fluorescent compounds were synthesized. All these compounds were characterized by elemental analysis and infrared spectroscopy. The excitation wavelength of the two compounds is about 441-446 nm. The fluorescence spectra show that the compounds have good blue and green fluorescence. The characteristic peaks of t he absorption spectra of these compounds werefound by IR spectral analysis, which can be used to deduce the structures of chemical compounds. PMID:16013314

  16. Fluorescence spectroscopy for rapid detection and classification of bacterial pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study deals with the rapid detection and classification of three bacteria, Escherichia coli, Salmonella, and Campylobacter, using fluorescence spectroscopy and multivariative analysis. Each bacterial sample was diluted in physiologic saline for analysis. Fluoroscence spectra were collected ...

  17. Effect of probe pressure on cervical fluorescence spectroscopy measurements

    E-print Network

    Nath, Audrey

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

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

    ERIC Educational Resources Information Center

    Healy, Eamonn F.

    2007-01-01

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

  19. Assaying protein import into mitochondria using fluorescence spectroscopy

    E-print Network

    Cargill, Holly Beth

    2006-08-16

    chemical crosslinking of Su9- DHFR Cys mutants. The use of fluorescence spectroscopy, in association with chemical crosslinking, to analyze the mitochondrial protein import pathways will prove a useful tool to probe the environment of the nascent chain...

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

    NASA Astrophysics Data System (ADS)

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

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

  1. In vivo validation of quantitative frequency domain fluorescence tomography

    NASA Astrophysics Data System (ADS)

    Lin, Yuting; Ghijsen, Michael; Nalcioglu, Orhan; Gulsen, Gultekin

    2012-12-01

    We have developed a hybrid frequency domain fluorescence tomography and magnetic resonance imaging system (MRI) for small animal imaging. The main purpose of this system is to obtain quantitatively accurate fluorescence concentration and lifetime images using a multi-modality approach. In vivo experiments are undertaken to evaluate the system. We compare the recovered fluorescence parameters with and without MRI structural a priori information. In addition, we compare two optical background heterogeneity correction methods: Born normalization and utilizing diffuse optical tomography (DOT) functional a priori information. The results show that the concentration and lifetime of a 4.2-mm diameter indocyanine green inclusion located 15 mm deep inside a rat can be recovered with less than a 5% error when functional a priori information from DOT and structural a priori information from MRI are utilized.

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

    NASA Astrophysics Data System (ADS)

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

    2010-07-01

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

  3. In vivo validation of quantitative frequency domain fluorescence tomography.

    PubMed

    Lin, Yuting; Ghijsen, Michael; Nalcioglu, Orhan; Gulsen, Gultekin

    2012-12-01

    We have developed a hybrid frequency domain fluorescence tomography and magnetic resonance imaging system (MRI) for small animal imaging. The main purpose of this system is to obtain quantitatively accurate fluorescence concentration and lifetime images using a multi-modality approach. In vivo experiments are undertaken to evaluate the system. We compare the recovered fluorescence parameters with and without MRI structural a priori information. In addition, we compare two optical background heterogeneity correction methods: Born normalization and utilizing diffuse optical tomography (DOT) functional a priori information. The results show that the concentration and lifetime of a 4.2-mm diameter indocyanine green inclusion located 15 mm deep inside a rat can be recovered with less than a 5% error when functional a priori information from DOT and structural a priori information from MRI are utilized. PMID:23323291

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

    PubMed Central

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

    2010-01-01

    A fully integrated trimodality fluorescence, diffuse optical, and x-ray computed tomography (FT?DOT?XCT) system for small animal imaging is reported in this work. The main purpose of this system is to obtain quantitatively accurate fluorescence concentration images using a multimodality approach. XCT offers anatomical information, while DOT provides the necessary background optical property map to improve FT image accuracy. The quantitative accuracy of this trimodality system is demonstrated in vivo. In particular, we show that a 2-mm-diam fluorescence inclusion located 8 mm deep in a nude mouse can only be localized when functional a priori information from DOT is available. However, the error in the recovered fluorophore concentration is nearly 87%. On the other hand, the fluorophore concentration can be accurately recovered within 2% error when both DOT functional and XCT structural a priori information are utilized together to guide and constrain the FT reconstruction algorithm. PMID:20799770

  5. In vivo validation of quantitative frequency domain fluorescence tomography

    PubMed Central

    Lin, Yuting; Ghijsen, Michael; Nalcioglu, Orhan; Gulsen, Gultekin

    2012-01-01

    Abstract. We have developed a hybrid frequency domain fluorescence tomography and magnetic resonance imaging system (MRI) for small animal imaging. The main purpose of this system is to obtain quantitatively accurate fluorescence concentration and lifetime images using a multi-modality approach. In vivo experiments are undertaken to evaluate the system. We compare the recovered fluorescence parameters with and without MRI structural a priori information. In addition, we compare two optical background heterogeneity correction methods: Born normalization and utilizing diffuse optical tomography (DOT) functional a priori information. The results show that the concentration and lifetime of a 4.2-mm diameter indocyanine green inclusion located 15 mm deep inside a rat can be recovered with less than a 5% error when functional a priori information from DOT and structural a priori information from MRI are utilized. PMID:23323291

  6. Ex Vivo Fluorescence Molecular Tomography of the Spine

    PubMed Central

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

    2012-01-01

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

  7. Recent Advances in Fluorescence Cross-correlation Spectroscopy

    Microsoft Academic Search

    Ling Chin Hwang; Thorsten Wohland

    2007-01-01

    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

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

    NASA Astrophysics Data System (ADS)

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

    1999-07-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2000-05-01

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

  10. Unfolding Features of Bovine Testicular Hyaluronidase Studied by Fluorescence Spectroscopy and Fourier Transformed Infrared Spectroscopy

    Microsoft Academic Search

    Nina Pan; Xiaoqiang Cai; Kai Tang; Guolin Zou

    2005-01-01

    Chemical unfolding of bovine testicular hyaluronidase (HAase) has been studied by fluorescence spectroscopy and Fourier transformed\\u000a infrared spectroscopy (FTIR). Thermodynamic parameters were determined for unfolding HAase from changes in the intrinsic fluorescence\\u000a emission intensity and the formations of several possible unfolding intermediates have been identified. This was further confirmed\\u000a by representation of fluorescence data in terms of ‘phase diagram’. The

  11. Fluorescence Correlation Spectroscopy: A Review of Biochemical and Microfluidic Applications

    PubMed Central

    Tian, Yu; Martinez, Michelle M.

    2011-01-01

    Over the years fluorescence correlation spectroscopy (FCS) has proven to be a useful technique that has been utilized in several fields of study. Although FCS initially suffered from poor signal to noise ratios, the incorporation of confocal microscopy has overcome this drawback and transformed FCS into a sensitive technique with high figures of merit. In addition, tandem methods have evolved to include dual-color cross-correlation, total internal reflection fluorescence correlation, and fluorescence lifetime correlation spectroscopy combined with time-correlated single photon counting. In this review, we discuss several applications of FSC for biochemical, microfluidic, and cellular investigations. PMID:21396180

  12. In vivo lipidomics using single-cell Raman spectroscopy

    PubMed Central

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

    2011-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-07-01

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

  14. Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

    PubMed Central

    Paredes, Jose M.; Casares, Salvador; Ruedas-Rama, Maria J.; Fernandez, Elena; Castello, Fabio; Varela, Lorena; Orte, Angel

    2012-01-01

    Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of ?-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies. PMID:22949804

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

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

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

  16. Deep tissue fluorescence imaging and in vivo biological applications

    NASA Astrophysics Data System (ADS)

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

    2012-11-01

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

  17. Laser-induced fluorescence spectroscopy at endoscopy

    NASA Astrophysics Data System (ADS)

    Qu, Jianan Y.; MacAulay, Calum E.; Lam, Stephen; Palcic, Branko

    1994-07-01

    A spectrofluorometry system has been developed for the collection of laser induced fluorescense spectra of tissue during endoscopy. In this system, a catheter with seven optical fibers was used to deliver the excitation light and collect the emitted fluorescence. The system enables one to switch from regular endoscopy into fluorescence measurement in 50 ms using a computerized shutter system. The fluorescence spectra can be recorded in 100 ms. This spectrofluorometry system has been used to obtain spectra from bronchial, larynx and nasopharyngeal tissues when employed with the appropriate endoscopes. The results demonstrate that laser induced fluorescence can be used to differentiate abnormal tissue from normal tissue. The illumination and fluorescence collection patterns of this system have been modeled using a Monte Carlo simulation. The Monte Carlo simulation data shows that the spectra recorded by our collection pattern is very close to the intrinsic spectra of tissue. The experimental results and the Monte Carlo simulation suggest that changes in fluorescence intensity are more robust for the detection of early cancers than the differences in spectral characteristics.

  18. In vivo infrared and Raman spectroscopy of human stratum corneum

    Microsoft Academic Search

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

    1998-01-01

    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

  19. In Vivo Blood Characterization From Bioimpedance Spectroscopy of Blood Pooling

    Microsoft Academic Search

    Tao Dai; Andy Adler

    2009-01-01

    Characterization of blood impedance properties is important to estimate clinical diagnostic indexes such as hematocrit, glucose level, and hydration. Current in vivo bioimpedance spectroscopy methods are performed on a body appendage and thus represent a combined measurement of all tissues in the measurement field, rather than the blood individually. This paper describes a novel in vivo measurement technique to calculate

  20. In Vivo Blood Characterization from Bioimpedance Spectroscopy of Blood

    E-print Network

    Adler, Andy

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

  1. [Rapid identification of hogwash oil by using synchronous fluorescence spectroscopy].

    PubMed

    Sun, Yan-Hui; An, Hai-Yang; Jia, Xiao-Li; Wang, Juan

    2012-10-01

    To identify hogwash oil quickly, the characteristic delta lambda of hogwash oil was analyzed by three dimensional fluorescence spectroscopy with parallel factor analysis, and the model was built up by using synchronous fluorescence spectroscopy with support vector machines (SVM). The results showed that the characteristic delta lambda of hogwash oil was 60 nm. Collecting original spectrum of different samples under the condition of characteristic delta lambda 60 nm, the best model was established while 5 principal components were selected from original spectrum and the radial basis function (RBF) was used as the kernel function, and the optimal penalty factor C and kernel function g were 512 and 0.5 respectively obtained by the grid searching and 6-fold cross validation. The discrimination rate of the model was 100% for both training sets and prediction sets. Thus, it is quick and accurate to apply synchronous fluorescence spectroscopy to identification of hogwash oil. PMID:23285875

  2. Use of NADH fluorescence to determine mitochondrial function in vivo.

    PubMed

    Mayevsky, Avraham; Barbiro-Michaely, Efrat

    2009-10-01

    Normal mitochondrial function is a critical factor in maintaining cellular homeostasis in various organs of the body. Due to the involvement of mitochondrial dysfunction in many pathological states, the real-time in vivo monitoring of the mitochondrial metabolic state is crucially important. This type of monitoring in animal models as well as in patients provides real-time data that can help interpret experimental results or optimize patient treatment. In this paper we are summarizing the following items: (1) presenting the solid scientific ground underlying nicotine amide adenine dinucleotide (NADH) NADH fluorescence measurements based on published materials. (2) Presenting NADH fluorescence monitoring and its physiological significance. (3) Providing the reader with basic information on the methodologies of the fluorometers reflectometers. (4) Clarifying various factors affecting the monitored signals, including artifacts. (5) Presenting the potential use of monitoring mitochondrial function in vivo for the evaluation of drug development. The large numbers of publications by different groups testify to the valuable information gathered in various experimental conditions. The monitoring of NADH levels in the tissue provides the most important information on the metabolic state of the mitochondria in terms of energy production and intracellular oxygen levels. Although NADH signals are not calibrated in absolute units, their trend monitoring is important for the interpretation of physiological or pathological situations. To better understand the tissue function, the multiparametric approach has been developed where NADH serves as the key parameter to be monitored. PMID:19703658

  3. Fluorescence Imaging In Vivo up to 1700 nm

    E-print Network

    Diao, Shuo; Hong, Guosong; Antaris, Alexander L; Chang, Junlei; Wu, Justin Z; Zhang, Bo; Kuo, Calvin J; Dai, Hongjie

    2015-01-01

    Compared to visible and near-infrared regions below ~ 900 nm, imaging in the second near-infrared window beyond 1000 nm (NIR-II, 1000-1700 nm) is promising for deep-tissue high-resolution optical imaging in vivo owing to reduced scattering of photons traversing through tissues. Here, we succeeded fluorescence imaging in vivo in the long 1500-1700 nm (NIR-IIb) region using a novel, chemical separation enriched large-diameter semiconducting single-walled carbon nanotube material. Imaging in the 1500-1700 nm window resolved 3-4 um wide capillary blood vessels at ~ 3 millimeters depth through the intact body and brain of mice with the ability of blood-flow speed mapping in individual capillary vessels. Further, non-invasive single fluorophore imaging inside the tumor of a live mouse was achieved in the 1500-1700 nm window. NIR-IIb imaging can be generalized to a wide range of fluorophores emitting up to 1700 nm for a new paradigm of high performance in vivo optical imaging.

  4. Trp aporepressor engineered for fluorescence spectroscopy

    Microsoft Academic Search

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

    1992-01-01

    The tryptophan repressor from Escherichia coli binds to the trp operator in the presence of L- tryptophan, thereby inhibiting the biosynthesis of L-tryptophan. Site-directed mutagenesis was used to change tryptophan-19 and tryptophan-99 to leucine and methionine, respectively. This mutant protein without tryptophan in its amino acid sequence has wild-type repressor activity and is a suitable model for fluorescence studies of

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

    ERIC Educational Resources Information Center

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

    2014-01-01

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

  6. Development and Application of Optical Spectroscopies for In Vivo Cancer Diagnostics

    NASA Astrophysics Data System (ADS)

    Wilson, Brian C.

    2000-06-01

    Several complementary optical spectroscopies are under development for non-invasive tissue diagnostics, based on diffuse reflectance, fluorescence or Raman scattering. These techniques yield information on the surface or subsurface tissue microstructure and/or biochemical composition. For many clinical applications, minimally-invasive measurements can be obtained using fiberoptic-based instrumentation. This will be illustrated for the particular case of endoscopic detection of early cancer. In addition, for fluorescence, real-time imaging is possible and can be used either for cancer detection or to guide surgery. Significant progress has been made, using detailed microscopy of tissue samples ex vivo, in understanding the biological changes in tissue which can be exploited by non-invasive methods in vivo. However, significant challenges remain, both in further developing these biophysical techniques and in the design and construction of realistic clinical devices and systems.

  7. Fibre optic fluorescence spectroscopy for monitoring fish freshness

    NASA Astrophysics Data System (ADS)

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

    2012-01-01

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

  8. Emerging biomedical applications of time-resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    1994-07-01

    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.

  9. Sucrose monoester micelles size determined by Fluorescence Correlation Spectroscopy (FCS).

    PubMed

    Sanchez, Susana A; Gratton, Enrico; Zanocco, Antonio L; Lemp, Else; Gunther, German

    2011-01-01

    One of the several uses of sucrose detergents, as well as other micelle forming detergents, is the solubilization of different membrane proteins. Accurate knowledge of the micelle properties, including size and shape, are needed to optimize the surfactant conditions for protein purification and membrane characterization. We synthesized sucrose esters having different numbers of methylene subunits on the substituent to correlate the number of methylene groups with the size of the corresponding micelles. We used Fluorescence Correlation Spectroscopy (FCS) and two photon excitation to determine the translational D of the micelles and calculate their corresponding hydrodynamic radius, R(h). As a fluorescent probe we used LAURDAN (6-dodecanoyl-2-dimethylaminonaphthalene), a dye highly fluorescent when integrated in the micelle and non-fluorescent in aqueous media. We found a linear correlation between the size of the tail and the hydrodynamic radius of the micelle for the series of detergents measured. PMID:22216230

  10. Fluorescence fluctuation spectroscopy and its artifacts: simulations and tests

    NASA Astrophysics Data System (ADS)

    Meng, Fanbo; Chen, Bo; Liu, Guang; Ding, Jianying; Ma, Hui

    2005-05-01

    Fluorescence fluctuation spectroscopy (FFS) technique is capable of monitoring changes in concentration, mass, size and structure of fluorescent-labeled bio-molecules in microscopic volume and is suitable for measuring biological interactions in living cells. FFS data may be affected by many experimental factors in complicated biological systems. Using a Monte Carlo approach, we generate fluorescence fluctuation data for different experimental systems. This approach helps to separate the contributions by different experimental factors in a complicated fluorescence fluctuation spectrum. It also helps to validate new theoretical models and new fitting formulations. We describe the algorithm of the simulation program and tests on its statistical performance. The program is then used successfully to study the effects of several experimental factors on FFS detection.

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

    Microsoft Academic Search

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

    1999-01-01

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

  12. Fluorescence spectroscopy of tissue: recovery of intrinsic fluorescence from measured fluorescence

    NASA Astrophysics Data System (ADS)

    Gardner, Craig M.; Jacques, Steven L.; Welch, Ashley J.

    1996-04-01

    We present a method for recovering the intrinsic fluorescence coefficient, defined as the product of the fluorophore absorption coefficient and the fluorescence energy yield, of an optically thick, homogeneous, turbid medium from a surface measurement of fluorescence and from knowledge of medium optical properties. The measured fluorescence signal is related to the intrinsic fluorescence coefficient by an optical property dependent path-length factor. A simple expression was developed for the path-length factor, which characterizes the penetration of excitation light and the escape of fluorescence from the medium. Experiments with fluorescent tissue phantoms demonstrated that intrinsic fluorescence line shape could be recovered and that fluorophore concentration could be estimated within +/-15%, over a wide range of optical properties. transport, photodynamic therapy, photosensitizer.

  13. Laser induced fluorescence spectroscopy of ruthenium monoboride

    NASA Astrophysics Data System (ADS)

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

    2012-09-01

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

  14. Ultrasensitive molecular fluorescence spectroscopy in levitated microdroplets

    SciTech Connect

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

    1990-01-01

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

  15. Fluorescence correlation spectroscopy: Diagnostics for sparse?molecules

    PubMed Central

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

    1997-01-01

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

  16. Surface-plasmon field-enhanced fluorescence spectroscopy

    Microsoft Academic Search

    Thorsten Liebermann; Wolfgang Knoll

    2000-01-01

    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

  17. The spectroscopic basis of Fluorescence Triple Correlation Spectroscopy

    PubMed Central

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

    2012-01-01

    We have developed Fluorescence Triple Correlation Spectroscopy (F3CS) as an extension of the widely-used fluorescence microscopy technique Fluorescence Correlation Spectroscopy. F3CS correlates three signals at once and provides additional capabilities for the study of systems with complex stoichiometry, kinetic processes and irreversible reactions. A general theory of F3CS was developed to describe the interplay of molecular dynamics and microscope optics, leading to an analytical function to predict experimental triple correlations of molecules that freely diffuse through the tight focus of the microscope. Experimental correlations were calculated from raw fluorescence data using triple correlation integrals that extend multiple-tau correlation theory to delay times in two dimensions. The quality of experimental data was improved by tuning specific spectroscopic parameters and employing multiple independent detectors to minimize optoelectronic artifacts. Experiments with the reversible system of freely-diffusing 16S rRNA revealed that triple correlation functions contain symmetries predicted from time-reversal arguments. Irreversible systems are shown to break these symmetries and correlation strategies were developed to detect time-reversal asymmetries in a comprehensive way with respect to two delay times, each spanning many orders of magnitude in time. The correlation strategies, experimental approaches and theory developed here enable studies of the composition and dynamics of complex systems using F3CS. PMID:22229664

  18. Quantitative in vivo solubility and reconstitution of truncated circular permutants of green fluorescent protein

    PubMed Central

    Huang, Yao-Ming; Nayak, Sasmita; Bystroff, Christopher

    2011-01-01

    Several versions of split green fluorescent protein (GFP) fold and reconstitute fluorescence, as do many circular permutants, but little is known about the dependence of reconstitution on circular permutation. Explored here is the capacity of GFP to fold and reconstitute fluorescence from various truncated circular permutants, herein called “leave-one-outs” using a quantitative in vivo solubility assay and in vivo reconstitution of fluorescence. Twelve leave-one-out permutants are discussed, one for each of the 12 secondary structure elements. The results expand the outlook for the use of permuted split GFPs as specific and self-reporting gene encoded affinity reagents. PMID:21910151

  19. Ultrasensitive fluorescence correlation spectroscopy of highly parallelized microfluidic devices

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2003-07-01

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

  2. Fluorescence Fluctuation Spectroscopy of mCherry in Living Cells

    PubMed Central

    Wu, Bin; Chen, Yan; Müller, Joachim D.

    2009-01-01

    The red fluorescent protein mCherry is of considerable interest for fluorescence fluctuation spectroscopy (FFS), because the wide separation in color between mCherry and green fluorescent protein provides excellent conditions for identifying protein interactions inside cells. This two-photon study reveals that mCherry exists in more than a single brightness state. Unbiased analysis of the data needs to account for the presence of multiple states. We introduce a two-state model that successfully describes the brightness and fluctuation amplitude of mCherry. The properties of the two states are characterized by FFS and fluorescence lifetime experiments. No interconversion between the two states was observed over the experimentally probed timescales. The effect of fluorescence resonance energy transfer between enhanced green fluorescent protein (EGFP) and mCherry is incorporated into the two-state model to describe protein hetero-oligomerization. The model is verified by comparing the predicted and measured brightness and fluctuation amplitude of several fusion proteins that contain mCherry and EGFP. In addition, hetero-fluorescence resonance energy transfer between mCherry molecules in different states is detected, but its influence on FFS parameters is small enough to be negligible. Finally, the two-state model is applied to study protein oligomerization in living cells. We demonstrate that the model successfully describes the homodimerization of nuclear receptors. In addition, we resolved a mixture of interacting and noninteracting proteins labeled with EGFP and mCherry. These results provide the foundation for quantitative applications of mCherry in FFS studies. PMID:19289064

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

    Microsoft Academic Search

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

    2009-01-01

    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

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

    NASA Astrophysics Data System (ADS)

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

    2011-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2010-12-01

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

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

    NASA Astrophysics Data System (ADS)

    Zheng, Longjiang; Hu, Yuanting

    2009-07-01

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

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

    SciTech Connect

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

    2014-03-15

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

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

    PubMed

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

    2014-03-01

    The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 ?m diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8-7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence lifetime measurements of low quantum efficiency sub-nanosecond fluorophores. PMID:24689603

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

    Microsoft Academic Search

    Tammy E. Starr; Nancy L. Thompson

    2001-01-01

    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

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

    NASA Astrophysics Data System (ADS)

    Makoui, Anali

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

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2003-07-01

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

  13. Fluorescence spectroscopy for endogenous porphyrins in human facial skin

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

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

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

    NASA Astrophysics Data System (ADS)

    Harbater, Osnat; Gannot, Israel

    2015-03-01

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

  15. Real-time in vivo cancer diagnosis using raman spectroscopy.

    PubMed

    Wang, Wenbo; Zhao, Jianhua; Short, Michael; Zeng, Haishan

    2015-07-01

    Raman spectroscopy has becoming a practical tool for rapid in vivo tissue diagnosis. This paper provides an overview on the latest development of real-time in vivo Raman systems for cancer detection. Instrumentation, data handling, as well as oncology applications of Raman techniques were covered. Optic fiber probes designs for Raman spectroscopy were discussed. Spectral data pre-processing, feature extraction, and classification between normal/benign and malignant tissues were surveyed. Applications of Raman techniques for clinical diagnosis for different types of cancers, including skin cancer, lung cancer, stomach cancer, oesophageal cancer, colorectal cancer, cervical cancer, and breast cancer, were summarized. Schematic of a real-time Raman spectrometer for skin cancer detection. Without correction, the image captured on CCD camera for a straight entrance slit has a curvature. By arranging the optic fiber array in reverse orientation, the curvature could be effectively corrected. PMID:25220508

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

    NASA Astrophysics Data System (ADS)

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

    1999-04-01

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

  17. Time-domain laser-induced fluorescence spectroscopy apparatus for clinical diagnostics

    NASA Astrophysics Data System (ADS)

    Fang, Qiyin; Papaioannou, Thanassis; Jo, Javier A.; Vaitha, Russel; Shastry, Kumar; Marcu, Laura

    2004-01-01

    We report the design and development of a compact optical fiber-based apparatus for in situ time-resolved laser-induced fluorescence spectroscopy (tr-LIFS) of biological systems. The apparatus is modular, optically robust, and compatible with the clinical environment. It incorporates a dual output imaging spectrograph, a gated multichannel plate photomultiplier (MCP-PMT), an intensified charge-coupled-device (ICCD) camera, and a fast digitizer. It can accommodate various types of light sources and optical fiber probes for selective excitation and remote light delivery/collection as required by different applications. The apparatus allows direct recording of the entire fluorescence decay with high sensitivity (nM range fluorescein dye concentration with signal-to-noise ratio of 46) and with four decades dynamic range. It is capable of resolving a broad range of fluorescence lifetimes from hundreds of picoseconds (as low as 300 ps) using the MCP-PMT coupled to the digitizer to milliseconds using the ICCD. The data acquisition and analysis process is fully automated, enabling fast recording of fluorescence intensity decay across the entire emission spectrum (0.8 s per wavelength or ˜40 s for a 200 nm wavelength range at 5 nm increments). The spectral and temporal responses of the apparatus were calibrated and its performance was validated using fluorescence lifetime standard dyes (Rhodamin B, 9-cyanoanthracene, and rose Bengal) and tissue endogenous fluorophores (elastin, collagen, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide). Fluorescence decay lifetimes and emission spectra of all tested compounds measured with the current tr-LIFS apparatus were found in good agreement with the values reported in the literature. The design and performance of tr-LIFS apparatus have enabled in vivo studies of atherosclerotic plaques and brain tumors.

  18. Quantum process tomography by 2D fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

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

  19. Quantum process tomography by 2D fluorescence spectroscopy.

    PubMed

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

    2015-06-01

    Reconstruction of the dynamics (quantum process tomography) of the single-exciton manifold in energy transfer systems is proposed here on the basis of two-dimensional fluorescence spectroscopy (2D-FS) with phase-modulation. The quantum-process-tomography protocol introduced here benefits from, e.g., the sensitivity enhancement ascribed to 2D-FS. Although the isotropically averaged spectroscopic signals depend on the quantum yield parameter ? of the doubly excited-exciton manifold, it is shown that the reconstruction of the dynamics is insensitive to this parameter. Applications to foundational and applied problems, as well as further extensions, are discussed. PMID:26049462

  20. Optical fiber fluorescence spectroscopy for detecting AFM1 in milk

    NASA Astrophysics Data System (ADS)

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

    2008-04-01

    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.

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

    Microsoft Academic Search

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

    2009-01-01

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

  2. Identification of active fluorescence stained bacteria by Raman spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2008-04-01

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

  3. Detectors for single-molecule fluorescence imaging and spectroscopy

    PubMed Central

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

    2010-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-04-01

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

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

    PubMed Central

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

    2010-01-01

    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

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

    PubMed

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

    2015-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1997-12-01

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

  8. Fluorescence Spectroscopy and Chemometric Modeling for Bioprocess Monitoring

    PubMed Central

    Faassen, Saskia M.; Hitzmann, Bernd

    2015-01-01

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

  9. Fluorescence spectroscopy and chemometric modeling for bioprocess monitoring.

    PubMed

    Faassen, Saskia M; Hitzmann, Bernd

    2015-01-01

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

  10. Measurement of Formaldehyde by Laser Induced Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Cryer, D. R.; Ingham, T.; Whalley, L. K.; Heard, D. E.

    2014-12-01

    Gas phase formaldehyde (HCHO) is a key species in the troposphere. It is formed as an intermediate product during the removal of almost all volatile organic compounds (VOCs) by the hydroxyl radical (OH) and is a tracer of overall oxidising capacity. A new instrument has been developed for the measurement of [HCHO] by laser induced fluorescence (LIF) spectroscopy and deployed in the field. Ultra-violet (UV) radiation from a tuneable fibre laser was used to excite HCHO in a low pressure cell (~130 Torr) at ca. 353 nm with fluorescence collected between 390 - 550 nm. The resulting fluorescence was detected by a photomultiplier tube (PMT) and processed by photon counting techniques. The instrument performance will be described in detail in addition to a novel calibration method where a known quantity of HCHO was produced through photolysis of methanol (CH3OH) vapour in the presence of oxygen. The instrument was first deployed in June 2014 at a suburban site in York (UK). Data from this campaign and interpretation will be presented in addition to observations from more recent field measurements.

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

    PubMed Central

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

    2013-01-01

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

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

    Microsoft Academic Search

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

    2000-01-01

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

  13. Evaluation of fiber optic probes for in-vivo Raman spectroscopy

    Microsoft Academic Search

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

    1998-01-01

    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

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

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

    PubMed

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

    2014-01-01

    Fluorescence is a powerful tool for in-vivo imaging in living animals. The traditional in-vivo fluorescence imaging equipment is based on single-view two-dimensional imaging systems. However, they cannot meet the needs for accurate positioning during modern scientific research. A near-infrared in-vivo fluorescence imaging system is demonstrated, which has the capability of deep source signal detecting and three-dimensional positioning. A three-dimensional coordinates computing (TDCP) method including a preprocess algorithm is presented based on binocular stereo vision theory, to figure out the solution for diffusive nature of light in tissue and the emission spectra overlap of fluorescent labels. This algorithm is validated to be efficient to extract targets from multispectral images and determine the spot center of biological interests. Further data analysis indicates that this TDCP method could be used in three-dimensional positioning of the fluorescent target in small animals. The study also suggests that the combination of a large power laser and deep cooling charge-coupled device will provide an attractive approach for fluorescent detection from deep sources. This work demonstrates the potential of binocular stereo vision theory for three-dimensional positioning for living animal in-vivo imaging. PMID:25364949

  16. Deep tissue fluorescence imaging and in vivo biological applications

    E-print Network

    2012-01-01

    better adapted to image live small animals. The experimentalimage normal and diseased tissue in the whole live animal,animals. Using this system we were able to successfully obtain high resolution fluorescence images

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

    PubMed Central

    Palmer, A G; Thompson, N L

    1987-01-01

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

  18. Excited state dynamics of dGMP measured by steady-state and femtosecond fluorescence spectroscopy

    E-print Network

    Boyer, Edmond

    -state fluorescence spectrum of dGMP shows one band centered at 334 nm but has an extraordinary long red tail spectrocopy.7,13,16 In ref. 16 it was noted that the steady-state fluorescence spectrum of the guanine fluorescence spectroscopy Francois-Alexandre Miannay, Thomas Gustavsson, Akos Banyasz and Dimitra Markovitsi

  19. Fluorescence spectroscopy: an adjunct diagnostic tool to image-guided core needle biopsy of the breast.

    PubMed

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

    2009-10-01

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

  20. A new fluorescent imaging procedure in vivo for evaluation of the retinal microcirculation in rats.

    PubMed

    Kimura, H; Kiryu, J; Nishiwaki, H; Ogura, Y

    1995-03-01

    We investigated a new method for in vivo evaluation of the retinal microcirculation in rats using a cell-permeant fluorescent dye, acridine orange (AO), which stains cell nuclei and cytoplasm, and a scanning laser ophthalmoscope (SLO). AO, which binds and interacts with DNA and RNA, and thus stains cell nuclei and cytoplasm, was administered intravenously to rats. Fluorescein angiography was performed after administration of the AO, and fundus images were recorded on S-VHS videotape by means of an SLO. Argon laser was used as an exciter of the dye. The retinal vessels were stained with the dye, rendering the retinal microvasculature clearly visible. Cell nuclei and vessel walls were observed as greater fluorescence and lesser fluorescence, respectively. Leukocytes were also observed as highly fluorescent dots moving through the vessels. The results suggest that SLO visualization of AO uptake by cells may be a useful procedure for the evaluation of retinal microcirculation in vivo in rats. PMID:7796605

  1. Dynamic and reversible fluorescence imaging of superoxide anion fluctuations in live cells and in vivo.

    PubMed

    Zhang, Wen; Li, Ping; Yang, Fan; Hu, Xiufen; Sun, Chuanzhi; Zhang, Wei; Chen, Dezhan; Tang, Bo

    2013-10-01

    Overgeneration of reactive oxygen species (ROS) is closely associated with cellular damage and diseases. As superoxide anion (O2(•-)) is the precursor of other ROS, exploring O2(•-) fluctuations in cells and in vivo is of great significance. To address this critical need, we have developed a novel reversible fluorescent probe with one-photon and two-photon fluorescence properties, which is well suited for monitoring O2(•-) fluxes selectively and dynamically. Imaging results substantiate dynamic and reversible fluorescence responses of this probe to intracellular O2(•-) under apoptotic stimuli. Moreover, this probe can conveniently visualize changes in O2(•-) concentration during reperfusion injury in hepatocytes, zebrafish, and mice, by means of one-photon or two-photon imaging according to depths of various samples. The present study provides a powerful fluorescent imaging tool for dynamic tracking of O2(•-) in live cells and in vivo. PMID:24059644

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

    PubMed

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

    2012-02-14

    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

  3. Electrically induced microflows probed by fluorescence correlation spectroscopy.

    PubMed

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

    2005-03-01

    We report on the experimental characterisation of electrically induced flows at the micrometer scale through Fluorescence Correlation Spectroscopy (FCS) measurements. We stress the potential of FCS as a useful characterisation technique in microfluidics devices for transport properties cartography. The experimental results obtained in a model situation are in agreement with previous calculations (F. Nadal, F. Argoul, P. Kestener, B. Pouligny, C. Ybert, A. Ajdari, Eur. Phys. J. E 9, 387 (2002)) predicting the structure and electric-field dependency of the induced flow. Additionally, the present study evidences a complex behaviour of the probe nanobeads under electric field whose precise understanding might prove relevant for situations where nano-objects interact with an external electric field. PMID:15660186

  4. H4 tail interactions revealed by fluorescent fluctuation spectroscopy

    NASA Astrophysics Data System (ADS)

    Nurse, Nathan; Yuan, Chongli

    2015-03-01

    Post-translational modifications to histone tails nave been shown to play a large role in dictating the conformation of chromatin. Structural changes in chromatin can play a large role in gene expression as compact chromatin can occlude transcriptional machinery. The role of the flexible regions of H4 N terminal tails is investigated using fluorescent correlation spectroscopy and the photon counting histogram. The combination of these techniques allows for the distinction between intra-array and inter-array interactions, as well as reveals structural changes that result from these interactions. The H4 tail was found to partake in attractive intra-array interactions that compact the 6x167 nucleosome arrays but did not partake in inter-array interactions that lead to oligomerization.

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

    PubMed Central

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

    2013-01-01

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

  6. Autofluorescence spectroscopy of colorectal carcinoma: ex vivo study

    NASA Astrophysics Data System (ADS)

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

    2003-10-01

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

  7. Fluorescence correlation spectroscopy: Statistical analysis and biological applications

    NASA Astrophysics Data System (ADS)

    Saffarian, Saveez

    2002-01-01

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

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

    PubMed

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

    2015-03-01

    Organization and functions of the nucleolus is maintained by mobilities and interactions of nucleolar factors. Because the nucleolus is a densely packed structure, molecular crowding effects determined by the molecular concentrations and mobilities in the nucleolus should also be important for regulating nucleolar organization and functions. However, such molecular property of nucleolar organization is not fully understood. To understand the biophysical property of nucleolar organization, the diffusional behaviors of inert green fluorescent protein (GFP) oligomers with or without nuclear localization signals (NLSs) were analyzed under various conditions by fluorescence correlation spectroscopy. Our result demonstrates that the mobility of GFPs inside the nucleolus and the nucleoplasm can be represented by single free diffusion under normal conditions, even though the mobility in the nucleolus is considerably slower than that in the chromatin region. Moreover, the free diffusion of GFPs is found to be significantly size- and NLS-dependent only in the nucleolus. Interestingly, the mobility in the nucleolus is highly sensitive to ATP depletion, as well as actinomycin D (ActD) treatment. In contrast, the ultra-structure of the nucleolus was not significantly changed by ATP depletion but was changed by ActD treatment. These results suggest that the nucleolus behaves similarly to an open aqueous-phase medium with an increased molecular crowding effect that depends on both energy and transcription. PMID:25404711

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

    NASA Astrophysics Data System (ADS)

    Zettergren, Eric William

    2011-12-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

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

    Microsoft Academic Search

    Guowen Zhang; Qingmin Que; Junhui Pan; Jinbao Guo

    2008-01-01

    The interaction between icariin and human serum albumin (HSA) in physiological buffer (pH 7.4) was investigated by fluorescence and UV–Vis absorption spectroscopy. Results obtained from analysis of fluorescence spectrum and fluorescence intensity indicated that icariin has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The thermodynamic parameters, ?H? and ?S?, were calculated to

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

    PubMed

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

    2012-11-21

    We report here the soft nanomaterial-based targeting polymersomes for near-infrared (NIR) fluorescence imaging to carry out in vivo tumor detection. Two polymersome-based NIR fluorescent probes were prepared through the self-assembly of amphiphilic block copolymers, poly(butadiene-b-ethylene oxide) (PEO-b-PBD). Each of them was encapsulated with distinct hydrophobic near-infrared dyes (DiD and DiR) and modified with different targeting ligands (anti-CEA antibody and anti-EGFR antibody), respectively. After simultaneous injection of these two probes into the tumor-bearing mice via tail vein, multispectral near-infrared fluorescence images were obtained. The results indicate that both probes are successfully directed to the tumor foci, where two distinguishable fluorescent signals were detected through the unmixed fluorescence images. By taking advantage of two targeting polymersome-based probes with distinct fluorescent features, the proposed multispectral near-infrared fluorescence imaging method can greatly improve the specificity and accuracy for in vivo tumor detection. PMID:23069779

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

    NASA Astrophysics Data System (ADS)

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

    2012-10-01

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

  14. receptor dynamics in vivo Knockin mice expressing fluorescent {delta}-opioid receptors uncover G protein-coupled

    E-print Network

    Steinbach, Joe Henry

    receptor dynamics in vivo Knockin mice expressing fluorescent {delta}-opioid receptors uncover G.pnas.org/misc/reprints.shtml To order reprints, see: Notes: #12;Knockin mice expressing fluorescent -opioid receptors uncover G protein functional imaging of a G protein- coupled receptor (GPCR) in vivo. We created mice where the -opioid

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

    NASA Astrophysics Data System (ADS)

    Glassman, Wenling Sha

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

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

    PubMed Central

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

    2011-01-01

    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. PMID:21904601

  18. The effects of flow rate on in vivo fluorescence measurements

    E-print Network

    Sweet, Stephen Thomas

    1988-01-01

    adaptation period, as a function of illumination time (Kautsky and Hirsch, 1931). Kiefer (1973a) showed the diatom Lauderia borealis had a two phase photoinhibitory response to intense solar radiation as a result of self-shading by the chloroplasts... regimes on Lauderio borealis and found a photoinhibitory response which lacked the slow phase, Heaney (1978) observed photo-inhibition of in uivo fluorescence for laboratory cultures of Asterionello formosa and natural populations dominated by Cerotium...

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

    NASA Astrophysics Data System (ADS)

    Ma, Ke

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

  20. Biocompatible fluorescent nanoparticles for in vivo stem cell tracking.

    PubMed

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

    2013-06-21

    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

  1. Comparison between Visual Examination and a Laser Fluorescence System for in vivo Diagnosis of Occlusal Caries

    Microsoft Academic Search

    E. C. Sheehy; S. R. Brailsford; E. A. M. Kidd; D. Beighton; L. Zoitopoulos

    2001-01-01

    This study compared a laser fluorescence (LF) system (DIAGNOdent) with a visual caries scoring system for in vivo detection and diagnosis of occlusal caries under the conditions of an epidemiological study, in 132 mandibular and 38 maxillary first permanent molars in 170 children (mean age: 6.85 ± 0.58 years). The teeth were cleaned and occlusal caries status in a selected

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2003-10-01

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

  7. In Vivo Time-gated Fluorescence Imaging with Biodegradable Luminescent Porous Silicon Nanoparticles

    PubMed Central

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

    2014-01-01

    Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 ?s) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (< 10 ns) emission signals from organic chromophores or tissue autofluorescence.Here, using a conventional animal imaging system not optimized for such long-lived excited states, we demonstrate improvement of signal to background contrast ratio by > 50-fold in vitro and by > 20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed. PMID:23933660

  8. In vivo inflammation imaging using a CB2R-targeted near infrared fluorescent probe

    PubMed Central

    Zhang, Shaojuan; Shao, Pin; Ling, Xiaoxi; Yang, Ling; Hou, Weizhou; Thorne, Steve H; Beaino, Wissam; Anderson, Carolyn J; Ding, Ying; Bai, Mingfeng

    2015-01-01

    Chronic inflammation is considered as a critical cause of a host of disorders, such as cancer, rheumatoid arthritis, atherosclerosis, and neurodegenerative diseases, although the exact mechanism is yet to be explored. Imaging tools that can specifically target inflammation are therefore important to help reveal the role of inflammation in disease progression, and allows for developing new therapeutic strategies to ultimately improve patient care. The purpose of this study was to develop a new in vivo inflammation imaging approach by targeting the cannabinoid receptor type 2 (CB2R), an emerging inflammation biomarker, using a unique near infrared (NIR) fluorescent probe. Herein, we report the first in vivo CB2R-targeted NIR inflammation imaging study using a synthetic fluorescent probe developed in our laboratory, NIR760-mbc94. In vitro binding assay and fluorescence microscopy study indicate NIR760-mbc94 specifically binds towards CB2R in mouse RAW264.7 macrophage cells. Furthermore, in vivo imaging was performed using a Complete Freund’s Adjuvant (CFA)-induced inflammation mouse model. NIR760-mbc94 successfully identified inflamed tissues and the probe uptake was blocked by a CB2R ligand, SR144528. Additionally, immunofluorescence staining in cryosectioned tissues validated the NIR760-mbc94 uptake in inflamed tissues. In conclusion, this study reports the first in vivo CB2R-targeted inflammation imaging using an NIR fluorescent probe. Specific targeting of NIR760-mbc94 has been demonstrated in macrophage cells, as well as a CFA-induced inflammation mouse model. The combined evidence indicates that NIR760-mbc94 is a promising inflammation imaging probe. Moreover, in vivo CB2R-targeted fluorescence imaging may have potential in the study of inflammation-related diseases.

