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Sample records for vivo fluorescence spectroscopy

  1. 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.

  2. Bayesian model selection applied to the analysis of fluorescence correlation spectroscopy data of fluorescent proteins in vitro and in vivo.

    PubMed

    Sun, Guangyu; Guo, Syuan-Ming; Teh, Cathleen; Korzh, Vladimir; Bathe, Mark; Wohland, Thorsten

    2015-04-21

    Fluorescence correlation spectroscopy (FCS) is a powerful technique to investigate molecular dynamics with single molecule sensitivity. In particular, in the life sciences it has found widespread application using fluorescent proteins as molecularly specific labels. However, FCS data analysis and interpretation using fluorescent proteins remains challenging due to typically low signal-to-noise ratio of FCS data and correlated noise in autocorrelated data sets. As a result, naive fitting procedures that ignore these important issues typically provide similarly good fits for multiple competing models without clear distinction of which model is preferred given the signal-to-noise ratio present in the data. Recently, we introduced a Bayesian model selection procedure to overcome this issue with FCS data analysis. The method accounts for the highly correlated noise that is present in FCS data sets and additionally penalizes model complexity to prevent over interpretation of FCS data. Here, we apply this procedure to evaluate FCS data from fluorescent proteins assayed in vitro and in vivo. Consistent with previous work, we demonstrate that model selection is strongly dependent on the signal-to-noise ratio of the measurement, namely, excitation intensity and measurement time, and is sensitive to saturation artifacts. Under fixed, low intensity excitation conditions, physical transport models can unambiguously be identified. However, at excitation intensities that are considered moderate in many studies, unwanted artifacts are introduced that result in nonphysical models to be preferred. We also determined the appropriate fitting models of a GFP tagged secreted signaling protein, Wnt3, in live zebrafish embryos, which is necessary for the investigation of Wnt3 expression and secretion in development. Bayes model selection therefore provides a robust procedure to determine appropriate transport and photophysical models for fluorescent proteins when appropriate models are provided, to help detect and eliminate experimental artifacts in solution, cells, and in living organisms. PMID:25815704

  3. Fluorescence Correlation Spectroscopy: A Tool to Study Protein Oligomerization and Aggregation In Vitro and In Vivo.

    PubMed

    Sahoo, Bankanidhi; Drombosky, Kenneth W; Wetzel, Ronald

    2016-01-01

    Fluorescence correlation spectroscopy (FCS) is a highly sensitive analytical technique used to measure dynamic molecular parameters, such as diffusion time (from which particle size can be calculated), conformation, and concentration of fluorescent molecules. It has been particularly powerful in characterizing size distributions in molecular associations (e.g., dimer/multimer formation) both in well-behaved thermodynamically equilibrated systems in vitro as well as in more complex environments in vivo. Protein aggregation reactions like amyloid formation, in contrast, are complex, often involving a series of uniquely structured aggregation intermediates appearing at different time scales. Nonetheless, FCS can be used in appropriate cases to characterize the early stages of some aggregation reactions. Here are described step-by-step protocols and experimental procedures for the study of molecular complex formation in aggregation systems as observed in simple buffer systems, cell extracts, and living cells. The methods described are illustrated with examples from studies of the self-assembly of huntingtin fragments, but in principle can be adapted for any aggregating system. PMID:26453206

  4. Model-based analysis of clinical fluorescence spectroscopy for in vivo detection of cervical intraepithelial dysplasia

    NASA Astrophysics Data System (ADS)

    Chang, Sung K.; Marín, Nena; Follen, Michelle; Richards-Kortum, Rebecca R.

    2006-03-01

    We present a mathematical model to calculate the relative concentration of light scatterers, light absorbers, and fluorophores in the epithelium and stroma. This mathematical description is iteratively fit to the fluorescence spectra measured in vivo, yielding relative concentrations of each molecule. The mathematical model is applied to a total of 493 fluorescence measurements of normal and dysplastic cervical tissue acquired in vivo from 292 patients. The estimated parameters are compared with histopathologic diagnosis to evaluate their diagnostic potential. The mathematical model is validated using fluorescence spectra simulated with known sets of optical parameters. Subsequent application of the mathematical model to in vivo fluorescence measurements from cervical tissue yields fits that accurately describe measured data. The optical parameters estimated from 493 fluorescence measurements show an increase in epithelial flavin adenine dinucleotide (FAD) fluorescence, a decrease in epithelial keratin fluorescence, an increase in epithelial light scattering, a decrease in stromal collagen fluorescence, and an increase in stromal hemoglobin light absorption in dysplastic tissue compared to normal tissue. These changes likely reflect an increase in the metabolic activity and loss of differentiation of epithelial dysplastic cells, and stromal angiogenesis associated with dysplasia. The model presented here provides a tool to analyze clinical fluorescence spectra yielding quantitative information about molecular changes related to dysplastic transformation.