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

    PubMed Central

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

    2015-01-01

    Background Visceral leishmaniasis (VL) is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years. Methodology/Principal findings We generated two infrared fluorescent L. infantum strains, which stably overexpress the IFP 1.4 and iRFP reporter genes and performed comparative studies of their biophotonic properties at both promastigote and amastigote stages. To improve the fluorescence emission of the selected reporter in intracellular amastigotes, we engineered distinct constructs by introducing regulatory sequences of differentially-expressed genes (A2, AMASTIN and HSP70 II). The final strain that carries the iRFP gene under the control of the L. infantum HSP70 II downstream region (DSR), was employed to perform a phenotypic screening of a collection of small molecules by using ex vivo splenocytes from infrared-infected BALB/c mice. In order to further investigate the usefulness of this infrared strain, we monitored an in vivo infection by imaging BALB/c mice in a time-course study of 20 weeks. Conclusions/Significance The near-infrared fluorescent L. infantum strain represents an important step forward in bioimaging research of VL, providing a robust model of phenotypic screening suitable for HTS of small molecule collections in the mammalian parasite stage. Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL. This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic disease. PMID:25826250

  10. Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy

    E-print Network

    G. Meacci; J. Ries; E. Fischer-Friedrich; N. Kahya; P. Schwille; K. Kruse

    2007-01-29

    In the bacterium Escherichia coli, selection of the division site involves pole-to-pole oscillations of the proteins MinD and MinE. Different oscillation mechanisms based on cooperative effects between Min-proteins and on the exchange of Min-proteins between the cytoplasm and the cytoplasmic membrane have been proposed. The parameters characterizing the dynamics of the Min-proteins in vivo are not known. It has therefore been difficult to compare the models quantitatively with experiments. Here, we present in vivo measurements of the mobility of MinD and MinE using fluorescence correlation spectroscopy. Two distinct time-scales are clearly visible in the correlation curves. While the faster time-scale can be attributed to cytoplasmic diffusion, the slower time-scale could result from diffusion of membrane-bound proteins or from protein exchange between the cytoplasm and the membrane. We determine the diffusion constant of cytoplasmic MinD to be approximately 16\\mu^{2}/s, while for MinE we find about 10\\mu^{2}/s, independently of the processes responsible for the slower time-scale. Implications of the measured values for the oscillation mechanism are discussed.

  11. Investigation of adipose tissues in Zucker rats using in vivo and ex vivo magnetic resonance spectroscopy

    PubMed Central

    Mosconi, Elisa; Fontanella, Marco; Sima, Diana M.; Van Huffel, Sabine; Fiorini, Silvia; Sbarbati, Andrea; Marzola, Pasquina

    2011-01-01

    In vivo single-voxel magnetic resonance spectroscopy (MRS) at 4.7T and ex vivo high-resolution proton magnetic resonance spectroscopy (HR-NMR) at 500 MHz were used to study the composition of adipose tissues in Zucker obese and Zucker lean rats. Lipid composition was characterized by unsaturation and polyunsaturation indexes and mean chain lengths. In vitro experiments were conducted in known mixtures of triglycerides and oils in order to validate the method. To avoid inaccuracies due to partial peak overlapping in MRS, peak quantification was performed after fitting of spectral peaks by using the QUEST algorithm. The intensity of different spectral lines was also corrected for T2 relaxation. Albeit with different sensitivity and accuracy, both techniques revealed that white adipose tissue is characterized by lower unsaturation and polyunsaturation indexes in obese rats compared with controls. HR-NMR revealed similar differences in brown adipose tissue. The present findings confirm the hypothesis that obese and lean Zucker rats have different adipose tissue composition. PMID:21098380

  12. Fluorescence Correlation Spectroscopy and Nonlinear Stochastic Reaction-Diffusion

    E-print Network

    Mauricio J. Del Razo; Wenxiao Pan; Hong Qian; Guang Lin

    2014-06-16

    The currently existing theory of fluorescence correlation spectroscopy(FCS) is based on the linear fluctuation theory originally developed by Einstein, Onsager, Lax, and others as a phenomenological approach to equilibrium fluctuations in bulk solutions. For mesoscopic reaction-diffusion systems with nonlinear chemical reactions among a small number of molecules, a situation often encountered in single-cell biochemistry, it is expected that FCS time correlation functions of a reaction-diffusion system can deviate from the classic results of Elson and Magde. We first discuss this nonlinear effect for reaction systems without diffusion. For nonlinear stochastic reaction-diffusion systems here are no closed solutions; therefore, stochastic Monte-Carlo simulations are carried out. We show that the deviation is small for a simple bimolecular reaction; the most significant deviations occur when the number of molecules is small and of the same order. Our results show that current linear FCS theory could be adequate for measurements on biological systems that contain many other sources of uncertainties. At the same time it provides a framework for future measurements of nonlinear, fluctuating chemical reactions with high-precision FCS. Extending Delbr\\"uck-Gillespie's theory for stochastic nonlinear reactions with rapidly stirring to reaction-diffusion systems provides a mesoscopic model for chemical and biochemical reactions at nanometric and mesoscopic level such as a single biological cell.

  13. Fluorescence Correlation Spectroscopy and Nonlinear Stochastic Reaction-Diffusion

    SciTech Connect

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

    2014-05-30

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-07-01

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

  15. [Synthesis of new blue pyrazoline fluorescent compounds and study of infrared spectroscopy].

    PubMed

    Xian, Yuan-Fang; Li, Dong-Feng; Wang, Yu-Ming

    2008-07-01

    According to Schellhammer theory of the relation between chemical structure and fluorescent quality, and referring to the synthesized benzothiazolyl pyrazoline compounds, the authors designed 1-benzimidazolyl or 1-benzothiazolyl, 3-phenylic derivatives, which posses fluorescent property. The authors introduced 5-phenyl as auxochrome group which can make fluorescence spectrum bathochromic. If there were -NH2 in the benzene ring, the fluorescence would be increased. Two kinds of benzimidazolyl and benzothiazolyl compounds with -NH2 were synthesized which were not reported. The determination of fluorescence proved that its fluorescence strength is better. The fluorescence emission wavelength is in the region of green-blue light, and there are two kinds of blue light fluorescence compounds. All these compounds were characterized by elemental analysis and infrared spectroscopy. The characteristic peaks of the absorption spectra of these compounds were found by IR spectral analysis. The compound structure was determined. PMID:18844173

  16. Study of normal/tumorous tissue fluorescence using a pH-dependent fluorescent probe in vivo

    NASA Astrophysics Data System (ADS)

    Mordon, Serge R.; Maunoury, Vincent; Devoisselle, Jean-Marie; Abbas, Y.; Coustaut, Denise

    1992-04-01

    The pH of interstitial fluid of malignant tumors tends to be lower than that of normal tissue and depressed by glucose administration. This study aimed to evaluate the effectiveness of dual-wavelength ratio fluorometry using a pH-dependent indicator (5,6-carboxyfluorescein: 5,6-CF) for the characterization of normal and tumoral areas in vivo. 5,6-CF has two main characteristics: it has two wavelengths of maximum absorbance (465 and 490 nm) and its fluorescence emission (maximum at 515 nm) increases as a function of pH in the physiological 6 - 7.4 pH range. The experimental study was performed on 31 CDF mice bearing lymphoid leukaemia P388 grafted subcutaneously. The tissular pH values were evaluated from the ratio of the fluorescence intensities (I490/I465) on the basis of a calibration curve linking pH measurements performed intratissularly with a microelectrode and fluorescence intensities ratio values. The fluorescence intensity reached its maximum value at 60 min after 5,6-CF and glucose administration, followed by a plateau (90 min). The ratios remain constant at 1.79 +/- 0.06 for normal tissue and 1.61 +/- 0.07 (without glucose administration) for tumoral tissue. The tumoral tissue ratios decrease down to 1.35 +/- 0.04 after 6 g/kg glucose administration. These results were correlated to the pH measurements in accordance to the calibration curve. This study validates the relevance of dual-wavelength fluorometry using a pH-dependent indicator to characterize in-vivo normal and tumoral tissues after glucose administration.

  17. Probing Single-Stranded DNA Conformational Flexibility Using Fluorescence Spectroscopy

    E-print Network

    Lohman, Timothy M.

    , fluorescence lifetimes may act here as an internal clock that influences fluorescence signals depending on how metabolic processes such as replication, re- combination, repair, and transcription and is specifically

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

    E-print Network

    Dabir, Aditi Sandeep

    2010-07-14

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

  19. Development of a Time Resolved Fluorescence Spectroscopy System for Near Real-Time Clinical Diagnostic Applications 

    E-print Network

    Trivedi, Chintan A.

    2010-07-14

    The design and development of a versatile time resolved fluorescence spectroscopy (TRFS) system capable of near real time data acquisition and processing for potential clinical diagnostic applications is reported. The TRFS apparatus is portable...

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

    SciTech Connect

    Gong, S.; Labanca, I.; Rech, I.; Ghioni, M. [Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano (Italy)

    2014-10-15

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

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

    PubMed

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

    2014-10-01

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

  3. In vivo magnetic resonance spectroscopy of liver tumors and metastases

    PubMed Central

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

    2011-01-01

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

  4. In Vivo Imaging of GLP-1R with a Targeted Bimodal PET/Fluorescence Imaging Agent

    PubMed Central

    2015-01-01

    Accurate visualization and quantification of ?-cell mass is critical for the improved understanding, diagnosis, and treatment of both type 1 diabetes (T1D) and insulinoma. Here, we describe the synthesis of a bimodal imaging probe (PET/fluorescence) for imaging GLP-1R expression in the pancreas and in pancreatic islet cell tumors. The conjugation of a bimodal imaging tag containing a near-infrared fluorescent dye, and the copper chelator sarcophagine to the GLP-1R targeting peptide exendin-4 provided the basis for the bimodal imaging probe. Conjugation was performed via a novel sequential one-pot synthetic procedure including 64Cu radiolabeling and copper-catalyzed click-conjugation. The bimodal imaging agent 64Cu-E4-Fl was synthesized in good radiochemical yield and specific activity (RCY = 36%, specific activity: 141 ?Ci/?g, >98% radiochemical purity). The agent showed good performance in vivo and ex vivo, visualizing small xenografts (<2 mm) with PET and pancreatic ?-cell mass by phosphor autoradiography. Using the fluorescent properties of the probe, we were able to detect individual pancreatic islets, confirming specific binding to GLP-1R and surpassing the sensitivity of the radioactive label. The use of bimodal PET/fluorescent imaging probes is promising for preoperative imaging and fluorescence-assisted analysis of patient tissues. We believe that our procedure could become relevant as a protocol for the development of bimodal imaging agents. PMID:24856928

  5. Biocompatible near-infrared fluorescent nanoparticles for macro and microscopic in vivo functional bioimaging.

    PubMed

    Chu, Liliang; Wang, Shaowei; Li, Kanghui; Xi, Wang; Zhao, Xinyuan; Qian, Jun

    2014-11-01

    Near-infrared (NIR) imaging technology has been widely used for biomedical research and applications, since it can achieve deep penetration in biological tissues due to less absorption and scattering of NIR light. In our research, polymer nanoparticles with NIR fluorophores doped were synthesized. The morphology, absorption/emission features and chemical stability of the fluorescent nanoparticles were characterized, separately. NIR fluorescent nanoparticles were then utilized as bright optical probes for macro in vivo imaging of mice, including sentinel lymph node (SLN) mapping, as well as distribution and excretion monitoring of nanoparticles in animal body. Furthermore, we applied the NIR fluorescent nanoparticles in in vivo microscopic bioimaging via a confocal microscope. Under the 635 nm-CW excitation, the blood vessel architecture in the ear and the brain of mice, which were administered with nanoparticles, was visualized very clearly. The imaging depth of our one-photon microscopy, which was assisted with NIR fluorescent nanoprobes, can reach as deep as 500 ?m. Our experiments show that NIR fluorescent nanoparticles have great potentials in various deep-tissue imaging applications. PMID:25426331

  6. Ultraviolet fluorescence spectroscopy of blood plasma in the discrimination of cancer from normal

    Microsoft Academic Search

    S. Madhuri; P. Aruna; M. I. Summiya Bibi; V. S. Gowri; D. Koteeswaran; S. Ganesan

    1997-01-01

    Native fluorescence spectroscopy of biomolecules has emerged as an intrinsic parameter in the characterization of the physiological state and the discrimination of pathological from normal conditions of cells and tissues. The key fluorescing biomolecules inc ells and tissues ar tryptophan, tyrosine, phenylalanine, collagen, elastin, NADH, flavin and porphyrin. Extensive studies were made on tissues of various origin to discriminate the

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

    Microsoft Academic Search

    Austin Nevin; Sharon Cather; Demetrios Anglos; Costas Fotakis

    2006-01-01

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

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

    NSDL National Science Digital Library

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

    2005-02-01

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

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

    E-print Network

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  11. Fluorescence spectroscopy in combination with reflectance measurements in human skin examination: what for and how

    Microsoft Academic Search

    Sergei R. Utz; Yuri P. Sinichkin; Igor V. Meglinsky; Helena A. Pilipenko

    1995-01-01

    Reflectance and fluorescence spectroscopy are successfully used for skin disease diagnostics. Human skin optical parameters are defined by its turbid, scattering properties with nonuniform absorption and fluorescence chromophores distribution, its multilayered structure, and variability under different physiological and pathological conditions. Theoretical modeling of light propagation in skin could improve the understanding of these conditions and may be useful in the

  12. Characterization of dissolved organic matter in fogwater by excitation–emission matrix fluorescence spectroscopy

    Microsoft Academic Search

    Justin E. Birdwell; Kalliat T. Valsaraj

    2010-01-01

    Dissolved organic matter (DOM) present in fogwater samples collected in southeastern Louisiana and central-eastern China has been characterized using excitation–emission matrix fluorescence spectroscopy. The goal of the study was to illustrate the utility of fluorescence for obtaining information on the large fraction of organic carbon in fogwaters (typically >40% by weight) that defies characterization in terms of specific chemical compounds

  13. Fluorescence spectroscopy of single-walled carbon nanotubes synthesized from alcohol

    E-print Network

    Maruyama, Shigeo

    I&EC 221 Fluorescence spectroscopy of single-walled carbon nanotubes synthesized from alcohol fluorescence measurements of single-walled carbon nanotubes (SWNTs) catalytically synthesized from alcohol (Alcohol catalytic CVD method, ACCVD) in various experimental conditions were performed. The chirality

  14. An Activatable Near Infrared Fluorescent Probe for In Vivo Imaging of Fibroblast Activation Protein-alpha

    PubMed Central

    Li, Jinbo; Chen, Kai; Liu, Hongguang; Cheng, Kai; Yang, Meng; Zhang, Jiping; Cheng, Jonathan D.; Zhang, Yan; Cheng, Zhen

    2012-01-01

    Fibroblast activation protein-alpha (FAP?) is a cell surface glycoprotein which is selectively expressed by tumor-associated fibroblasts in malignant tumors but rarely on normal tissues. FAP? has also been reported to promote tumor growth and invasion and therefore has been of increasing interest as a promising target for designing tumor-targeted drugs and imaging agents. Although medicinal study on FAP? inhibitors has led to the discovery of many FAP?-targeting inhibitors including a drug candidate in a phase II clinical trial, the development of imaging probes to monitor the expression and activity of FAP? in vivo has largely lagged behind. Herein we report an activatable near infrared (NIR) fluorescent probe (ANPFAP) for in vivo optical imaging of FAP?. The ANPFAP consists of a NIR dye (Cy5.5) and a quencher dye (QSY21) which are linked together by a short peptide sequence (KGPGPNQC) specific for FAP? cleavage. Because of the efficient fluorescence resonance energy transfer (FRET) between Cy5.5 and QSY21 in ANPFAP, high contrast on the NIR fluorescence signal can be achieved after the cleavage of the peptide sequence by FAP? both in vitro and in vivo. In vitro assay on ANPFAP indicated the specificity of the probe to FAP?. The in vivo optical imaging using ANPFAP showed fast tumor uptake as well as high tumor to background contrast on U87MG tumor models with FAP? expression, while much lower signal and tumor contrast were observed in the C6 tumor without FAP? expression, demonstrating the in vivo targeting specificity of the ANPFAP. Ex vivo imaging also demonstrated ANPFAP had high tumor uptake at 4 h post injection. Collectively, these results indicated that ANPFAP could serve as a useful NIR optical probe for early detection of FAP? expressing tumors. PMID:22812530

  15. Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy.

    PubMed

    Wachsmuth, M; Waldeck, W; Langowski, J

    2000-05-12

    We have investigated spatial variations of the diffusion behavior of the green fluorescent protein mutant EGFP (F64L/S65T) and of the EGFP-beta-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 lines expressing EGFP are caused by molecular diffusion as well as a possible internal and a pH-dependent external protonation process of the EGFP chromophore. The latter processes result in two apparent nonfluorescent states that have to be taken into account when evaluating the fluorescence correlation spectroscopy data. The diffusional contribution deviates from ideal behavior and depends on the position in the cell. The fluorescence correlation spectroscopy data can either be evaluated as a two component model with one fraction of the molecules undergoing free Brownian motion with a diffusion coefficient approximately five times smaller than in aqueous solution, and another fraction diffusing one or two orders of magnitude slower. This latter component is especially noticeable in the nuclei. Alternatively, we can fit the data to an anomalous diffusion model where the time dependence of the diffusion serves as a measure for the degree of obstruction, which is large especially in nuclei. Possible mechanisms for this long tail behavior include corralling, immobile obstacles, and binding with a broad distribution of binding affinities. The results are consistent with recent numerical models of the chromosome territory structure in the cell nucleus. PMID:10788329

  16. The Mobility of Phytochrome within Protonemal Tip Cells of the Moss Ceratodon purpureus, Monitored by Fluorescence Correlation Spectroscopy

    Microsoft Academic Search

    Guido Böse; Petra Schwille; Tilman Lamparterz

    2004-01-01

    Fluorescence correlation spectroscopy (FCS) is a versatile tool for investigating the mobilities of fluorescent molecules in cells. In this article, we show that it is possible to distinguish between freely diffusing and membrane-bound forms of biomolecules involved in signal transduction in living cells. Fluorescence correlation spectroscopy was used to measure the mobility of phytochrome, which plays a role in phototropism

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

    PubMed Central

    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

    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. PMID:18316743

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Punj, Deep; Rigneault, Hervé; Wenger, Jérôme

    2014-05-01

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

  20. Fluorescence lifetime spectroscopy for breast cancer margins assessment

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

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

    PubMed Central

    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

    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. PMID:19566223

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

    NASA Astrophysics Data System (ADS)

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

    2009-06-01

    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.

  3. Knockin mice expressing fluorescent ?-opioid receptors uncover G protein-coupled receptor dynamics in vivo

    PubMed Central

    Scherrer, Grégory; Tryoen-Tóth, Petra; Filliol, Dominique; Matifas, Audrey; Laustriat, Delphine; Cao, Yu Q.; Basbaum, Allan I.; Dierich, Andrée; Vonesh, Jean-Luc; Gavériaux-Ruff, Claire; Kieffer, Brigitte L.

    2006-01-01

    The combination of fluorescent genetically encoded proteins with mouse engineering provides a fascinating means to study dynamic biological processes in mammals. At present, green fluorescent protein (GFP) mice were mainly developed to study gene expression patterns or cell morphology and migration. Here we used enhanced GFP (EGFP) to achieve functional imaging of a G protein-coupled receptor (GPCR) in vivo. We created mice where the ?-opioid receptor (DOR) is replaced by an active DOR-EGFP fusion. Confocal imaging revealed detailed receptor neuroanatomy throughout the nervous system of knockin mice. Real-time imaging in primary neurons allowed dynamic visualization of drug-induced receptor trafficking. In DOR-EGFP animals, drug treatment triggered receptor endocytosis that correlated with the behavioral response. Mice with internalized receptors were insensitive to subsequent agonist administration, providing evidence that receptor sequestration limits drug efficacy in vivo. Direct receptor visualization in mice is a unique approach to receptor biology and drug design. PMID:16766653

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2011-01-01

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

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

    PubMed Central

    Chiara Munisso, Maria; Yamaoka, Tetsuji

    2014-01-01

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

  7. FRONTIERS ARTICLE Fluorescence correlation spectroscopy as a tool for measuring the rotational

    E-print Network

    Enderlein, Jörg

    FRONTIERS ARTICLE Fluorescence correlation spectroscopy as a tool for measuring the rotational correlation spectroscopy (FCS) for measuring rotational diffu- sion of macromolecules, and present a new experimental scheme, pulsed-interleaved excitation or PIE-FCS, which allows for measuring all conceivable

  8. Antibody-labeled fluorescence imaging of dendritic cell populations in vivo

    PubMed Central

    Cummings, Ryan J.; Mitra, Soumya; Lord, Edith M.; Foster, Thomas H.

    2008-01-01

    We report an optical molecular imaging technique that exploits local administration of fluorophore-conjugated antibodies and confocal fluorescence microscopy to achieve high contrast imaging of host cell populations in normal and tumor tissue in living mice. The method achieves micron-scale spatial resolution to depths greater than 100 ?m. We illustrate the capabilities of this approach by imaging two dendritic cell populations in the skin and normal and tumor vasculature in vivo. PMID:19021368

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

    Microsoft Academic Search

    Yasuharu Hirai; Akio Yoneyama; Akiko Hisada; Kenko Uchida

    2007-01-01

    We have observed in vivo three-dimensional distributions of constituent elements of single-celled spores of the fern Adiantum capillus-veneris using an X-ray fluorescence computed microtomography method. The images of these distributions are generated from a series of slice data, each of which is acquired by a sample translation-rotation method. An incident X-ray microbeam irradiates the sample with a spot size of

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

    PubMed

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

    2014-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-04-01

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

  13. Quantum dots: bright and versatile in vitro and in vivo fluorescence imaging biosensors.

    PubMed

    Wegner, K David; Hildebrandt, Niko

    2015-07-01

    Semiconductor quantum dots (QDs) have become important fluorescent probes for in vitro and in vivo bioimaging research. Their nanoparticle surfaces for versatile bioconjugation, their adaptable photophysical properties for multiplexed detection, and their superior stability for longer investigation times are the main advantages of QDs compared to other fluorescence imaging agents. Here, we review the recent literature dealing with the design and application of QD-bioconjugates for advanced in vitro and in vivo imaging. After a short summary of QD preparation and their most important properties, different QD-based imaging applications will be discussed from the technological and the biological point of view, ranging from super-resolution microscopy and single-particle tracking over in vitro cell and tissue imaging to in vivo investigations. A substantial part of the review will focus on multifunctional applications, in which the QD fluorescence is combined with drug or gene delivery towards theranostic approaches or with complementary technologies for multimodal imaging. We also briefly discuss QD toxicity issues and give a short outlook on future directions of QD-based bioimaging. PMID:25777768

  14. Metabolism-enhanced tumor localization by fluorescence imaging: in vivo animal studies

    NASA Astrophysics Data System (ADS)

    Chen, Y.; Zheng, G.; Zhang, Z. H.; Blessington, D.; Zhang, M.; Li, H.; Liu, Q.; Zhou, L.; Intes, X.; Achilefu, S.; Chance, B.

    2003-11-01

    We present a high-sensitivity near-infrared optical imaging system for noninvasive cancer detection and localization based on molecularly labeled fluorescent contrast agents. This frequency-domain system utilizes the interferencelike pattern of diffuse photon density waves to achieve high detection sensitivity and localization accuracy for the fluorescent heterogeneity embedded inside the scattering media. A two-dimensional localization map is obtained through reflectance probe geometry and goniometric reconstruction. In vivo measurements with a tumor-bearing mouse model by use of the novel Cypate-mono-2-deoxy-glucose fluorescent contrast agent, which targets the enhanced tumor glycolysis, demonstrate the feasibility of detection of a 2-cm-deep subsurface tumor in the tissuelike medium, with a localization accuracy within 2-3 mm.

  15. Fluorescent Nanorods and Nanospheres for Real-Time In Vivo Probing of Nanoparticle Shape-Dependent Tumor Penetration

    E-print Network

    Chauhan, Vikash P.

    Shape dependent: Fluorescent quantum-dot-based nanospheres and nanorods with identical hydrodynamic size and surface properties but different aspect ratios were developed for real-time in vivo tumor imaging. The nanorods ...

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

    SciTech Connect

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

    1995-12-31

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

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

    PubMed

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

    2009-11-15

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

  18. Combining in vivo reflectance with fluorescence confocal microscopy provides additive information on skin morphology

    PubMed Central

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

    2012-01-01

    Background: Within the last decade, confocal microscopy has become a valuable non-invasive diagnostic tool in imaging human skin in vivo. Of the two different methods that exist, reflectance confocal microscopy (RCM) displays the backscattering signal of naturally occurring skin components, whereas fluorescence confocal microscopy (FCM) provides contrast by using an exogenously applied fluorescent dye. Methodology: A newly developed multilaser device, in which both techniques are implemented, has been used to combine both methods and allows to highlight different information in one image. In our study, we applied the fluorophore sodium fluorescein (SFL) intradermally on forearm skin of 10 healthy volunteers followed by fluorescence and reflectance imaging. Results: In fluorescence mode the intercellular distribution of SFL clearly outlines every single cell in the epidermis, whereas in reflectance mode keratin and melanin-rich cells and structures provide additional information. The combination of both methods enables a clear delineation between the cell border, the cytoplasm and the nucleus. Imaging immediately, 20, 40 and 60 minutes after SFL injection, represents the dynamic distribution pattern of the dye. Conclusion: The synergism of RCM and FCM in one device delivering accurate information on skin architecture and pigmentation will have a great impact on in vivo diagnosis of human skin in the future. PMID:24765544

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

    NASA Astrophysics Data System (ADS)

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

    2013-01-01

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

  20. The Design and Development of Fluorescent Nano-Optodes for in Vivo Glucose Monitoring

    PubMed Central

    Balaconis, Mary K.; Billingsley, Kelvin; Dubach, J. Matthew; Cash, Kevin J.; Clark, Heather A.

    2011-01-01

    Background The advent of fluorescent nanosensors has enabled intracellular monitoring of several physiological analytes, which was previously not possible with molecular dyes or other invasive techniques. We have extended the capability of these sensors to include the detection of small molecules with the development of glucose-sensitive nano-optodes. Herein, we discuss the design and development of glucose-sensitive nano-optodes, which have been proven functional both in vitro and in vivo. Methods Throughout the design process, each of the sensor formulations was evaluated based on their response to changes in glucose levels. The percent change in signal, sensor reversibility, and the overall fluorescence intensity were the specific parameters used to assess each formulation. Results A hydrophobic boronic acid was selected that yielded a fully reversible fluorescence response to glucose in accordance with the sensor mechanism. The change in fluorescence signal in response to glucose was approximately 11%. The use of different additives or chromophores did not improve the response; however, modifications to the plasticized polymeric membrane extended sensor lifetime. Conclusions Sensors were developed that yielded a dynamic response to glucose and through further modification of the components, sensor lifetime was improved. By following specific design criteria for the macrosensors, the sensors were miniaturized into nano-optodes that track changes in glucose levels in vivo. PMID:21303627

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  3. Different analysis techniques for fluorescence excitation-emission matrix spectroscopy to assess compost maturity.

    PubMed

    Tang, Zhu; Yu, Guanghui; Liu, Dongyang; Xu, Dabing; Shen, Qirong

    2011-02-01

    Assessment of compost maturity is essential for achieving high quality compost. In this study, fluorescence excitation-emission matrix spectroscopy combined with different analysis techniques was applied to improve the sensitivity of compost maturity assessment. Results showed that composts in two parallel piles could be believed mature after 37d when combined with the evolution of temperature, chemical and biological indices in the two piles. Pearson correlation between the common maturity indices and fluorescence analysis parameters demonstrated that fluorescence regional integration (FRI) had a higher correlation coefficient than that of fluorescence intensities and the ratios of peaks, suggesting that FRI technique is more suitable to characterize the maturity of compost than the other two analysis techniques, i.e., peak intensity and peak ratio. Furthermore, the fluorescence spectroscopy combined with FRI analysis could be used as a valuable industrial and research tool for assessing compost maturity. PMID:21129765

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

    PubMed Central

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

    2009-01-01

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

  5. Fluorescence spectroscopy of the retina for diagnosis of transmissible spongiform encephalopathies.

    PubMed

    Adhikary, Ramkrishna; Mukherjee, Prasun; Krishnamoorthy, Govindarajan; Kunkle, Robert A; Casey, Thomas A; Rasmussen, Mark A; Petrich, Jacob W

    2010-05-15

    The feasibility of exploiting fluorescence spectra of the eye for diagnosis of transmissible spongiform encephalopathies (TSEs) was examined. Retinas from scrapie-positive sheep were compared with scrapie-negative sheep using fluorescence spectroscopy, and distinct differences in the fluorescence intensity and spectroscopic signatures were observed. The characteristic fluorescent signatures are thought to be the result of an accumulation of lipofuscin in the retina. It appears that the eye, in particular the retina, is a useful tissue for noninvasive examination of some neurological pathologies such as scrapie. The development of procedures based on examinations of the eye that permit the detection of neurological disorders in animals holds great promise. PMID:20411920

  6. Diffuse reflectance spectroscopy for in vivo pediatric brain tumor detection

    NASA Astrophysics Data System (ADS)

    Lin, Wei-Chiang; Sandberg, David I.; Bhatia, Sanjiv; Johnson, Mahlon; Oh, Sanghoon; Ragheb, John

    2010-11-01

    The concept of using diffuse reflectance spectroscopy to distinguish intraoperatively between pediatric brain tumors and normal brain parenchyma at the edge of resection cavities is evaluated using an in vivo human study. Diffuse reflectance spectra are acquired from normal and tumorous brain areas of 12 pediatric patients during their tumor resection procedures, using a spectroscopic system with a handheld optical probe. A total of 400 spectra are acquired at the rate of 33 Hz from a single investigated site, from which the mean spectrum and the standard deviation are calculated. The mean diffuse reflectance spectra collected are divided into the normal and the tumorous categories in accordance with their corresponding results of histological analysis. Statistical methods are used to identify those spectral features that effectively separated the two tissue categories, and to quantify the spectral variations induced by the motion of the handheld probe during a single spectral acquisition procedure. The results show that diffuse reflectance spectral intensities between 600 and 800 nm are effective in terms of differentiating normal cortex from brain tumors. Furthermore, probe movements induce large variations in spectral intensities (i.e., larger standard deviation) between 400 and 600 nm.

  7. In vivo impedance spectroscopy of deep brain stimulation electrodes

    NASA Astrophysics Data System (ADS)

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

    2009-08-01

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

  8. In Vivo Blood Glucose Quantification Using Raman Spectroscopy

    PubMed Central

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

    2012-01-01

    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

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

    NASA Astrophysics Data System (ADS)

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

    2011-02-01

    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.

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

    PubMed

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

    2012-08-21

    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

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

    PubMed Central

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

    2011-01-01

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

  12. Fluorescence spectra of blood plasma treated with ultraviolet irradiation in vivo

    NASA Astrophysics Data System (ADS)

    Zalesskaya, G. A.; Maslova, T. O.

    2010-09-01

    We have studied the fluorescence spectra of blood plasma from patients with acute coronary syndrome, and also the effect of therapeutic doses of in vivo ultraviolet blood irradiation (UBI) on the spectra. We have established that the maxima in the fluorescence spectra of the original plasma samples, obtained from unirradiated blood, are located in the wavelength interval 330-340 nm, characteristic for the fluorescence of tryptophan residues. In extracorporeal UBI ( ? = 254 nm), we observed changes in the shape and also both a blue and a red shift in the maxima of the fluorescence spectra, differing in magnitude for blood plasma samples from different patients in the test group. We show that UBI-initiated changes in the fluorescence spectra of the plasma depend on the original pathological disturbances of metabolite levels, and also on the change in the oxygen-transport function of the blood and the acid-base balance, affecting the oxidative stability of the plasma. We have concluded that UV irradiation, activating buffer systems in the blood, has an effect on the universal and specific interactions of the tryptophan residue with the amino acid residues and water surrounding it.

  13. Fluorescence spectroscopy: A promising tool for carbonate petrology

    SciTech Connect

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

    1992-01-01

    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.

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

    NASA Technical Reports Server (NTRS)

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

    1993-01-01

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

  15. Improved Diffuse Fluorescence Flow Cytometer Prototype for High Sensitivity Detection of Rare Circulating Cells In Vivo

    NASA Astrophysics Data System (ADS)

    Pestana, Noah Benjamin

    Accurate quantification of circulating cell populations is important in many areas of pre-clinical and clinical biomedical research, for example, in the study of cancer metastasis or the immune response following tissue and organ transplants. Normally this is done "ex-vivo" by drawing and purifying a small volume of blood and then analyzing it with flow cytometry, hemocytometry or microfludic devices, but the sensitivity of these techniques are poor and the process of handling samples has been shown to affect cell viability and behavior. More recently "in vivo flow cytometry" (IVFC) techniques have been developed where fluorescently-labeled cells flowing in a small blood vessel in the ear or retina are analyzed, but the sensitivity is generally poor due to the small sampling volume. To address this, our group recently developed a method known as "Diffuse Fluorescence Flow Cytometry" (DFFC) that allows detection and counting of rare circulating cells with diffuse photons, offering extremely high single cell counting sensitivity. In this thesis, an improved DFFC prototype was designed and validated. The chief improvements were three-fold, i) improved optical collection efficiency, ii) improved detection electronics, and iii) development of a method to mitigate motion artifacts during in vivo measurements. In combination, these improvements yielded an overall instrument detection sensitivity better than 1 cell/mL in vivo, which is the most sensitive IVFC system reported to date. Second, development and validation of a low-cost microfluidic device reader for analysis of ocular fluids is described. We demonstrate that this device has equivalent or better sensitivity and accuracy compared a fluorescence microscope, but at an order-of-magnitude reduced cost with simplified operation. Future improvements to both instruments are also discussed.

  16. Interstitial Fluorescence Spectroscopy in the Human Prostate During Motexafin Lutetium–Mediated Photodynamic Therapy

    PubMed Central

    Zhu, Timothy C.; Dimofte, Andreea; Stripp, Diana; Malkowicz, S. Bruce; Busch, Theresa M.; Hahn, Stephen M.

    2015-01-01

    The in vivo fluorescence emission from human prostates was measured before and after motexafin lutetium (MLu)-mediated photodynamic therapy (PDT). A single side-firing optical fiber was used for both the delivery of 465 nm light-emitting diode excitation light and the collection of emitted fluorescence. It was placed interstitially within the prostate via a closed transparent plastic catheter. Fitting of the collected fluorescence emission spectra using the known fluorescence spectrum of 1 mg/kg MLu in an intralipid phantom yields a quantitative measure of the local MLu concentration. We found that an additional correction factor is needed to account for the reduction of the MLu fluorescence intensity measured in vivo due to strong optical absorption in the prostate. We have adopted an empirical correction formula given by C=(3.1cm?1/µs?) exp (µeff · 0.97 cm), which ranges from approximately 3 to 16, with a mean of 9.3 ± 4.8. Using a computer-controlled step motor to move the probe incrementally along parallel tracks within the prostate we can determine one-dimensional profiles of the MLu concentration. The absolute MLu concentration and the shape of its distribution are confirmed by ex vivo assay and by diffuse absorption measurements, respectively. We find significant heterogeneity in photosensitizer concentration within and among five patients. These variations occur over large enough spatial scales compared with the sampling volume of the fluorescence emission that mapping the distribution in three dimensions is possible. PMID:16808592

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

    SciTech Connect

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

    2003-11-15

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

  18. In vivo fluorescence lifetime imaging monitors binding of specific probes to cancer biomarkers.

    PubMed

    Ardeshirpour, Yasaman; Chernomordik, Victor; Zielinski, Rafal; Capala, Jacek; Griffiths, Gary; Vasalatiy, Olga; Smirnov, Aleksandr V; Knutson, Jay R; Lyakhov, Ilya; Achilefu, Samuel; Gandjbakhche, Amir; Hassan, Moinuddin

    2012-01-01

    One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the "image and treat" concept, especially for early evaluation of the efficacy of the therapy. PMID:22384092

  19. Anabaena cell ageing monitored with confocal fluorescence spectroscopy.

    PubMed

    Ke, Shan; Bindokas, Vytas; Haselkorn, Robert

    2015-01-01

    Cyanobacteria use a sophisticated system of pigments to collect light energy across the visible spectrum for photosynthesis. The pigments are assembled in structures called phycobilisomes, composed of phycoerythrocyanin, phycocyanin and allophycocyanin, which absorb energy and transfer it to chlorophyll in photosystem II reaction centres. All of the components of this system are fluorescent, allowing sensitive measurements of energy transfer using single cell confocal fluorescence microscopy. The native pigments can be interrogated without the use of reporters. Here, we use confocal fluorescence microscopy to monitor changes in the efficiency of energy transfer as single cells age, between the time they are born at cell division until they are ready to divide again. Alteration of fluorescence was demonstrated to change with the age of the cyanobacterial cell. PMID:25378560

  20. Ultrafast Fluorescence Spectroscopy via Upconversion: Applications to Biophysics

    PubMed Central

    Xu, Jianhua; Knutson, Jay R.

    2012-01-01

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

  1. DNA biosensor using fluorescence microscopy and impedance spectroscopy

    Microsoft Academic Search

    Daniel Berdat; Annick Marin; Fernando Herrera; Martin A. M. Gijs

    2006-01-01

    Two types of DNA biosensors are presented. Both sensing principles are demonstrated using synthetic oligomer single-stranded DNA (ssDNA) with concentrations in the micromolar range. A first sensor type is based on the detection of fluorescently labeled ssDNA to a complementary probe that is bound to a silicon substrate by a disuccinimidyl terephtalate and aminosilane immobilization procedure. An enhanced fluorescent response

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

    PubMed

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

    2015-01-10

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

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

    Microsoft Academic Search

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

    2003-01-01

    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

  4. Fluorescence spectroscopy of anisole at elevated temperatures and pressures

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

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

    PubMed Central

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

    2013-01-01

    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. PMID:20595964

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

    PubMed Central

    Todd, A C; Chettle, D R

    1994-01-01

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

  7. Noninvasive and Quantitative Assessment of In Vivo Fetomaternal Interface Angiogenesis Using RGD-Based Fluorescence

    PubMed Central

    Keramidas, M.; Lavaud, J.; Sergent, F.; Hoffmann, P.; Brouillet, S.; Feige, J.-J.; Coll, J.-L.; Alfaidy, N.

    2014-01-01

    Angiogenesis is a key process for proper placental development and for the success of pregnancy. Although numerous in vitro methods have been developed for the assessment of this process, relatively few reliable in vivo methods are available to evaluate this activity throughout gestation. Here we report an in vivo technique that specifically measures placental neovascularization. The technique is based on the measurement of a fluorescent alpha v beta 3 (?v?3) integrin-targeting molecule called Angiolone-Alexa-Fluor 700. The ?v?3 integrin is highly expressed by endothelial cells during the neovascularization and by trophoblast cells during their invasion of the maternal decidua. Angiolone was injected to gravid mice at 6.5 and 11.5 days post coitus (dpc). The fluorescence was analyzed one day later at 7.5 and 12.5?dpc, respectively. We demonstrated that (i) Angiolone targets ?v?3 protein in the placenta with a strong specificity, (ii) this technique is quantitative as the measurement was correlated to the increase of the placental size observed with increasing gestational age, and (iii) information on the outcome is possible, as abnormal placentation could be detected early on during gestation. In conclusion, we report the validation of a new noninvasive and quantitative method to assess the placental angiogenic activity, in vivo. PMID:25110672

  8. TOPICAL REVIEW: Prospects for in vivo Raman spectroscopy

    Microsoft Academic Search

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

    2000-01-01

    Raman spectroscopy is a potentially important clinical tool for real-time diagnosis of disease and in situ evaluation of living tissue. The purpose of this article is to review the biological and physical basis of Raman spectroscopy of tissue, to assess the current status of the field and to explore future directions. The principles of Raman spectroscopy and the molecular level

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

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

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

    PubMed Central

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

    2013-01-01

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

  12. High-Resolution In Vivo Imaging of Fluorescent Proteins Using Window Chamber Models

    PubMed Central

    Palmer, Gregory M.; Fontanella, Andrew N.; Shan, Siqing; Dewhirst, Mark W.

    2013-01-01

    Fluorescent proteins enable in vivo characterization of a wide and growing array of morphological and functional biomarkers. To fully capitalize on the spatial and temporal information afforded by these reporter proteins, a method for imaging these proteins at high resolution longitudinally is required. This chapter describes the use of window chamber models as a means of imaging fluorescent proteins and other optical parameters. Such models essentially involve surgically implanting a window through which tumor or normal tissue can be imaged using existing microscopy techniques. This enables acquisition of high-quality images down to the cellular or subcellular scale, exploiting the diverse array of optical contrast mechanisms, while also maintaining the native microenvironment of the tissue of interest. This makes these techniques applicable to a wide array of problems in the biomedical sciences. PMID:22700402

  13. Time-resolved Hyperspectral Fluorescence Spectroscopy using Frequency Modulated Excitation

    SciTech Connect

    ,; Neill, M

    2012-07-01

    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).

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

    PubMed

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

    2013-06-01

    Raman spectroscopy is a unique optical technique that can probe the changes of vibrational modes of biomolecules associated with tissue premalignant transformation. This study evaluates the clinical utility of confocal Raman spectroscopy over near-infrared (NIR) autofluorescence (AF) spectroscopy and composite NIR AF/Raman spectroscopy for improving early diagnosis of cervical precancer in vivo at colposcopy. A rapid NIR Raman system coupled with a ball-lens fiber-optic confocal Raman probe was utilized for in vivo NIR AF/Raman spectral measurements of the cervix. A total of 1240 in vivo Raman spectra [normal (n=993), dysplasia (n=247)] were acquired from 84 cervical patients. Principal components analysis (PCA) and linear discriminant analysis (LDA) together with a leave-one-patient-out, cross-validation method were used to extract the diagnostic information associated with distinctive spectroscopic modalities. The diagnostic ability of confocal Raman spectroscopy was evaluated using the PCA-LDA model developed from the significant principal components (PCs) [i.e., PC4, 0.0023%; PC5, 0.00095%; PC8, 0.00022%, (p<0.05)], representing the primary tissue Raman features (e.g., 854, 937, 1095, 1253, 1311, 1445, and 1654 cm(-1)). Confocal Raman spectroscopy coupled with PCA-LDA modeling yielded the diagnostic accuracy of 84.1% (a sensitivity of 81.0% and a specificity of 87.1%) for in vivo discrimination of dysplastic cervix. The receiver operating characteristic curves further confirmed that the best classification was achieved using confocal Raman spectroscopy compared to the composite NIR AF/Raman spectroscopy or NIR AF spectroscopy alone. This study illustrates that confocal Raman spectroscopy has great potential to improve early diagnosis of cervical precancer in vivo during clinical colposcopy. PMID:23797897

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

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

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

    NASA Astrophysics Data System (ADS)

    Carstea, Elfrida M.; Bridgeman, John

    2015-04-01

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

  17. Improved preparation of acellular nerve scaffold and application of PKH26 fluorescent labeling combined with in vivo fluorescent imaging system in nerve tissue engineering.

    PubMed

    Zhao, Bin; Sun, Xiaolei; Li, Xiulan; Yang, Qiang; Li, Yanjun; Zhang, Yang; Li, Bing; Ma, Xinlong

    2013-11-27

    Acellular nerve scaffold has been widely used for peripheral nerve defect treatment. However, the structure of traditional acellular nerve scaffold is dense; the interval porosity and void diameter are too small to meet the requirement of cell seeding, which limits the application. This study was designed to prepare a novel acellular nerve scaffold by the technique of hypotonic buffer combined with freeze-drying, and use PKH26 fluorescent labeling combined with in vivo fluorescent imaging system to evaluate the biological behavior of tissue-engineered nerve in vitro and in vivo. According to light and electron microscopy, the scaffold, which microarchitecture was similar to the fibrous framework of rabbit sciatic nerves, was cell-free and rich in laminin, collagen I and collagen III. In vitro experiment showed that the novel acellular nerve scaffold could provide a 3-D environment to support the attachment, proliferation and migration of adipose-derived stem cells (ADSCs). ADSCs labeled with fluorescent dye PKH26 were then seeded on scaffolds and implanted subcutaneously into nude mice. After 4 weeks, nerve-like tissue rounded by vessels formed. Cells in the tissue seemed to confirm that they originated from the labeled ADSCs, as confirmed by in vivo fluorescent imaging. In conclusion, the prepared novel acellular nerve scaffold can be used as a new kind of nerve scaffold material, which is more conducible for seeding cells; And PKH26 fluorescent labeling and in vivo fluorescent imaging can be useful for cell tracking and analyzing cell-scaffold constructs in vivo. PMID:24148304

  18. Fluorescence spectroscopy of Rhodamine 6G: concentration and solvent effects.

    PubMed

    Zehentbauer, Florian M; Moretto, Claudia; Stephen, Ryan; Thevar, Thangavel; Gilchrist, John R; Pokrajac, Dubravka; Richard, Katherine L; Kiefer, Johannes

    2014-01-01

    Rhodamine 6G (R6G), also known as Rhodamine 590, is one of the most frequently used dyes for application in dye lasers and as a fluorescence tracer, e.g., in the area of environmental hydraulics. Knowing the spectroscopic characteristics of the optical emission is key to obtaining high conversion efficiency and measurement accuracy, respectively. In this work, solvent and concentration effects are studied. A series of eight different organic solvents (methanol, ethanol, n-propanol, iso-propanol, n-butanol, n-pentanol, acetone, and dimethyl sulfoxide (DMSO)) are investigated at constant dye concentration. Relatively small changes of the fluorescence spectrum are observed for the different solvents; the highest fluorescence intensity is observed for methanol and lowest for DMSO. The shortest peak wavelength is found in methanol (568 nm) and the longest in DMSO (579 nm). Concentration effects in aqueous R6G solutions are studied over the full concentration range from the solubility limit to highly dilute states. Changing the dye concentration provides tunability between ?550 nm in the dilute case and ?620 nm at high concentration, at which point the fluorescence spectrum indicates the formation of R6G aggregates. PMID:24239710

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

    Microsoft Academic Search

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

    2009-01-01

    Fiber optic probes are designed, developed and fabricated in the laboratories for remote fluorescence spectroscopic studies in various fields such as investigation of tissues, environmental monitoring, and analysis of samples in hostile environment. Optimized probe design is very much important for efficient transport and collection of photons, which ultimately helps in quantifying resultant emission and understanding light-matter interaction. Instead of

  20. Assaying protein import into mitochondria using fluorescence spectroscopy 

    E-print Network

    Cargill, Holly Beth

    2006-08-16

    of the Outer Membrane (TOM) complex, and the inner mitochondrial membrane (IM) via the Translocase of the Inner Membrane 23 (TIM23) complex. A novel system was set up to examine the import of matrix-targeted preproteins into mitochondria using fluorescence...

  1. Fluorescence Spectroscopy of Conformational Changes of Single LH2 Complexes

    PubMed Central

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

    2005-01-01

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

  2. Integrated optical measurement system for fluorescence spectroscopy in microfluidic channels

    Microsoft Academic Search

    Jörg Hübner; Klaus B. Mogensen; Anders M. Jorgensen; Peter Friis; Pieter Telleman; Jörg P. Kutter

    2001-01-01

    A transportable miniaturized fiber-pigtailed measurement system is presented which allows quantitative fluorescence detection in microliquid handling systems. The microliquid handling chips are made in silica on silicon technology and the optical functionality is monolithically integrated with the microfluidic channel system. This results in inherent stability and photolithographic alignment precision. Permanently attached optical fibers provide a rugged connection to the light

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

    E-print Network

    Rossman. George R.

    NEW MICROSCOPIC LASER-COUPLED SPECTROSCOPY INSTRUMENT COMBINING RAMAN, LIBS, AND FLUORESCENCE (LIBS, Raman) have been the subject of increasing attention and development [e.g., 1, 2, 3] because simultaneously collect spectra from Raman, Laser Induced Breakdown Spectroscopy (LIBS), and fluorescence

  4. Front face fluorescence spectroscopy and multiway analysis for process control and NFC prediction in industrially processed cookies

    Microsoft Academic Search

    Jad Rizkallah; Francisco J. Morales; Lamia Ait-ameur; Vincenzo Fogliano; Alexia Hervieu; Mathilde Courel; Inès Birlouez Aragon

    2008-01-01

    The aim of this work was to evaluate the potential of using front face fluorescence spectroscopy for rapid quantitative estimation of neoformed contaminants in industrially processed cookies. Two dimensional synchronous front face fluorescence spectra were acquired on cookies to assess the industrial process impact on the fluorescence signal and predict the neoformed contaminants content in cookies. The signal was recorded

  5. In Vivo Stable Tumor-Specific Painting in Various Colors Using Dehalogenase-Based Protein-Tag Fluorescent Ligands

    PubMed Central

    Kosaka, Nobuyuki; Ogawa, Mikako; Choyke, Peter L.; Karassina, Natasha; Corona, Cesear; McDougall, Mark; Lynch, David; Hoyt, Clifford; Levenson, Richard; Los, Georgyi V.; Kobayashi, Hisataka

    2010-01-01

    In vivo fluorescence cancer imaging is an important tool in understanding tumor growth and therapeutic monitoring and can be performed either with endogenously produced fluorescent proteins or exogenously introduced fluorescent probes bound to targeting molecules. However, endogenous fluorescence proteins cannot be altered after transfection, thus requiring rederivation of cell lines for each desired color, while exogenously targeted fluorescence probes are limited by the heterogeneous expression of naturally occurring cellular targets. In this study, we adapted the dehalogenase-based protein-Tag (HaloTag) system to in vivo cancer imaging. By introducing highly expressed HaloTag receptors (HaloTagR) in cancer cells coupled with an externally injected a range of fluorophore-conjugated dehalogenase-reactive sequences. Tumor nodules arising from a single transfected cell line were stably labeled with fluorescence varying in emission spectra from green to near infrared. After establishing and validating a SHIN3 cell line stably transfected with HaloTagR (HaloTagR-SHIN3), in vivo spectral fluorescence imaging studies were performed in live animals using a peritoneal dissemination model. The tumor nodules arising from HaloTagR-SHIN3 could be successfully labeled by 4 different fluorophore-conjugated HaloTag-ligands each emitting light at different wavelengths. These fluorophores could be alternated on serial imaging sessions permitting assessment of interval growth. Fluorescence was retained in histological specimens after fixation. Thus, this tagging system proves versatile both for in vivo and in vitro imaging without requiring modification of the underlying cell line. Thus, this strategy can overcome some of the limitations associated with the use of endogenous fluorescent proteins and exogenous targeted optical agents in current use. PMID:19514716

  6. Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins

    PubMed Central

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

    2014-01-01

    Non-invasive imaging of biological processes in vivo is invaluable in advancing biology. Photoacoustic tomography is a scalable imaging technique that provides higher resolution at greater depths in tissue than achievable by purely optical methods. Here we report the application of two spectrally distinct near-infrared fluorescent proteins, iRFP670 and iRFP720, engineered from bacterial phytochromes, as photoacoustic contrast agents. iRFPs provide tissue-specific contrast without the need for delivery of any additional substances. Compared to conventional GFP-like red-shifted fluorescent proteins, iRFP670 and iRFP720 demonstrate stronger photoacoustic signals at longer wavelengths, and can be spectrally resolved from each other and hemoglobin. We simultaneously visualized two differently labeled tumors, one with iRFP670 and the other with iRFP720, as well as blood vessels. We acquired images of a mouse as 2D sections of a whole animal, and as localized 3D volumetric images with high contrast and sub-millimeter resolution at depths up to 8?mm. Our results suggest iRFPs are genetically-encoded probes of choice for simultaneous photoacoustic imaging of several tissues or processes in vivo. PMID:24487319

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

    NASA Astrophysics Data System (ADS)

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

    2011-09-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1999-07-01

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

  9. Naphthenic acids quantification in organic solvents using fluorescence spectroscopy.

    PubMed

    Martin, Nancy; Burkus, Zvonko; McEachern, Preston; Yu, Tong

    2014-01-01

    Quantification of naphthenic acids in water has been traditionally performed after extraction with organic solvents followed by analytic methods that are complex and costly for preliminary research or for continuous monitoring purposes. This study examines the application of fluorescence in organic solvents as an effective alternative, and the role of organic solvents on quantification results. Nine organic solvents were used: three polar protic alcohols (methanol, ethanol, and propanol), three polar aprotic (dichloromethane, acetone, and acetonitrile) and three non-polar (hexane, toluene, and diethyl ether). The calibration curves of the polar protic solvents performed the best; they had lower light scattering and higher method sensitivity than polar aprotic and non-polar. Methanol was selected for further experiments having a strong linearity for concentrations lower than 250 mg/L (R(2) > 0.99), and a low relative standard deviation (< 10%). The method sensitivity was improved by 70% using a methanol-deionized water mixture (50:50) as a solvent. The synchronous fluorescence mode with a reduced offset value of ?? = 10 nm demonstrated potential for fingerprinting. The fluorescence technique for quantifying total naphthenic acids directly in organic solvents is a cost-effective analytical method compatible with the solid phase extraction of the sample. PMID:24279621

  10. Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy

    PubMed Central

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

    2014-01-01

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

  11. Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy.

    PubMed

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

    2014-12-01

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

  12. Solidphase fluorescence spectroscopy for the determination of acetylsalicylic acid in powdered pharmaceutical samples

    Microsoft Academic Search

    Altair B. Moreira; Iara L. T. Dias; Graciliano Oliveira Neto; Elias A. G. Zagatto; Lauro T. Kubota

    2004-01-01

    A rapid, simple and rugged procedure without requiring any prior sample treatment was developed for the determination of acetylsalicylic acid (ASA) in tablets formulations by solid-phase fluorescence spectroscopy. The method was carried out on powdered samples, consisting of an active substance dispersed in lactose, maize starch, talc and magnesium stearate. Previous knowledge of the sample bulk composition is needed for

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

    Microsoft Academic Search

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

    2005-01-01

    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

  14. Performance evaluation of fiber optic probes for tissue lifetime fluorescence spectroscopy

    Microsoft Academic Search

    Thanassis Papaioannou; Norris W. Preyer Jr.; Qiyin Fang; Hamza Kurt; Michael Carnohan; Russel Ross; Adam Brightwell; Greg Cottone; Linda R. Jones; Laura Marcu

    2003-01-01

    The design of fiber-optic probes plays an important role in optical spectroscopic studies, including fluorescence spectroscopy of biological tissues. It can affect the light delivery and propagation into the tissue, the collection efficiency (total number of photons collected vs. total number of photons launched) and the origin of collected light. This in turn affects the signal to noise ratio (SNR)

  15. Recombinant phytochrome of the moss Ceratodon purpureus (CP2): fluorescence spectroscopy and photochemistry

    Microsoft Academic Search

    V Sineshchekov; L Koppel’; J Hughes; T Lamparter; M Zeidler

    2000-01-01

    The recombinant phytochrome of the moss Ceratodon purpureus (CP2) expressed in Saccharomyces cerevisiae and reconstituted with phycocyanobilin (PCB) was investigated using fluorescence spectroscopy. The pigment had an emission maximum at 670 nm at low temperature (85 K) and at 667 nm at room temperature (RT) and an excitation maximum at 650–652 nm at 85 K (excitation spectra could not be

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

    SciTech Connect

    McGown, L.B.

    1990-01-01

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

  17. Discrepancy between fluorescence correlation spectroscopy and fluorescence recovery after photobleaching diffusion measurements of G-protein-coupled receptors.

    PubMed

    Calizo, Rhodora Cristina; Scarlata, Suzanne

    2013-09-01

    Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) are the two most direct methods to measure the diffusion of molecules in intact living cells. Ideally, these methods should produce similar results for an identical system. We have used these methods to monitor the diffusion of two G-protein-coupled receptors and their associated proteins in the plasma membranes of cells that do not or do contain invaginated protein domains called caveolae. FRAP studies show that caveolae domains increase the immobile fraction of receptors without significantly changing their mobility. On the other hand, FCS studies show an unexpected increase the mobility of caveolae-associated proteins. Our data suggest that the geometry of caveolae domains gives rise to a confined diffusion of its attached proteins, resulting in an apparent increase in mobility. PMID:23748145

  18. Discrepancy between fluorescence correlation spectroscopy and fluorescence recovery after photobleaching diffusion measurements of G-protein-coupled receptors

    PubMed Central

    Calizo, Rhodora Cristina; Scarlata, Suzanne

    2013-01-01

    Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) are the two most direct methods to measure the diffusion of molecules in intact living cells. Ideally, these methods should produce similar results for an identical system. We have used these methods to monitor the diffusion of two G-protein-coupled receptors and their associated proteins in the plasma membranes of cells that do not or do contain invaginated protein domains called caveolae. FRAP studies show that caveolae domains increase the immobile fraction of receptors without significantly changing their mobility. On the other hand, FCS studies show an unexpected increase the mobility of caveolae-associated proteins. Our data suggest that the geometry of caveolae domains gives rise to a confined diffusion of its attached proteins, resulting in an apparent increase in mobility. PMID:23748145

  19. High-throughput single-molecule fluorescence spectroscopy using parallel detection

    PubMed Central

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

    2011-01-01

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

  20. Target-cancer-cell-specific activatable fluorescence imaging probes: rational design and in vivo applications.

    PubMed

    Kobayashi, Hisataka; Choyke, Peter L

    2011-02-15

    Conventional imaging methods, such as angiography, computed tomography (CT), magnetic resonance imaging (MRI), and radionuclide imaging, rely on contrast agents (iodine, gadolinium, and radioisotopes, for example) that are "always on." Although these indicators have proven clinically useful, their sensitivity is lacking because of inadequate target-to-background signal ratio. A unique aspect of optical imaging is that fluorescence probes can be designed to be activatable, that is, only "turned on" under certain conditions. These probes are engineered to emit signal only after binding a target tissue; this design greatly increases sensitivity and specificity in the detection of disease. Current research focuses on two basic types of activatable fluorescence probes. The first developed were conventional enzymatically activatable probes. These fluorescent molecules exist in the quenched state until activated by enzymatic cleavage, which occurs mostly outside of the cells. However, more recently, researchers have begun designing target-cell-specific activatable probes. These fluorophores exist in the quenched state until activated within targeted cells by endolysosomal processing, which results when the probe binds specific receptors on the cell surface and is subsequently internalized. In this Account, we present a review of the rational design and in vivo applications of target-cell-specific activatable probes. In engineering these probes, researchers have asserted control over a variety of factors, including photochemistry, pharmacological profile, and biological properties. Their progress has recently allowed the rational design and synthesis of target-cell-specific activatable fluorescence imaging probes, which can be conjugated to a wide variety of targeting molecules. Several different photochemical mechanisms have been utilized, each of which offers a unique capability for probe design. These include self-quenching, homo- and hetero-fluorescence resonance energy transfer (FRET), H-dimer formation, and photon-induced electron transfer (PeT). In addition, the repertoire is further expanded by the option for reversibility or irreversibility of the signal emitted through these mechanisms. Given the wide range of photochemical mechanisms and properties, target-cell-specific activatable probes have considerable flexibility and can be adapted to specific diagnostic needs. A multitude of cell surface molecules, such as overexpressed growth factor receptors, are directly related to carcinogenesis and thus provide numerous targets highly specific for cancer. This discussion of the chemical, pharmacological, and biological basis of target-cell-specific activatable imaging probes, and methods for successfully designing them, underscores the systematic, rational basis for further developing in vivo cancer imaging. PMID:21062101

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

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

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

  2. Compensation of optical heterogeneity-induced artifacts in fluorescence molecular tomography: theory and in vivo validation

    NASA Astrophysics Data System (ADS)

    Mohajerani, Pouyan; Adibi, Ali; Kempner, Joshua; Yared, Wael

    2009-05-01

    We present a method for reduction of image artifacts induced by the optical heterogeneities of tissue in fluorescence molecular tomography (FMT) through identification and compensation of image regions that evidence propagation of emission light through thin or low-absorption tunnels in tissue. The light tunneled as such contributes to the emission image as spurious components that might substantially overwhelm the desirable fluorescence emanating from the targeted lesions. The proposed method makes use of the strong spatial correlation between the emission and excitation images to estimate the tunneled components and yield a residual image that mainly consists of the signal due to the desirable fluorescence. This residual image is further refined using a coincidence mask constructed for each excitation-emission image pair. The coincidence mask is essentially a map of the ``hot spots'' that occur in both excitation and emission images, as such areas are often associated with tunneled emission. In vivo studies are performed on a human colon adenocarcinoma xenograft tumor model with subcutaneous tumors and a murine breast adenocarcinoma model with aggressive tumor cell metastasis and growth in the lungs. Results demonstrate significant improvements in the reconstructions achieved by the proposed method.

  3. Dual-Color Fluorescence Cross-Correlation Spectroscopy on a

    E-print Network

    Garbe, Christoph S.

    spectroscopy. II. an experimental realization." Biopolymers 13, 29­61 (1974). 3. K. M. Berland, P. T. So, and E, Germany jl@dkfz.de http://www.dkfz.de/Macromol Abstract: Single plane illumination microscopy based, 1416­1420 (1998). 6. K. G. Heinze, M. Jahnz, and P. Schwille, "Triple-color coincidence analysis: One

  4. Fluorescence kinetics of Trp-Trp dipeptide and its derivatives in water via ultrafast fluorescence spectroscopy.

    PubMed

    Jia, Menghui; Yi, Hua; Chang, Mengfang; Cao, Xiaodan; Li, Lei; Zhou, Zhongneng; Pan, Haifeng; Chen, Yan; Zhang, Sanjun; Xu, Jianhua

    2015-08-01

    Ultrafast fluorescence dynamics of Tryptophan-Tryptophan (Trp-Trp/Trp2) dipeptide and its derivatives in water have been investigated using a picosecond resolved time correlated single photon counting (TCSPC) apparatus together with a femtosecond resolved upconversion spectrophotofluorometer. The fluorescence decay profiles at multiple wavelengths were fitted by a global analysis technique. Nanosecond fluorescence kinetics of Trp2, N-tert-butyl carbonyl oxygen-N'-aldehyde group-l-tryptophan-l-tryptophan (NBTrp2), l-tryptophan-l-tryptophan methyl ester (Trp2Me), and N-acetyl-l-tryptophan-l-tryptophan methyl ester (NATrp2Me) exhibit multi-exponential decays with the average lifetimes of 1.99, 3.04, 0.72 and 1.22ns, respectively. Due to the intramolecular interaction between two Trp residues, the "water relaxation" lifetime was observed around 4ps, and it is noticed that Trp2 and its derivatives also exhibit a new decay with a lifetime of ?100ps, while single-Trp fluorescence decay in dipeptides/proteins shows 20-30ps. The intramolecular interaction lifetime constants of Trp2, NBTrp2, Trp2Me and NATrp2Me were then calculated to be 3.64, 0.93, 11.52 and 2.40ns, respectively. Candidate mechanisms (including heterogeneity, solvent relaxation, quasi static self-quenching or ET/PT quenching) have been discussed. PMID:26111991

  5. Changes in yield of in-vivo fluorescence of chlorophyll a as a tool for selective herbicide monitoring

    Microsoft Academic Search

    Roswitha Conrad; Claudia Bilchel; Christian Wilhelml; Wafa Arsalane; Claire Berkaloff; Jean-Claude Duval

    1993-01-01

    Triazines and derivatives of phenylurea, which are often found in outdoor water samples, induce specific changes in the yield of thein-vivo chlorophyll ?-fluorescence of PSII. These changes are correlated quantitatively with the concentration of the herbicides and can therefore be used to set-up a low-price monitor system. In order to detect selectively the herbicide-sensitive part of the fluorescence emission a

  6. Dual-color fluorescence cross-correlation spectroscopy on a planar optofluidic chip.

    PubMed

    Chen, A; Eberle, M M; Lunt, E J; Liu, S; Leake, K; Rudenko, M I; Hawkins, A R; Schmidt, H

    2011-04-21

    Fluorescence cross-correlation spectroscopy (FCCS) is a highly sensitive fluorescence technique with distinct advantages in many bioanalytical applications involving interaction and binding of multiple components. Due to the use of multiple beams, bulk optical FCCS setups require delicate and complex alignment procedures. We demonstrate the first implementation of dual-color FCCS on a planar, integrated optofluidic chip based on liquid-core waveguides that can guide liquid and light simultaneously. In this configuration, the excitation beams are delivered in predefined locations and automatically aligned within the excitation waveguides. We implement two canonical applications of FCCS in the optofluidic lab-on-chip environment: particle colocalization and binding/dissociation dynamics. Colocalization is demonstrated in the detection and discrimination of single-color and double-color fluorescently labeled nanobeads. FCCS in combination with fluorescence resonance energy transfer (FRET) is used to detect the denaturation process of double-stranded DNA at nanomolar concentration. PMID:21340094

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

    SciTech Connect

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

    2006-12-01

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

  8. Use of a Microscope Photometer To Analyze In Vivo Fluorescence Intensity of Epilithic Microalgae Grown on Artificial Substrata

    PubMed Central

    Becker, G.; Holfeld, H.; Hasselrot, A. T.; Fiebig, D. M.; Menzler, D. A.

    1997-01-01

    An epifluorescence microscope photometer was used to develop a new, in vivo fluorimetric method for analyzing fluorescence intensities of epilithic microalgae grown on clay tiles in the field. This enabled a nondestructive, direct quantification of algal biomass on the substratum surface. Measurements of a chlorophyll a standard in ethanol (90%) with our fluorimetric method (exitation at 546 nm; emission, >590 nm) correlated well with those from conventional spectrofluorimetric and spectrophotometric methods. Biofilms were analyzed with the microscope photometer by measuring the in vivo fluorescence intensity of 70 spots distributed randomly over the tile surface. They were then analyzed by the two in vitro methods after photopigment extraction. Chlorophyll a content and in vivo fluorescence intensity correlated well. The regression curves were linear up to 6 (mu)g cm(sup-2) but were quadratic or hyperbolic at higher concentrations of up to 28 (mu)g cm(sup-2). The degree of scatter among individual measurements was higher in biofilms than chlorophyll a standards. This in vivo analysis is well suited to ecological experiments and has the advantage of measuring on an extremely small scale, which enables direct analysis of the microdistribution of epilithic microalgae in live biofilms. We demonstrated this by comparing fluorescence intensities of the grazing tracks of the snail Ancylus fluviatilis with those of ungrazed areas. Our in vivo analysis is also unique in enabling biofilms on artificial substrata to be removed, analyzed, and then returned intact in field or laboratory experiments. PMID:16535568

  9. A novel indocyanine green nanoparticle probe for non invasive fluorescence imaging in vivo

    NASA Astrophysics Data System (ADS)

    Navarro, Fabrice P.; Berger, Michel; Goutayer, Mathieu; Guillermet, Stéphanie; Josserand, Véronique; Rizo, Philippe; Vinet, Françoise; Texier, Isabelle

    2009-02-01

    Fluorescence imaging (FLI) allows the in vivo monitoring of biological events associated with disease and represents a new promising tool for drug discovery. In particular, it speeds up the development and assessment of new therapies in oncology, helps in diagnosis, and improves surgery by fluorescence-guided tumor resection. This technique is highly sensitive, non-ionizing, easy to use and relatively inexpensive. Nevertheless, the main limitation of FLI lies in the optical properties of biological tissues. Mainly because of haemoglobin and water absorption, only near-infrared (NIR) light is adapted to image tissues in depth. Using a contrasting agent absorbing and emitting in the NIR region is therefore necessary to improve the background signal ratio, and thus the image contrast. Among many commercially available NIR optical contrast agents, only indocyanine green (ICG), has been approved by the United State Food and Drug Administration (FDA) for various medical applications. However, its instability (photo-degradation, thermal-degradation and low aqueous solubility) limits its applications as a fluorescent probe for imaging purposes. In order to improve the effectiveness of ICG, we engineered ICG-doped lipid nanoparticles (LNP). In this communication, we will report the design of these novel fluorescent nanoparticle probes. These low cost nanocarriers have numerous advantages, including their high chemical stability and biocompatibility. The characterization of the optical properties of the nanoparticles entrapping ICG will also be discussed. Finally, the biodistribution in mice of ICG when delivered through nanoparticles in comparison to free ICG in solution is presented. It demonstrates the efficient accumulation of ICG-doped nanoparticles in the tumor site.

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

    NASA Astrophysics Data System (ADS)

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

    2008-06-01

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

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

    PubMed Central

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

    2010-01-01

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

  12. Far-field infrared super-resolution microscopy using picosecond time-resolved transient fluorescence detected IR spectroscopy

    NASA Astrophysics Data System (ADS)

    Sakai, Makoto; Kawashima, Yasutake; Takeda, Akihiro; Ohmori, Tsutomu; Fujii, Masaaki

    2007-05-01

    A new far-field infrared super-resolution microscopy combining laser fluorescence microscope and picosecond time-resolved transient fluorescence detected IR (TFD-IR) spectroscopy is proposed. TFD-IR spectroscopy is a kind of IR-visible/UV double resonance spectroscopy, and detects IR transitions by the transient fluorescence due to electronic transition originating from vibrationally excited level populated by IR light. IR images of rhodamine-6G solution and of fluorescent beads were clearly observed by monitoring the transient fluorescence. Super-resolution twice higher than the diffraction limit for IR light was achieved. The IR spectrum due to the transient fluorescence was also measured from spatial domains smaller than the diffraction limit.

  13. In vivo soft tissue differentiation by diffuse reflectance spectroscopy: preliminary results

    NASA Astrophysics Data System (ADS)

    Zam, Azhar; Stelzle, Florian; Tangermann-Gerk, Katja; Adler, Werner; Nkenke, Emeka; Neukam, Friedrich Wilhelm; Schmidt, Michael; Douplik, Alexandre

    Remote laser surgery does not provide haptic feedback to operate layer by layer and preserve vulnerable anatomical structures like nerve tissue or blood vessels. The aim of this study is identification of soft tissue in vivo by diffuse reflectance spectroscopy to set the base for a feedback control system to enhance nerve preservation in oral and maxillofacial laser surgery. Various soft tissues can be identified by diffuse reflectance spectroscopy in vivo. The results may set the base for a feedback system to prevent nerve damage during oral and maxillofacial laser surgery.

  14. Laguerre-based method for analysis of time-resolved fluorescence data: application to in-vivo characterization and diagnosis of atherosclerotic lesions

    NASA Astrophysics Data System (ADS)

    Jo, Javier A.; Fang, Qiyin; Papaioannou, Thanassis; Baker, J. Dennis; Dorafshar, Amir; Reil, Todd; Qiao, Jianhua; Fishbein, Michael C.; Freischlag, Julie A.; Marcu, Laura

    2006-03-01

    We report the application of the Laguerre deconvolution technique (LDT) to the analysis of in-vivo time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data and the diagnosis of atherosclerotic plaques. TR-LIFS measurements were obtained in vivo from normal and atherosclerotic aortas (eight rabbits, 73 areas), and subsequently analyzed using LDT. Spectral and time-resolved features were used to develop four classification algorithms: linear discriminant analysis (LDA), stepwise LDA (SLDA), principal component analysis (PCA), and artificial neural network (ANN). Accurate deconvolution of TR-LIFS in-vivo measurements from normal and atherosclerotic arteries was provided by LDT. The derived Laguerre expansion coefficients reflected changes in the arterial biochemical composition, and provided a means to discriminate lesions rich in macrophages with high sensitivity (>85%) and specificity (>95%). Classification algorithms (SLDA and PCA) using a selected number of features with maximum discriminating power provided the best performance. This study demonstrates the potential of the LDT for in-vivo tissue diagnosis, and specifically for the detection of macrophages infiltration in atherosclerotic lesions, a key marker of plaque vulnerability.

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

    PubMed Central

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

    2014-01-01

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

  16. Noninvasive Fluorescence Resonance Energy Transfer Imaging of in vivo Premature Drug Release from Polymeric Nanoparticles

    PubMed Central

    Zou, Peng; Chen, Hongwei; Paholak, Hayley J.; Sun, Duxin

    2013-01-01

    Understanding in vivo drug release kinetics is critical for the development of nanoparticle-based delivery systems. In this study, we developed a fluorescence resonance energy transfer (FRET) imaging approach to noninvasively monitor in vitro and in vivo cargo release from polymeric nanoparticles. The FRET donor dye (DiO or DiD) and acceptor dye (DiI or DiR) were individually encapsulated into poly(ethylene oxide)-b-polystyrene (PEO-PS) nanoparticles. When DiO (donor) nanoparticles and DiI (acceptor) nanoparticles were co-incubated with cancer cells for 2 h, increased FRET signals were observed from cell membranes, suggesting rapid release of DiO and DiI to cell membranes. Similarly, increased FRET ratios were detected in nude mice after intravenous co-administration of DiD (donor) nanoparticles and DiR (acceptor) nanoparticles. In contrast, another group of nude mice i.v. administrated with DiD/DiR co-loaded nanoparticles showed decreased FRET ratios. Based on the difference in FRET ratios between the two groups, in vivo DiD/DiR release half-life from PEO-PS nanoparticles was determined to be 9.2 min. In addition, it was observed that the presence of cell membranes facilitated burst release of lipophilic cargos while incorporation of oleic acid-coated iron oxide into PEO-PS nanoparticles slowed the release of DiD/DiR to cell membranes. The developed in vitro and in vivo FRET imaging techniques can be used to screening stable nano-formulations for lipophilic drug delivery. PMID:24033270

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

    NASA Astrophysics Data System (ADS)

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

    1998-07-01

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

  18. Method for rapid multidiameter single-fiber reflectance and fluorescence spectroscopy through a fiber bundle.

    PubMed

    Hoy, Christopher L; Gamm, Ute A; Sterenborg, Henricus J C M; Robinson, Dominic J; Amelink, Arjen

    2013-10-01

    We have recently demonstrated a means for quantifying the absorption and scattering properties of biological tissue through multidiameter single-fiber reflectance (MDSFR) spectroscopy. These measurements can be used to correct single-fiber fluorescence (SFF) spectra for the influence of optical properties, enabling quantification of intrinsic fluorescence. In our previous work, we have used a series of pinholes to show that selective illumination and light collection using a coherent fiber bundle can simulate a single solid-core optical fiber with variable diameter for the purposes of MDSFR spectroscopy. Here, we describe the construction and validation of a clinical MDSFR/SFF spectroscopy system that avoids the limitations encountered with pinholes and free-space optics. During one measurement, the new system acquires reflectance spectra at the effective diameters of 200, 600, and 1000 ?m, and a fluorescence spectrum at an effective diameter of 1000 ?m. From these spectra, we measure the absolute absorption coefficient, ?(a), reduced scattering coefficient, ?'(s'), phase function parameter, ?, and intrinsic fluorescence, Q?(a,x)(f), across the measured spectrum. We validate the system using Intralipid- and polystyrene sphere-based scattering phantoms, with and without the addition of the absorber Evans Blue. Finally, we demonstrate the combined MDSFR/SFF of phantoms with varying concentrations of Intralipid and fluorescein, wherein the scattering properties are measured by MDSFR and used to correct the SFF spectrum for accurate quantification of Q?(a,x)(f). PMID:24126725

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

    PubMed

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

    2014-12-01

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

  20. Towards in situ fluorescence spectroscopy and microscopy investigations of asphaltene precipitation kinetics.

    PubMed

    Franco, Juliana C; Gonçalves, Grasiele; Souza, Monique S; Rosa, Samantha B C; Thiegue, Larissa M; Atvars, Teresa D Z; Rosa, Paulo T V; Nome, René A

    2013-12-16

    We perform a spectroscopic analysis of asphaltene in solution and in crude oil with the goal of designing an optical probe of asphaltene precipitation inside high-pressure cells. Quantitative analysis of steady-state spectroscopic data is employed to identify fluorescence and Raman contributions to the observed signals. Time-resolved fluorescence spectroscopy indicates that fluorescence lifetime can be used as a spectroscopic probe of asphaltene in crude oil. Quantitative confocal laser-scanning microscopy studies of asphaltene in n-heptane are used to calculate particle-size distributions as a function of time, both at the sample surface and asphaltene interior. The resulting precipitation kinetics is well described by stochastic numerical simulations of diffusion-limited aggregation. Based on these results, we present the design and construction of an apparatus to optically probe the in situ precipitation of asphaltene suitable for studies inside high pressure cells. Design considerations include the use of a spatial light modulator for aberration correction in microscopy measurements, together with the design of epi-fluorescence spectrometer, both fiber-based and for remote sensing fluorescence spectroscopy. PMID:24514660

  1. Doppler-free Yb spectroscopy with the fluorescence spot technique

    SciTech Connect

    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

    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.

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

    PubMed

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

    2014-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

    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.

  4. In vivo detection of cancer cells with immunoconjugated fluorescent probes by macro zoom microscopy and two-photon microscopy

    NASA Astrophysics Data System (ADS)

    Koga, Shigehiro; Oshima, Yusuke; Hikita, Atsuhiko; Sato, Koichi; Yoshida, Motohira; Yamamoto, Yuji; Iimura, Tadahiro; Watanabe, Yuji; Imamura, Takeshi

    2015-03-01

    We developed a near infrared fluorophore-conjugated anti-Carcinoembryonic antigen (CEA) antibody for mice bearing tumor of CEA expressing cells, and demonstrated in vivo optical imaging by macro zoom microscopy. In the result, tumors of CEA-expressing cancer cells were specifically detected in vivo. Furthermore, cancer-specific fluorescence images were acquired at subcellular level in vivo by two-photon microscopy. In preclinical applications, the lymph node micrometastasis was also successfully visualized by two-photon microscopy. These results suggest that two-photon excitation microscopy in combination with an immunoconjugated probe could be widely adapted to cancer detection in clinical settings.

  5. In vivo nanoparticle-mediated radiopharmaceutical-excited fluorescence molecular imaging.

    PubMed

    Hu, Zhenhua; Qu, Yawei; Wang, Kun; Zhang, Xiaojun; Zha, Jiali; Song, Tianming; Bao, Chengpeng; Liu, Haixiao; Wang, Zhongliang; Wang, Jing; Liu, Zhongyu; Liu, Haifeng; Tian, Jie

    2015-01-01

    Cerenkov luminescence imaging utilizes visible photons emitted from radiopharmaceuticals to achieve in vivo optical molecular-derived signals. Since Cerenkov radiation is weak, non-optimum for tissue penetration and continuous regardless of biological interactions, it is challenging to detect this signal with a diagnostic dose. Therefore, it is challenging to achieve useful activated optical imaging for the acquisition of direct molecular information. Here we introduce a novel imaging strategy, which converts ? and Cerenkov radiation from radioisotopes into fluorescence through europium oxide nanoparticles. After a series of imaging studies, we demonstrate that this approach provides strong optical signals with high signal-to-background ratios, an ideal tissue penetration spectrum and activatable imaging ability. In comparison with present imaging techniques, it detects tumour lesions with low radioactive tracer uptake or small tumour lesions more effectively. We believe it will facilitate the development of nuclear and optical molecular imaging for new, highly sensitive imaging applications. PMID:26123615

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

    SciTech Connect

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

    2007-01-19

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

  7. In vivo nanoparticle-mediated radiopharmaceutical-excited fluorescence molecular imaging

    PubMed Central

    Hu, Zhenhua; Qu, Yawei; Wang, Kun; Zhang, Xiaojun; Zha, Jiali; Song, Tianming; Bao, Chengpeng; Liu, Haixiao; Wang, Zhongliang; Wang, Jing; Liu, Zhongyu; Liu, Haifeng; Tian, Jie

    2015-01-01

    Cerenkov luminescence imaging utilizes visible photons emitted from radiopharmaceuticals to achieve in vivo optical molecular-derived signals. Since Cerenkov radiation is weak, non-optimum for tissue penetration and continuous regardless of biological interactions, it is challenging to detect this signal with a diagnostic dose. Therefore, it is challenging to achieve useful activated optical imaging for the acquisition of direct molecular information. Here we introduce a novel imaging strategy, which converts ? and Cerenkov radiation from radioisotopes into fluorescence through europium oxide nanoparticles. After a series of imaging studies, we demonstrate that this approach provides strong optical signals with high signal-to-background ratios, an ideal tissue penetration spectrum and activatable imaging ability. In comparison with present imaging techniques, it detects tumour lesions with low radioactive tracer uptake or small tumour lesions more effectively. We believe it will facilitate the development of nuclear and optical molecular imaging for new, highly sensitive imaging applications. PMID:26123615

  8. Temporal changes in microvessel leakiness during wound healing discriminated by in vivo fluorescence recovery after photobleaching

    PubMed Central

    Machado, Maria J C; Mitchell, Christopher A

    2011-01-01

    Abstract Regeneration of injured tissue is a dynamic process, critically dependent on the formation of new blood vessels and restructuring of the nascent plexus. Endothelial barrier function, a functional correlate of vascular restructuring and maturation, was quantified via intravital microscopic analysis of 150 kDa FITC-dextran-perfused blood vessels within discrete wounds created in the panniculus carnosus (PC) muscle of dorsal skinfold chamber (DSC) preparations in mice. Time to recovery of half-peak fluorescence intensity (t1/2) within individual vessel segments in three functional regions of the wound (pre-existing vessels, angiogenic plexus and blind-ended vessels (BEVs)) was quantified using in vivo fluorescence recovery after photobleaching (FRAP) and linear regression analysis of recovery profiles. Plasma flux across the walls of new vessel segments, particularly BEVs, was greater than that of pre-existing vessels at days 5–7 after injury (P < 0.05). TNP-470 reduced the permeability of BEVs at the leading edge of the advancing vascular plexus as measured by the decrease in luminal t1/2 (P < 0.05), confirming the utility of FRAP as a quantitative measure of endothelial barrier function. Furthermore, these data are suggestive of a role for TNP-470 in selection for less leaky vascular segments within healing wounds. Increased FITC-dextran leakage was observed from pre-existing vessels after treatment with TNP-470 (P < 0.05), consistent with induction of transient vascular damage, although the significance of this finding is unclear. Using in vivo FRAP this study demonstrates the relationship between temporal changes in microvascular macromolecular flux and the morphology of maturing vascular segments. This combination of techniques may be useful to assess the therapeutic potential of angiogenic agents in restoring pre-injury levels of endothelial barrier function, following the establishment of a functional vascular plexus such as in models of wounding or tumour development. PMID:21768268

  9. Detection of Non-Melanoma Skin Cancer by in vivo Fluorescence Imaging with Fluorocoxib A

    PubMed Central

    Ra, Hyejun; González-González, Emilio; Uddin, Md. Jashim; King, Bonnie L.; Lee, Alex; Ali-Khan, Irfan; Marnett, Lawrence J.; Tang, Jean Y.; Contag, Christopher H.