  5. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

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

    PubMed Central

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

    2012-01-01

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

  8. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

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

  11. In vivo detection of macrophages in a rabbit atherosclerotic model by time-resolved laser-induced fluorescence spectroscopy.

    PubMed

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

    2005-08-01

    Accumulation of numerous macrophages in the fibrous cap is a key identifying feature of plaque inflammation and vulnerability. This study investigates the use of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as a potential tool for detection of macrophage foam cells in the intima of atherosclerotic plaques. Experiments were conducted in vivo on 14 New Zealand rabbits (6 control, 8 hypercholesterolemic) following aortotomy to expose the intimal luminal surface of the aorta. Tissue autofluorescence was induced with a nitrogen pulse laser (337 nm, 1 ns). Lesions were histologically classified by the percent of collagen or macrophage foam cells as well as thickness of the intima. Using parameters derived from the time-resolved fluorescence emission of plaques, we determined that intima rich in macrophage foam cells can be distinguished from intima rich in collagen with high sensitivity (>85%) and specificity (>95%). This study demonstrates, for the first time, that a time-resolved fluorescence-based technique can differentiate and demark macrophage content versus collagen content in vivo. Our results suggest that TR-LIFS technique can be used in clinical applications for identification of inflammatory cells important in plaque formation and rupture. PMID:16039283

  12. Quantitative diffuse reflectance and fluorescence spectroscopy: tool to monitor tumor physiology in vivo

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-06-01

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

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

    PubMed Central

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

    2014-01-01

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

  16. Gallstone identification by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Pradhan, Asima; Laxmi, B. V.; Jena, Sidhartha S.; Khulbe, P. K.; Bist, Hari D.; Agarwal, Asha

    1998-04-01

    Gallstones have been classified as being cholesterol type and pigment type. The classification is important for diet control of the patient to avoid recurrence of the stone. Spectroscopy is a sensitive technique to determine the composition of the gallstone both in-vitro and in-vivo. this work deals with the fluorescence spectroscopy of gallstone. For fluorescence spectroscopic studies of gallstone, samples were excited with 5 mw of 488 nm line of argon-ion laser and spectra were recorded with a SPEX 1877E triplemate attached with a cooled PMT and DM3000R data acquisition system. Fluorescence spectra from pure cholesterol and bilirubin were also recorded for comparison. Different types of gallstones: mixed, cholesterol, pigment type were studied. All spectra exhibited a very broad band, 500 to 800 nm and sometimes two bands, depending on type of stone. Pure cholesterol shows three prominent fluorescence peaks at 513, 550, 583 nm along with two peaks at approximately 568 and 586 nm. Pure bilirubin shows prominent peak at 628 nm, without any Raman line. From fluorescence spectra different types of stones are identified. Different gallstones studied show a mixture of cholesterol and bilirubin types and the ratio of the two varies from one sample type to another.

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-11-01

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

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

    PubMed Central

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

    2012-01-01

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

  20. Smartphone fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  1. 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.

  2. Noise on Fluorescence Correlation Spectroscopy.

    PubMed

    Starchev; Ricka; Buffle

    2001-01-01

    The time dependence of the noise and the signal-to-noise (SN) ratio of the fluorescence correlation spectroscopy (FCS) autocorrelation function is obtained from replica measurements of standard dextran solutions. The noise dependence on the delay time is fitted by a hyperbolic function with two fitting parameters. The dependence of these parameters on concentration, fluorescence intensity, and accumulation time is obtained experimentally. The behavior of SN at zero delay time agrees well with the theoretical predictions reported in the literature. The obtained data are useful for the quantitative evaluation of the FCS data fits, as well as for simulation of the FCS autocorrelation functions. Copyright 2001 Academic Press. PMID:11112305

  3. Supercritical Angle Fluorescence Correlation Spectroscopy

    PubMed Central

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

    2008-01-01

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

  4. CHICKEN DISEASE CHARACTERIZATION BY FLUORESCENCE SPECTROSCOPY

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. CHICKEN DISEASE CHARACTERIZATION BY FLUORESCENCE SPECTROSCOPY

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Ultrafast Nonlinear Spectroscopy of Red Fluorescent Proteins