    2015-01-01

    Non-melanoma skin cancer (NMSC) is the most common form of cancer in the US and its incidence is increasing. The current standard of care is visual inspection by physicians and/or dermatologists, followed by skin biopsy and pathologic confirmation. We have investigated the use of in vivo fluorescence imaging using fluorocoxib A as a molecular probe for early detection and assessment of skin tumors in mouse models of NMSC. Fluorocoxib A targets the cyclooxygenase-2 (COX-2) enzyme that is preferentially expressed by inflamed and tumor tissue, and therefore has potential to be an effective broadly active molecular biomarker for cancer detection. We tested the sensitivity of fluorocoxib A in a BCC allograft SCID hairless mouse model using a wide-field fluorescence imaging system. Subcutaneous allografts comprised of 1000 BCC cells were detectable above background. These BCC allograft mice were imaged over time and a linear correlation (R2 = 0.8) between tumor volume and fluorocoxib A signal levels was observed. We also tested fluorocoxib A in a genetically engineered spontaneous BCC mouse model (Ptch1+/? K14-Cre-ER2 p53fl/fl), where sequential imaging of the same animals over time demonstrated that early, microscopic lesions (100 ?m size) developed into visible macroscopic tumor masses over 11 to 17 days. Overall, for macroscopic tumors, the sensitivity was 88% and the specificity was 100%. For microscopic tumors, the sensitivity was 85% and specificity was 56%. These results demonstrate the potential of fluorocoxib A as an in vivo imaging agent for early detection, margin delineation and guided biopsies of NMSCs. PMID:25748239

  10. Effects of a topically applied wound ointment on epidermal wound healing studied by in vivo fluorescence laser scanning microscopy analysis

    Microsoft Academic Search

    Bernhard Lange-Asschenfeldt; Alena Alborova; Daniela Krüger-Corcoran; Alexa Patzelt; Heike Richter; Wolfram Sterry; Axel Kramer; Eggert Stockfleth; Jürgen Lademann

    2009-01-01

    Epidermal wound healing is a complex and dynamic regenerative process necessary to reestablish skin integrity. Fluorescence confocal laser scanning microscopy (FLSM) is a noninvasive imaging technique that has previously been used for evaluation of inflammatory and neoplastic skin disorders in vivo and at high resolution. We employed FLSM to investigate the evolution of epidermal wound healing noninvasively over time and

  11. Fabrication of folate bioconjugated near-infrared fluorescent silver nanoclusters for targeted in vitro and in vivo bioimaging.

    PubMed

    Wang, Yong; Dai, Cong; Yan, Xiu-Ping

    2014-11-28

    Thiolpolyethyleneimine stabilized silver nanoclusters (SH-PEI-AgNCs) with intense NIR fluorescence and chemical stability were fabricated in aqueous solution. The SH-PEI-AgNCs were subsequently bioconjugated with folate for targeted in vitro and in vivo bioimaging. PMID:25285944

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

    NASA Astrophysics Data System (ADS)

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

    2006-07-01

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

  13. Assessment of humification degree of dissolved organic matter from different composts using fluorescence spectroscopy technology.

    PubMed

    Wei, Zimin; Zhao, Xinyu; Zhu, Chaowei; Xi, Beidou; Zhao, Yue; Yu, Xue

    2014-01-01

    This study was conducted to assess the degree of humification in dissolved organic matter (DOM) from different composts, and their environmental impact after soil amending based on fluorescence measurements (emission, excitation, synchronous scan, and excitation-emission matrix [EEM]). The compost sources studied included dairy cattle manure (DCM), kitchen waste (KW), cabbage waste (CW), tomato stem waste (TSW), municipal solid waste (MSW), green waste (GW), chicken manure (CM), and peat (P). Conventional and EEM fluorescence spectroscopy indicated that the DOM of these composts contained compounds similar in structure but comparisons between conventional fluorescence parameters and fluorescence regional integration of EEM fluorescence spectra showed that the DOM was different in degree of humification. Regression analysis demonstrated significant corrections between major fluorescence parameters. In hierarchical cluster analysis, these composts were clustered into 2 groups and 4 subgroups, and projection pursuit regression analysis further ranked the compost sources as KW, CW, P>CM, DCM, TW, GW>MSW in their degree of humification in DOM. PMID:24188626

  14. Moving in on the Action: An Experimental Comparison of Fluorescence Excitation and Photodissociation Action Spectroscopy.

    PubMed

    Wellman, Sydney M J; Jockusch, Rebecca A

    2015-06-18

    Photodissociation action spectroscopy is often used as a proxy for measuring gas-phase absorption spectra of ions in a mass spectrometer. Although the potential discrepancy between linear optical and photodissociation spectra is generally acknowledged, direct experimental comparisons are lacking. In this work, we use a quadrupole ion trap that has been modified to enable both photodissociation and laser-induced fluorescence to assess how closely the visible photodissociation action spectrum of a fluorescent dye reflects its fluorescence excitation spectrum. Our results show the photodissociation action spectrum of gaseous rhodamine 110 is both substantially narrower and slightly red-shifted (?120 cm(-1)) compared to its fluorescence excitation spectrum. Power dependence measurements reveal that the photodissociation of rhodamine 110 requires, on average, the absorption of three photons whereas fluorescence is a single-photon process. These differing power dependences are the key to interpreting the differences in the measured spectra. The experimental results provide much-needed quantification and insight into the differences between action spectra and linear optical spectra, and emphasize the utility of fluorescence excitation spectra to provide a more reliable benchmark for comparison with theory. PMID:26020810

  15. TOPICAL REVIEW: Prospects for in vivo Raman spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2000-02-01

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

  16. Volatile fractions of landfill leachates and their effect on Chlamydomonas reinhardtii: In vivo chlorophyll a fluorescence

    SciTech Connect

    Brack, W.; Rottler, H.; Frank, H. [Univ. of Bayreuth (Germany)

    1998-10-01

    Volatile organic compounds such as short-chain halogenated hydrocarbons and alkylated benzenes are widely used as solvents or as intermediates in the chemical industry, and some of them are fuel components. Dichloromethane, trichloroethene, 1,1,1-trichloroethane, and tetrachloroethene have been produced in amounts of 500,000 to 1 million t/year, 80 to 100% of which are released to the environment. The production of toluene, a major component of fuels for internal combustion engines, amounts to about 30 million t/year. A method for identification of toxic volatile constituents of landfill leachates is presented that combines bioassay-compatible sample preparation, chemical analysis, and a bioassay based on in vivo chlorophyll a fluorescence of the green alga Chlamydomonas reinhardtii. Two major pathways of toxicity were identified by comparing fluorescence patterns: specific toxicity of hydrogen sulfide, and narcotic action of nonreactive organic compounds. For quantification, the contributions of identified compounds were calculated using toxic units. The ecotoxicologic relevance of volatile fractions from hazardous waste leachates was shown.

  17. In vivo EPR spectroscopy: biomedical and potential diagnostic applications.

    PubMed

    Jackson, Simon K; Thomas, Matthew P; Smith, Sam; Madhani, Melanie; Rogers, Stephen C; James, Philip E

    2004-01-01

    EPR spectroscopic techniques have been developed for the measurement of oxygen and nitric oxide in vivo. Specifically, the methods for in vivo measurement of these molecules has been applied to the study of septic shock, utilising an experimental murine model developed in our laboratory. Oxygen was measured as pO2 by the particlulate probes Gloxy and LiPc, which were surgically implanted at specific sites in tissues, and the soluble probe Trityl, which was administered intravenously. Nitric oxide was measured as the NO-Fe-(DETC)2 complex after administration of Fe2+ and DETC. LPS was seen to significantly decrease liver oxygen measured across the lobule and at the sinusoids by the Gloxy probe; there was a corresponding increase in nitric oxide both in the liver and systemically. The nitric oxide most likely originated from increased iNOS enzyme in the liver as demonstrated by Western blotting and the localisation of nitric oxide to the liver was confirmed with EPR imaging. LPS also caused a decrease in cerebral blood and tissue oxygenation, the rate of which was found to be dependent on the blood oxygenation. The development and applications of these in vivo EPR techniques for biomedical research and diagnostics is discussed. PMID:14992402

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2013-01-01

    Superoxide anion is the key radical that causes intracellular oxidative stress. The lack of a method to directly monitor superoxide concentration in vivo in real time has severely hindered our understanding on its pathophysiology. We made transgenic zebrafish to specifically express yellow fluorescent proteins, a reversible superoxide-specific indicator, in the liver and used a fiber-optic fluorescent probe to noninvasively monitor the superoxide concentration in real time. Several superoxide-inducing and scavenging reagents were administrated onto the fish to alter superoxide concentrations. The distinct biochemical pathways of the reagents can be discerned from the transient behaviors of fluorescence time courses. These results demonstrate the feasibility of this method for analyzing superoxide dynamics and its potential as an in vivo pharmaceutical screening platform. PMID:24049691

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

    PubMed Central

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

    2010-01-01

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

  1. In vivo deep tissue fluorescence imaging of the murine small intestine and colon

    NASA Astrophysics Data System (ADS)

    Crosignani, Viera; Dvornikov, Alexander; Aguilar, Jose S.; Stringari, Chiara; Edwards, Roberts; Mantulin, Williams; Gratton, Enrico

    2012-03-01

    Recently we described a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. This technique was applied to in vivo imaging of murine small intestine and colon. Individuals with Inflammatory Bowel Disease (IBD), commonly presenting as Crohn's disease or Ulcerative Colitis, are at increased risk for developing colorectal cancer. We have developed a Gi?2 gene knock out mouse IBD model that develops colitis and colon cancer. The challenge is to study the disease in the whole animal, while maintaining high resolution imaging at millimeter depth. In the Gi?2-/- mice, we have been successful in imaging Lgr5-GFP positive stem cell reporters that are found in crypts of niche structures, as well as deeper structures, in the small intestine and colon at depths greater than 1mm. In parallel with these in vivo deep tissue imaging experiments, we have also pursued autofluorescence FLIM imaging of the colon and small intestine-at more shallow depths (roughly 160?m)- on commercial two photon microscopes with excellent structural correlation (in overlapping tissue regions) between the different technologies.

  2. In vivo wound healing diagnosis with second harmonic and fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Deka, Gitanjal; Wu, Wei-Wen; Kao, Fu-Jen

    2013-06-01

    Skin wounds heal when a series of cell lineages are triggered, followed by collagen deposition, to reconstruct damaged tissues. This study evaluates the regeneration of collagen and change in cellular metabolic rate in vivo during wound healing in rats, with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy respectively. The metabolic rate of cells is reflected through the lifetime of the autofluorescence from the co-enzyme protein, reduced nicotinamide adenine dinucleotide, due to its change in the relative concentration of bound and free forms. A higher than normal cellular metabolic rate is observed during the first week of healing, which decreases gradually after eight days of wound formation. SHG signal intensity change indicates the net degradation of collagen during the inflammatory phase, and net regeneration begins on day five. Eventually, the quantity of collagen increases gradually to form a scar tissue as the final product. Importantly, this work demonstrates the feasibility of an in vivo imaging approach for a normal wound on rat skin, which has the potential to supplement the noninvasive clinical diagnosis of wounds.

  3. Percutaneous Penetration Enhancement in Vivo Measured by Attenuated Total Reflectance Infrared Spectroscopy

    Microsoft Academic Search

    Vivien H. W. Mak; Russell O. Potts; Richard H. Guy

    1990-01-01

    A novel application of attenuated total reflectance IR Spectroscopy (ATR-IR) was used to monitor the outer several microns of the stratum corneum (SC) and, thereby, demonstrate enhanced percutaneous absorption in vivo in man. 4-Cyanophenol (CP) as a model permeant yielded a unique IR signal, distinct from those of the stratum corneum and the vehicle components. CP was administered for 1,

  4. Ultrafast 2D NMR Spectroscopy Using Sinusoidal Gradients: Principles and Ex Vivo Brain Investigations

    E-print Network

    Frydman, Lucio

    Ultrafast 2D NMR Spectroscopy Using Sinusoidal Gradients: Principles and Ex Vivo Brain ultrafast acquisitions of 2D NMR spectra with suitable spectral widths on a microimaging probe (for both Wiley-Liss, Inc. Key words: ultrafast 2D NMR; magnetic resonance spectros- copy; brain metabolites; 2D

  5. Subcellular In Vivo 1 HM R Spectroscopy ofXenopus laevis Oocytes

    Microsoft Academic Search

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

    2006-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-07-01

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

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

    PubMed Central

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

    2011-01-01

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

  8. Electronic Resonance Enhancement in Raman and CARS Spectroscopy: Surface Enhanced Scattering of Highly Fluorescent Molecules

    NASA Astrophysics Data System (ADS)

    Lawhead, Carlos; Ujj, Laszlo

    2015-03-01

    Surface enhanced Raman spectroscopy (SERS) is an extremely useful tool in increasing sensitivity of Raman spectroscopy; this technique significantly increases the signal from vibrational resonances which can overcome background fluoresces. Silver nanoparticles coated substrates and the silver nanoparticles in solution were used on a variety of fluorescent molecules in order to overcome sample complexities and measure the vibrational spectra. The possible enhancement of SERS using a coherent Raman (CARS) method was investigated, but enhancement factors due to Surface Enhanced CARS have yet to be verified. The instrument used was developed in the University of West Florida Physics Department utilized the second harmonic of a Nd:YAG laser to provide the excitation wavelength at 532 nm and is capable of both transmission and reflection Raman measurements. Special thanks to the UWF Office of Undergraduate Research.

  9. An in vivo multiwell-based fluorescent screen for monitoring vertebrate thyroid hormone disruption.

    PubMed

    Fini, Jean-Baptiste; Le Mevel, Sebastien; Turque, Nathalie; Palmier, Karima; Zalko, Daniel; Cravedi, Jean-Pierre; Demeneix, Barbara A

    2007-08-15

    There is a pressing need for high throughput methods to assess potential effects of endocrine disrupting chemicals (EDCs). released into the environment. Currently our ability to identify effects in vitro exceeds that for in vivo monitoring. However, only in vivo analysis provides the full spectrum of physiological impacts exerted by a given chemical. With the aim of finding a physiological system compatible with automatic plate reading we tested the capacity of early embryonic stage Xenopus laevis tadpoles to monitor thyroid hormone (TH) disruption. Fluorescent transgenic X. laevis embryos bearing a TH/bZIP-eGFP construct, placed in 96 well plates, were used for a physiological-based screen for potential TH signaling disruptors. Using stage NF-45 embryos (time of thyroid gland formation) allowed rapid detection of chemical interference with both peripheral TR signaling and production of endogenous TH. Nanomolar concentrations of TH receptor agonists could be detected within 72 h. Moreover, when testing against a 5nM T3 challenge, the effects of inhibitors of TH production were revealed, including inhibitors of TH synthesis, (methimazole: 1 mM or sodium perchlorate: 3.56 microM), as well as antagonists acting at the receptor level (NH3: 2 microM) and a deiodinase inhibitor (iopanoic acid: 10 microM). Finally, we show that the thyroid disrupting activities of BPA (10 microM) and TBBPA (1 microM) can also be detected in this rapid screening protocol. Finally, this noninvasive technology using an automatic reading system shows low variability (around 5%) and permits detection of subtle changes in signaling by EDCs that either inhibit or activate TH signaling in vivo. PMID:17874805

  10. Optical phantoms with variable properties and geometries for diffuse and fluorescence optical spectroscopy.

    PubMed

    Leh, Barbara; Siebert, Rainer; Hamzeh, Hussein; Menard, Laurent; Duval, Marie-Alix; Charon, Yves; Abi Haidar, Darine

    2012-10-01

    Growing interest in optical instruments for biomedical applications has increased the use of optically calibrated phantoms. Often associated with tissue modeling, phantoms allow the characterization of optical devices for clinical purposes. Fluorescent gel phantoms have been developed, mimicking optical properties of healthy and tumorous brain tissues. Specific geometries of dedicated molds offer multiple-layer phantoms with variable thicknesses and monolayer phantoms with cylindrical inclusions at various depths and diameters. Organic chromophores are added to allow fluorescence spectroscopy. These phantoms are designed to be used with 405 nm as the excitation wavelength. This wavelength is then adapted to excite large endogenous molecules. The benefits of these phantoms in understanding fluorescence tissue analysis are then demonstrated. In particular, detectability aspects as a function of geometrical and optical parameters are presented and discussed. PMID:23224016

  11. Visible-super-resolution infrared microscopy using saturated transient fluorescence detected infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Bokor, Nándor; Inoue, Keiichi; Kogure, Satoshi; Fujii, Masaaki; Sakai, Makoto

    2010-02-01

    A scanning visible-super-resolution microscope based on the saturation behaviour of transient fluorescence detected infrared (TFD-IR) spectroscopy is proposed. A Gaussian IR beam, a Gaussian visible beam and a Laguerre-Gaussian (LG) visible beam are used to obtain two separate two-color excitation fluorescence (2CF) images of the sample. The final image is obtained as the difference between the two recorded images. If the peak intensity of the LG beam is high enough to induce saturation in the fluorescence signal, the image can, in principle, have unlimited spatial resolution. A ˜3-fold improvement in transverse resolution over the visible diffraction limit (and far exceeding the IR diffraction limit) is easily achievable in present experimental setups.

  12. The Detection of Quality Deterioration of Apple Juice by Near Infrared and Fluorescence Spectroscopy

    Microsoft Academic Search

    Dazhou Zhu; Baoping Ji; Zhaoshen Qing; Cheng Wang; Manuela Zude

    2010-01-01

    \\u000a Processing and storage of apple juice often triggers quality deterioration regarding nutritional valuable compounds and unfavourable\\u000a color changes resulting from browning. Fluorescence and near-infrared (NIR) spectroscopy were applied to detect such quality\\u000a loss in apple juice. Juice samples were produced from Malus x domestica ’Pinova’, stored at 20 °C for 4 days or heated at\\u000a 80 °C for 10 min

  13. Polymer Dynamics, Fluorescence Correlation Spectroscopy, and the Limits of Optical Resolution Jorg Enderlein*

    E-print Network

    Enderlein, Jörg

    and diffusional dynamics. We find that fluorescence correlation spectroscopy cannot reliably elucidate processes of the capability of FCS to elucidate polymer dynamics on the nanometer scale. If FCS is indeed capable of measuringÞ are nonzero only for jxj a and where they are 2jðxÞ ¼ cosðjx=aÞ for even indices and 2jþ1ðxÞ ¼ sin½ðj þ 1=2Þx

  14. Spoilage of foods monitored by native fluorescence spectroscopy with selective excitation wavelength

    NASA Astrophysics Data System (ADS)

    Pu, Yang; Wang, Wubao; Alfano, Robert R.

    2015-03-01

    The modern food processing and storage environments require the real-time monitoring and rapid microbiological testing. Optical spectroscopy with selective excitation wavelengths can be the basis of a novel, rapid, reagent less, noncontact and non-destructive technique for monitoring the food spoilage. The native fluorescence spectra of muscle foods stored at 2-4°C (in refrigerator) and 20-24°C (in room temperature) were measured as a function of time with a selective excitation wavelength of 340nm. The contributions of the principal molecular components to the native fluorescence spectra of meat were measured spectra of each fluorophore: collagen, reduced nicotinamide adenine dinucleotide (NADH), and flavin. The responsible components were extracted using a method namely Multivariate Curve Resolution with Alternating Least-Squares (MCR-ALS). The native fluorescence combined with MCR-ALS can be used directly on the surface of meat to produce biochemically interpretable "fingerprints", which reflects the microbial spoilage of foods involved with the metabolic processes. The results show that with time elapse, the emission from NADH in meat stored at 24°C increases much faster than that at 4°C. This is because multiplying of microorganisms and catabolism are accompanied by the generation of NADH. This study presents changes of relative content of NADH may be used as criterion for detection of spoilage degree of meat using native fluorescence spectroscopy.

  15. Freshness estimation of intact frozen fish using fluorescence spectroscopy and chemometrics of excitation-emission matrix.

    PubMed

    ElMasry, Gamal; Nagai, Hiroto; Moria, Keisuke; Nakazawa, Naho; Tsuta, Mizuki; Sugiyama, Junichi; Okazaki, Emiko; Nakauchi, Shigeki

    2015-10-01

    The current study attempted to provide a convenient, non-invasive and time-saving method to estimate the freshness of intact horse mackerel (Trachurus japonicus) fish in a frozen state using autofluorescence spectroscopy in tandem with multivariate analysis of fluorescence excitation-emission matrices (EEM). The extracted fluorescence data from different freshness conditions were pretreated, masked and reorganized to resolve fish fluorescence spectra from overlapping signals and scattering profiles for detecting and characterizing freshness changes. The real freshness values of the examined fish samples were then traditionally determined by the hard chemical analysis using the high performance liquid chromatography (HPLC) method and expressed as K-values. The fluorescence EEM data and the real freshness values were modeled using partial least square (PLS) regression and a novel algorithm was proposed to identify the ideal combinations of excitation and emission wavelengths being used as perfect predictors. The results revealed that freshness of frozen fish could be accurately predicted with R(2) of 0.89 and root mean square error estimated by cross validation (RMSECV) of 9.66%. This work substantially demonstrated that the autofluorescence spectroscopy associated with the proposed technical approaches has a high potential in non-destructive sensing of fish freshness in the frozen state. PMID:26078142

  16. New photon-counting detectors for single-molecule fluorescence spectroscopy and imaging

    PubMed Central

    Michalet, X.; Colyer, R. A.; Scalia, G.; Weiss, S.; Siegmund, Oswald H. W.; Tremsin, Anton S.; Vallerga, John V.; Villa, F.; Guerrieri, F.; Rech, I.; Gulinatti, A.; Tisa, S.; Zappa, F.; Ghioni, M.; Cova, S.

    2013-01-01

    Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. Two typical geometries can be used for these experiments: point-like and widefield excitation and detection. In point-like geometries, the basic concept is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements. In widefield geometries, the same issues of background reduction and single-molecule concentration apply, but the duration of the experiment is fixed by the time scale of the process studied and the survival time of the fluorescent probe. Temporal resolution on the other hand, is limited by signal-to-noise and/or detector resolution, which calls for new detector concepts. We will briefly present our recent results in this domain. PMID:24729836

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

    PubMed Central

    2012-01-01

    How to find early gastric cancer cells in vivo is a great challenge for the diagnosis and therapy of gastric cancer. This study is aimed at investigating the feasibility of using fluorescent magnetic nanoparticle (FMNP)-labeled mesenchymal stem cells (MSCs) to realize targeted imaging and hyperthermia therapy of in vivo gastric cancer. The primary cultured mouse marrow MSCs were labeled with amino-modified FMNPs then intravenously injected into mouse model with subcutaneous gastric tumor, and then, the in vivo distribution of FMNP-labeled MSCs was observed by using fluorescence imaging system and magnetic resonance imaging system. After FMNP-labeled MSCs arrived in local tumor tissues, subcutaneous tumor tissues in nude mice were treated under external alternating magnetic field. The possible mechanism of MSCs targeting gastric cancer was investigated by using a micro-multiwell chemotaxis chamber assay. Results show that MSCs were labeled with FMNPs efficiently and kept stable fluorescent signal and magnetic properties within 14?days, FMNP-labeled MSCs could target and image in vivo gastric cancer cells after being intravenously injected for 14?days, FMNP-labeled MSCs could significantly inhibit the growth of in vivo gastric cancer because of hyperthermia effects, and CCL19/CCR7 and CXCL12/CXCR4 axis loops may play key roles in the targeting of MSCs to in vivo gastric cancer. In conclusion, FMNP-labeled MSCs could target in vivo gastric cancer cells and have great potential in applications such as imaging, diagnosis, and hyperthermia therapy of early gastric cancer in the near future. PMID:22709686

  18. Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways.

    PubMed

    Shirshin, Evgeny; Cherkasova, Olga; Tikhonova, Tatiana; Berlovskaya, Elena; Priezzhev, Alexander; Fadeev, Victor

    2015-05-01

    We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation. PMID:25692658

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

    NASA Astrophysics Data System (ADS)

    Lodwick, Jeffrey C.

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

  20. Evaluating aggregation of gold nanoparticles and humic substances using fluorescence spectroscopy.

    PubMed

    Pallem, Vasanta L; Stretz, Holly A; Wells, Martha J M

    2009-10-01

    The fate and transport of diagnostic gold nanoparticles in surface waters would significantly depend on their interactions with humic substances, which are ubiquitously found in natural aquatic systems. The current study employs UV-visible absorbance and fluorescence spectroscopy to investigate the interactions of commercial humic acid (HA) with gold nanoparticles having a core size of 5 nm and coated with two different stabilizers, beta-D-glucose and citrate. Humic substances (HS) are fluorescent in nature, providing a unique probe of nanometer-scale morphological changes for interactions between these natural polyelectrolytes and water-soluble gold nanoparticles. Quenching of fluorescence intensity was observed with beta-D-glucose-coated gold nanoparticles, whereas an enhancement effect was noticed with the citrate-coated particles when mixed with HA having concentrations of 2 and 8 ppm (surface waters typically may contain approximately 10 ppm HS). Examining the quenching and enhancement of fluorescence provides insight into the structural changes taking place at the coated gold nanoparticle-HA interface. The quenching behavior suggested ligand exchange due to nanometer-scale contact between the HA and beta-D-glucose-coated gold nanoparticles, whereas the enhancement effect with citrate particles would indicate overcoating, leading to increased transfer distances for fluorescence resonance energy transfer. PMID:19848172

  1. Quantifying interactions between propranolol and dissolved organic matter (DOM) from different sources using fluorescence spectroscopy.

    PubMed

    Peng, Na; Wang, Kaifeng; Liu, Guoguang; Li, Fuhua; Yao, Kun; Lv, Wenying

    2014-04-01

    Beta blockers are widely used pharmaceuticals that have been detected in the environment. Interactions between beta blockers and dissolved organic matter (DOM) may mutually alter their environmental behaviors. To assess this potential, propranolol (PRO) was used as a model beta blocker to quantify the complexation with DOM from different sources using the fluorescence quenching titration method. The sources of studied DOM samples were identified by excitation-emission matrix spectroscopy (EEMs) combined with fluorescence regional integration analysis. The results show that PRO intrinsic fluorescence was statically quenched by DOM addition. The resulting binding constants (log K oc) ranged from 3.90 to 5.20, with the surface-water-filtered DOM samples claiming the lower log K oc and HA having the highest log K oc. Log K oc is negatively correlated with the fluorescence index, biological index, and the percent fluorescence response (P i,n) of protein-like region (P I,n) and the P i,n of microbial byproduct-like region (P II,n) of DOM EEMs, while it is correlated positively with humification index and the P i,n of UVC humic-like region (P III,n). These results indicate that DOM samples from allochthonous materials rich in aromatic and humic-like components would strongly bind PRO in aquatic systems, and autochthonous DOM containing high protein-like components would bind PRO more weakly. PMID:24390196

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

    NASA Astrophysics Data System (ADS)

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

    1993-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

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

    USGS Publications Warehouse

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

    2012-01-01

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

  5. Single-molecule spectroscopy to probe competitive fluorescence resonance energy transfer pathways in bichromophoric synthetic systems

    NASA Astrophysics Data System (ADS)

    Cotlet, Mircea; Vosch, Tom; Masuo, Sadahiro; Sauer, Marcus; Muellen, Klaus; Hofkens, Johan; De Schryver, Frans

    2004-07-01

    Using single molecule fluorescence spectroscopy we have investigated fluorescence resonance energy transfer (FRET) occurring between two peryleneimide (PI) chromophores in a series of synthetic systems: PI end-capped fluorene trimers, hexamers and polymers for which the interchromophoric distance vary from 3.4 to 5.9 and 42 nm, respectively. By monitoring in parallel the fluorescence intensity and the number of independent emitting chromophores from each molecule, we could discriminate between competitive Foerster-type energy transfer processes such as energy hopping, singlet-singlet annihilation and singlet-triplet annihilation for the PI end-capped fluorine compounds. Due to different energy transfer efficiencies, variations in the interchromophoric distance enable switching between these processes. The single molecule fluorescence data reported here suggest that similar energy transfer pathways have to be considered in the analysis of single molecule trajectories of donor/acceptor pairs, as well as in the case of more complex systems like natural multichromophoric systems, such as light harvesting antennas or oligomeric fluorescent proteins.

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

    PubMed Central

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

    2010-01-01

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

  7. On the performance of bioanalytical fluorescence correlation spectroscopy measurements in a multiparameter photon-counting microscope.

    PubMed

    Mazouchi, Amir; Liu, Baoxu; Bahram, Abdullah; Gradinaru, Claudiu C

    2011-02-28

    Fluorescence correlation spectroscopy (FCS) data acquisition and analysis routines were developed and implemented in a home-built, multiparameter photon-counting microscope. Laser excitation conditions were investigated for two representative fluorescent probes, Rhodamine110 and enhanced green fluorescent protein (EGFP). Reliable local concentrations and diffusion constants were obtained by fitting measured FCS curves, provided that the excitation intensity did not exceed 20% of the saturation level for each fluorophore. Accurate results were obtained from FCS measurements for sample concentrations varying from pM to ?M range, as well as for conditions of high background signals. These experimental constraints were found to be determined by characteristics of the detection system and by the saturation behavior of the fluorescent probes. These factors actually limit the average number of photons that can be collected from a single fluorophore passing through the detection volume. The versatility of our setup and the data analysis capabilities were tested by measuring the mobility of EGFP in the nucleus of Drosophila cells under conditions of high concentration and molecular crowding. As a bioanalytical application, we studied by FCS the binding affinity of a novel peptide-based drug to the cancer-regulating STAT3 protein and corroborated the results with fluorescence polarization analysis derived from the same photon data. PMID:21296206

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

    NASA Astrophysics Data System (ADS)

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

    2006-03-01

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

  9. Uranyl sorption onto gibbsite studied by time-resolved laser-induced fluorescence spectroscopy (TRLFS).

    PubMed

    Baumann, Nils; Brendler, Vinzenz; Arnold, Thuro; Geipel, Gerhard; Bernhard, Gert

    2005-10-15

    Time-resolved laser-induced fluorescence spectroscopy (TRLFS) was combined with batch experiments to study the sorption of uranium(VI) onto gibbsite (gamma-Al(OH)3). The experiments were performed under ambient conditions in 0.1 M NaClO4 solution in the pH range from 5.0 to 8.5 using a total uranium concentration of 1x10(-5) M, and a solid concentration of 0.5 g/40 ml. Two uranyl surface species with fluorescence lifetimes of 330+/-115 and 5600+/-1640 ns, respectively, were identified. The first species was dominating the more acid pH region whereas the second one became gradually more prominent towards higher pH values. The fluorescence spectra of both adsorbed uranyl(VI) surface species were described with six characteristic fluorescence emission bands situated at 479.5+/-1.1, 497.4+/-0.8, 518.7+/-1.0, 541.6+/-0.7, 563.9+/-1.2, and 585.8+/-2.1 nm. The surface species with the short-lived fluorescence lifetime of 330 ns is attributed to a bidentate mononuclear inner-sphere surface complex in which the uranyl(VI) is bound to two reactive OH- groups at the broken edge linked to one Al. The second surface species with the significant longer fluorescence lifetime of 5600 ns was attributed to small sorbed clusters of polynuclear uranyl(VI) surface species. The longer fluorescence lifetime of the long-lived uranyl surface species at pH 8.5 is explained with the growing average size of the adsorbed polynuclear uranyl surface species. PMID:16129445

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

    NASA Astrophysics Data System (ADS)

    Birdwell, Justin E.; Valsaraj, Kalliat T.

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

  11. Structural changes of yellow Cameleon domains observed by quantitative FRET analysis and polarized fluorescence correlation spectroscopy.

    PubMed

    Borst, J W; Laptenok, S P; Westphal, A H; Kühnemuth, R; Hornen, H; Visser, N V; Kalinin, S; Aker, J; van Hoek, A; Seidel, C A M; Visser, A J W G

    2008-12-01

    Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand binding. PMID:18790855

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

    USGS Publications Warehouse

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

    2010-01-01

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

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

    PubMed

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

    2009-09-01

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

  14. Time-Resolved Fluorescence Spectroscopy as a Diagnostic Technique of Oral Carcinoma

    PubMed Central

    Farwell, D. Gregory; Meier, Jeremy D.; Park, Jesung; Sun, Yang; Coffman, Heather; Poirier, Brian; Phipps, Jennifer; Tinling, Steve; Enepekides, Danny J.; Marcu, Laura

    2014-01-01

    Objective To investigate the benefit of using time-resolved, laser-induced fluorescence spectroscopy for diagnosing malignant and premalignant lesions of the oral cavity. Design The carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) was applied to 1 cheek pouch of 19 hamsters. The contralateral pouch and the cheek pouches of 3 hamsters without DMBA exposure served as controls. Setting University of California, Davis. Participants Twenty-two golden/Syrian hamsters. Intervention A nitrogen pulse laser was used to induce tissue autofluorescence between the wavelengths of 360 and 650 nm. Main Outcome Measures Spectral intensities and time-domain measurements were obtained and compared with the histopathologic findings at each corresponding site. Results Spectral intensities and lifetime values at 3 spectral bands (SBs; SB1=380±10 nm; SB2=460±10 nm, and SB3 = 635 ± 10 nm) allowed for discrimination among healthy epithelium, dysplasia, carcinoma in situ, and invasive carcinoma. The lifetime values at SB2 were the most important when distinguishing the lesions using only time-resolved parameters. An algorithm combining spectral fluorescence parameters derived from both spectral and time-domain parameters (peak intensities, average fluorescence lifetimes, and the Laguerre coefficient [zero-order]) for healthy epithelium, dysplasia, carcinoma in situ, and invasive carcinoma provided the best diagnostic discrimination, with 100%, 100%, 69.2%, and 76.5% sensitivity and 100%, 92.2%, 97.1%, and 96.2% specificity, respectively. Conclusions The addition of time-resolved fluorescence-derived parameters significantly improves the capability of fluorescence spectroscopy–based diagnostics in the hamster buccal pouch. This technique provides a potential non-invasive diagnostic instrument for head and neck cancer. PMID:20157056

  15. Percutaneous absorption of flurbiprofen in the hairless rat measured in vivo using 19F magnetic resonance spectroscopy.

    PubMed

    Lee, D J; Burt, C T; Koch, R L

    1992-10-01

    The objective of this investigation was to develop a new methodology using 19F-magnetic resonance spectroscopy (MRS) to measure the in vivo percutaneous absorption of flurbiprofen through hairless rat skin. A 2% W/V flurbiprofen gel (Klucel HF, hydroxypropyl cellulose 1.5% to 2% W/V) containing isopropyl alcohol, water, and propylene glycol (55:35:10 v/v/v) was prepared. A 2-mg dose (100 mg of gel) was applied to the skin of the lower back of an anesthetized hairless rat, contained with a rubber o-ring, and occluded with a lexan plastic cover slip. The animal was placed on an MR surface coil (3.5-cm diameter tuned to 19F) and measurements taken continuously over approximately 3 h in 10-min intervals with a 2-tesla GE CSI nuclear magnetic resonance (NMR) spectrometer. One measures the disappearance of MR signal intensity per interval, which directly relates to the percent of drug disappearance over time, which in turn was converted to a flux value. The flux of flurbiprofen in vivo was found to be 95 +/- 22 micrograms/cm2/h. This is approximately four times greater than the flux of flurbiprofen through excised human skin reported by Akhter and Barry (22 +/- 14 micrograms/cm2/h). This new in vivo method measures drug disappearance and can be readily transferred to man. This method may be adapted to study other fluorine compounds or other nuclei with magnetic properties. It avoids exposure of a patient or animal to the radiation used in x-ray fluorescence methods or to 14C- or 3H-radiolabeled drugs. PMID:1402001

  16. Fluorescence Fluctuation Spectroscopy Enables Quantitative Imaging of Single mRNAs in Living Cells

    PubMed Central

    Wu, Bin; Chao, Jeffrey A.; Singer, Robert H.

    2012-01-01

    Imaging mRNA with single-molecule sensitivity in live cells has become an indispensable tool for quantitatively studying RNA biology. The MS2 system has been extensively used due to its unique simplicity and sensitivity. However, the levels of the coat protein needed for consistent labeling of mRNAs limits the sensitivity and quantitation of this technology. Here, we applied fluorescence fluctuation spectroscopy to quantitatively characterize and enhance the MS2 system. Surprisingly, we found that a high fluorescence background resulted from inefficient dimerization of fluorescent protein (FP)-labeled MS2 coat protein (MCP). To mitigate this problem, we used a single-chain tandem dimer of MCP (tdMCP) that significantly increased the uniformity and sensitivity of mRNA labeling. Furthermore, we characterized the PP7 coat protein and the binding to its respective RNA stem loop. We conclude that the PP7 system performs better for RNA labeling. Finally, we used these improvements to study endogenous ?-actin mRNA, which has 24xMS2 binding sites inserted into the 3? untranslated region. The tdMCP-FP allowed uniform RNA labeling and provided quantitative measurements of endogenous mRNA concentration and diffusion. This work provides a foundation for quantitative spectroscopy and imaging of single mRNAs directly in live cells. PMID:22735544

  17. Oligomeric interface modifiers in hybrid polymer solar cell prototypes investigated by fluorescence voltage spectroscopy.

    PubMed

    Reeja-Jayan, B; Koen, Katherine A; Ono, Robert J; Vanden Bout, David A; Bielawski, Christopher W; Manthiram, Arumugam

    2015-04-28

    Carboxylated oligothiophenes were evaluated as interfacial modifiers between the organic poly(3-hexylthiophene) (P3HT) and inorganic TiO2 layers in bilayer hybrid polymer solar cells. Carboxylated oligothiophenes can be isolated using conventional purification techniques resulting in pure, monodisperse molecules with 100% carboxylation. Device prototypes using carboxylated oligothiophenes as interfacial modifiers showed improved performance in the open-circuit voltage and fill factor over devices using unmodified oligothiophenes as interfacial modifiers. X-ray photoelectron spectroscopy (XPS) studies supported the idea that interface layer adhesion was improved by functionalizing oligothiophenes with a carboxyl moiety. Wide-field fluorescence images revealed that devices made using carboxylated oligothiophenes had fewer aggregates in the P3HT layers atop the modified TiO2 surface. Hysteresis seen in the fluorescence intensity as a function of applied bias, obtained from In-Device Fluorescence Voltage Spectroscopy (ID-FVS), was found to be a diagnostic criterion of the quality of the hybrid interface modification. The best interfaces were found using oligothiophenes functionalized with carboxylates, which created smooth layers on TiO2, and showed no hysteresis, suggesting elimination of interfacial charge traps. However, this hysteresis could be re-introduced by increasing the scan rate of the applied bias, suggesting that smooth P3HT layers created by carboxylated oligothiophene interface modifiers were necessary but not sufficient for sustaining improved photovoltaic properties especially during long-term device operation. PMID:25804286

  18. Tissue classification and diagnostics using a fiber probe for combined Raman and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

    Two different optical fiber probes for combined Raman and fluorescence spectroscopic measurements were designed, developed and used for tissue diagnostics. Two visible laser diodes were used for fluorescence spectroscopy, whereas a laser diode emitting in the NIR was used for Raman spectroscopy. The two probes were based on fiber bundles with a central multimode optical fiber, used for delivering light to the tissue, and 24 surrounding optical fibers for signal collection. Both fluorescence and Raman spectra were acquired using the same detection unit, based on a cooled CCD camera, connected to a spectrograph. The two probes were successfully employed for diagnostic purposes on various tissues in a good agreement with common routine histology. This study included skin, brain and bladder tissues and in particular the classification of: malignant melanoma against melanocytic lesions and healthy skin; urothelial carcinoma against healthy bladder mucosa; brain tumor against dysplastic brain tissue. The diagnostic capabilities were determined using a cross-validation method with a leave-one-out approach, finding very high sensitivity and specificity for all the examined tissues. The obtained results demonstrated that the multimodal approach is crucial for improving diagnostic capabilities. The system presented here can improve diagnostic capabilities on a broad range of tissues and has the potential of being used for endoscopic inspections in the near future.

  19. Tomography of epidermal growth factor receptor binding to fluorescent Affibody in vivo studied with magnetic resonance guided fluorescence recovery in varying orthotopic glioma sizes

    NASA Astrophysics Data System (ADS)

    Holt, Robert W.; Demers, Jennifer-Lynn H.; Sexton, Kristian J.; Gunn, Jason R.; Davis, Scott C.; Samkoe, Kimberley S.; Pogue, Brian W.

    2015-02-01

    The ability to image targeted tracer binding to epidermal growth factor receptor (EGFR) was studied in vivo in orthotopically grown glioma tumors of different sizes. The binding potential was quantified using a dual-tracer approach, which employs a fluorescently labeled peptide targeted to EGFR and a reference tracer with similar pharmacokinetic properties but no specific binding, to estimate the relative bound fraction from kinetic compartment modeling. The recovered values of binding potential did not vary significantly as a function of tumor size (1 to 33 mm3), suggesting that binding potential may be consistent in the U251 tumors regardless of size or stage after implantation. However, the fluorescence yield of the targeted fluorescent tracers in the tumor was affected significantly by tumor size, suggesting that dual-tracer imaging helps account for variations in absolute uptake, which plague single-tracer imaging techniques. Ex vivo analysis showed relatively high spatial heterogeneity in each tumor that cannot be resolved by tomographic techniques. Nonetheless, the dual-tracer tomographic technique is a powerful tool for longitudinal bulk estimation of receptor binding.

  20. [Studies on laser induced dispersive fluorescence spectroscopy of SO2 molecule excited by two-photon].

    PubMed

    Zhao, Zhan-Long; Li, Ming; Zhang, Lian-Shui; Chen, Si-Yuan; Zhao, Kui-Fang

    2012-11-01

    The processes of excitation and complicated de-excitation of A-symmetric state in the first-excited band of SO2 molecule were studied experimentally with the techniques of two-photon laser induced dispersive fluorescence spectroscopy where a pulsed dye laser (579 nm) was used as excitation sources. The SO2 molecule which were excited from ground state X1A1 to the high vibrational levels of A1 A2 state by absorbing two photons, will realize the repopulation in several vibration-rotational energy levels of A1 A2, B1 B1 and alpha3 B1 states by internal energy conversion and collision relaxation. Because of transitions to the different vibrational levels of ground electronic state X1 A1 from the ground vibrational levels of A1 A2, B1 B1, and alpha3 B1, the fluorescence spectrum envelopes centered at 305 and 425 nm and the regular fluorescence lines centered at 347.2 nm were formed in the fluorescence spectra. In addition, the process of tri-photon excitation X1 A1 --> C1 B2 of SO2 molecule was observed, and the result of the process was the fluorescence spectrum envelope in 200-278 nm and the overlapping fluorescence lines centered at 425 nm. The harmonic frequencies of the symmetry stretch vibration and the bendvibration and the anharmonic constants of stretch vibration mode and the bend vibration mode of related states were calculated from the experimental data. PMID:23387180

  1. Fluorescence Lifetime Imaging and Intravascular Ultrasound: Co-Registration Study Using Ex Vivo Human Coronaries

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2013-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  4. Detection of Malondialdehyde in vivo Using Microdialysis Sampling with CE-Fluorescence

    PubMed Central

    Cooley, Justin Carl; Lunte, Craig Edward

    2012-01-01

    Oxidative damage is a naturally occurring process where reactive oxygen species (ROS) attack and disrupt normal cellular function, however, these effects become elevated during a stress event, such as ischemia/reperfusion or seizure. One result of oxidative stress is lipid peroxidation, where ROS attack free unsaturated fatty acids forming lipid hydorperoxides, which then breakdown to form secondary products acrolein, 4-hydroxynonenal, and malondialdehyde (MDA) resulting in irreversible membrane damage and ultimately cell death. Described here is a CE – fluorescence method for the determination of MDA in conjunction with in vivo microdialysis sampling. MDA was derivatized with thiobarbituric acid under acidic conditions for 20 minutes and injected directly onto the capillary without any pretreatment. This method provided a limit of detection of 25 nM (S/N = 3) and a linear range of 25-2400 nM (1.8-174 ng/ml). This method was used to quantify MDA in rat heart, muscle, liver, and brain dialysate. PMID:22034011

  5. A near-infrared fluorescent probe for the detection of hydrogen polysulfides biosynthetic pathways in living cells and in vivo.

    PubMed

    Gao, Min; Wang, Rui; Yu, Fabiao; You, Jinmao; Chen, Lingxin

    2015-05-18

    Hydrogen polysulfides (H2Sn, n > 1), derived from hydrogen sulfide (H2S), have been considered to be involved in cytoprotective processes and redox signaling. The emerging evidences imply that the actual signaling molecule is H2Sn rather than H2S. In this work, we present a near-infrared fluorescent probe BD-ss for the selective detection of H2Sn biosynthetic pathways in living cells and in vivo. The probe is constructed by equipping a bis-electrophilic H2Sn capture group p-nitrofluorobenzoate to a near-infrared fluorophore azo-BODIPY. BD-ss can provide a remarkable turn-on fluorescence response for assessing endogenous H2Sn formation ways in serum, in living cells and in vivo. PMID:25811591

  6. High pressure sample cell for total internal reflection fluorescence spectroscopy at pressures up to 2500 bar

    NASA Astrophysics Data System (ADS)

    Koo, Juny; Czeslik, Claus

    2012-08-01

    Total internal reflection fluorescence (TIRF) spectroscopy is a surface sensitive technique that is widely used to characterize the structure and dynamics of molecules at planar liquid-solid interfaces. In particular, biomolecular systems, such as protein adsorbates and lipid membranes can easily be studied by TIRF spectroscopy. Applying pressure to molecular systems offers access to all kinds of volume changes occurring during assembly of molecules, phase transitions, and chemical reactions. So far, most of these volume changes have been characterized in bulk solution, only. Here, we describe the design and performance of a high pressure sample cell that allows for TIRF spectroscopy under high pressures up to 2500 bar (2.5 × 108 Pa), in order to expand the understanding of volume effects from the bulk phase to liquid-solid interfaces. The new sample cell is based on a cylindrical body made of Nimonic 90 alloy and incorporates a pressure transmitting sample cuvette. This cuvette is composed of a fused silica prism and a flexible rubber gasket. It contains the sample solution and ensures a complete separation of the sample from the liquid pressure medium. The sample solution is in contact with the inner wall of the prism forming the interface under study, where fluorescent molecules are immobilized. In this way, the new high pressure TIRF sample cell is very useful for studying any biomolecular layer that can be deposited at a planar water-silica interface. As examples, high pressure TIRF data of adsorbed lysozyme and two phospholipid membranes are presented.

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

    PubMed

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

    2010-10-01

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

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

    PubMed Central

    Ray, Krishanu; Lakowicz, Joseph R.

    2013-01-01

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

  9. Ultraviolet fluorescence spectroscopy of blood plasma in the discrimination of cancer from normal

    NASA Astrophysics Data System (ADS)

    Madhuri, S.; Aruna, Prakasa R.; Summiya Bibi, M. I.; Gowri, V. S.; Koteeswaran, D.; Ganesan, Singaravelu

    1997-05-01

    Native fluorescence spectroscopy of biomolecules has emerged as an intrinsic parameter in the characterization of the physiological state and the discrimination of pathological from normal conditions of cells and tissues. The key fluorescing biomolecules inc ells and tissues ar tryptophan, tyrosine, phenylalanine, collagen, elastin, NADH, flavin and porphyrin. Extensive studies were made on tissues of various origin to discriminate the malignancy from normal. The differences in the fluorescence emission spectra have been shown to separate benign and malignant tissues. In the present work, a pilot study was carried out on the characterization of blood plasma of both normal and cancerous subjects. The blood plasma was separated by centrifuging the blood and it was diluted in PBS by adjusting the O.D. to 0.5 at 280 nm. This diluted sample as excited in the UV region between 250-340 nm. Among the various excitation wavelengths, emission spectrum at 300 nm excitation has considerable difference between blood plasma of normal subjects and cancer patients. To quantify these differences and to verify if there is any diagnostic potential exists, the ratio of fluorescence intensities at 340 and 440 nm was calculated. It is found that the ratio value of normal blood plasma is less than 11 and for tumor, it is greater than 11. Besides, it is found that the ratio value of blood plasma from patients with cancer varies from 11 to 28, depending upon the stage of malignancy.

  10. Fluorescence spectroscopy of collagen crosslinking: non-invasive and in situ evaluation of corneal stiffness

    NASA Astrophysics Data System (ADS)

    Franco, Walfre; Ortega-Martinez, Antonio; Zhu, Hong; Wang, Ruisheng; Kochevar, Irene E.

    2015-03-01

    Collagen is a long fibrous structural protein that imparts mechanical support, strength and elasticity to many tissues. The state of the tissue mechanical environment is related to tissue physiology, disease and function. In the cornea, the collagen network is responsible for its shape and clarity; disruption of this network results in degradation of visual acuity, for example in the keratoconus eye disease. The objective of the present study is to investigate the feasibility of using the endogenous fluorescence of collagen crosslinks to evaluate variations in the mechanical state of tissue, in particular, the stiffness of cornea in response to different degrees of photo-crosslinking or RGX treatment—a novel keratoconus treatment. After removing the epithelium, rabbit corneas were stained with Rose Bengal and then irradiated with a 532 nm solid-state laser. Analysis of the excitation spectra obtained by fluorescence spectroscopy shows a correlation between the fluorescence intensity at 370/460 nm excitation/emission wavelengths and the mechanical properties. In principle, it may be feasible to use the endogenous fluorescence of collagen crosslinks to evaluate the mechanical stiffness of cornea non-invasively and in situ.

  11. Substrate-Supported Phospholipid Membranes Studied by Surface Plasmon Resonance and Surface Plasmon Fluorescence Spectroscopy

    PubMed Central

    Tawa, Keiko; Morigaki, Kenichi

    2005-01-01

    Substrate-supported planar lipid bilayer membranes are attractive model cellular membranes for biotechnological applications such as biochips and sensors. However, reliable fabrication of the lipid membranes on solid surfaces still poses significant technological challenges. In this study, simultaneous surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) measurements were applied to the monitoring of adsorption and subsequent reorganization of phospholipid vesicles on solid substrates. The fluorescence intensity of SPFS depends very sensitively on the distance between the gold substrate and the fluorophore because of the excitation energy transfer to gold. By utilizing this distance dependency, we could obtain information about the topography of the adsorbed membranes: Adsorbed vesicles could be clearly distinguished from planar bilayers due to the high fluorescence intensity. SPSF can also incorporate various analytical techniques to evaluate the physicochemical properties of the adsorbed membranes. As an example, we demonstrated that the lateral mobility of lipid molecules could be estimated by observing the recovery of fluorescence after photobleaching. Combined with the film thickness information obtained by SPR, SPR-SPFS proved to be a highly informative technique to monitor the lipid membrane assembly processes on solid substrates. PMID:16040759

  12. Measurement of Fuel Dilution of Oil in a Diesel Engine using Laser-Induced Fluorescence Spectroscopy

    SciTech Connect

    Parks, II, James E [ORNL; Partridge Jr, William P [ORNL] [ORNL

    2007-01-01

    A technique for measuring the fuel dilution of oil in a diesel engine is presented. Fuel dilution can occur when advanced in-cylinder fuel injection techniques are employed for the purpose of producing rich exhaust for lean NOx trap catalyst regeneration. Laser-induced fluorescence (LIF) spectroscopy is used to monitor the oil in a Mercedes 1.7-liter engine operated on a dynamometer platform. A fluorescent dye suitable for use in diesel fuel and oil systems is added to the engine fuel. The LIF spectra are monitored to detect the growth of the dye signal relative to the background fluorescence of the oil; fuel mass concentration is quantified based on a known sample set. The technique was implemented with fiber optic probes which can be inserted at various points in the oil system of the engine. A low cost 532-nm laser diode was used for excitation of the fluorescence. Measurements of fuel dilution of oil are presented for various in-cylinder injection strategies for rich operation of the diesel engine. Rates of fuel dilution increase for all strategies relative to normal lean operation, and higher fuel dilution rates are observed when extra fuel injection occurs later in the combustion cycle when fuel penetration into the cylinder wall oil film is more likely.

  13. Bioimaging of Fluorescence-Labeled Mitochondria in Subcutaneously Grafted Murine Melanoma Cells by the “In Vivo Cryotechnique”

    PubMed Central

    Lei, Ting; Huang, Zheng; Ohno, Nobuhiko; Wu, Bao; Sakoh, Takashi; Saitoh, Yurika; Saiki, Ikuo

    2014-01-01

    The microenvironments of organs with blood flow affect the metabolic profiles of cancer cells, which are influenced by mitochondrial functions. However, histopathological analyses of these aspects have been hampered by technical artifacts of conventional fixation and dehydration, including ischemia/anoxia. The purpose of this study was to combine the in vivo cryotechnique (IVCT) with fluorescent protein expression, and examine fluorescently labeled mitochondria in grafted melanoma tumors. The intensity of fluorescent proteins was maintained well in cultured B16-BL6 cells after cryotechniques followed by freeze-substitution (FS). In the subcutaneous tumors of mitochondria-targeted DsRed2 (mitoDsRed)-expressing cells, a higher number of cancer cells were found surrounding the widely opened blood vessels that contained numerous erythrocytes. Such blood vessels were immunostained positively for immunoglobulin M and ensheathed by basement membranes. MitoDsRed fluorescence was detected in scattering melanoma cells using the IVCT-FS method, and the total mitoDsRed volume in individual cancer cells was significantly decreased with the expression of markers of hypoxia. MitoDsRed was frequently distributed throughout the cytoplasm and in processes extending along basement membranes. IVCT combined with fluorescent protein expression is a useful tool to examine the behavior of fluorescently labeled cells and organelles. We propose that the mitochondrial volume is dynamically regulated in the hypoxic microenvironment and that mitochondrial distribution is modulated by cancer cell interactions with basement membranes. PMID:24394469

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

    Microsoft Academic Search

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

    1996-01-01

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

  15. Structure and dynamics of a DNA: polymerase complex by time-resolved fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Millar, David P.; Benkovic, Stephen J.

    1990-05-01

    The interaction of a fluorescent DNA primer:template with the Klenow fragment of DNA polymerase I has been studied in solution using time-resolved fluorescence spectroscopy. The excited-state decay behavior and internal reorientation dynamics of a dansyl sulfonamide probe connected by a propyl chain to a modified uridine base in the primer strand were very sensitive to the local probe environment and exhibited characteristic changes upon binding of Kienow fragment to the DNA and elongation of the primer strand. Between 5 and 7 bases of duplex DNA upstream of the 3' primer terminus were protected from the solvent by the Kienow fragment and the strength of DNA:protein contacts varied within this region, being strongest at the 3' primer terminus. About 5% of the substrates were bound in a second spatially distinct site on the enzyme. Site-directed mutagenesis of the Kienow fragment was consistent with this being the active site for 3'->5' exonuclease activity.

  16. Fluorescence spectroscopy to study dissolved organic matter interactions with agrochemicals applied in Swiss vineyards.

    PubMed

    Daouk, Silwan; Frege, Carla; Blanc, Nicolas; Mounier, Stéphane; Redon, Roland; Merdy, Patricia; Lucas, Yves; Pfeifer, Hans-Rudolf

    2015-06-01

    UV/Vis fluorescence spectroscopy was used to study the possible interactions of dissolved organic matter (DOM) with the herbicide glyphosate and copper-based fungicide used in vineyards. The study focused on the role of DOM in the transport of these micropollutants from parcels to surface waters (river, lake). Soil solution and river water samples were collected in the Lavaux vineyard area, western Switzerland. Their fluorescence excitation emission matrices (EEM) were decomposed using parallel factor (PARAFAC) analysis, and compared to their content in glyphosate and copper. PARAFAC analysis of EEM of both types of samples showed the contribution of protein-like and humic-like fluorophores. In soil water samples, complexes between fulvic-like and humic-like fluorophores of DOM, copper, and glyphosate were likely formed. In surface water, DOM-copper and glyphosate-copper interactions were observed, but not between glyphosate and DOM. PMID:25592914

  17. Probe diffusion in polymer solutions and hydrogels using fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Michelman-Ribeiro, Ariel; Boukari, Hacene; Horkay, Ferenc; Nossal, Ralph

    2006-03-01

    We apply fluorescence correlation spectroscopy (FCS) to measure the diffusion of small fluorescent probes (TAMRA, Mw = 430 Da; dextran, Mw = 10 kDa) in poly(vinyl alcohol) (PVA) solutions and hydrogels. PVA is a linear, neutral, biocompatible polymer, whose hydrogels have many biotechnology applications, such as drug-delivery devices and tissue scaffolds. The FCS measurements indicate that the probe diffusion decreases when the polymer solution is cross-linked. Further, the more the polymer chains are cross-linked, the slower the particles diffuse. These results suggest that the cross-link density, which is often ignored in the analysis of probe diffusion data in gels, must be taken into account. Remarkably, we find that the apparent diffusion time and the elastic modulus of the gels show a linear correlation.

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

    PubMed Central

    Prabhakaran, Venkateshkumar; Arges, Christopher G.; Ramani, Vijay

    2012-01-01

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

  19. In-vivo autofluorescence of nasopharyngeal carcinoma and normal tissue

    NASA Astrophysics Data System (ADS)

    Qu, Jianan Y.; Yuen, Po W.; Huang, Zhijian; Wei, William I.

    2000-04-01

    An optical imaging and spectroscopy system has been developed for the study of in vivo fluorescence of nasopharyngeal tissue through an endoscope. The system records the fluorescence signal in the imaging plane of the endoscopic system. This allows analyze the characteristics of the light induced fluorescence (LIF) spectra recorded by each pixel of the 2D detector which may be used for fluorescence endoscopic imaging. If the endoscope for fluorescence endoscopy is the same as one employed for the in vivo fluorescence study, the algorithms developed to distinguish the diseased tissue from normal tissue based on the in vivo fluorescence study should be highly reliable for fluorescence imaging of lesions. In this work, fluorescence spectra were collected from 27 full term patients. Different algorithms were tested for separation of cancerous lesions from normal tissue. High sensitivity and specificity were achieved.

  20. Simultaneous recording of fluorescence and electrical signals by photometric patch electrode in deep brain regions in vivo.

    PubMed

    Hirai, Yasuharu; Nishino, Eri; Ohmori, Harunori

    2015-06-01

    Despite its widespread use, high-resolution imaging with multiphoton microscopy to record neuronal signals in vivo is limited to the surface of brain tissue because of limited light penetration. Moreover, most imaging studies do not simultaneously record electrical neural activity, which is, however, crucial to understanding brain function. Accordingly, we developed a photometric patch electrode (PME) to overcome the depth limitation of optical measurements and also enable the simultaneous recording of neural electrical responses in deep brain regions. The PME recoding system uses a patch electrode to excite a fluorescent dye and to measure the fluorescence signal as a light guide, to record electrical signal, and to apply chemicals to the recorded cells locally. The optical signal was analyzed by either a spectrometer of high light sensitivity or a photomultiplier tube depending on the kinetics of the responses. We used the PME in Oregon Green BAPTA-1 AM-loaded avian auditory nuclei in vivo to monitor calcium signals and electrical responses. We demonstrated distinct response patterns in three different nuclei of the ascending auditory pathway. On acoustic stimulation, a robust calcium fluorescence response occurred in auditory cortex (field L) neurons that outlasted the electrical response. In the auditory midbrain (inferior colliculus), both responses were transient. In the brain-stem cochlear nucleus magnocellularis, calcium response seemed to be effectively suppressed by the activity of metabotropic glutamate receptors. In conclusion, the PME provides a powerful tool to study brain function in vivo at a tissue depth inaccessible to conventional imaging devices. PMID:25761950

  1. Fluorescence lifetime imaging to differentiate bound from unbound ICG-cRGD both in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Stegehuis, Paulien L.; Boonstra, Martin C.; de Rooij, Karien E.; Powolny, François E.; Sinisi, Riccardo; Homulle, Harald; Bruschini, Claudio; Charbon, Edoardo; van de Velde, Cornelis J. H.; Lelieveldt, Boudewijn P. F.; Vahrmeijer, Alexander L.; Dijkstra, Jouke; van de Giessen, Martijn

    2015-03-01

    Excision of the whole tumor is crucial, but remains difficult for many tumor types. Fluorescence lifetime imaging could be helpful intraoperative to differentiate normal from tumor tissue. In this study we investigated the difference in fluorescence lifetime imaging of indocyanine green coupled to cyclic RGD free in solution/serum or bound to integrins e.g. in tumors. The U87-MG glioblastoma cell line, expressing high integrin levels, was cultured to use in vitro and to induce 4 subcutaneous tumors in a-thymic mice (n=4). Lifetimes of bound and unbound probe were measured with an experimental time-domain single-photon avalanche diode array (time resolution <100ps). In vivo measurements were taken 30-60 minutes after intravenous injection, and after 24 hours. The in vitro lifetime of the fluorophores was similar at different concentrations (20, 50 and 100?M) and showed a statistically significant higher lifetime (p<0.001) of bound probe compared to unbound probe. In vivo, lifetimes of the fluorophores in tumors were significantly higher (p<0.001) than at the control site (tail) at 30-60 minutes after probe injection. Lifetimes after 24 hours confirmed tumor-specific binding (also validated by fluorescence intensity images). Based on the difference in lifetime imaging, it can be concluded that it is feasible to separate between bound and unbound probes in vivo.

  2. In vivo Raman spectroscopy integrated with multimodal endoscopic imaging for early diagnosis of gastric dysplasia

    NASA Astrophysics Data System (ADS)

    Bergholt, Mads Sylvest; Zheng, Wei; Lin, Kan; Ho, Khek Yu; Teh, Ming; Yeoh, Khay Guan; Huang, Zhiwei

    2010-02-01

    A near-infrared Raman spectroscopy system integrated with multimodal endoscopic imaging has been developed for the early noninvasive in vivo diagnosis and detection of gastric malignancies. High-quality in vivo Raman spectra in the range 800-1800 cm-1 can be acquired from gastric normal and premalignant (dysplastic) mucosal tissue within 1 second under the guidance of white-light and narrow-band gastroscopic imaging during clinical gastroscopy. Prominent differences in Raman spectral shapes and intensities are observed between normal and dysplastic gastric mucosal tissue, particularly in the spectral ranges 800-900, 1250-1450 and 1600-1800 cm-1, which primarily contain signals related to proteins, nucleic acids and lipids. The empirical intensity ratio algorithm I875/I1450 classifies in vivo Raman spectra of dysplasia with a sensitivity of 100% and specificity of 100%. Our initial investigations show that in vivo Raman spectroscopy in conjunction with multimodal endoscopic imaging modalities holds a great promise for improving the early diagnosis of gastric malignancies.

  3. A MEMS based handheld confocal microscope with Raman spectroscopy for in-vivo skin cancer diagnosis

    NASA Astrophysics Data System (ADS)

    Arrasmith, Christopher L.; Patil, Chetan A.; Dickensheets, David L.; Mahadevan-Jansen, Anita

    2009-02-01

    Both Confocal Microscopy and Raman Spectroscopy have shown potential for diagnosis and differentiation of cancerous and normal skin. Many current studies utilizing these techniques use large bench-top microscopes, and are not suited for in-vivo diagnosis in a clinical setting. We have developed a microscope which combines confocal reflectance imaging with Raman spectroscopy into a compact handheld probe, allowing images and Raman spectra to be taken in-vivo. The compact design of this handheld unit is largely due to the use of a MEMS mirror which scans the illumination laser light in two dimensions to produce the confocal reflectance image of the skin. An integrated CCD camera provides a large area view of the skin surface which helps to guide the location of the confocal reflectance image area. Using this probe, in-vivo confocal reflectance images and Raman spectra of normal skin have been obtained with axial resolutions of 4 ?m for the confocal channel and 10 ?m for the Raman channel. This paper presents the instrument design and optical characteristics, including representative in-vivo images and Raman data from normal skin tissue.

  4. Detection of Non-Cavitated Occlusal Caries with Impedance Spectroscopy and Laser Fluorescence: an In Vitro Study

    PubMed Central

    Mortensen, Diana; Dannemand, Katrine; Twetman, Svante; Keller, Mette Kirstine

    2014-01-01

    Objective: To evaluate the performance of an impedance spectroscopy technology for detecting non-cavitated occlusal caries lesions in permanent teeth in vitro. The method was compared with a commonly used laser fluorescence device and validated against histology. Material and Methodology: A non-cavitated sample of 100 extracted posterior teeth was randomly selected and assessed for caries on enamel and dentin level with aid of CarioScan PRO (ACIS) and DIAGNOdent pen (LF pen) by three examiners. After the measurements, the extension of the lesion was histologically determined as gold standard. Sensitivity, specificity, accuracy and receiver-operating curves were calculated. Intra- and inter-examiner reproducibility was expressed by intra class correlation coefficients. Results: The histological caries prevalence was 99% and 41% exhibited dentin caries. The ACIS technique displayed high specificities but almost negligible sensitivities at readings >50. A similar pattern was noted for the LF pen at readings >30. The intra- and inter-examiner reproducibility varied between 0.47 and 0.98 and the values were generally lower for the ACIS technique than for the LF pen. The inter-examiner agreement reached excellent levels with both methods. Conclusions: In vitro,the ACIS technique showed a low ability to disclose occlusal caries lesions in the enamel and/or dentin of non-cavitated permanent molars. However, further in vivo studies of permanent occlusal surfaces are needed to mirror the clinical situation. PMID:24799965

  5. Detection of orange juice frauds using front-face fluorescence spectroscopy and Independent Components Analysis.

    PubMed

    Ammari, Faten; Redjdal, Lamia; Rutledge, Douglas N

    2015-02-01

    The aim of this study was to find simple objective analytical methods to assess the adulteration of orange juice by grapefruit juice. The adulterations by addition of grapefruit juice were studied by 3D-front-face fluorescence spectroscopy followed by Independent Components Analysis (ICA) and by classical methods such as free radical scavenging activity and total flavonoid content. The results of this study clearly indicate that frauds by adding grapefruit juice to orange juice can be detected at percentages as low as 1%. PMID:25172702

  6. Effect of different agents onto multidrug resistant cells revealed by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Boutin, C.; Roche, Y.; Jaffiol, R.; Millot, J.-M.; Millot, C.; Plain, J.; Deturche, R.; Jeannesson, P.; Manfait, M.; Royer, P.

    Fluorescence correlation spectroscopy (FCS), which is a sensitive and non invasive technique, has been used to characterize the plasma membrane fluidity and heterogeneity of multidrug resistant living cells. At the single cell level, the effects of different membrane agents present in the extra-cellular medium have been analyzed. Firstly, we reveal a modification of plasma membrane microviscosity according to the addition of a fluidity modulator, benzyl alcohol. In the other hand, revertant such as verapamil and cyclosporin-A appears to act more specifically on the slow diffusion sites as microdomains.

  7. Direct measurements of neutral density depletion by two-photon absorption laser-induced fluorescence spectroscopy

    SciTech Connect

    Aanesland, A.; Liard, L.; Leray, G.; Jolly, J.; Chabert, P. [Laboratoire de Physique et Technologie des Plasmas, Ecole Polytechnique, 91128 Palaiseau Cedex (France)

    2007-09-17

    The ground state density of xenon atoms has been measured by spatially resolved laser-induced fluorescence spectroscopy with two-photon excitation in the diffusion chamber of a magnetized Helicon plasma. This technique allows the authors to directly measure the relative variations of the xenon atom density without any assumptions. A significant neutral gas density depletion was measured in the core of the magnetized plasma, in agreement with previous theoretical and experimental works. It was also found that the neutral gas density was depleted near the radial walls.

  8. Ultrasensitive detection of waste products in water using fluorescence emission cavity-enhanced spectroscopy.

    PubMed

    Bixler, Joel N; Cone, Michael T; Hokr, Brett H; Mason, John D; Figueroa, Eleonora; Fry, Edward S; Yakovlev, Vladislav V; Scully, Marlan O

    2014-05-20

    Clean water is paramount to human health. In this article, we present a technique for detection of trace amounts of human or animal waste products in water using fluorescence emission cavity-enhanced spectroscopy. The detection of femtomolar concentrations of urobilin, a metabolic byproduct of heme metabolism that is excreted in both human and animal waste in water, was achieved through the use of an integrating cavity. This technique could allow for real-time assessment of water quality without the need for expensive laboratory equipment. PMID:24799690

  9. Study on the interaction of anticancer drug mitoxantrone with DNA by fluorescence and Raman spectroscopies

    NASA Astrophysics Data System (ADS)

    Tang, Lingjuan; Sun, Zhenrong; Guo, Jianyu; Wang, Zugeng

    2006-02-01

    Mitoxantrone, a clinically useful antitumour antibiotic for leukaemia and breast cancer, has received more attentions. In this paper, the interaction between mitoxantrone and calf thymus DNA is investigated by Raman and fluorescence spectroscopies, and the binding site of mitoxantrone to calf thymus DNA is explored. The results showed that mitoxantrone interacts with calf thymus DNA bases by the intercalation of anthracycline into the base pair plane of adenine (A) and thymine (T), and it results in the disruption of the hydrogen bonds between calf thymus DNA bases, and thus the calf thymus DNA double-strand can be disrupted into the B-form DNA double-strand segments.

  10. Sequentially shifted excitation Raman spectroscopy: novel algorithm and instrumentation for fluorescence-free Raman spectroscopy in spectral space.

    PubMed

    Cooper, John B; Abdelkader, Mohamed; Wise, Kent L

    2013-08-01

    A novel Raman spectrometer is presented in a handheld format. The spectrometer utilizes a temperature-controlled, distributed Bragg reflector diode laser, which allows the instrument to operate in a sequentially shifted excitation mode to eliminate fluorescence backgrounds, fixed pattern noise, and room lights, while keeping the Raman data in true spectral space. The cost-efficient design of the instrument allows rapid acquisition of shifted excitation data with a shift time penalty of less than 2 s. The Raman data are extracted from the shifted excitation spectra using a novel algorithm that is typically three orders of magnitude faster than conventional shifted-excitation algorithms operating in spectral space. The superiority of the instrument and algorithm in terms of background removal and signal-to-noise ratio is demonstrated by comparison to FT-Raman, standard deviation spectra, shifted excitation Raman difference spectroscopy (SERDS), and conventional multiple-shift excitation methods. PMID:23876736

  11. Tracking Biological Organic Compounds In Atmospheric Deposition In Alpine Environments With Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Mladenov, N.; Oldani, K. M.; Williams, M. W.; Schmidt, S. K.; Darcy, J.; Lemons, S.; Reche, I.

    2013-12-01

    Alpine environments, such as those of the Colorado Rocky Mountains, USA and the Sierra Nevada Mountains, Spain, contain undeveloped, barren soils that are carbon-limited. Atmospheric wet and dry deposition of organic carbon (OC) represents a substantial fraction of the OC load available to alpine soils, and includes contributions from atmospheric pollutants, dust, and biological aerosols, such as bacteria, algae, fungi, and plant debris. To evaluate the seasonal variability and sources of atmospheric deposition at these alpine sites, we measured the chemical characteristics of weekly wet and dry deposition and snowpack samples, including characterization of dissolved organic matter (DOM) and water soluble organic matter (WSOM) with fluorescence spectroscopy. The excitation-emission matrix (EEM) spectra we acquired show the presence of recurring peaks at low excitation and emission wavelengths typically associated with highly biodegradable organic carbon, presumably derived from the aromatic amino acids, tyrosine and tryptophan. Solar simulation experiments demonstrated that amino acid-like fluorescent components were more resistant to photo-degradation than humic- and fulvic-like fluorescent components. Our results also reveal the presence of a unique fluorophore, not previously described, that is found in both Rocky Mountains and the Sierra Nevada snowpack, wet deposition, and dry deposition and may be attributed to fluorescent pigments in bacteria. Biological aerosols may represent a labile source of carbon for alpine soil microbes, and consequently their deposition has important consequences for biogeochemical processes occurring in barren, alpine soils. Excitation emission matrix image of 24 Aug 2010 wet deposition sample from the Soddie site at Niwot Ridge, Colorado showing a unique fluorescent component with dual excitation peaks (285 nm and 340 nm) at 410 nm emission.

  12. TECHNICAL COMMUNICATION A new setup for in vivo fluorescence imaging of photosynthetic

    E-print Network

    of CCD, we have opted for a 12-bits high frame rate [150 fps (frames per second)] at the expense Plastoquinol Introduction Chlorophyll fluorescence detection is a non-invasive tech- nique for the imaging material is not used for energy conversion, but reemitted as fluorescence photons: the fluorescence signal

  13. Nitrate and nitrite as ‘in vivo’ quenchers of chlorophyll fluorescence in blue-green algae

    Microsoft Academic Search

    A. Serrano; J. Rivas; M. Losada

    1981-01-01

    The effect of nitrate and nitrite on long-term chlorophyll fluorescence has been studied in filamentous blue-green algae. Cells grown autotrophically with nitrate as nitrogen source show, under argon atmosphere, a high level of fluorescence. The addition of either nitrete or nitrite induces a significant fluorescence quenching, but, whereas in the case of nitrite no previous treatment is required, in the

  14. Fluorescent properties and spontaneous Raman spectroscopy of new ketocyanine probes in organic solvents

    NASA Astrophysics Data System (ADS)

    Nemkovich, N. A.; Sobchuk, A. N.; Khodasevich, I. A.

    2006-11-01

    We have used fluorescence spectroscopy and spontaneous Raman spectroscopy to study the characteristics of two ketocyanine dyes: 2,5-di[(E)-1-(4-diethylaminophenyl)methylidene]-1-cyclopentanone (CPET) and 2-[(E)-1-(4-diethylaminophenyl)methylidene]-5-{(E)-1-[4-(4,7,10,13-tetraoxa-1-azacyclopentadecalin) phenyl]methylidene}-1-cyclopentanone (CPMR) in organic solvents. The position of their electronic spectra depends strongly on the polarity of the solvent. We measured the dipole moments of the dyes in the equilibrium ground state and the Franck-Condon excited state. In mixtures of neutral nonpolar toluene with aprotic polar dimethylsulfoxide, we observe inhomogeneous broadening of the electronic spectra for the indicated compounds, due to fluctuations in solution of the intermolecular interaction energy. The time-resolved characteristics of fluorescence obtained suggest formation of an intermolecular hydrogen bond between the dye and the surrounding medium in a toluene-ethanol mixture. We measured the Raman spectra of CPET and CPMR in different organic solvents. The most intense lines in the 1582 1591 cm-1 region can be assigned to stretching of the phenyl rings of the molecules; the lines in the 831 842 cm-1 region can be assigned to a cyclopentanone ring mode; the lines at 1186 1195 cm-1 can be assigned to stretching of the =C-C-bond of the phenyl ring and rocking of the H atoms of the phenyl ring. We have observed that the position and width of the lines for the stretching vibrations of the ketocyanines depend substantially on the polarity of the surrounding medium. The studied dyes can be used as probes for studying different biological systems by site-selective laser spectroscopy and Raman spectroscopy. The fact that these two methods can be used simultaneously for diagnostics of biosystems is an important advantage of ketocyanine dyes compared with other known probes.

  15. What might be the impact on neurology of the analysis of brain metabolism by in vivo magnetic resonance spectroscopy?

    Microsoft Academic Search

    J. Vion-Dury; D. J. Meyerhoff; P. J. Cozzone; M. W. Weiner

    1994-01-01

    In vivo nuclear magnetic resonance spectroscopy (MRS) of the human brain is a recently developed technique which allows to assay noninvasively in vivo key molecules of brain metabolism. After a review of the origin of the signals detected by phosphorus and proton MRS of human brain, the impact of MRS on clinical neurology is examined. MRS of the brain does

  16. Diffusion of organic dyes in a niosome immobilized on a glass surface using fluorescence correlation spectroscopy.

    PubMed

    Ghosh, Shirsendu; Mandal, Amit Kumar; Das, Atanu Kumar; Mondal, Tridib; Bhattacharyya, Kankan

    2012-07-21

    Giant multilameller niosomes containing cholesterol and triton X-100 are studied using fluorescence correlation spectroscopy (FCS). Dynamic light scattering (DLS) data indicates formation of niosomes of broadly two different sizes (diameter)--~150 nm and ~1300 nm. This is confirmed by field emission scanning electron microscopy (FE-SEM) and confocal microscopy. The diffusion coefficient (D(t)) of three organic dyes in the niosome immobilized on a glass surface is studied using fluorescence correlation spectroscopy. On addition of the room temperature ionic liquids (RTIL) (1-methyl-3-pentylimidazolium bromide, [pmim][Br] and 1-methyl- 3-pentylimidazolium tetra-fluoroborate, [pmim][BF(4)]) the size of the niosome particles increases. The D(t) of all the organic dyes (DCM, C343 and C480) increases on addition of RTILs, indicating faster diffusion. The viscosity calculated from the D(t) of the three dyes exhibits weak probe dependence. Unlike lipid or catanionic vesicle, the D(t) values in a niosome exhibit very narrow distribution. This indicates that the niosomes are fairly homogeneous with small variation of viscosity. PMID:22692627

  17. Organic dye penetration quantification into a dental composite resin cured by LED system using fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Lizarelli, Rosane de Fátima Zanirato; Silva, Maciel E., Jr.; Lins, Emery C. C. C.; Costa, Mardoqueu M.; Pelino, José Eduardo P.; Bagnato, Vanderlei S.

    2007-02-01

    A major characteristic of LEDs systems is the lower heat emission related with the kind of light generation and spectral emission band. Material temperature during photoactivation can promote different photocuring performance. Organic dye penetration could be a trace to identify the efficacy of photocured composite resin. A new method using fluorescent spectroscopy through digital image evaluation was developed in this study. In order to understand if there is a real influence of material temperature during the photoactivation procedure of a dental restorative material, a hybrid composite resin (Z250, 3M-Espe, USA) and 3 light sources, halogen lamp (510 mW/cm2) and two LED systems 470+/-10nm (345 and 1000 mW/cm2) under different temperatures and intensities were used. One thousand and five hundred samples under different associations between light sources and temperatures (0, 25, 50, 75 and 100 °C were tested and immediately kept in 6G rodamin dye solution. Dye penetration was evaluated through fluorescent spectroscopy recorded by digital image data. Pixels in gray scale showed the percentage penetration of organic dye into the composite resin mass. Time and temperature were statistically significant (p<0.05) through the ANOVA statistical test. The lowest penetration value was with 60 seconds and 25 °C. Time and temperature are important factors to promote a homogeneous structure polymerized composite resin more than the light source type, halogen or LEDs system.

  18. The use of solid-phase fluorescence spectroscopy in the characterisation of organic matter transformations.

    PubMed

    Albrecht, R; Verrecchia, E; Pfeifer, H-R

    2015-03-01

    Given its high sensitivity and non-destructive nature, fluorescence excitation-emission matrix (EEM) spectroscopy is widely used to differentiate changes and transformations of dissolved or water-extracted organic matter (OM) in natural environments. The same technique applied directly on solid samples (solid-phase fluorescence spectroscopy, SPF-EEM) provides accurate results when used with pharmaceutical products or food samples, but only a few studies have considered natural OM. This study reports on the use of SPF-EEM on solid compost samples and emphasises the way the different maturation phases can be distinguished with fluorophores closely resembling those found in dissolved samples. A very good correlation has been found with data from Rock-Eval pyrolysis, nuclear magnetic resonance ((13)C CPMAS NMR), and humic-fulvic acid ratios determined by conventional NaOH-extraction. SPF-EEM appears as a much simpler method than the conventional ones to detect transformations in natural OM samples with low mineral contents. However, direct application to soil samples requires some additional studies. PMID:25618693

  19. Seven-color fluorescence imaging of tissue samples based on Fourier spectroscopy and singular value decomposition.

    PubMed

    Tsurui, H; Nishimura, H; Hattori, S; Hirose, S; Okumura, K; Shirai, T

    2000-05-01

    Seven-color analyses of immunofluorescence-stained tissue samples were accomplished using Fourier spectroscopy-based hyperspectral imaging and singular value decomposition. This system consists of a combination of seven fluorescent dyes, three filtersets, an epifluorescence microscope, a spectral imaging system, a computer for data acquisition, and data analysis software. The spectra of all pixels in a multicolor image were taken simultaneously using a Sagnac type interferometer. The spectra were deconvolved to estimate the contribution of each component dye, and individual dye images were constructed based on the intensities of assigned signals. To obtain mixed spectra, three filter sets, i.e., Bl, Gr, and Rd for Alexa488 and Alexa532, for Alexa546, Alexa568, and Alexa594, and for Cy5 and Cy5.5, respectively, were used for simultaneous excitation of two or three dyes. These fluorophores have considerable spectral overlap which precludes their separation by conventional analysis. We resolved their relative contributions to the fluorescent signal by a method involving linear unmixing based on singular value decomposition of the matrices consisting of dye spectra. Analyses of mouse thymic tissues stained with seven different fluorescent dyes provided clear independent images, and any combination of two or three individual dye images could be used for constructing multicolor images. PMID:10769049

  20. Correlation Spectroscopy of Minor Fluorescent Species: Signal Purification and Distribution Analysis

    PubMed Central

    Laurence, Ted A.; Kwon, Youngeun; Yin, Eric; Hollars, Christopher W.; Camarero, Julio A.; Barsky, Daniel

    2007-01-01

    We are performing experiments that use fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) to monitor the movement of an individual donor-labeled sliding clamp protein molecule along acceptor-labeled DNA. In addition to the FRET signal sought from the sliding clamp-DNA complexes, the detection channel for FRET contains undesirable signal from free sliding clamp and free DNA. When multiple fluorescent species contribute to a correlation signal, it is difficult or impossible to distinguish between contributions from individual species. As a remedy, we introduce “purified FCS”, which uses single molecule burst analysis to select a species of interest and extract the correlation signal for further analysis. We show that by expanding the correlation region around a burst, the correlated signal is retained and the functional forms of FCS fitting equations remain valid. We demonstrate the use of purified FCS in experiments with DNA sliding clamps. We also introduce “single-molecule FCS”, which obtains diffusion time estimates for each burst using expanded correlation regions. By monitoring the detachment of weakly-bound 30-mer DNA oligomers from a single-stranded DNA plasmid, we show that single-molecule FCS can distinguish between bursts from species that differ by a factor of 5 in diffusion constant. PMID:17189306

  1. Intramolecular vibrations of the phenol dimer revealed by spectral hole burning (SHB) and dispersed fluorescence spectroscopy

    SciTech Connect

    Schmitt, M.; Henrichs, U.; Mueller, H.; Kleinermanns, K. [Universitaet Duesseldorf (Germany)

    1995-12-31

    In the hydrogen bridge bonded phenol dimer one phenol moiety is the proton donor, while the other acts as proton acceptor. The donor absorbs at longer, the acceptor at shorter wavelength than the monomer. The authors performed hole burning spectroscopy to rule out the existence of different conformers, absorbing in the investigated region. Holes burned in the population of the dimer are analyzed via fluorescence from the donor origin. Even acceptor vibrations could be analyzed this way, because acceptor and donor moiety belong to one species in the electronic ground state and share a common set of ground state levels. This is favorable, because the excited donor phenol fluoresces much more efficiently than the acceptor part. 10 low frequency modes of the donor and 8 of the acceptor moiety could be observed. In order to assign the low frequency vibrational bands to distinct normal modes dispersed fluorescence spectra have been taken, by pumping all intermolecular vibrations. The Franck-Condon pattern then allows to assign unambiguously which level in the electronic ground state belongs to which excited state level.