    NASA Astrophysics Data System (ADS)

    Konold, Patrick Eugene

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

  7. New tools for in vivo fluorescence tagging.

    PubMed

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

    2005-12-01

    Engineering of fluorescent proteins continues to produce new tools for in vivo studies. The current selection contains brighter, monomeric, spectral variants that will facilitate multiplex imaging and FRET, and a collection of optical highlighter proteins that might replace photoactivatable-GFP. These new highlighter proteins, which include proteins that have photoswitchable fluorescence characteristics and a protein whose fluorescence can be repeatedly turned on and off, should simplify refined analyses of protein dynamics and kinetics. Fluorescent protein-based systems have also been developed to allow facile detection of protein-protein interactions in planta. In addition, new tags in the form of peptides that bind fluorescent ligands and quantum dots offer the prospect of overcoming some of the limitations of fluorescent proteins such as excessive size and insufficient brightness. PMID:16188488

  8. Glucose Recognition in Vitro Using Fluorescent Spectroscopy

    SciTech Connect

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

    2001-04-25

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

  9. Intraluminal fluorescence spectroscopy catheter with ultrasound guidance

    NASA Astrophysics Data System (ADS)

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

    2009-05-01

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

  10. Fluorescence spectroscopy applied to orange trees

    NASA Astrophysics Data System (ADS)

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

    2006-05-01

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

  11. Combined fiber probe for fluorescence lifetime and Raman spectroscopy.

    PubMed

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

    2015-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  13. Fluorescence spectroscopy of rhodopsins: Insights and approaches

    PubMed Central

    Alexiev, Ulrike; Farrens, David L.

    2014-01-01

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

  14. Colon cancer diagnosis using fluorescence spectroscopy and fluorescence imaging technique

    NASA Astrophysics Data System (ADS)

    Yova, Dido M.; Atlamazoglou, Vassilis; Davaris, P.; Kavantzas, Nikolaos; Loukas, Spyros

    1997-12-01

    It is well known that fluorescence spectroscopy can provide information about the differences in the concentration of chromophores in healthy and cancerous tissues. The tumor detection potential can be enhanced by using exogenous fluorescent agents with selective accumulation in cancerous tissue. In this study healthy and cancerous human colon tissue samples were obtained after colon surgery. Excitation-emission matrices were collected using a fluorescence spectrometer. The optimum excitation wavelength lied at 340 nm. After the acquisition of autofluorescence spectra, the samples were incubated in a solution of 4 (mu) g/ml of Rhodamine analogs. Rhodamine B, Rhodamine 6G and three recently synthesized analogs, were used. For the acquisition of fluorescence images, an endoscopic imaging system was developed. Fluorescence imaging with the concomitant use of Rhodamine analogs revealed a remarkable differentiation of cancerous from healthy colonic mucosa.

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

    PubMed

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

    2016-03-01

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

  16. Extraction of masked fluorescence peaks through synchronous fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  17. Fingerprinting of crude oil using fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Holmes-Smith, A. S.; Uttamlal, M.; McCormick, C.; Hepburn, D. M.; Graham, A.; Faichnie, D.

    2012-06-01

    Crude oil is a complex mixture of hydrocarbons (e.g. paraffins, aromatics, napthenes), sulphur compounds (e.g. sulphur, sulphides), amines, metals (e.g. Ni, Fe) and salts (e.g. NaCl, sand). Quantitative chemical analysis of such combinations is difficult and requires partial or complete separation of the components, challenging outside of the laboratory. Qualitative chemical analysis of oil is simpler using techniques such fluorescence spectroscopy. In this paper we will present fluorescence (spectra and lifetime) data for crude oil samples of varying (specific) API gravity and show how qualitative chemical information can be extracted from the spectra. This will include data obtained using synchronous scanning fluorescence spectrometry (SS) and time-resolved emission spectroscopy (TRES) and demonstrate the ability of utilising these methods to obtain better qualitative chemical information and hence the ability to "fingerprint" crude oil.