  2. Plasmon-Enhanced Raman and Fluorescence Spectroscopy with Gold and Silver Nanoparticles

    NASA Astrophysics Data System (ADS)

    Guerrero Hernandez, Ariel Rodrigo

    This thesis contains five major contributions to the field of plasmon-enhanced spectroscopy. We start with the report of a unique SERS study of the amino acid hydroxyproline and a deuterated analogue. Later, we move on to the exploration of a major new research path known as shell-isolated nanoparticle-enhanced fluorescence (SHINEF), consisting in the application of silica-shelled noble metal nanoparticles to achieve surface-enhanced fluorescence. The proof of concept of this technique is explained in one chapter. The two following chapters are devoted to the exploration of the plasmonic properties of SHINEF: spectral profile modification showing the close relationship between the observed enhanced fluorescence and the nanoparticle scattering. The SHIN particles are employed to experimentally prove the relationship between the SEF and SERS enhancement factors, theoretically predicted before, but never verified experimentally until now. The thesis ends with an investigation, in aqueous solutions, of several different factors that play a role in the origin of SEF, showing greater enhancement for SHINEF after inducing nanoparticle aggregation.

  3. Frequency-domain fluorescence spectroscopy: instrumentation and applications to the biosciences

    NASA Astrophysics Data System (ADS)

    Lakowicz, Joseph R.; Gryczynski, Ignacy; Malak, Henryk M.; Johnson, Michael L.; Laczko, Gabor; Wiczk, Wieslaw M.; Szmacinski, Henryk; Kusba, Jozef

    1991-07-01

    Measurements of time-resolved fluorescence are increasingly used for research in biophysics, biochemistry, cell biology and medicine. Advances in the technology of light sources and detectors are resulting in more reliable and/or advanced instrumentation, which is resulting in the expanding applications of fluorescence spectroscopy. Time-resolved measurements are often performed by direct measurements in the time-domain. In this article the authors describe the alternative method of frequency-domain fluorometry. The frequency-response of the emission to intensity-modulated excitation can be used to recover the time-dependent decay. Commercial instrumentation now allows measurements to an upper light modulation frequency limit of 200 MHz. This laboratory has developed second and third generation instruments which allows measurements to 2 GHz and subsequently to 10 GHz. The frequency-domain data from such instrumentation provides excellent resolution of picosecond decays of intensity and anisotropy. Additionally, the frequency-domain method appears to provide remarkable resolution of complex decays which are often observed for biochemical samples. In this article the authors describe this instrumentation and applications of this method. Examples are shown using probes with ps decay and correlation times, the intrinsic fluorescence of proteins, and the measurement of end-to-end diffusion in proteins and/or flexible molecules.

  4. Sub-diffusion decays in fluorescence correlation spectroscopy: dye photophysics or protein dynamics?

    PubMed

    Mazouchi, Amir; Bahram, Abdullah; Gradinaru, Claudiu C

    2013-09-26

    Transitions between bright and dark fluorescent states of several rhodamine dyes were investigated by fluorescence correlation spectroscopy. We resolved two sub-diffusion exponential decays for free rhodamines in aqueous solutions, of which the slower component scales linearly with the viscosity of the solution. Correlation data for proteins and DNA labeled with tetramethylrhodamine were fitted with three to four exponential decays describing flickering dynamics on a time scale between 0.5 and 100 ?s. We investigated the nature of these processes by performing experiments under different experimental conditions and for different samples. On the basis of how their population and lifetime change with viscosity, the oxygen content of the solution, the laser irradiance, and the detection geometry, we assigned these states, in the order of increasing lifetimes, to a triplet state, a hybrid between twisted-intramolecular-charge-transfer state and a ground state lactonic state, a lactonic state, and a photoionized state, respectively. Our data suggests that none of the observed sub-diffusion correlation decays can be directly assigned to the intramolecular dynamics of the labeled biomolecules. However, we found evidence that the intrinsic conformational dynamics of the biomolecule appears in the correlation curves as a modulation of the photophysics of the dye label. This shows the importance of accurate control measurements and appropriate modeling of the dye photophysics in fluorescence correlation studies, and it cautions against direct assignments of dark-state relaxation times to folding kinetics in proteins and nucleic acids. PMID:23675915

  5. Use of time-resolved fluorescence spectroscopy to evaluate diagnostic value of collagen degradation products.

    PubMed

    Sikora, Joanna; Cyrankiewicz, Micha?; Wybranowski, Tomasz; Ziomkowska, Blanka; O?mia?owski, Borys; Obo?ska, Ewa; Augusty?ska, Beata; Kruszewski, Stefan; Kubica, Jacek

    2015-05-01

    The concentration of collagen degradation products (CDPs) may reflect the process of left ventricular remodeling (LVR). The aim of this study was to evaluate the potential diagnostic usefulness of time-resolved fluorescence spectroscopy (TRFS) in assessment of CDPs. The preliminary experiment was designed to establish if CDPs’ characteristics might be visible by mean fluorescence lifetime (FLT) in determined conditions. The in vitro model of CDPs was prepared by conducting the hydrolysis of type III collagen. The FLT of samples was measured by the time-resolved spectrometer Life Spec II with the subnanosecond pulsed 360-nm EPLED diode. The FLTs were obtained by deconvolution analysis of the data using a multiexponential model of fluorescence decay. In order to determine the limit of traceability of CDPs, a comparison of different collagen/plasma ratio in samples was performed. The results of our study showed that the increase of added plasma to hydrolyzed collagen extended the mean FLT. Thus, the diagnosis of LVR based on measurements using TRFS is possible. However, it is important to point out the experiment was preliminary and further investigation in this field of research is crucial. PMID:25764396

  6. [Combining ultrafiltration, fluorescence spectroscopy and HPSEC to characterize dissolved organic matter in surface waters].

    PubMed

    Wang, Jing; Wu, Feng-Chang; Wang, Li-Ying; Liao, Hai-Qing; Li, Wen

    2008-11-01

    The combination of ultrafiltration, three-dimensional excitation/emission matrix (3DEEM) fluorescence spectroscopy and high-performance size-exclusion chromatography (HPSEC) was used to characterize the molecular weight distribution properties of different fluorescence materials, further revealing the differences in the sources and components. Dissolved organic matter (DOM) from different lakes fractionated by ultrafiltration with a nominal 1,000 molecular weight cut-off regenerated cellulose membrane, then 3DEEM spectrophotometry and HPSEC were applied to investigate the characteristics of high molecular weight materials in retentate and low molecular weight materials in permeate. The result indicated that the flulvic acid-like (Ex/Em approximately equal to 260 nm/450 nm of peak A and Ex/Em approximately equal to 320 nm/439 nm of peak C) and protein-like (Ex/Em approximately equal to 275 nm/312 nm of peak B and Ex/Em approximately equal to 220 nm/308 nm of peak D) fluorophores in permeate were detected after ultrafiltration which is covered by high absorbing peak of humic-like (Ex/Em approximately equal to 360 nm/462 nm of peak E) fluorescence fluorophores in the original Shennonajia bog water, Hubei province. They permeated the membrane because of their low molecular weight distribution properties. The content of autochthonous protein-like (Ex/Em approximately equal to 280 nm/334 nm of peak B and Ex/Em approximately equal to 225 nm/328 nm of peak D) component was too low to be measured by high-sensitivity fluorescence spectrophotometer in the original water of Lake Hongfeng, Guizhou province. But they can be concentrated attribute to their high molecular weight distribution properties. The 3DEEM fluorescence spectroscopy of retentate exhibited evident protein-like fluorophores. Moreover, there had obviously difference in molecular weight between different sources of fulvic acid-like and protein-like components. It has been shown that the molecular weight distribution of autochthonous protein-like matter in Hongfeng lake is larger than allochthonous protein-like matter in Aha lake, Guizhou province. One was retained by membrane and another was permeated. Therefore, these techniques together will offer direct and convenient qualitative information about DOM in lake waters. PMID:19186797

  7. In vivo spectroscopic photoacoustic tomography imaging of a far red fluorescent protein expressed in the exocrine pancreas of adult zebrafish

    NASA Astrophysics Data System (ADS)

    Liu, Mengyang; Schmitner, Nicole; Sandrian, Michelle G.; Zabihian, Behrooz; Hermann, Boris; Salvenmoser, Willi; Meyer, Dirk; Drexler, Wolfgang

    2014-03-01

    Fluorescent proteins brought a revolution in life sciences and biological research in that they make a powerful tool for researchers to study not only the structural and morphological information, but also dynamic and functional information in living cells and organisms. While green fluorescent proteins (GFP) have become a common labeling tool, red-shifted or even near infrared fluorescent proteins are becoming the research focus due to the fact that longer excitation wavelengths are more suitable for deep tissue imaging. In this study, E2-Crimson, a far red fluorescent protein whose excitation wavelength is 611 nm, was genetically expressed in the exocrine pancreas of adult zebrafish. Using spectroscopic all optical detection photoacoustic tomography, we mapped the distribution of E2-Crimson in 3D after imaging the transgenic zebrafish in vivo using two different wavelengths. With complementary morphological information provided by imaging the same fish using a spectral domain optical coherence tomography system, the E2-Crimson distribution acquired from spectroscopic photoacoustic tomography was confirmed in 2D by epifluorescence microscopy and in 3D by histology. To the authors' knowledge, this is the first time a far red fluorescent protein is imaged in vivo by spectroscopic photoacoustic tomography. Due to the regeneration feature of zebrafish pancreas, this work preludes the longitudinal studies of animal models of diseases such as pancreatitis by spectroscopic photoacoustic tomography. Since the effective penetration depth of photoacoustic tomography is beyond the transport mean free path length, other E2-Crimson labeled inner organs will also be able to be studied dynamically using spectroscopic photoacoustic tomography.

  8. Hyperspectral fluorescence lifetime fibre probe spectroscopy for use in the study and diagnosis of osteoarthritis and skin cancer

    NASA Astrophysics Data System (ADS)

    Thompson, Alex; Manning, Hugh; Brydegaard, Mikkel; Coda, Sergio; Kennedy, Gordon; Patalay, Rakesh; Waitong-Braemming, Ulrika; De Beule, Pieter; Neil, Mark; Andersson-Engels, Stefan; Itoh, Yoshifumi; Bendsøe, Niels; Dunsby, Christopher; Svanberg, Katarina; French, Paul M.

    2011-03-01

    We present the application of two fibre-optic-coupled time-resolved spectrofluorometers and a compact steady-state diffuse reflected light/fluorescence spectrometer to in vivo and ex vivo studies of skin cancer and osteoarthritis. In a clinical study of skin cancer, 27 lesions on 25 patients were investigated in vivo before surgical excision of the region measured. Preliminary analysis reveals a statistically significant decrease in the autofluorescence lifetime of basal cell carcinomas compared to neighbouring healthy tissue. A study of autofluorescence signals associated with the onset of osteoarthritis indicates autofluorescence lifetime changes associated with collagen degradation.

  9. Spectral decomposition of NAD(P)H fluorescence components recorded by multi-wavelength fluorescence lifetime spectroscopy in living cardiac cells

    NASA Astrophysics Data System (ADS)

    Chorvatova, Alzbeta; Mateasik, Anton; Chorvat, Dusan, Jr.

    2013-12-01

    We report a novel analytical approach to identify individual components of a cell’s endogenous fluorescence, recorded by spectrally-resolved time-correlated single photon counting (TCSPC). Time-resolved area-normalized emission spectroscopy (TRANES) and principal component analysis (PCA) were applied to estimate the number of spectral components after metabolic modulation of cardiac cells following excitation with a 375 nm picosecond laser. Linear unmixing of TCSPC data spectrally decomposed individual components in living cells, while using characteristics of endogenously fluorescing molecules in solvents as a reference spectral database. Our data demonstrate the presence of three individual components, corresponding to the nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in organic and inorganic solvents and to the residual flavoprotein fluorescence. The presented analytical approach offers a new alternative for the spectral separation of multi-wavelength fluorescence lifetime spectroscopy data to the conventional analysis, and opens a new possibility for the use of pattern recognition for fast resolution of components in 2D fluorescence lifetime microscopy images.

  10. Local conformations and excited state dynamics of porphyrins and nucleic acids by 2-dimensional fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Widom, Julia R.

    Biological systems present many challenges to researchers attempting to study them using spectroscopy. Low specificity, low sensitivity, and broad and overlapping lineshapes limit the amount of information that can be obtained in experiments. Two-dimensional fluorescence spectroscopy (2D FS) is a highly sensitive and information-rich spectroscopic technique that was developed to study the conformations and excited state dynamics of systems exhibiting exciton coupling. In this dissertation, I describe a variety of extensions of 2D FS that further increase its utility for the study of biological systems. I describe experiments on a dimer of zinc tetraphenylporphyrin embedded in a membrane, in which the signals from two conformational subpopulations were separated in order to study the thermodynamics of their interconversion. I present proof-of-principle experiments on nucleic acids that utilize fluorescence resonance energy transfer to separate signals from different subpopulations. I also describe experiments in which 2D FS was performed using ultraviolet excitation to determine the conformation of a dinucleotide of a fluorescent analogue of the nucleic acid base adenine. I discuss experiments on porphyrin dimers in which 2D FS was used as a probe of excited state dynamics. Finally, I present model calculations for a proposed variation of 2D FS in which entangled photons would be used as the excitation source. These calculations suggest that this approach has the potential to yield significantly narrower spectral lineshapes than conventional 2D FS. These experiments and calculations yield new insight into the systems investigated and establish a `toolbox' of variations of 2D FS that can be used to gain as much information as possible from experiments on challenging systems such as protein-DNA complexes.

  11. Imaging fluorescence-correlation spectroscopy for measuring fast surface diffusion at liquid/solid interfaces.

    PubMed

    Cooper, Justin T; Harris, Joel M

    2014-08-01

    The development of techniques to probe interfacial molecular transport is important for understanding and optimizing surface-based analytical methods including surface-enhanced spectroscopies, biological assays, and chemical separations. Single-molecule-fluorescence imaging and tracking has been used to measure lateral diffusion rates of fluorescent molecules at surfaces, but the technique is limited to the study of slower diffusion, where molecules must remain relatively stationary during acquisition of an image in order to build up sufficient intensity in a spot to detect and localize the molecule. Although faster time resolution can be achieved by fluorescence-correlation spectroscopy (FCS), where intensity fluctuations in a small spot are related to the motions of molecules on the surface, long-lived adsorption events arising from surface inhomogeneity can overwhelm the correlation measurement and mask the surface diffusion of the moving population. Here, we exploit a combination of these two techniques, imaging-FCS, for measurement of fast interfacial transport at a model chromatographic surface. This is accomplished by rapid imaging of the surface using an electron-multiplied-charged-coupled-device (CCD) camera, while limiting the acquisition to a small area on the camera to allow fast framing rates. The total intensity from the sampled region is autocorrelated to determine surface diffusion rates of molecules with millisecond time resolution. The technique allows electronic control over the acquisition region, which can be used to avoid strong adsorption sites and thus minimize their contribution to the measured autocorrelation decay and to vary the acquisition area to resolve surface diffusion from adsorption and desorption kinetics. As proof of concept, imaging-FCS was used to measure surface diffusion rates, interfacial populations, and adsorption-desorption rates of 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine (DiI) on planar C18- and C1-modified surfaces. PMID:24975169

  12. Characterization of dissolved organic matter in urban sewage using excitation emission matrix fluorescence spectroscopy and parallel factor analysis

    Microsoft Academic Search

    Weidong Guo; Jing Xu; Jiangping Wang; Yingrou Wen; Jianfu Zhuo; Yuchao Yan

    2010-01-01

    Wastewater dissolved organic matter (DOM) from different processing stages of a sewage treatment plant in Xiamen was characterized using fluorescence and absorption spectroscopy. Parallel factor analysis modeling of excitation-emission matrix spectra revealed five fluorescent components occurring in sewage DOM: one protein-like (C1), three humic-like (C2, C4 and C5) and one xenobiotic-like (C3) components. During the aerated grit chamber and primary

  13. Fluorescence imaging for a noninvasive in vivo toxicity-test using a transgenic silkworm expressing green fluorescent protein.

    PubMed

    Inagaki, Yoshinori; Matsumoto, Yasuhiko; Ishii, Masaki; Uchino, Keiro; Sezutsu, Hideki; Sekimizu, Kazuhisa

    2015-01-01

    In drug development, the toxicity of candidate chemicals must be carefully examined in an animal model. Here we developed a live imaging technique using silkworms for a noninvasive toxicity test applicable for drug screening. Injection of carbon tetrachloride, a tissue-injuring chemical, into transgenic silkworms expressing green fluorescent protein (GFP) induced leakage of GFP from the tissues into the hemolymph. The leakage of GFP was suppressed by pre-administration of either cimetidine, a cytochrome P450 inhibitor, or N-acetyl cysteine, a free-radical scavenger. The transgenic silkworm was made transparent by feeding a diet containing chemicals that inhibit uric acid deposition in the epithelial cells. In the transparent silkworms, GFP fluorescence in the fat body could be observed from outside the body. Injection of salicylic acid or iron sulfate, tissue-injuring chemicals, into the transparent silkworms decreased the fluorescence intensity of the GFP in the fat body. These findings suggest that the transparent GFP-expressing silkworm model is useful for evaluating the toxicity of chemicals that induce tissue injury. PMID:26061948

  14. Fluorescence imaging for a noninvasive in vivo toxicity-test using a transgenic silkworm expressing green fluorescent protein

    PubMed Central

    Inagaki, Yoshinori; Matsumoto, Yasuhiko; Ishii, Masaki; Uchino, Keiro; Sezutsu, Hideki; Sekimizu, Kazuhisa

    2015-01-01

    In drug development, the toxicity of candidate chemicals must be carefully examined in an animal model. Here we developed a live imaging technique using silkworms for a noninvasive toxicity test applicable for drug screening. Injection of carbon tetrachloride, a tissue-injuring chemical, into transgenic silkworms expressing green fluorescent protein (GFP) induced leakage of GFP from the tissues into the hemolymph. The leakage of GFP was suppressed by pre-administration of either cimetidine, a cytochrome P450 inhibitor, or N-acetyl cysteine, a free-radical scavenger. The transgenic silkworm was made transparent by feeding a diet containing chemicals that inhibit uric acid deposition in the epithelial cells. In the transparent silkworms, GFP fluorescence in the fat body could be observed from outside the body. Injection of salicylic acid or iron sulfate, tissue-injuring chemicals, into the transparent silkworms decreased the fluorescence intensity of the GFP in the fat body. These findings suggest that the transparent GFP-expressing silkworm model is useful for evaluating the toxicity of chemicals that induce tissue injury. PMID:26061948

  15. Characterization of the BAC Id3-enhanced green fluorescent protein transgenic mouse line for in vivo imaging of astrocytes

    PubMed Central

    Lamantia, Cassandra; Tremblay, Marie-Eve; Majewska, Ania

    2014-01-01

    Abstract. Astrocytes are highly ramified glial cells with critical roles in brain physiology and pathology. Recently, breakthroughs in imaging technology have expanded our understanding of astrocyte function in vivo. The in vivo study of astrocytic dynamics, however, is limited by the tools available to label astrocytes and their processes. Here, we characterize the bacterial artificial chromosome transgenic Id3-EGFP knock-in mouse to establish its usefulness for in vivo imaging of astrocyte processes. Using fixed brain sections, we observed enhanced green fluorescent protein expression in astrocytes and blood vessel walls throughout the brain, although the extent and cell type specificity of expression depended on the brain area and developmental age. Using in vivo two-photon imaging, we visualized astrocytes in cortical layers 1–3 in both thin skull and window preparations. In adult animals, astrocytic cell bodies and fine processes could be followed over many hours. Our results suggest that Id3 mice could be used for in vivo imaging of astrocytes and blood vessels in development and adulthood.

  16. A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo

    PubMed Central

    Tada, Mayumi; Takeuchi, Atsuya; Hashizume, Miki; Kitamura, Kazuo; Kano, Masanobu

    2014-01-01

    Calcium imaging of individual neurons is widely used for monitoring their activity in vitro and in vivo. Synthetic fluorescent calcium indicator dyes are commonly used, but the resulting calcium signals sometimes suffer from a low signal-to-noise ratio (SNR). Therefore, it is difficult to detect signals caused by single action potentials (APs) particularly from neurons in vivo. Here we showed that a recently developed calcium indicator dye, Cal-520, is sufficiently sensitive to reliably detect single APs both in vitro and in vivo. In neocortical neurons, calcium signals were linearly correlated with the number of APs, and the SNR was > 6 for in vitro slice preparations and > 1.6 for in vivo anesthetised mice. In cerebellar Purkinje cells, dendritic calcium transients evoked by climbing fiber inputs were clearly observed in anesthetised mice with a high SNR and fast decay time. These characteristics of Cal-520 are a great advantage over those of Oregon Green BAPTA-1, the most commonly used calcium indicator dye, for monitoring the activity of individual neurons both in vitro and in vivo. PMID:24405482

  17. Characterization of the Pressure-induced Intermediate and Unfolded State of Red-shifted Green Fluorescent Protein—A Static and Kinetic FTIR, UV\\/VIS and Fluorescence Spectroscopy Study

    Microsoft Academic Search

    H. Herberhold; S. Marchal; R. Lange; C. H. Scheyhing; R. F. Vogel; R. Winter

    2003-01-01

    The green fluorescence proteins (GFP) are widely used as reporters in molecular and cell biology. For their use it in high-pressure microbiology and biotechnology studies, their structural properties, thermodynamic parameters and stability diagrams have to be known. We investigated the pressure stability of the red-shifted green fluorescent protein (rsGFP) using Fourier-transform infrared spectroscopy, fluorescence and UV\\/Vis spectroscopy. We found that

  18. A system for endoscopic mechanically scanned localized proton MR and light-induced fluorescence emission spectroscopies

    NASA Astrophysics Data System (ADS)

    Sonmez, Ahmet E.; Webb, Andrew G.; Spees, William M.; Ozcan, Alpay; Tsekos, Nikolaos V.

    2012-09-01

    Molecular and near-cellular modalities offer new opportunities in assessing living tissue in situ, and multimodality approaches, which offer complementary information, may lead to improved characterization of tissue pathophysiology benefiting diagnosis and focal therapy. However, many such modalities are limited by their low penetration through tissue, which has led to minimally invasive trans-cannula approaches to place the corresponding sensors locally at the area of interest. This work presents a system for performing localized fluorescence emission and proton magnetic resonance (MR) spectroscopies via endoscopic access. The in-house developed side-firing 1.9-mm wide dual-sensor integrates a three-fiber optical sensor for fluorescence emission optical spectroscopy and a 1-mm circular radiofrequency (RF) coil for localized MR proton spectroscopy. An MR-compatible manipulator was developed for carrying and mechanically translating the dual-sensor along a linear access channel. The hardware and software control of the system allows reconfigurable synchronization of the manipulator-assisted translation of the sensor, and MR and optical data collection. The manipulator serves as the mechanical link for the three modalities and MR images, MR spectra and optical spectra are inherently co-registered to the MR scanner coordinate system. These spectra were then used to generate spatio-spectral maps of the fluorophores and proton MR-signal sources in three-compartment phantoms with optically- and MR-visible, and distinguishable, materials. These data demonstrate a good spatial match between MR images, MR spectra and optical spectra along the scanned path. In addition to basic research, such a system may have clinical applications for assessing and characterizing cancer in situ, as well as guiding focal therapies.

  19. High pressure sample cell for total internal reflection fluorescence spectroscopy at pressures up to 2500 bar.

    PubMed

    Koo, Juny; Czeslik, Claus

    2012-08-01

    Total internal reflection fluorescence (TIRF) spectroscopy is a surface sensitive technique that is widely used to characterize the structure and dynamics of molecules at planar liquid-solid interfaces. In particular, biomolecular systems, such as protein adsorbates and lipid membranes can easily be studied by TIRF spectroscopy. Applying pressure to molecular systems offers access to all kinds of volume changes occurring during assembly of molecules, phase transitions, and chemical reactions. So far, most of these volume changes have been characterized in bulk solution, only. Here, we describe the design and performance of a high pressure sample cell that allows for TIRF spectroscopy under high pressures up to 2500 bar (2.5 × 10(8) Pa), in order to expand the understanding of volume effects from the bulk phase to liquid-solid interfaces. The new sample cell is based on a cylindrical body made of Nimonic 90 alloy and incorporates a pressure transmitting sample cuvette. This cuvette is composed of a fused silica prism and a flexible rubber gasket. It contains the sample solution and ensures a complete separation of the sample from the liquid pressure medium. The sample solution is in contact with the inner wall of the prism forming the interface under study, where fluorescent molecules are immobilized. In this way, the new high pressure TIRF sample cell is very useful for studying any biomolecular layer that can be deposited at a planar water-silica interface. As examples, high pressure TIRF data of adsorbed lysozyme and two phospholipid membranes are presented. PMID:22938334

  20. Fluorescence Correlation Spectroscopy Analysis of Serotonin, Adrenergic, Muscarinic, and Dopamine Receptor Dimerization: The Oligomer Number Puzzle

    PubMed Central

    Grinde, Ellinor; Cowan, Ann; Mazurkiewicz, Joseph E.

    2013-01-01

    The issue of G protein–coupled receptor (GPCR) oligomer status has not been resolved. Although many studies have provided evidence in favor of receptor-receptor interactions, there is no consensus as to the exact oligomer size of class A GPCRs. Previous studies have reported monomers, dimers, tetramers, and higher-order oligomers. In the present study, this issue was examined using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH) analysis, a sensitive method for monitoring diffusion and oligomer size of plasma membrane proteins. Six different class A GPCRs were selected from the serotonin (5-HT2A), adrenergic (?1b-AR and ?2-AR), muscarinic (M1 and M2), and dopamine (D1) receptor families. Each GPCR was C-terminally labeled with green fluorescent protein (GFP) or yellow fluorescent protein (YFP) and expressed in human embryonic kidney 293 cells. FCS provided plasma membrane diffusion coefficients on the order of 7.5 × 10?9 cm2/s. PCH molecular brightness analysis was used to determine the GPCR oligomer size. Known monomeric (CD-86) and dimeric (CD-28) receptors with GFP and YFP tags were used as controls to determine the molecular brightness of monomers and dimers. PCH analysis of fluorescence-tagged GPCRs revealed molecular brightness values that were twice the monomeric controls and similar to the dimeric controls. Reduced ?2 analyses of the PCH data best fit a model for a homogeneous population of homodimers, without tetramers or higher-order oligomers. The homodimer configuration was unaltered by agonist treatment and was stable over a 10-fold range of receptor expression level. The results of this study demonstrate that biogenic amine receptors freely diffusing within the plasma membrane are predominantly homodimers. PMID:23907214

  1. Fluorescence spectroscopy: a powerful technique for the noninvasive characterization of artwork.

    PubMed

    Romani, Aldo; Clementi, Catia; Miliani, Costanza; Favaro, Gianna

    2010-06-15

    After electronic excitation by ultraviolet or visible radiation, atoms and molecules can undergo thermal or radiative deactivation processes before relaxing to the ground state. They can emit photons with longer wavelengths than the incoming exciting radiation, that is, they can fluoresce in the UV-vis-near-infrared (NIR) range. The study of fluorescence relaxation processes is one of the experimental bases on which modern theories of atomic and molecular structure are founded. Over the past few decades, technological improvements in both optics and electronics have greatly expanded fluorimetric applications, particularly in analytical fields, because of the high sensitivity and specificity afforded by the methods. Using fluorimetry in the study and conservation of cultural heritage is a recent innovation. In this Account, we briefly summarize the use of fluorescence-based techniques in examining the constituent materials of a work of art in a noninvasive manner. Many chemical components in artwork, especially those of an organic nature, are fluorescent materials, which can be reliably used for both diagnostic and conservative purposes. We begin by examining fluorimetry in the laboratory setting, considering the organic dyes and inorganic pigments that are commonly studied. For a number of reasons, works of art often cannot be moved into laboratories, so we continue with a discussion of portable instruments and a variety of successful "field applications" of fluorimetry to works of cultural heritage. These examples include studies of mural paintings, canvas paintings, tapestries, and parchments. We conclude by examining recent advances in treating the data that are generated in fluorescence studies. These new perspectives are focused on the spectral shape and lifetime of the emitted radiation. Recent developments have provided the opportunity to use various spectroscopic techniques on an increasing number of objects, as well as the ability to fully characterize very small amounts of sample, either in a laboratory setting or on site. Thus, a new technological highway is open to scientists; it is still difficult to navigate but offers an enormous potential for investigating objects without touching them. Fluorescence spectroscopy is one of the most important of these techniques. PMID:20415498

  2. A fiber-based single-unit dual-mode optical Imaging system: Swept source optical coherence tomography and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Ryu, Seon Young; Choi, Hae Young; Chang, Ki Soo; Kim, Geon Hee; Choi, Woo June; Ahn, Sang-Gun; Kim, Yong Chul; Yoon, Jung-Hoon; Lee, Byeong Ha

    2012-05-01

    We propose a fiber optic single-unit but dual-mode optical imaging system that can provide fast cross-sectional imaging capabilities of swept-source optical coherence tomography (SS-OCT) and functional capabilities of fluorescence spectroscopy (FS). By adopting a fiber optic FS system into a fiber-based SS-OCT system, a compact and effective multimodal single-unit SSOCT-FS system is achieved. Here, the key element of the proposed multimodal imaging system is a specially designed fiber coupler based on double-clad fiber (DCF), which has only cladding-mode coupling capability. The DCF couplers are fabricated with home-drawn DCF by several fabrication methods; a twisting method, a side-polishing method and a fused biconical tapered (FBT) method. Experimentally, the FBT method provides rather flat cladding mode coupling efficiency over 40% in a wide wavelength range. With this specially designed DCF coupler, the OCT signal and the fluorescence signal is measured independently but with a single-unit system. The performance of the SSOCT-FS system is confirmed by measuring the cross-sectional image and the fluorescence signal of a photosensitizer chlorin e6 injected in-vivo rat tumor model.

  3. In Vivo Confocal Fluorescence Imaging of the Intratumor Distribution of the Photosensitizer Mono-l-Aspartylchlorin-e61

    PubMed Central

    Mitra, Soumya; Foster, Thomas H

    2008-01-01

    We present an in vivo fluorescence microscopic evaluation of intratumor distribution of the photosensitizer mono-l-aspartylchlorin-e6 (NPe6) in an intradermal mouse EMT6 tumor model. Although the identification of favorable photophysical and pharmacological properties has led to the development of new photosensitizers in photodynamic therapy, their intratumor distribution kinetics have remained relatively understudied. In this study, we used confocal fluorescence microscopy to follow the transport of NPe6 in vivo after systemic administration through the tail vein. Labeling of vasculature using fluorophore-conjugated anti-CD31 antibodies allows visualization of the uptake of NPe6 in tumor and normal vessels and its partitioning kinetics into the adjacent parenchyma for 3 hours after injection. During the initial 60 minutes after injection, the drug is predominantly confined to the vasculature. Subsequently, it significantly redistributes throughout the extravascular regions with no discernable difference in its extravasation rate between tumor and normal tissues. Further, we investigate the sensitizer's altered intratumor distribution in response to photodynamic therapy irradiation and observe that treatment-induced changes in vessel permeability caused enhanced accumulation of NPe6 in the extravascular space. Our findings are of immediate clinical relevance and demonstrate the importance of an in vivo imaging approach to examine the dynamic process of intratumor drug distribution. PMID:18472960

  4. Noninvasive tissue fluorescence study of a fluorescent dye (calcein) on intraperitoneal glucose administration

    NASA Astrophysics Data System (ADS)

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

    1995-01-01

    The diagnostic exploitation of fluorescence spectroscopy and imaging has been largely used in experimental and clinical oncology. Only a few studies concern the ability of these techniques to study the in vivo behavior of fluorescent drugs or dyes. We have proposed recently a spectroscopic and imaging method to monitor pH in living tissues using pH-sensitive dyes. The tumor pH was depressed by previous glucose injection. We describe in this study the effect of glucose injection on the pharmacokinetic behavior of a fluorescent dye (calcein) by noninvasive fluorescence spectroscopy.

  5. In vivo cytochrome P450 drug metabolizing enzyme characterization using surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Li, Yanfang; Bachmann, Kenneth A.; Cameron, Brent D.

    2003-07-01

    The development of a rapid, inexpensive, and accurate in vivo phenotyping methodology for characterizing drug-metabolizing phenotypes with reference to the cytochrome P450 (CYP450) enzymes would be very beneficial. In terms of application, in the wake of the human genome project, considerable interest is focused on the development of new drugs whose uses will be tailored to specific genetic polymorphisms, and on the individualization of dosing regimens that are also tailored to meet individual patient needs depending upon genotype. In this investigation, chemical probes for CYP450 enzymes were characterized and identified with Raman spectroscopy. Furthermore, gold-based metal colloid clusters were utilized to generate surface enhanced Raman spectra for each of the chemical probes. Results will be presented demonstrating the ability of SERS to identify minute quantities of these probes on the order needed for in vivo application.

  6. In vivo proton magnetic resonance spectroscopy of breast cancer: a review of the literature

    PubMed Central

    2012-01-01

    An emerging clinical modality called proton magnetic resonance spectroscopy (1H-MRS) enables the non-invasive in vivo assessment of tissue metabolism and is demonstrating applications in improving the specificity of MR breast lesion diagnosis and monitoring tumour responsiveness to neoadjuvant chemotherapies. Variations in the concentration of choline-based cellular metabolites, detectable with 1H-MRS, have shown an association with malignant transformation of tissue in in vivo and in vitro studies. 1H-MRS exists as an adjunct to the current routine clinical breast MR examination. This review serves as an introduction to the field of breast 1H-MRS, discusses modern high-field strength and quantitative approaches and technical considerations, and reviews the literature with respect to the application of 1H-MRS for breast cancer. PMID:22515594

  7. Leaf water dynamics of Arabidopsis thaliana monitored in-vivo using terahertz time-domain spectroscopy

    PubMed Central

    Castro-Camus, E.; Palomar, M.; Covarrubias, A. A.

    2013-01-01

    The declining water availability for agriculture is becoming problematic for many countries. Therefore the study of plants under water restriction is acquiring extraordinary importance. Botanists currently follow the dehydration of plants comparing the fresh and dry weight of excised organs, or measuring their osmotic or water potentials; these are destructive methods inappropriate for in-vivo determination of plants' hydration dynamics. Water is opaque in the terahertz band, while dehydrated biological tissues are partially transparent. We used terahertz spectroscopy to study the water dynamics of Arabidopsis thaliana by comparing the dehydration kinetics of leaves from plants under well-irrigated and water deficit conditions. We also present measurements of the effect of dark-light cycles and abscisic acid on its water dynamics. The measurements we present provide a new perspective on the water dynamics of plants under different external stimuli and confirm that terahertz can be an excellent non-contact probe of in-vivo tissue hydration. PMID:24105302

  8. Fluorescence yield X-ray absorption spectroscopy for OK ? with elimination of NK ? background using superconducting tunnel junction detectors

    Microsoft Academic Search

    S. Shiki; M. Ukibe; Y. Kitajima; M. Ohkubo

    2011-01-01

    Fluorescent yield X-ray absorption fine structure (XAFS) spectroscopy is widely used for measuring chemical or physical states of particular elements in materials. A superconducting tunnel junction (STJ) detector is promising for X-ray absorption spectroscopy, especially in a soft X-ray region below 1keV, because of an excellent energy resolution of 10–20eV, which is crucial for resolving the characteristic X-ray lines from

  9. Evaluating fluorescence spectroscopy as a tool to characterize cyanobacteria intracellular organic matter upon simulated release and oxidation in natural water.

    PubMed

    Korak, Julie A; Wert, Eric C; Rosario-Ortiz, Fernando L

    2015-01-01

    Intracellular organic matter (IOM) from cyanobacteria may be released into natural waters following cell death in aquatic ecosystems and during oxidation processes in drinking water treatment plants. Fluorescence spectroscopy was evaluated to identify the presence of IOM from three cyanobacteria species during simulated release into natural water and following oxidation processes (i.e. ozone, free chlorine, chloramine, chlorine dioxide). Peak picking and the fluorescence index (FI) were explored to determine which IOM components (e.g., pigments) provide unique and persistent fluorescence signatures with minimal interferences from the background dissolved organic matter (DOM) found in Colorado River water (CRW). When IOM was added to ultrapure water, the fluorescence signature of the three cyanobacteria species showed similarities to each other. Each IOM exhibited a strong protein-like fluorescence and fluorescence at Ex 370 nm and Em 460 nm (FDOM), where commercial fluorescence sensors monitor. All species also had strong phycobiliprotein fluorescence (i.e. phycocyanin or phycoerythrin) in the higher excitation range (500-650 nm). All three IOM isolates had FI values greater than 2. When IOM was added to CRW, phycobiliprotein fluorescence was quenched through interactions between IOM and CRW-DOM. Mixing IOM and CRW demonstrated that protein-like and FDOM intensity responses were not a simple superposition of the starting material intensities, indicating that interactions between IOM and CRW-DOM fluorescing moieties were important. Fluorescence intensity in all regions decreased with exposure to ozone, free chlorine, and chlorine dioxide, but the FI still indicated compositional differences compared to CRW-DOM. The phycobiliproteins in IOM are not promising as a surrogate for IOM release, because their fluorescence intensity is quenched by interactions with DOM and decreased during oxidation processes. Increases in both FDOM intensity and FI are viable qualitative indicators of IOM release in natural waters and following oxidation and may provide a more robust real-time indication of the presence of IOM than conventional dissolved organic carbon or UV absorbance measurements. PMID:25462750

  10. In vivo pharmacokinetic analysis for fluorescently labeled RGD peptide targeted to the ?v?3 integrin in Kaposi"s sarcoma

    NASA Astrophysics Data System (ADS)

    Kwon, Sunkuk; Ke, Shi; Houston, Jessica P.; Wang, Wei; Wu, Qingping; Li, Chun; Sevick Muraca, Eva M.

    2005-04-01

    The dose dependence of near-infrared (NIR) fluorescent labeled RGD peptide targeted to the ?v?3 integrin was assessed from xenografts bearing a subcutaneous human Kaposi"s sarcoma (KS1767) with dynamic NIR fluorescence optical imaging. The three-compartment pharmacokinetic (PK) model was used to determine PK parameters from fluorescence images acquired with an intensified charge-coupled device (ICCD) system. Dynamic imaging of Kaposi"s sarcoma bearing animals was conducted with i.v. administration of Cy5.5-c(KRGDf) at doses of 0.75 to 6 nmol/animal and at the doses of 300 or 600 nmol of c(KRGDf) administered 1 hour before the injection of 3 nmol dose of the conjugate. The results show early and rapid uptake of Cy5.5-c(KRGDf), which was mediated by the administration of c(KRGDf) 1 hour before administration at the conjugate agent. From the results we found a linear increase in PK uptake rates at doses of 0.75 to 1.5 nmol, reflecting unsaturated binding to the integrin receptor. However, the results show the dose independence at large dose amounts from 3 to 6 nmol per animal. The effects of cancer treatments as well as diagnostics may be evaluated by in vivo PK analysis with NIR fluorescence optical imaging.

  11. Rapid proton fat-water spectroscopy for the characterization of non-CNS lesions in vivo.

    PubMed

    Riedy, Gerard

    2003-01-01

    The presence or absence of fat in lesions can have important diagnostic implications. Current MR techniques for the evaluation of fat within lesions in the body rely on indirect imaging methods. The goal of this study was to develop a rapid clinically practical proton spectroscopy procedure for the direct observation of a localized fat-water signal within the body. The technique developed reliably determined fat-water ratios in phantoms and from lesions in vivo in 6 s with single voxel sizes as small as 0.125 cc. PMID:12727049

  12. Characterization of human cervical remodeling throughout pregnancy using in vivo Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    O'Brien, Christine M.; Vargis, Elizabeth; Slaughter, Chris; Rudin, Amy P.; Herington, Jennifer L.; Bennett, Kelly A.; Reese, Jeff; Mahadevan-Jansen, Anita

    2015-02-01

    Globally, fifteen million babies are born preterm each year, affecting 1 in 8 pregnancies in the US alone. Cervical remodeling includes a biochemical cascade of changes that ultimately result in the thinning and dilation of the cervix for passage of a fetus. This process is poorly understood and is the focus of this study. Our group is utilizing Raman spectroscopy to evaluate biochemical changes occurring in the human cervix throughout pregnancy. This technique has high molecular specificity and can be performed in vivo, with the potential to unveil new molecular dynamics essential for cervical remodeling.

  13. Enzyme-Directed Assembly of Nanoparticles in Tumors Monitored by In Vivo Whole Animal and Ex Vivo Super Resolution Fluorescence Imaging

    PubMed Central

    Chien, Miao-Ping; Carlini, Andrea S.; Hu, Dehong; Barback, Christopher V.; Rush, Anthony M.; Hall, David J.; Orr, Galya; Gianneschi, Nathan C.

    2014-01-01

    Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block, and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo, and in ex vivo organ analysis following intratumoral injection into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micron-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls. PMID:24308273

  14. In vivo and in vitro characterization of ?70 constitutive promoters by real-time PCR and fluorescent measurements.

    PubMed

    Chappell, James; Freemont, Paul

    2013-01-01

    The characterization of DNA regulatory elements such as ribosome binding sites and transcriptional promoters is a fundamental aim of synthetic biology. Characterization of such DNA regulatory elements by monitoring the synthesis of fluorescent proteins is a commonly used technique to resolve the relative or absolute strengths. These measurements can be used in combination with mathematical models and computer simulation to rapidly assess performance of DNA regulatory elements both in isolation and in combination, to assist predictable and efficient engineering of complex novel biological devices and systems. Here we describe the construction and relative characterization of Escherichia coli (E. coli) ?(70) transcriptional promoters by monitoring the synthesis of green fluorescent protein (GFP) both in vivo in E. coli and in vitro in a E. coli cell-free transcription and translation reaction. PMID:23996440

  15. Laser Fluorescence Measurements Compared to Electrical Resistance of Residual Dentine in Excavated Cavities in vivo

    Microsoft Academic Search

    F. Krause; A. Braun; J. Eberhard; S. Jepsen

    2007-01-01

    It has been suggested that laser fluorescence close to the dental pulp shows higher values than more distant measurements. The aim of this study was to assess fluorescence on the cavity floor and to correlate these measurements with electrical resistance as a measure of residual dentine thickness. Thirty carious lesions were excavated with a bur. The endpoint of caries removal

  16. In Vivo Fluorescence of the Ocular Fundus Exhibits Retinal Pigment Epithelium Lipofuscin Characteristics

    Microsoft Academic Search

    Frangois C. Delori; C. Kathleen Dorey; Giovanni Staurenghi; Oliver Arend; Douglas G. Goger; John J. Writer

    1995-01-01

    Purpose. To characterize the intrinsic fluorescence (autofluorescence) of the human ocular fundus with regard to its excitation and emission spectra, age relationship, retinal location, and topography, and to identify the dominant fluorophore among the fundus layers. Methods. Using a novel fundus spectrophotometer, fluorescence measurements were made at 7° temporal to the fovea and at the fovea in 30 normal subjects

  17. Elucidating Structure and Function In Vivo With Hybrid Fluorescence and Magnetic Resonance Imaging

    Microsoft Academic Search

    Mark Niedre; Vasilis Ntziachristos

    2008-01-01

    While the mathematics, physics, and technology behind magnetic resonance (MR) and fluorescence image formation are distinctively different, the two modalities have significant complementary features to impart strong preclinical and clinical application synergies. Traditionally, hybrid MR and fluorescence imaging implied the use of a system where optical and MR signals can be concurrently acquired. In this case, the common geometry allows

  18. In vivo fluorescence spectra unmixing and autofluorescence removal by sparse Non-negative

    E-print Network

    Paris-Sud XI, Université de

    of targeting allows researchers to detect and localize cancer cells in patients. Once injected, markers. Mars2 Abstract--Fluorescence imaging locates fluorescent markers that specifically bind to targets markers detection, we suggest a new constrained NMF algorithm which takes sparsity constraints

  19. The Response of RIF-1 Fibrosarcomas to the Vascular-Disrupting Agent ZD6126 Assessed by In Vivo and Ex Vivo 1H Magnetic Resonance Spectroscopy1

    PubMed Central

    Madhu, Basetti; Waterton, John C; Griffiths, John R; Ryan, Anderson J; Robinson, Simon P

    2006-01-01

    Abstract The response of radiation-induced fibrosarcoma 1 (RIF-1) tumors treated with the vascular-disrupting agent (VDA) ZD6126 was assessed by in vivo and ex vivo 1H magnetic resonance spectroscopy (MRS) methods. Tumors treated with 200 mg/kg ZD6126 showed a significant reduction in total choline (tCho) in vivo 24 hours after treatment, whereas control tumors showed a significant increase in tCho. This response was investigated further within both ex vivo unprocessed tumor tissues and tumor tissue metabolite extracts. Ex vivo high-resolution magic angle spinning (HRMAS) and 1H MRS of metabolite extracts revealed a significant reduction in phosphocholine and glycerophosphocholine in biopsies of ZD6126-treated tumors, confirming in vivo tCho response. ZD6126-induced reduction in choline compounds is consistent with a reduction in cell membrane turnover associated with necrosis and cell death following disruption of the tumor vasculature. In vivo tumor tissue water diffusion and lactate measurements showed no significant changes in response to ZD6126. Spin-spin relaxation times (T2) of water and metabolites also remained unchanged. Noninvasive 1H MRS measurement of tCho in vivo provides a potential biomarker of tumor response to VDAs in RIF-1 tumors. PMID:16867218

  20. Cherenkov flashes and fluorescence flares on telescopes: New lights on UHECR spectroscopy while unveiling neutrinos astronomy

    NASA Astrophysics Data System (ADS)

    Fargion, D.; Oliva, P.; Massa, F.; Moreno, G.

    2008-04-01

    Multi-GeV and TeVs gamma sources are currently observed by their Cherenkov flashes on telescopes (as Magic, Hess and Veritas), looking vertically up into the sky. These detectors while pointing horizontally should also reveal the fluorescence flare tails of nearby down-going air-showers. Such air-showers, born at higher (tens of km) altitudes, are growing and extending up to lowest atmospheres (EeVs) or up to higher (few km) quotas (PeVs). These fluorescence signals extend the Cherenkov telescopes to a much higher cosmic ray spectroscopy. Vice versa, as it has been foreseen and only recently observed, the opposite takes place. Fluorescence telescopes made for UHECR detection (as AUGER ones) may be blazed by inclined Cherenkov lights: less energetic but frequent (PeVs) CR are expected to be often detected. Nearly dozens of blazing Cherenkov at EeV should be already found each year in AUGER, possibly in hybrid mode (FD SD, fluorescence and/or surface detector). Many more CR events (tens of thousands or hundreds of thousands) at PeVs energies should blaze Cherenkov lights each year on the AUGER fluorescence telescopes. Their UV filter may partially hide their signals and they cannot, unfortunately, be seen in any hybrid mode. At these comparable energies, the rarest UHE resonant anti-neutrino ?+e interactions in air at MW2/2me=6.3PeV energy offer enhanced W neutrino astronomy showering at air horizon, at ˜90, while crossing deep atmosphere column depth or Earth (Ande) boundaries. However, AUGER fluorescence detector (FD) are facing opposite way. An additional decay channel also rises (after resonant neutrino skimming Earth) via their secondary ? exit in air, by decay in flight via amplified showering: ?+e?W??+?. Moreover, expected horizontal UHE GZK neutrinos ??? at EeVs energy, powered by guaranteed cosmogenic GZK [K.Greisen, Phys. Rev. Lett. 16 (1966) 748; G.T. Zatsepin, V.A. Kuz’min, Zh. Eks. Teor. Fiz., Pis’ma Red. 4 (1966) 144], ??? flavor conversions (in cosmic distances), are also producing penetrating UHE EeV lepton taus that could sample, better and deeper than PeVs ones, the Earth skin. Such almost horizontal and up-going tau showers, originated by UHE astronomical neutrino, may shower and flash by fluorescence and/or Cherenkov diffused lights at AUGER sky in a few years (nearly three). Vice versa, at Hess, MAGIC and VERITAS horizons, at tens or a hundred km distances, the same up-going ??¯ air-showers might rise via fluorescence. On axis they might blaze (rarely) as a Cherenkov flashes below the horizons, possibly correlated to BL Lac or GRB activity. Also UHE (1 0.1 EeV) GZK ? showering can be observed upward once reflected onto clouds. The geomagnetic splitting may tag the energy as well as the inclined shower footprint as seen in a recent peculiar event in AUGER. Additional stereoscopic detection may define the event origination distance and its consequent primary composition, extending our understanding on UHECR composition.

  1. Impaired In Vivo Mitochondrial Krebs Cycle Activity After Myocardial Infarction Assessed Using Hyperpolarized Magnetic Resonance Spectroscopy

    PubMed Central

    Carr, Carolyn A.; Stuckey, Daniel J.; West, James A.; Griffin, Julian L.; Radda, George K.; Clarke, Kieran; Heather, Lisa C.; Tyler, Damian J.

    2015-01-01

    Background Myocardial infarction (MI) is one of the leading causes of heart failure. An increasing body of evidence links alterations in cardiac metabolism and mitochondrial function with the progression of heart disease. The aim of this work was to, therefore, follow the in vivo mitochondrial metabolic alterations caused by MI, thereby allowing a greater understanding of the interplay between metabolic and functional abnormalities. Methods and Results Using hyperpolarized carbon-13 (13C)-magnetic resonance spectroscopy, in vivo alterations in mitochondrial metabolism were assessed for 22 weeks after surgically induced MI with reperfusion in female Wister rats. One week after MI, there were no detectable alterations in in vivo cardiac mitochondrial metabolism over the range of ejection fractions observed (from 28% to 84%). At 6 weeks after MI, in vivo mitochondrial Krebs cycle activity was impaired, with decreased 13C-label flux into citrate, glutamate, and acetylcarnitine, which correlated with the degree of cardiac dysfunction. These changes were independent of alterations in pyruvate dehydrogenase flux. By 22 weeks, alterations were also seen in pyruvate dehydrogenase flux, which decreased at lower ejection fractions. These results were confirmed using in vitro analysis of enzyme activities and metabolomic profiles of key intermediates. Conclusions The in vivo decrease in Krebs cycle activity in the 6-week post-MI heart may represent an early maladaptive phase in the metabolic alterations after MI in which reductions in Krebs cycle activity precede a reduction in pyruvate dehydrogenase flux. Changes in mitochondrial metabolism in heart disease are progressive and proportional to the degree of cardiac impairment. PMID:25201905

  2. Analysis of photographs and photo-paintings by energy-dispersive X-ray fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Neiva, Augusto Camara; Marcondes, Marli A.; Pinto, Herbert Prince Favero; Almeida, Paula Aline Durães

    2014-02-01

    A collection of Brazilian family photographs and photo-paintings from the beginning of the XX Century was analyzed by portable EDXRF (Energy-Dispersive X-Ray Fluorescence) spectroscopy. The spectrometer uses a Si-drift Amptek detector and an Oxford Cr-tube or an Oxford W-tube. For every region under analysis, spectra obtained with the W-tube were used to detect all the elements above Al, while the Cr-tube was used to obtain more accurate results for elements between Al and V. Thirty nine elements were identified in the photos, and the origin of the most important ones was discussed. These results can be used for cataloging, preservation and restoring procedures.

  3. Fluorescence and polarization spectroscopy of single silicon vacancy centers in heteroepitaxial nanodiamonds on iridium

    NASA Astrophysics Data System (ADS)

    Neu, Elke; Fischer, Martin; Gsell, Stefan; Schreck, Matthias; Becher, Christoph

    2011-11-01

    We introduce an advanced material system for the production and spectroscopy of single silicon vacancy (SiV) color centers in diamond. We use microwave plasma chemical vapor deposition to synthesize heteroepitaxial nanodiamonds of approximately 160 nm in lateral size with a thickness of approximately 75 nm. These oriented “nanoislands” combine the enhanced fluorescence extraction from subwavelength-sized nanodiamonds with defined crystal orientation. The investigated SiV centers display narrow zero-phonon lines down to 0.7 nm in the wavelength range 730-750 nm. We investigate in detail the phonon coupling and vibronic sidebands of single SiV centers, revealing significant inhomogeneous effects. Polarization measurements reveal polarized luminescence and preferential absorption of linearly polarized light.

  4. A fluorescence lifetime spectroscopy study of matrix metalloproteinases -2 and -9 in human atherosclerotic plaque

    PubMed Central

    Phipps, Jennifer E.; Hatami, Nisa; Galis, Zorina S.; Baker, J. Dennis; Fishbein, Michael C.; Marcu, Laura

    2011-01-01

    Matrix metalloproteinase (MMP) -2 and -9 play important roles in the progression of atherosclerosis. This study aims to determine whether MMP-2 and -9 content in the fibrotic caps of atherosclerotic plaque is correlated with plaque autofluorescence. A time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) system was used to measure the autofluorescence and assess the biochemical composition of human plaques obtained from carotid endarterectomy. Results presented here demonstrate for the first time the ability to characterize the biochemical composition as it relates to MMP-2 and -9 content in the atherosclerotic plaque cap using a label-free imaging technique implemented with a fiberoptic TR-LIFS system. PMID:21770037

  5. Diffusion and segmental dynamics of rodlike molecules by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Winkler, Roland G.

    2007-08-01

    The dynamics of weakly bending polymers is analyzed on the basis of a Gaussian semiflexible chain model and the fluorescence correlation spectroscopy (FCS) correlation function is determined. Particular attention is paid to the influence of the rotational motion on the decay of the FCS correlation function. An analytical expression for the correlation function is derived, from which the averaged segmental mean square displacement can be determined independent of any specific model for the polymer dynamcis. The theoretical analysis exhibits a strong dependence of the correlation function on the rotational motion for semiflexible polymers with typical lengths and persistence lengths of actin filaments or fd viruses. Hence, FCS allows for a measurement of the rotational motion of such semiflexible polymers. The theoretical results agree well with experimental measurements on actin filaments and confirm the importance of large relaxation times.

  6. Probing the photoluminescence properties of gold nanoclusters by fluorescence lifetime correlation spectroscopy

    SciTech Connect

    Yuan, C. T., E-mail: ctyuan@cycu.edu.tw; Lin, T. N.; Shen, J. L. [Department of Physics, Chung Yuan Christian University, Chung-Li, Taiwan (China) [Department of Physics, Chung Yuan Christian University, Chung-Li, Taiwan (China); Center for Biomedical Technology, Chung Yuan Christian University, Taiwan (China); Lin, C. A.; Chang, W. H. [Center for Biomedical Technology, Chung Yuan Christian University, Taiwan (China) [Center for Biomedical Technology, Chung Yuan Christian University, Taiwan (China); Department of Biomedical Engineering, Chung Yuan Christian University, Taiwan (China); Cheng, H. W. [Department of Engineering and system science, National Tsing Hua University, Taiwan (China)] [Department of Engineering and system science, National Tsing Hua University, Taiwan (China); Tang, J., E-mail: jautang@gate.sinica.edu.tw [Research Center for Applied Sciences, Academia Sinica, Taiwan (China)

    2013-12-21

    Gold nanoclusters (Au NCs) have attracted much attention for promising applications in biological imaging owing to their tiny sizes and biocompatibility. So far, most efforts have been focused on the strategies for fabricating high-quality Au NCs and then characterized by conventional ensemble measurement. Here, a fusion single-molecule technique combining fluorescence correlation spectroscopy and time-correlated single-photon counting can be successfully applied to probe the photoluminescence (PL) properties for sparse Au NCs. In this case, the triplet-state dynamics and diffusion process can be observed simultaneously and the relevant time constants can be derived. This work provides a complementary insight into the PL mechanism at the molecular levels for Au NCs in solution.

  7. Fluorescence and polarization spectroscopy of single silicon vacancy centers in heteroepitaxial nanodiamonds on iridium

    E-print Network

    Neu, Elke; Gsell, Stefan; Schreck, Matthias; Becher, Christoph

    2011-01-01

    We introduce an advanced material system for the production and spectroscopy of single silicon vacancy (SiV) color centers in diamond. We use microwave plasma chemical vapor deposition to synthesize heteroepitaxial nanodiamonds of approx. 160 nm in lateral size with a thickness of approx. 75 nm. These oriented 'nanoislands' combine the enhanced fluorescence extraction from subwavelength sized nanodiamonds with defined crystal orientation. The investigated SiV centers display narrow zero-phonon-lines down to 0.7 nm in the wavelength range 730-750 nm. We investigate in detail the phonon-coupling and vibronic sidebands of single SiV centers, revealing significant inhomogeneous effects. Polarization measurements reveal polarized luminescence and preferential absorption of linearly polarized light.

  8. Fluorescence and polarization spectroscopy of single silicon vacancy centers in heteroepitaxial nanodiamonds on iridium

    E-print Network

    Elke Neu; Martin Fischer; Stefan Gsell; Matthias Schreck; Christoph Becher

    2011-10-24

    We introduce an advanced material system for the production and spectroscopy of single silicon vacancy (SiV) color centers in diamond. We use microwave plasma chemical vapor deposition to synthesize heteroepitaxial nanodiamonds of approx. 160 nm in lateral size with a thickness of approx. 75 nm. These oriented 'nanoislands' combine the enhanced fluorescence extraction from subwavelength sized nanodiamonds with defined crystal orientation. The investigated SiV centers display narrow zero-phonon-lines down to 0.7 nm in the wavelength range 730-750 nm. We investigate in detail the phonon-coupling and vibronic sidebands of single SiV centers, revealing significant inhomogeneous effects. Polarization measurements reveal polarized luminescence and preferential absorption of linearly polarized light.

  9. Photosystem II antenna phosphorylation-dependent protein diffusion determined by fluorescence correlation spectroscopy

    PubMed Central

    Iwai, Masakazu; Pack, Chan-Gi; Takenaka, Yoshiko; Sako, Yasushi; Nakano, Akihiko

    2013-01-01

    Flexibility of chloroplast thylakoid membrane proteins is essential for plant fitness and survival under fluctuating light environments. Phosphorylation of light-harvesting antenna complex II (LHCII) is known to induce dynamic protein reorganization that fine-tunes the rate of energy conversion in each photosystem. However, molecular details of how LHCII phosphorylation causes light energy redistribution throughout thylakoid membranes still remain unclear. By using fluorescence correlation spectroscopy, we here determined the LHCII phosphorylation-dependent protein diffusion in thylakoid membranes isolated from the green alga Chlamydomonas reinhardtii. As compared to the LHCII dephosphorylation-induced condition, the diffusion coefficient of LHCII increased nearly twofold under the LHCII phosphorylation-induced condition. We also verified the results by using the LHCII phosphorylation-deficient mutant. Our observation suggests that LHCII phosphorylation-dependent protein reorganization occurs along with the changes in the rate of protein diffusion, which would have an important role in mediating light energy redistribution throughout thylakoid membranes. PMID:24088948

  10. Oxidatively modified low-density lipoprotein in mononuclear cells detected by laser-induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Glenn, Tami N.; Oraevsky, Alexander A.; Tittel, Frank K.; Thomsen, Sharon L.; Jacques, Steven L.; Henry, Philip D.

    1995-05-01

    Hyperlipidemic states are associated with focal accumulations in arterial walls of oxidatively modified low density lipoprotein (LDL) and monocyte-derived liquid-laden macrophages, biochemical and cellular hallmarks of atheromatous lesions. Mechanisms underlying the generation and cellular uptake of oxidized LDL are still incompletely understood. We have used laser-induced fluorescence spectroscopy to monitor the formation, intracellular accumulation, and tissue distribution of oxidized LDL. Oxidatively modified LDL excited by a XeCl excimer laser (308 nm) exhibits unique spectral characteristics that distinguish it from native (non-oxidized) LDL. The same spectral characteristics were demonstrated in lipid-rich atheromatous lesions, macrophages after incubation with oxidized LDL, and peripheral blood monocytes from hyperlipidemic, but not normolipidemic subjects. Detection of oxidized LDL in peripheral blood monocytes and arterial tissue may provide information on the atherogenic activity of hyperlipidemic states and serve as a novel risk factor for the assessment of atherosclerosis.

  11. Experimental study and verification of the residence time distribution using fluorescence spectroscopy and color measurement

    NASA Astrophysics Data System (ADS)

    Aigner, Michael; Lepschi, Alexander; Aigner, Jakob; Garmendia, Izaro; Miethlinger, Jürgen

    2015-05-01

    We report on the inline measurement of residence time (RT) and residence time distribution (RTD) by means of fluorescence spectroscopy [1] and optical color measurements [2]. Measurements of thermoplastics in a variety of single-screw extruders were conducted. To assess the influence of screw configurations, screw speeds and mass throughput on the RT and RTD, tracer particles were introduced into the feeding section and the RT was measured inline in the plasticization unit. Using special measurement probes that can be inserted into 1/2? - 20 UNF (unified fine thread) bore holes, the mixing ability of either the whole plasticization unit or selected screw regions, e.g., mixing parts, can be validated during the extrusion process. The measurement setups complement each other well, and their combined use can provide further insights into the mixing behavior of single-screw plasticization units.

  12. A sensitive and microscale method for drug screening combining affinity probes and single molecule fluorescence correlation spectroscopy.

    PubMed

    Ruan, Lingao; Su, Di; Shao, Chang; Wang, Jinjie; Dong, Chaoqing; Huang, Xiangyi; Ren, Jicun

    2015-02-21

    In this paper, a sensitive and microscale method for drug screening is described using single molecule spectroscopy fluorescence correlation spectroscopy (FCS). The principle of this method is mainly based on the competition of candidate drugs to the fluorescent probe-target complexes and the excellent capacity of FCS for sensitively distinguishing the free fluorescent probes and the fluorescent probe-target complexes in solution. In this study, the screening of protein kinase inhibitors was used as a model, tyrosine-protein kinase ABL1 was used as a target and a known inhibitor dasatinib derivative labeled with a fluorescent dye was used as a fluorescent affinity probe. We firstly established the theoretical model of drug screening based on the binding process of fluorescent probes and targets, the competition of candidate drugs to the fluorescent probe-target complexes and FCS theory. Then, the dasatinib derivatives were synthesized and labeled with the fluorescent dye Alexa 488, and the binding and dissociation processes of Alexa 488-dasatinib and ABL1 were systematically investigated. The dissociation constant and the dissociation rate for the Alexa 488-dasatinib-ABL1 complex were determined. Finally, the established method was used to screen candidate drugs. The dissociation constants of ABL1 kinase to six known drugs for treating chronic myeloid leukemia (CML) were evaluated and the results obtained are well consistent with the reported values. Furthermore, a homemade chip with micro-wells was successfully utilized in FCS measurements as the carrier of samples, and the sample requirements were only 1-2 ?L in this case. Our results demonstrated that the drug screening method described here is universal, sensitive and shows small sample and reagent quantity requirements. We believe that this method will become a high throughput platform for screening of small molecule drugs. PMID:25526365

  13. An electrochemical enrichment procedure for the determination of heavy metals by total-reflection X-ray fluorescence spectroscopy

    Microsoft Academic Search

    A. Ritschel; P Wobrauschek; E Chinea; F Grass; Ch Fabjan

    1999-01-01

    An electrolytic separation and enrichment technique was developed for the determination of trace elements by total-reflection X-ray fluorescence spectroscopy (TXRF). The elements of interest are electrodeposited out of the sample solution onto a solid, polished disc of pure niobium which is used as sample carrier for the TXRF measurement. The electrochemical deposition leads to a high enrichment of the analytes

  14. Characterization of DNA immobilization and subsequent hybridization using in situ quartz crystal microbalance, fluorescence spectroscopy, and surface plasmon resonance

    Microsoft Academic Search

    Yoon-Kyoung Cho; Sunhee Kim; Young A Kim; Hee Kyun Lim; Kyusang Lee; DaeSung Yoon; Geunbae Lim; Y. Eugene Pak; Tai Hwan Ha; Kwan Kim

    2004-01-01

    We have characterized the immobilization of thiol-modified oligomers on Au surfaces and subsequent hybridization with a perfectly matched or single-base mismatched target using a quartz crystal microbalance (QCM) and fluorescence spectroscopy. The surface density of immobilized probe molecules and the hybridization efficiency depending on the type of buffer and salt concentration were investigated. We observed some ambiguities in surface coverage

  15. Detection of Pharmaceutical and Personal Care Products (PPCPs) in 3 Maine Lakes by Synchronous-Scan Fluorescence Spectroscopy (SFS)

    Microsoft Academic Search

    Anne Marie Lausier

    2009-01-01

    The occurrence of pharmaceutical and personal care products (PPCP's) and their byproducts in the environment is of growing concern due to their potential harmful effects on environmental and human health. Synchronous-scan fluorescence spectroscopy (SFS) was used to detect caffeine, 17?-ethynylestradiol, and the PPCP additive Triclosan levels in Pushaw Lake, Branch Lake, and Sebasticook Lake. These lakes are three heavily populated

  16. Excitation energy dependence of excited states dynamics in all- trans-carotenes determined by femtosecond absorption and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Kosumi, Daisuke; Yanagi, Kazuhiro; Nishio, Tomohiro; Hashimoto, Hideki; Yoshizawa, Masayuki

    2005-06-01

    Ultrafast relaxation kinetics in ?-carotene and lycopene has been investigated by femtosecond absorption and fluorescence spectroscopies using tunable excitation pulses. The transient signals induced by the photoexcitation with larger excess energy have broader bands and longer lifetimes both in the 11Bu+and21Ag- excited states. The excess vibrational energy remains longer than several picoseconds and slows the relaxation kinetics in carotenoids.

  17. Fluorescence spectroscopy of single molecules at room temperature and its applications

    SciTech Connect

    Ha, Taekjip

    1996-12-01

    We performed fluorescence spectroscopy of single and pairs of dye molecules on a surface at room temperature. Near field scanning optical microscope (NSOM) and far field scanning optical microscope with multi-color excitation/detection capability were built. The instrument is capable of optical imaging with 100nm resolution and has the sensitivity necessary for single molecule detection. A variety of dynamic events which cannot be observed from an ensemble of molecules is revealed when the molecules are probed one at a time. They include (1) spectral jumps correlated with dark states, (2) individually resolved quantum jumps to and from the meta-stable triplet state, (3) rotational jumps due to desorption/readsorption events of single molecules on the surface. For these studies, a computer controlled optical system which automatically and rapidly locates and performs spectroscopic measurements on single molecules was developed. We also studied the interaction between closely spaced pairs of molecules. In particular, fluorescence resonance energy transfer between a single resonant pair of donor and acceptor molecules was measured. Photodestruction dynamics of the donor or acceptor were used to determine the presence and efficiency of energy transfer Dual molecule spectroscopy was extended to a non-resonant pair of molecules to obtain high resolution differential distance information. By combining NSOM and dual color scheme, we studied the co-localization of parasite proteins and host proteins on a human red blood cell membrane infected with malaria. These dual-molecule techniques can be used to measure distances, relative orientations, and changes in distances/orientations of biological macromolecules with very good spatial, angular and temporal resolutions, hence opening new capabilities in the study of such systems.

  18. Planetary Surface Analysis Using Fast Laser Spectroscopic Techniques: Combined Microscopic Raman, LIBS, and Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Blacksberg, J.; Rossman, G. R.; Maruyama, Y.; Charbon, E.

    2011-12-01

    In situ exploration of planetary surfaces has to date required multiple techniques that, when used together, yield important information about their formation histories and evolution. We present a time-resolved laser spectroscopic technique that could potentially collect complementary sets of data providing information on mineral structure, composition, and hydration state. Using a picosecond-scale pulsed laser and a fast time-resolved detector we can simultaneously collect spectra from Raman, Laser Induced Breakdown Spectroscopy (LIBS), and fluorescence emissions that are separated in time due to the unique decay times of each process. The use of a laser with high rep rate (40 KHz) and low pulse energy (1 ?J/pulse) allows us to rapidly collect high signal to noise Raman spectra while minimizing sample damage. Increasing the pulse energy by about an order of magnitude creates a microscopic plasma near the surface and enables the collection of LIBS spectra at an unusually high rep rate and low pulse energy. Simultaneously, broader fluorescence peaks can be detected with lifetimes varying from nanosecond to microsecond. We will present Raman, LIBS, and fluorescence spectra obtained on natural mineral samples such as sulfates, clays, pyroxenes and carbonates that are of interest for Mars mineralogy. We demonstrate this technique using a photocathode-based streak camera detector as well as a newly-developed solid state Single Photon Avalanche Diode (SPAD) sensor array based on Complementary Metal-Oxide Semiconductor (CMOS) technology. We will discuss the impact of system design and detector choice on science return of a potential planetary surface mission, with a specific focus on size, weight, power, and complexity. The research described here was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration (NASA).

  19. High resolution x-ray fluorescence spectroscopy - a new technique for site- and spin-selectivity

    SciTech Connect

    Wang, Xin [Univ. of California, Davis, CA (United States). Dept. of Applied Science

    1996-12-01

    X-ray spectroscopy has long been used to elucidate electronic and structural information of molecules. One of the weaknesses of x-ray absorption is its sensitivity to all of the atoms of a particular element in a sample. Through out this thesis, a new technique for enhancing the site- and spin-selectivity of the x-ray absorption has been developed. By high resolution fluorescence detection, the chemical sensitivity of K emission spectra can be used to identify oxidation and spin states; it can also be used to facilitate site-selective X-ray Absorption Near Edge Structure (XANES) and site-selective Extended X-ray Absorption Fine Structure (EXAFS). The spin polarization in K fluorescence could be used to generate spin selective XANES or spin-polarized EXAFS, which provides a new measure of the spin density, or the nature of magnetic neighboring atoms. Finally, dramatic line-sharpening effects by the combination of absorption and emission processes allow observation of structure that is normally unobservable. All these unique characters can enormously simplify a complex x-ray spectrum. Applications of this novel technique have generated information from various transition-metal model compounds to metalloproteins. The absorption and emission spectra by high resolution fluorescence detection are interdependent. The ligand field multiplet model has been used for the analysis of K{alpha} and K{beta} emission spectra. First demonstration on different chemical states of Fe compounds has shown the applicability of site selectivity and spin polarization. Different interatomic distances of the same element in different chemical forms have been detected using site-selective EXAFS.

  20. Recombinant phytochrome of the moss Ceratodon purpureus (CP2): fluorescence spectroscopy and photochemistry.

    PubMed

    Sineshchekov, V; Koppel, L; Hughes, J; Lamparter, T; Zeidler, M

    2000-07-01

    The recombinant phytochrome of the moss Ceratodon purpureus (CP2) expressed in Saccharomyces cerevisiae and reconstituted with phycocyanobilin (PCB) was investigated using fluorescence spectroscopy. The pigment had an emission maximum at 670 nm at low temperature (85 K) and at 667 nm at room temperature (RT) and an excitation maximum at 650-652 nm at 85 K (excitation spectra could not be measured at RT). Both spectra had a half-band width of approx. 30-35 nm at 85 K. The fluorescence intensity revealed a steep temperature dependence with an activation energy of fluorescence decay (Ea) of 5.9-6.4 and 12.6-14.7 kJ mol(-1) in the interval from 85 to 210 K and from 210 to 275 K, respectively. The photochemical properties of CP2/PCB were characterised by the extent of the red-induced (lambda(a) = 639 nm) Pr conversion into the first photoproduct lumi-R at 85 K (gamma1) of approximately 0.07 and into Pfr at RT (gamma2) of approximately 0.7. From these characteristics, CP2/PCB can be attributed to the Pr" photochemical type with gamma1 < or = 0.05, which comprises the minor phyA fraction (phyA"), phyB, Adiantum phy1 and Synechocystis Cph1 in contrast to the major phyA' fraction (Pr' type with gamma1 = 0.5). Within the Pr" type, it is closer to phyA" than to phyB and Cph1. PMID:11079475

  1. Fluorescence spectroscopy of graphene quantum dots: temperature effect at different excitation wavelengths.

    PubMed

    Li, Changzheng; Yue, Yanan

    2014-10-31

    This paper reports a comprehensive study of temperature dependence of fluorescence spectroscopy of graphene quantum dots at different excitation wavelengths. Very significant (more than 50%) and similar decrease of normalized spectrum intensity is observed within temperature range less than 80 °C for excitation wavelengths of 310 nm, 340 nm and 365 nm. Besides, the temperature dependence of the red-shift of spectrum peak shows different wavelength dependence characteristic with coefficient as high as 0.062 nm K(-1) for the same temperature range, which gives us a hint about selecting the right excitation wavelength by compromising the excitation efficiency for fluorescence intensity and the temperature coefficient for peak shift in thermal applications. Temperature dependence of peak width is in a weakly linear relationship with a coefficient of 0.026 nm K(-1). Regarding the excellent stability and reversibility during thermal measurement, graphene quantum dot is a good candidate for the implementation in the nanoscale thermometry, especially in the bio-thermal field considering its superior biocompatibility and low cytotoxicity. PMID:25299977

  2. Fluorescence spectroscopy of graphene quantum dots: temperature effect at different excitation wavelengths

    NASA Astrophysics Data System (ADS)

    Li, Changzheng; Yue, Yanan

    2014-10-01

    This paper reports a comprehensive study of temperature dependence of fluorescence spectroscopy of graphene quantum dots at different excitation wavelengths. Very significant (more than 50%) and similar decrease of normalized spectrum intensity is observed within temperature range less than 80 °C for excitation wavelengths of 310 nm, 340 nm and 365 nm. Besides, the temperature dependence of the red-shift of spectrum peak shows different wavelength dependence characteristic with coefficient as high as 0.062 nm K?1 for the same temperature range, which gives us a hint about selecting the right excitation wavelength by compromising the excitation efficiency for fluorescence intensity and the temperature coefficient for peak shift in thermal applications. Temperature dependence of peak width is in a weakly linear relationship with a coefficient of 0.026 nm K?1. Regarding the excellent stability and reversibility during thermal measurement, graphene quantum dot is a good candidate for the implementation in the nanoscale thermometry, especially in the bio-thermal field considering its superior biocompatibility and low cytotoxicity.

  3. Correcting for Spectral Cross-Talk in Dual-Color Fluorescence Cross-Correlation Spectroscopy

    PubMed Central

    Bacia, Kirsten; Petrášek, Zden?k; Schwille, Petra

    2012-01-01

    Dual-color fluorescence cross-correlation spectroscopy (dcFCCS) allows one to quantitatively assess the interactions of mobile molecules labeled with distinct fluorophores. The technique is widely applied to both reconstituted and live-cell biological systems. A major drawback of dcFCCS is the risk of an artifactual false-positive or overestimated cross-correlation amplitude arising from spectral cross-talk. Cross-talk can be reduced or prevented by fast alternating excitation, but the technology is not easily implemented in standard commercial setups. An experimental strategy is devised that does not require specialized hardware and software for recognizing and correcting for cross-talk in standard dcFCCS. The dependence of the cross-talk on particle concentrations and brightnesses is quantitatively confirmed. Moreover, it is straightforward to quantitatively correct for cross-talk using quickly accessible parameters, that is, the measured (apparent) fluorescence count rates and correlation amplitudes. Only the bleed-through ratio needs to be determined in a calibration measurement. Finally, the limitations of cross-talk correction and its influence on experimental error are explored. PMID:22344749

  4. Detection and characterization of stomach cancer and atrophic gastritis with fluorescence and Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Li, Xiaozhou; Lin, Junxiu; Jia, Chunde; Wang, Rong

    2003-12-01

    In this paper, we attempt to find a valid method to distinguish gastric cancer and atrophic gastritis. Auto-fluorescence and Raman spectroscopy of laser induced (514.5 nm and 488.0 nm) was measured. The serum spectrum is different between normal and cancer. Average value of diagnosis parameter for normal serum, red shift is less than 12 nm and Raman relative intensity of peak C by 514.5 nm excited is stronger than that of 488.0 nm. To gastric cancer, its red shift of average is bigger than 12 nm and relative intensity of Raman peak C by 514.5 nm excited is weaker than that by 488.0 nm. To atrophic gastritis, the distribution state of Raman peaks is similar with normal serum and auto-fluorescence spectrum's shape is similar to that of gastric cancer. Its average Raman peak red shift is bigger than 12 nm and the relative intensity of peak C by 514.5 excited is stronger than that of by 488.0. We considered it as a criterion and got an accuracy of 85.6% for diagnosis of gastric cancer compared with the result of clinical diagnosis.

  5. Fluorescence correlation spectroscopy to measure the metabolism of high-density lipoprotein

    NASA Astrophysics Data System (ADS)

    Deitrick, Russell; Gibson, Emily; Razzaghi, Hamid

    2009-10-01

    High-density lipoprotein (HDL), referred to as the ``good cholesterol'', carries free cholesterol to the liver to be filtered from the bloodstream and is important to our understanding of atherosclerosis. HDL is metabolized in part by the enzyme Endothelial Lipase (EL). With this project we will use fluorescence correlation spectroscopy (FCS) to study the metabolism of HDL by EL comparing wild type with different genetic mutations. FCS is an advanced microscopy technique in which we record fluctuations in the fluorescence of dye-labeled molecules (in this case, HDL labeled with Nile Red) as they freely diffuse through a small focal volume. This data can be analyzed mathematically using the cross-correlation function, from which we can ultimately ascertain much information. In our case, we are interested in the diffusion coefficient which, via the Stokes-Einstein relation for a sphere, we can determine the size of HDL as it undergoes the process of metabolism. Preliminary results seem to indicate that the metabolic process occurs very quickly, that the final size of HDL depends primarily on the concentration of EL, and that the wild and mutant variants of EL have a similar effectiveness. In following experiments, we hope to investigate these relationships further.

  6. Distance measurements near the myosin head-rod junction using fluorescence spectroscopy.

    PubMed Central

    Kekic, M; Huang, W; Moens, P D; Hambly, B D; dos Remedios, C G

    1996-01-01

    We reacted a fluorescent probe, N-methyl-2-anilino-6-naphthalenesulfonyl chloride (MNS-Ci), with a specific lysine residue of porcine cardiac myosin located in the S-2 region of myosin. We performed fluorescence resonance energy transfer (FRET) spectroscopy measurements between this site and three loci (Cys109, Cys125, and Cys154) located within different myosin light-chain 2s (LC2) bound to the myosin "head". We used LC2s from rabbit skeletal muscle myosin (Cys125), chicken gizzard smooth muscle myosin (Cys109), or a genetically engineered mutant of chicken skeletal muscle myosin (Cys154). The atomic coordinates of these LC2 loci can be closely approximated, and the FRET measurements were used to determine the position of the MNS-labeled lysine with respect to the myosin head. The C-terminus of myosin subfragment-1 determined by Rayment et al. ends abruptly after a sharp turn of its predominantly alpha-helical structure. We have constructed a model based on our FRET distance data combined with the known structure of chicken skeletal muscle myosin subfragment-1. This model suggests that the loci that bracket the head-rod junction will be useful for evaluating dynamic changes in this region. Images FIGURE 4 FIGURE 5 PMID:8804587

  7. Fluorescence excitation-emission spectroscopy with regional integration analysis for assessment of compost maturity.

    PubMed

    Yu, Guang-Hui; Wu, Min-Jie; Luo, Yi-Hong; Yang, Xing-Ming; Ran, Wei; Shen, Qi-Rong

    2011-08-01

    Composting of animal manures is believed as an alternative way for directly recycling them in farms, and therefore assessment of compost maturity is crucial for achieving high quality compost. Fluorescence excitation-emission matrices (EEMs) combined with regional integration analysis is presented to assess compost maturity. The results showed that the EEM contours of water-extract organic matter (WEOM) from immature composts exhibited four peaks at excitation/emission (Ex/Em) of 220/340nm, 280/340nm, 220/410nm, and 330/410nm, whereas EEM contour of WEOM from mature composts had only two peaks at Ex/Em of 230/420nm and 330/420nm. Pearson correlation demonstrated that peaks intensity rather than their ratios had a significantly correlation with the common indices assessing compost maturity, whereas the normalized excitation-emission area volumes (?(i,n)s) from regional integration analysis had a stronger correlation with the common indices assessing compost maturity than peaks intensity. It is concluded that the ?(i,n)s from regional integration analysis are more suitable to assess the maturity of compost than the intensities of peaks. Therefore, the fluorescence spectroscopy combined with regional integration analysis can be used as a valuable industrial and research tool for assessing compost maturity, given its high sensitivity and selectivity. PMID:21546234

  8. Fluorescence and Circular Dichroism Spectroscopy of Cytochrome c in Alkylammonium Formate Ionic Liquids

    PubMed Central

    Wei, Wenjun; Danielson, Neil D.

    2012-01-01

    The structural stability of cytochrome c has been studied in alkylammonium formate (AAF) ionic liquids such as methylammonium formate (MAF) and ethylammonium formate (EAF) by fluorescence and circular dichroism (CD) spectroscopy. At room temperature, the native structure of cytochrome c is maintained in relatively high ionic liquid concentrations (50%–70% AAF/water or AAF/phosphate buffer pH 7.0) in contrast to denaturation of cytochrome c in similar solutions of methanol or acetonitrile, with water or buffer co-solvents. Fluorescence and CD spectra indicate the conformation of cytochrome c is maintained in 20% AAF-80% water from 30 – 50 °C. No such temperature stability is found in 80% AAF-20% water. About one third of the enzyme activity of cytochrome c in 80% AAF-20% water can be maintained as compared to phosphate buffer and this is greater than the activities measured in corresponding methanol and acetonitrile aqueous solutions. This biophysical study shows that AAFs have potential application as organic solvent replacements at moderate temperature in the mobile phase for the separation of proteins in their native form by reversed phase liquid chromatography. PMID:21210672

  9. Light adaptation of the unicellular red alga, Cyanidioschyzon merolae, probed by time-resolved fluorescence spectroscopy.

    PubMed

    Ueno, Yoshifumi; Aikawa, Shimpei; Kondo, Akihiko; Akimoto, Seiji

    2015-08-01

    Photosynthetic organisms change the quantity and/or quality of their pigment-protein complexes and the interactions among these complexes in response to light conditions. In the present study, we analyzed light adaptation of the unicellular red alga Cyanidioschyzon merolae, whose pigment composition is similar to that of cyanobacteria because its phycobilisomes (PBS) lack phycoerythrin. C. merolae were grown under different light qualities, and their responses were measured by steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopies. Cells were cultivated under four monochromatic light-emitting diodes (blue, green, yellow, and red), and changes in pigment composition and energy transfer were observed. Cells grown under blue and green light increased their relative phycocyanin levels compared with cells cultured under white light. Energy-transfer processes to photosystem I (PSI) were sensitive to yellow and red light. The contribution of direct energy transfer from PBS to PSI increased only under yellow light, while red light induced a reduction in energy transfer from photosystem II to PSI and an increase in energy transfer from light-harvesting chlorophyll protein complex I to PSI. Differences in pigment composition, growth, and energy transfer under different light qualities are discussed. PMID:25577254

  10. Characterization of self-assembling copolymers in aqueous solutions using Electron Paramagnetic Resonance and Fluorescence spectroscopy.

    PubMed

    Beghein, N; Rouxhet, L; Dinguizli, M; Brewster, M E; Ariën, A; Préat, V; Habib, J L; Gallez, B

    2007-02-12

    Electron Paramagnetic Resonance and fluorescence spectroscopy have been used to determine the micropolarity and microviscosity of self-assembling systems based on mmePEG-p(CL-co-TMC) having different PEG chain lengths and different CL/TMC ratios and PEG/MOG/SA (45/5/50) polymers with different PEG chain lengths. Four reporter probes have been used: two spin probes, 16-doxyl stearic acid and 5-doxylstearic acid, and two fluorescent probes, pyrene and 1,3-bis(1-pyrenyl) propane (P3P). We found that the micelles based on mmePEG-p(CL-co-TMC) polymers are of a biphasic nature. The micelles are made of a hydrophilic corona with low viscosity while the core of the micelle is more hydrophobic and more viscous. The outer shell is made up of PEG chains, the hydrophobic part of the chains making the core. The partial hydration of the shell seems to lead to a looser chain network than that associated with deeper domains in the micelles. By contrast, in micelles composed of PEG/MOG/SA, there is no clear domain separation. This is consistent with a spatial configuration of random polymeric chains forming a loose network. In these micelles, the microviscosity is low and the hydrophobicity is high. PMID:17196699

  11. THE USE OF FLUORESCENCE CORRELATION SPECTROSCOPY TO PROBE CHROMATIN IN THE CELL NUCLEUS

    SciTech Connect

    Sorscher, Stanley M.; Bartholemew, James C.; Klein, Melvin P.

    1980-03-01

    All systems in thermodynamic equilibrium are subject to spontaneous fluctuations from equilibrium. For very small systems, the fluctuations can be made apparent, and can be used to study the behavior of the system without introducing any external perturbations. The mean squared amplitude of these fluctuations contains information about the absolute size of the system. The characteristic time of the fluctuation autocorrelation function contains kinetic information. In the experiments reported here, these concepts are applied to the binding equilibrium between ethidium bromide and DNA, a system where the fluorescence properties of the dye greatly enhance the effect of spontaneous fluctuations in the binding equilibrium. Preliminary experiments employ well characterized DNA preparations, including calf thymus DNA, SV40 DNA, and calf thymus nucleohistone particles. Additional measurements are described which have been made in small regions of individual nuclei, isolated from green monkey kidney cells, observing as few as 5000 dye molecules. The data indicate that the strength of dye binding increases in nuclei isolated from cells which have been stimulated to enter the cell growth cycle. The viscosity of nuclear material is inferred to be between one and two orders of magnitude greater than that of water, and decreases as the cells leave the resting state, and enter the cell growth cycle. Washing the nuclei also lowers the viscosity. These experiments demonstrate that fluorescence correlation spectroscopy can provide information at the subnuclear level that is otherwise unavailable.

  12. Dynamics of water-in-oil nanoemulsions revealed by fluorescence lifetime correlation spectroscopy.

    PubMed

    Orte, Angel; Ruedas-Rama, Maria J; Paredes, Jose M; Crovetto, Luis; Alvarez-Pez, Jose M

    2011-11-01

    The size, diffusional properties, and dynamics of reverse water-in-oil nanoemulsions, or reverse micelles (RMs), have been widely investigated because of interest in this system as a model for biological compartmentalization. Here, we have employed fluorescence lifetime correlation spectroscopy (FLCS) to reveal the dynamics and sizes of aerosol-OT (AOT)/isooctane RMs using a fluorescent xanthene derivative called Tokyo Green II (TG-II). The dye undergoes a partition and a shift in its tautomeric equilibrium such that the TG-II anion remains in the inner micellar aqueous core, and the neutral quinoid form lies in the interfacial region. By applying FLCS, we specifically obtained the lifetime filtered autocorrelation curves of the anionic TG-II, which shows a characteristic lifetime of approximately 4 ns. Analysis of the FLCS curves provides the diffusion coefficient and hydrodynamic radius of the RMs as well as micelle dynamics in the same experiment. The FLCS curves show dynamics in the microsecond time range, which represents an interconversion rate that changes the distribution of the TG-II neutral and anionic forms in the hydrophobic interface and the water core. PMID:21913723

  13. Fluorescence correlation spectroscopy to study antibody binding and stoichiometry of complexes

    NASA Astrophysics Data System (ADS)

    Swift, Kerry M.; Matayoshi, Edmund D.

    2008-02-01

    FCS (fluorescence correlation spectroscopy) was used to study the association at the single molecule level of tumor necrosis factor alpha (TNF-?) and two of its protein antagonists Humira (TM) (adalimumab), a fully humanized monoclonal antibody, and Enbrel (TM) (etanercept), a soluble form of the TNF receptor. Single molecule approaches potentially have the advantage not only of enhanced sensitivity, but also of observing at equilibrium the details that would otherwise be lost in classical ensemble experiments where heterogeneity is averaged. We prepared fluorescent conjugates of the protein drugs and their biological target, the trimeric soluble form of TNF-?. The bivalency of adalimumab and the trimeric nature of TNF-? potentially allow several forms of associative complexes that may differ in stoichiometry. Detailed knowledge of this reaction may be relevant to understanding adalimumab's pharmacological properties. Our FCS data showed that a single trimeric TNF-? can bind up to three adalimumab molecules. Under some conditions even larger complexes are formed, apparently the result of cross-linking of TNF-? trimers by adalimumab. In addition, distinct differences between Humira and Enbrel were observed in their association with TNF-?.

  14. A multichannel monolithic Ge detector system for fluorescence x-ray absorption spectroscopy

    NASA Astrophysics Data System (ADS)

    Bucher, J. J.; Allen, P. G.; Edelstein, N. M.; Shuh, D. K.; Madden, N. W.; Cork, C.; Luke, P.; Pehl, D.; Malone, D.

    1996-09-01

    The construction and performance characteristics of a monolithic quad-pixel Ge detector designed specifically for fluorescence x-ray absorption spectroscopy (XAS) at synchrotron radiation sources is described. The detector semiconductor element has an active surface area of 4.0 cm2 that is electrically separated into four 1.0 cm2 pixels, with little interfacial dead volume. The spatial response of the array demonstrates that cross-talk between adjacent pixels is less than 10% for 5.9-keV photons that fall within 0.5 mm of the pixel boundaries. The detector electronics system utilizes preamplifiers built at LBNL with commercial Tennelec Model TC 244 amplifiers. Employing an 55Fe test source (Mn K? , 5.9 keV), energy resolution of better than 200 eV is achieved with a 4 msec peaking time. At 0.5 msec peaking time, pulse pileup results in a 75% throughput efficiency for an incoming count rate of 100 kHz. Initial XAS fluorescence measurements at the beamline 4 wiggler end stations at SSRL show that the detector system has several advantages over commercially available x-ray spectrometers for low-concentration counting applications.

  15. The citrate carrier CitS probed by single-molecule fluorescence spectroscopy.

    PubMed

    Kästner, Christopher N; Prummer, Michael; Sick, Beate; Renn, Alois; Wild, Urs P; Dimroth, Peter

    2003-03-01

    A prominent region of the Na(+)-dependent citrate carrier (CitS) from Klebsiella pneumoniae is the highly conserved loop X-XI, which contains a putative citrate binding site. To monitor potential conformational changes within this region by single-molecule fluorescence spectroscopy, the target cysteines C398 and C414 of the single-Cys mutants (CitS-sC398, CitS-sC414) were selectively labeled with the thiol-reactive fluorophores AlexaFluor 546/568 C(5) maleimide (AF(546), AF(568)). While both single-cysteine mutants were catalytically active citrate carriers, labeling with the fluorophore was only tolerated at C398. Upon citrate addition to the functional protein fluorophore conjugate CitS-sC398-AF(546), complete fluorescence quenching of the majority of molecules was observed, indicating a citrate-induced conformational change of the fluorophore-containing domain of CitS. This quenching was specific for the physiological substrate citrate and therefore most likely reflecting a conformational change in the citrate transport mechanism. Single-molecule studies with dual-labeled CitS-sC398-AF(546/568) and dual-color detection provided strong evidence for a homodimeric association of CitS. PMID:12609868

  16. Analyzing the Homeostasis of Signaling Proteins by a Combination of Western Blot and Fluorescence Correlation Spectroscopy

    PubMed Central

    Chung, Yi-Da; Sinzinger, Michael D.; Bovee-Geurts, Petra; Krause, Marina; Dinkla, Sip; Joosten, Irma; Koopman, Werner J.; Adjobo-Hermans, Merel J.W.; Brock, Roland

    2011-01-01

    The determination of intracellular protein concentrations is a prerequisite for understanding protein interaction networks in systems biology. Today, protein quantification is based either on mass spectrometry, which requires large cell numbers and sophisticated measurement protocols, or on quantitative Western blotting, which requires the expression and purification of a recombinant protein as a reference. Here, we present a method that uses a transiently expressed fluorescent fusion protein of the protein-of-interest as an easily accessible reference in small volumes of crude cell lysates. The concentration of the fusion protein is determined by fluorescence correlation spectroscopy, and this concentration is used to calibrate the intensity of bands on a Western blot. We applied this method to address cellular protein homeostasis by determining the concentrations of the plasma membrane-located transmembrane scaffolding protein LAT and soluble signaling proteins in naïve T cells and transformed T-cell lymphoma (Jurkat) cells (with the latter having nine times the volume of the former). Strikingly, the protein numbers of soluble proteins scaled with the cell volume, whereas that of the transmembrane protein LAT scaled with the membrane surface. This leads to significantly different stoichiometries of signaling proteins in transformed and naïve cells in concentration ranges that may translate directly into differences in complex formation. PMID:22261070

  17. Implantable semiconductor biosensor for continuous in vivo sensing of far-red fluorescent molecules.

    PubMed

    O'Sullivan, Thomas; Munro, Elizabeth A; Parashurama, Natesh; Conca, Christopher; Gambhir, Sanjiv S; Harris, James S; Levi, Ofer

    2010-06-01

    We have fabricated miniature implantable fluorescence sensors for continuous fluorescence sensing applications in living subjects. These monolithically integrated GaAs-based sensors incorporate a 675 nm vertical-cavity surface-emitting laser (VCSEL), a GaAs PIN photodiode, and a fluorescence emission filter. We demonstrate high detection sensitivity for Cy5.5 far-red dye (50 nanoMolar) in living tissue, limited by the intrinsic background autofluorescence. These low cost, sensitive and scalable sensors are promising for long-term continuous monitoring of molecular dynamics for biomedical studies in freely moving animals. PMID:20588377

  18. [Study on the effects of organic removal by traditional purification process with three-dimensional excitation emission matrix fluorescence spectroscopy].

    PubMed

    Ouyang, Er-Ming; Zhang, Xi-Hui; Wang, Wei

    2007-07-01

    Fluorescence spectroscopy is widely used to elucidate the origin and structure of organic matters in water substance. Due to its high sensitivity, good selectivity and nondestructive nature, fluorescence technique is suitable to the study of chemical and physical properties of organic matters. Three-dimensional excitation emission matrix fluorescence spectroscopy (3DEEM) was applied to analyze the effects of organic removal by traditional purification process. The results show that 3DEEM is able to disclosure the changes of organic matters in the treatment processes effectively. Traditional purification process can remove some fulvic-like organic matter, but cannot remove it completely. Coagulant sedimentation basically does not have an effect of fulvic-like organic matter removal. The removal proportion of fulvic-like organic matter through filtration is 5%-15%. PMID:17944417

  19. In vivo fluorescence imaging and urinary monoamines as surrogate biomarkers of disease progression in a mouse model of pheochromocytoma.

    PubMed

    Ullrich, Martin; Bergmann, Ralf; Peitzsch, Mirko; Cartellieri, Marc; Qin, Nan; Ehrhart-Bornstein, Monika; Block, Norman L; Schally, Andrew V; Pietzsch, Jens; Eisenhofer, Graeme; Bornstein, Stefan R; Ziegler, Christian G

    2014-11-01

    Pheochromocytoma (PHEO) is a rare but potentially lethal neuroendocrine tumor arising from catecholamine-producing chromaffin cells. Especially for metastatic PHEO, the availability of animal models is essential for developing novel therapies. For evaluating therapeutic outcome in rodent PHEO models, reliable quantification of multiple organ lesions depends on dedicated small-animal in vivo imaging, which is still challenging and only available at specialized research facilities. Here, we investigated whether whole-body fluorescence imaging and monitoring of urinary free monoamines provide suitable parameters for measuring tumor progression in a murine allograft model of PHEO. We generated an mCherry-expressing mouse PHEO cell line by lentiviral gene transfer. These cells were injected subcutaneously into nude mice to perform whole-body fluorescence imaging of tumor development. Urinary free monoamines were measured by liquid chromatography with tandem mass spectrometry. Tumor fluorescence intensity and urinary outputs of monoamines showed tumor growth-dependent increases (P < .001) over the 30 days of monitoring post-tumor engraftment. Concomitantly, systolic blood pressure was increased significantly during tumor growth. Tumor volume correlated significantly (P < .001) and strongly with tumor fluorescence intensity (rs = 0.946), and urinary outputs of dopamine (rs = 0.952), methoxytyramine (rs = 0.947), norepinephrine (rs = 0.756), and normetanephrine (rs = 0.949). Dopamine and methoxytyramine outputs allowed for detection of lesions at diameters below 2.3 mm. Our results demonstrate that mouse pheochromocytoma (MPC)-mCherry cell tumors are functionally similar to human PHEO. Both tumor fluorescence intensity and urinary outputs of free monoamines provide precise parameters of tumor progression in this sc mouse model of PHEO. This animal model will allow for testing new treatment strategies for chromaffin cell tumors. PMID:25137029

  20. Correlation of in vivo photosensitizer fluorescence and photodynamic-therapy-induced depth of necrosis

    E-print Network

    Yodh, Arjun G.

    with radiation-induced fibrosarcoma (RIF) and were in- jected with 0, 5, or 10 mg/kg Photofrin. 630-nm light (30-induced fluorescence; ne- crosis; photofrin; radiation-induced fibrosarcoma tumor; dosimetry. Paper 02019 received Mar

  1. Molecular IR Spectroscopy: New Trends and Methods of Noninvasive Diagnostics of Tissue IN VIVO

    NASA Astrophysics Data System (ADS)

    Afanasyeva, Natalia; Bruch, Reinhard

    1998-05-01

    Fiberoptic evanescent wave Fourier transform infrared (FEW-FTIR) spectroscopy using fiberoptic sensors operated in the attenuated total reflection (ATR) regime in the middle infrared (IR) region of the spectrum (850-1850 cm-1) has recently been applied to the diagnostics of tissues. The method is suitable for noninvasive and rapid (seconds) direct measurements of the spectra of normal and pathological tissues in vitro, ex vivo and in vivo. The aim of our studies is the express testing of various tumor tissues at the early stages of their development. The method is expected to be further developed for endoscopic and biopsy applications. We measured the normal skin and malignant tissues in vivo on the surface (directly on patients) in various cases of basaloma, melanoma and nevus. The experiments were performed in the operating room to measure the skin in the depth (under/in the layers of epidermis) of human breast, stomach, lung, and kidney tissues. The breast and skin tissues at different stages of tumor or cancer were distinguished very clearly in spectra of amide, side cyclic and noncyclic hydrogen bonded fragments of aminoacid residuals, phosphate groups and sugars. Computer monitoring is being developed for diagnostics.

  2. Endoscopy-coupled Raman spectroscopy for in vivo discrimination of inflammatory bowel disease

    NASA Astrophysics Data System (ADS)

    Pence, I. J.; Nguyen, Q. T.; Bi, X.; Herline, A. J.; Beaulieu, D. M.; Horst, S. N.; Schwartz, D. A.; Mahadevan-Jansen, A.

    2014-03-01

    Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's colitis (CC), affects nearly 2 million Americans, and the incidence is increasing worldwide. It has been established that UC and CC are distinct forms of IBD and require different medical care, however the distinction made between UC and CC is based upon inexact clinical, radiological, endoscopic, and pathologic features. A diagnosis of indeterminate colitis occurs in up to 15% of patients when UC and CC features overlap and cannot be differentiated; in these patients, diagnosis relies on long term followup, success or failure of existing treatment, and recurrence of the disease. Thus, there is need for a tool that can improve the sensitivity and specificity for fast, accurate and automated diagnosis of IBD. Here we present colonoscopy-coupled fiber probe-based Raman spectroscopy as a novel in vivo diagnostic tool for IBD. This in vivo study of both healthy control (NC, N=10) and diagnosed IBD patients with UC (N=15) and CC (N=26) aims to characterize spectral signatures of NC, UC, and CC. Samples are correlated with tissue pathology markers and endoscopic evaluation. Optimal collection parameters for detection have been identified based upon the new, application specific instrument design. The collected spectra are processed and analyzed using multivariate statistical techniques to identify spectral markers and discriminate NC, UC, and CC. Development of spectral markers to discriminate disease type is a necessary first step in the development of real-time, accurate and automated in vivo detection of IBD during colonoscopy procedures.

  3. Metabolism-enhanced tumor localization by fluorescence imaging: in vivo animal studies

    Microsoft Academic Search

    Y. Chen; G. Zheng; Z. H. Zhang; D. Blessington; M. Zhang; H. Li; Q. Liu; L. Zhou; X. Intes; S. Achilefu; B. Chance

    2003-01-01

    We present a high-sensitivity near-infrared optical imaging system for noninvasive cancer detection and localization based on molecularly labeled fluorescent contrast agents. This frequency-domain system utilizes the interferencelike pattern of diffuse photon density waves to achieve high detection sensitivity and localization accuracy for the fluorescent heterogeneity embedded inside the scattering media. A two-dimensional localization map is obtained through reflectance probe geometry

  4. Ultra-Fast Fluorescence Imaging in Vivo with Conjugated Polymer Fluorophores in the Second Near-Infrared Window

    E-print Network

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

    2014-01-01

    In vivo fluorescence imaging in the second near-infrared window (1.0-1.7 microns) can afford deep tissue penetration and high spatial resolution, owing to the reduced scattering of long-wavelength photons. Here, we synthesize a series of low-bandgap donor/acceptor copolymers with tunable emission wavelengths of 1050-1350 nm in this window. Non-covalent functionalization with phospholipid-polyethylene glycol results in water-soluble and biocompatible polymeric nanoparticles, allowing for live cell molecular imaging at > 1000 nm with polymer fluorophores for the first time. Importantly, the high quantum yield of the polymer allows for in vivo, deep-tissue and ultrafast imaging of mouse arterial blood flow with an unprecedented frame rate of > 25 frames per second. The high time resolution results in spatially and time resolved imaging of the blood flow pattern in cardiogram waveform over a single cardiac cycle (~ 200 ms) of a mouse, which has not been observed with fluorescence imaging in this window before.

  5. Fluorescence in vivo imaging of live tumor cells with pH-activatable targeted probes via receptor-mediated endocytosis

    NASA Astrophysics Data System (ADS)

    Asanuma, Daisuke; Urano, Yasuteru; Nagano, Tetsuo; Hama, Yukihiro; Koyama, Yoshinori; Kobayashi, Hisataka

    2009-02-01

    One goal of molecular imaging is to establish a widely applicable technique for specific detection of tumors with minimal background. Here, we achieve specific in vivo tumor visualization with a newly-designed "activatable" targeted fluorescence probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel acidic pH-activatable probes based on the BODIPY fluorophore were synthesized, and then conjugated to a cancer-targeting monoclonal antibody, Trastuzumab, or galactosyl serum albumin (GSA). As proof of concept, ex and in vivo imaging of two different tumor mouse models was performed: HER2-overexpressed lung metastasis tumor with Trastuzumab-pH probe conjugates and lectin-overexpressed i.p. disseminated tumor with GSA-pH probe conjugates. These pH-activatable targeted probes were highly specific for tumors with minimal background signal. Because the acidic pH in lysosomes is maintained by the energy-consuming proton pump, only viable cancer cells were successfully visualized. Furthermore, this strategy was also applied to fluorescence endoscopy in tumor mouse models, resulting in specific visualization of tumors as small as submillimeter in size that could hardly detected by naked eyes because of their poor contrast against normal tissues. The design concept can be widely adapted to cancer-specific cell-surface-targeting molecules that result in cellular internalization.

  6. Radiopaque fluorescence-transparent TaOx decorated upconversion nanophosphors for in vivo CT/MR/UCL trimodal imaging.

    PubMed

    Xiao, Qingfeng; Bu, Wenbo; Ren, Qingguo; Zhang, Shengjian; Xing, Huaiyong; Chen, Feng; Li, Ming; Zheng, Xiangpeng; Hua, Yanqing; Zhou, Liangping; Peng, Weijun; Qu, Haiyun; Wang, Zheng; Zhao, Kuaile; Shi, Jianlin

    2012-10-01

    To address the intractable issues such as the low performance or biocompatibility frequently encountered in previous CT, magnetic resonance (MR) and fluorescence trimodal imaging nanoprobes, a nanocomposite has been constructed by decorating gadolinium ions doped upconversion nanoparticle (Gd-doped UCNP) with radiopaque but fluorescence-transparent tantalum oxide (TaO(x), x ? 1). The as-synthesized water-soluble nanoparticle showed a litchi-like shape with an average size of ~30 nm and demonstrated extraordinarily high longitudinal and transverse relaxivity values (r(1) = 11.45 mM(-1)s(-1) and r(2) = 147.3 mM(-1)s(-1)) compared with the reported Gd-doped UCNPs to date. Obvious CT contrast enhancement was obtained by the combined effect between the radiopaque TaO(x) shell and the Gd-doped UCNP inner core. Strong upconversion luminescence (UCL) signal could unobstructedly penetrate out in virtue of high transparency of the TaO(x) shell. No mutual interference among different modalities of the upconversion nanolitchi (UCNL) was found, which ensured that the individual merits of every imaging modality could be brought into full play, demonstrated by in vitro and in vivo imagings. Furthermore, UCNLs showed only a slight effect on macrophages and RBCs in vitro and tissue in vivo. PMID:22840224

  7. Stress Tolerance and Stress-Induced Injury in Crop Plants Measured by Chlorophyll Fluorescence In Vivo1

    PubMed Central

    Smillie, Robert M.; Hetherington, Suzan E.

    1983-01-01

    The proposition is examined that measurements of chlorophyll fluorescence in vivo can be used to monitor cellular injury caused by environmental stresses rapidly and nondestructively and to determine the relative stress tolerances of different species. Stress responses of leaf tissue were measured by FR, the maximal rate of the induced rise in chlorophyll fluorescence. The time taken for FR to decrease by 50% in leaves at 0°C was used as a measure of chilling tolerance. This value was 4.3 hours for chilling-sensitive cucumber. In contrast, FR decreased very slowly in cucumber leaves at 10°C or in chilling-tolerant cabbage leaves at 0°C. Long-term changes in FR of barley, wheat, and rye leaves kept at 0°C were different in frost-hardened and unhardened material and in the latter appeared to be correlated to plant frost tolerance. To simulate damage caused by a thick ice cover, wheat leaves were placed at 0°C under N2. Kharkov wheat, a variety tolerant of ice encapsulation, showed a slower decrease in FR than Gatcher, a spring wheat. Relative heat tolerance was also indicated by the decrease in FR in heated leaves while changes in vivo resulting from photoinhibition, ultraviolet radiation, and photobleaching can also be measured. PMID:16663118

  8. Multi-spectral fluorescent reporter influenza viruses (Color-flu) as powerful tools for in vivo studies

    PubMed Central

    Fukuyama, Satoshi; Katsura, Hiroaki; Zhao, Dongming; Ozawa, Makoto; Ando, Tomomi; Shoemaker, Jason E.; Ishikawa, Izumi; Yamada, Shinya; Neumann, Gabriele; Watanabe, Shinji; Kitano, Hiroaki; Kawaoka, Yoshihiro

    2015-01-01

    Seasonal influenza A viruses cause annual epidemics of respiratory disease; highly pathogenic avian H5N1 and the recently emerged H7N9 viruses cause severe infections in humans, often with fatal outcomes. Although numerous studies have addressed the pathogenicity of influenza viruses, influenza pathogenesis remains incompletely understood. Here we generate influenza viruses expressing fluorescent proteins of different colours (‘Color-flu’ viruses) to facilitate the study of viral infection in in vivo models. On adaptation to mice, stable expression of the fluorescent proteins in infected animals allows their detection by different types of microscopy and by flow cytometry. We use this system to analyse the progression of viral spread in mouse lungs, for live imaging of virus-infected cells, and for differential gene expression studies in virus antigen-positive and virus antigen-negative live cells in the lungs of Color-flu-infected mice. Collectively, Color-flu viruses are powerful tools to analyse virus infections at the cellular level in vivo to better understand influenza pathogenesis. PMID:25807527

  9. The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy with Non-Ideal Photodetectors

    PubMed Central

    Hillesheim, Lindsey N.; Müller, Joachim D.

    2003-01-01

    Fluorescence fluctuation spectroscopy utilizes the signal fluctuations of single molecules for studying biological processes. Information about the biological system is extracted from the raw data by statistical methods such as used in fluctuation correlation spectroscopy or photon counting histogram (PCH) analysis. Since detectors are never ideal, it is crucial to understand the influence of photodetectors on signal statistics to correctly interpret the experimental data. Here we focus on the effects of afterpulsing and detector dead-time on PCH statistics. We determine the dead-time and afterpulse probability for our detectors experimentally and show that afterpulsing can be neglected for most experiments. Dead-time effects on the PCH are concentration-dependent and become significant when more than one molecule is present in the excitation volume. We develop a new PCH theory that includes dead-time effects and verify it experimentally. Additionally, we derive a simple analytical expression that accurately predicts the effect of dead-time on the molecular brightness. Corrections for non-ideal detector effects extend the useful concentration range of PCH experiments and are crucial for the interpretation of titration and dilution experiments. PMID:12944307

  10. Interaction of curcumin with intravenous immunoglobulin: a fluorescence quenching and Fourier transformation infrared spectroscopy study.

    PubMed

    Liu, Yongchun; Yang, Zhengyin; Du, Juan; Yao, Xiaojun; Lei, Ruixia; Zheng, Xudong; Liu, Jianning; Hu, Huaisheng; Li, Hong

    2008-01-01

    The interaction of curcumin with intravenous immunoglobulin (IVIG) mainly composed of immune gamma globulin (IgG) was studied in vitro by spectroscopic methods including fluorescence spectroscopy and Fourier transformation infrared (FTIR) spectroscopy. Docking was used to calculate the interaction mode between curcumin and IVIG. The binding parameters for the reaction were calculated according to the Sips equation, which suggested that the binding of IVIG to curcumin was characterized by two binding sites with the average affinity constant K(o) at 1.170 x 10(4) M(-1) (296 K), and it was a non-specific and weak drug-protein interaction. The secondary structure compositions of free IVIG and its curcumin complexes were calculated by the FTIR difference spectra, self-deconvolution, second derivative resolution enhancement and the curve-fitting procedures of amide I band. The observed spectral changes indicate a partial unfolding of the protein structure, but the typical beta structural conformation of IVIG is still retained. The average binding distance between curcumin and the chromophore of IVIG (5.57 nm) was obtained using the theory of Förster energy transfer. IVIG can serve as transport protein (carrier) for curcumin. PMID:18950593

  11. Conformation of self-assembled porphyrin dimers in liposome vesicles by phase-modulation 2D fluorescence spectroscopy

    E-print Network

    Lott, Geoffrey A; Utterback, James K; Widom, Julia R; Aspuru-Guzik, Alán; Marcus, Andrew H

    2011-01-01

    By applying a phase-modulation fluorescence approach to 2D electronic spectroscopy, we studied the conformation-dependent exciton-coupling of a porphyrin dimer embedded in a phospholipid bilayer membrane. Our measurements specify the relative angle and separation between interacting electronic transition dipole moments, and thus provide a detailed characterization of dimer conformation. Phase-modulation 2D fluorescence spectroscopy (PM-2D FS) produces 2D spectra with distinct optical features, similar to those obtained using 2D photon-echo spectroscopy (2D PE). Specifically, we studied magnesium meso tetraphenylporphyrin dimers, which form in the amphiphilic regions of 1,2-distearoyl-sn-glycero-3-phosphocholine liposomes. Comparison between experimental and simulated spectra show that while a wide range of dimer conformations can be inferred by either the linear absorption spectrum or the 2D spectrum alone, consideration of both types of spectra constrains the possible structures to a "T-shaped" geometry. The...

  12. Time-resolved spectroscopy of the probe fluorescence in the study of human blood protein dynamic structure on SR beam

    NASA Astrophysics Data System (ADS)

    Dobretsov, G. E.; Kurek, N. K.; Syrejshchikova, T. I.; Yakimenko, M. N.; Clarke, D. T.; Jones, G. R.; Munro, I. H.

    2000-06-01

    Time-resolved spectroscopy on the SRS of the Daresbury Laboratory was used for the study of the human serum lipoproteins and human blood albumins with fluorescent probes K-37 and K-35, developed in Russia. The probe K-37 was found sensitive to the difference in dynamic properties of the lipid objects. Two sets of the parameters were used for the description of lipid dynamic structure: (1) time-resolved fluorescence spectra and (2) time-resolved fluorescence depolarization as a function of rotational mobility of lipid molecules. Each measured dynamic parameter reflected the monotonous changes of dynamic properties in the range: lipid spheres-very low density lipoproteins-low density lipoproteins-high density lipoproteins-phospholipid liposomes. The range is characterized by the increase of the ratio polar/ nonpolar lipids. Thus, time-resolved fluorescence could be used to detect some structural modifications in lipoproteins related to atherosclerosis and subsequent cardiovascular diseases development.

  13. Two-photon scanning microscopy of in vivo sensory responses of cortical neurons genetically encoded with a fluorescent voltage sensor in rat

    PubMed Central

    Ahrens, Kurt F.; Heider, Barbara; Lee, Hanson; Isacoff, Ehud Y.; Siegel, Ralph M.

    2012-01-01

    A fluorescent voltage sensor protein “Flare” was created from a Kv1.4 potassium channel with YFP situated to report voltage-induced conformational changes in vivo. The RNA virus Sindbis introduced Flare into neurons in the binocular region of visual cortex in rat. Injection sites were selected based on intrinsic optical imaging. Expression of Flare occurred in the cell bodies and dendritic processes. Neurons imaged in vivo using two-photon scanning microscopy typically revealed the soma best, discernable against the background labeling of the neuropil. Somatic fluorescence changes were correlated with flashed visual stimuli; however, averaging was essential to observe these changes. This study demonstrates that the genetic modification of single neurons to express a fluorescent voltage sensor can be used to assess neuronal activity in vivo. PMID:22461770

  14. Optical fiber-based setup for in vivo measurement of the delayed fluorescence lifetime of oxygen sensors

    NASA Astrophysics Data System (ADS)

    Piffaretti, Filippo M.; Santhakumar, Kanappan; Forte, Eddy; van den Bergh, Hubert E.; Wagnières, Georges A.

    2011-03-01

    A new optical-fiber-based spectrofluorometer for in vivo or in vitro detection of delayed fluorescence is presented and characterized. This compact setup is designed so that it can be readily adapted for future clinical use. Optical excitation is done with a nitrogen laser-pumped, tunable dye laser, emitting in the UV-vis part of the spectrum. Excitation and luminescence signals are carried to and from the biological tissues under investigation, located out of the setup enclosure, by a single optical fiber. These measurements, as well as measurements performed without a fiber on in vitro samples in a thermostable quartz cell, in a controlled-atmosphere enclosure, are possible due to the efficient collection of the laser-induced luminescence light which is collected and focused on the detector with a high aperture parabolic mirror. The detection is based on a gated photomultiplier which allows for time-resolved measurements of the delayed fluorescence intensity. Thus, relevant luminescence lifetimes, typically in the sub-microsecond-to-millisecond range, can be measured with near total rejection of the sample's prompt fluorescence. The instrument spectral and temporal resolution, as well as its sensitivity, is characterized and measurement examples are presented. The primary application foreseen for this setup is the monitoring and adjustment of the light dose delivered during photodynamic therapy.

  15. In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair

    SciTech Connect

    Hsu, Y.-Y. [Faculty of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan (China); Liu, Y.-N. [Faculty of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan (China); Wang Wenyen [Incubation Center, National Yang-Ming University, Taipei, Taiwan (China); Kao, Fu-Jen [Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan (China); Kung, S.-H. [Faculty of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan (China)]. E-mail: szkung@ym.edu.tw

    2007-02-23

    An in vivo protease assay suitable for analysis by fluorescence resonance energy transfer (FRET) was developed on the basis of a novel FRET pair. The specifically designed fusion substrate consists of green fluorescent protein 2 (GFP{sup 2})-peptide-red fluorescent protein 2 (DsRed2), with a cleavage motif for the enterovirus 2A protease (2A{sup pro}) embedded within the peptide region. FRET can be readily visualized in real-time from cells expressing the fusion substrate until a proteolytic cleavage by 2A{sup pro} from the input virus. The level of FRET decay is a function of the amount and infection duration of the inoculated virus as measured by a fluorometer assay. The FRET biosensor also responded well to other related enteroviruses but not to a phylogenetically distant virus. Western blot analysis confirmed the physical cleavage of the fusion substrate upon the infections. The study provides proof of principle for applying the FRET technology to diagnostics, screening procedures, and cell biological research.

  16. Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

    SciTech Connect

    Al-Gubory, Kais H. [Unite de Biologie du Developpement et de la Reproduction, Departement de Physiologie Animale, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex (France)]. E-mail: kais.algubory@jouy.inra.fr

    2005-11-01

    The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals.

  17. In vivo terahertz spectroscopy of pigmentary skin nevi: Pilot study of non-invasive early diagnosis of dysplasia

    NASA Astrophysics Data System (ADS)

    Zaytsev, Kirill I.; Kudrin, Konstantin G.; Karasik, Valeriy E.; Reshetov, Igor V.; Yurchenko, Stanislav O.

    2015-02-01

    In vivo terahertz (THz) spectroscopy of pigmentary skin nevi is performed. The in vivo THz dielectric characteristics of healthy skin and dysplastic and non-dysplastic skin nevi are reconstructed and analyzed. The dielectric permittivity curves of these samples in the THz range exhibit significant differences that could allow non-invasive early diagnosis of dysplastic nevi, which are melanoma precursors. An approach for differentiating dysplastic and non-dysplastic skin nevi using the THz dielectric permittivity is proposed. The results demonstrate that THz pulsed spectroscopy is potentially an effective tool for non-invasive early diagnosis of dysplastic nevi and melanomas of the skin.

  18. ALA-PpIX variability quantitatively imaged in A431 epidermoid tumors using in vivo ultrasound fluorescence tomography and ex vivo assay

    NASA Astrophysics Data System (ADS)

    DSouza, Alisha V.; Flynn, Brendan P.; Gunn, Jason R.; Samkoe, Kimberley S.; Anand, Sanjay; Maytin, Edward V.; Hasan, Tayyaba; Pogue, Brian W.

    2014-03-01

    Treatment monitoring of Aminolevunilic-acid (ALA) - Photodynamic Therapy (PDT) of basal-cell carcinoma (BCC) calls for superficial and subsurface imaging techniques. While superficial imagers exist for this purpose, their ability to assess PpIX levels in thick lesions is poor; additionally few treatment centers have the capability to measure ALA-induced PpIX production. An area of active research is to improve treatments to deeper and nodular BCCs, because treatment is least effective in these. The goal of this work was to understand the logistics and technical capabilities to quantify PpIX at depths over 1mm, using a novel hybrid ultrasound-guided, fiber-based fluorescence molecular spectroscopictomography system. This system utilizes a 633nm excitation laser and detection using filtered spectrometers. Source and detection fibers are collinear so that their imaging plane matches that of ultrasound transducer. Validation with phantoms and tumor-simulating fluorescent inclusions in mice showed sensitivity to fluorophore concentrations as low as 0.025?g/ml at 4mm depth from surface, as presented in previous years. Image-guided quantification of ALA-induced PpIX production was completed in subcutaneous xenograft epidermoid cancer tumor model A431 in nude mice. A total of 32 animals were imaged in-vivo, using several time points, including pre-ALA, 4-hours post-ALA, and 24-hours post-ALA administration. On average, PpIX production in tumors increased by over 10-fold, 4-hours post-ALA. Statistical analysis of PpIX fluorescence showed significant difference among all groups; p<0.05. Results were validated by exvivo imaging of resected tumors. Details of imaging, analysis and results will be presented to illustrate variability and the potential for imaging these values at depth.

  19. Two-photon excited endogenous fluorescence for label-free in vivo imaging ingestion of disease-causing bacteria by human leukocytes

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

    Real time and in vivo monitoring leukocyte behavior provides unique information to understand the physiological and pathological process of infection. In this study, we demonstrate that two-photon excited reduced nicotinamide adenine dinucleotide (NADH) fluorescence provides imaging contrast to distinguish granulocyte and agranulocyte. By using spectral and time-resolved NADH fluorescence, we study the immune response of human neutrophils against bacterial infection (Escherichia coli). The two-photon excited NADH fluorescence images clearly review the morphological changes from resting neutrophils (round shape) to activated neutrophils (ruffle shape) during phagocytosis. The free-tobound NADH ratio of neutrophils decreases after ingesting disease-causing pathogen: Escherichia coli. This finding may provide a new optical tool to investigate inflammatory processes by using NADH fluorescence in vivo.

  20. Characterization of AB74, ARS, AO7, and DR28 During the Electro-Fenton Process by Using Three-Dimensional Excitation and Emission Matrix Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Y. H.; Lai, B.; Zhou, Y. X.; Wang, J. L.; Yang, P.

    2013-11-01

    The characteristics of Acid Blue 74 (AB74), Alizarin Red S (ARS), Acid Orange 7 (AO7) and Direct Red 28 (DR28) were investigated by using EEM fluorescence spectroscopy and UV-vis spectroscopy. The results suggest that the azo and anthraquinone dyes can be quickly distinguished from other dyes (such as AB74) according to their different total fluorescence intensity. During the decolorization process by the electro-Fenton technique, the total fluorescence intensity of the four dyes all would increase to their maximum value, but the fluorescence intensity increase in multiples of 12 (ARS), 61 (AO7), and 13 (DR28) times was much higher than that of AB74 (3 times). Furthermore, the different fluorescence intensities of the four dyes in the electro-Fenton process resulted mainly from their different chemical structure characteristics. Additionally, EEM spectroscopy can support much more information about the chemical structure characteristics of the dyes than the conventional UV-vis spectroscopy.