  18. Fluorescence correlation spectroscopy: novel variations of an established technique.

    PubMed

    Haustein, Elke; Schwille, Petra

    2007-01-01

    Fluorescence correlation spectroscopy (FCS) is one of the major biophysical techniques used for unraveling molecular interactions in vitro and in vivo. It allows minimally invasive study of dynamic processes in biological specimens with extremely high temporal and spatial resolution. By recording and correlating the fluorescence fluctuations of single labeled molecules through the exciting laser beam, FCS gives information on molecular mobility and photophysical and photochemical reactions. By using dual-color fluorescence cross-correlation, highly specific binding studies can be performed. These have been extended to four reaction partners accessible by multicolor applications. Alternative detection schemes shift accessible time frames to slower processes (e.g., scanning FCS) or higher concentrations (e.g., TIR-FCS). Despite its long tradition, FCS is by no means dated. Rather, it has proven to be a highly versatile technique that can easily be adapted to solve specific biological questions, and it continues to find exciting applications in biology and medicine. PMID:17477838

  19. Ultraviolet, Visible, and Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Penner, Michael H.

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

  20. Differentiating tissue by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Woessner, Stefan; Huen, Julien; Malthan, Dirk

    2004-03-01

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

  1. Membrane translocation assayed by fluorescence spectroscopy.

    PubMed

    Broecker, Jana; Keller, Sandro

    2010-01-01

    Assessing the ability of biomolecules or drugs to overcome lipid membranes in a receptor-independent way is of great importance in both basic research and applications involving the use of liposomes. A combination of uptake, release, and dilution experiments performed by steady-state fluorescence spectroscopy provides a powerful, straightforward, and inexpensive way of monitoring membrane translocation of fluorescent compounds. This is particularly true for peptides and proteins carrying intrinsic tryptophan residues, which eliminates the need for attaching extrinsic labeling moieties to the compound of interest. The approach encompasses three different kinds of fluorescence titrations and some simple calculations that can be carried out in a spreadsheet program. A complete set of experiments and data analyses can typically be completed within two days. PMID:20013403

  2. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2009-01-01

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

  6. 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.

  7. 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.

  8. Applications of Fluorescence Correlation Spectroscopy: Polydispersity Measurements.

    PubMed

    Starchev; Buffle; Pérez

    1999-05-15

    The method of histograms is applied to the determination of polydispersity of particles and molecules in solution from fluorescence correlation spectroscopy (FCS) data. This is an ill-posed problem, which can be overcome by using a common strategy for imposed regularization and constraint conditions. The method developed for evaluating the polydispersity is tested on both computer-generated correlation curves and real FCS data. The results obtained show that FCS measurements can be successfully used for the determination of polydispersity of suspensions, with an efficiency comparable to that of photon correlation spectroscopy (PCS). The advantage of FCS, however, is its better sensitivity to small particles (size <50 nm) and molecules in dilute solutions, as well as its better selectivity. The usefulness of FCS for environmental chemistry is discussed with regard to the obtained results. Copyright 1999 Academic Press. PMID:10222089

  9. The feasibility of using fluorescence spectroscopy as a rapid, non-invasive method for evaluating sunscreen performance.

    PubMed

    Stokes, R P; Diffey, B L

    1999-06-01

    We have carried out ex vivo studies to examine the feasibility of using fluorescence spectroscopy as an in vivo quantitative technique to assess sunscreen substantivity in terms of skin surface thickness and/or photoprotection. We found that the majority of sunscreens produced insufficient natural fluorescence and so we have attempted to increase the fluorescent signal by adding various fluorescing agents to the sunscreens. However, none of these substances is ideal; either they do not bind sufficiently strongly to sunscreen products, or their fluorescence is quenched by the active ingredients contained within sunscreens. The feasibility of using fluorescence spectroscopy for in vivo quantitative assessments of sunscreen substantivity therefore remains unproved and is dependent on a suitable fluorescent agent being found. Such an agent would have to be non-toxic, mix readily with sunscreens and be excited by visible wavelengths. PMID:10515077

  10. 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

  11. Ultrasensitive hybridization analysis using fluorescence correlation spectroscopy.

    PubMed Central

    Kinjo, M; Rigler, R

    1995-01-01

    The hybridization of fluorescently tagged 18mer deoxyribonucleotides with complementary DNA templates was analysed by fluorescence correlation spectroscopy (FCS) in a droplet under an epi-illuminated fluorescence microscope at the level of single molecules. The interaction can be monitored by the change in the translational diffusion time of the smaller (18mer) primer when binding to the bigger (7.5 kb) DNA containing the complementary sequence. The hybridization process in the presence of template M13mp18 ssDNA was monitored in a small volume (2 x 10(-16)I) at various temperatures. The Arrhenius plot of the association rate constant shows that the activation energy was 38.8 kcal/mol, but the hybridization process may involve several components. The titration experiment suggested that approximately 2 primers can be associated with one template DNA at 40 degrees C. Results of a simple homology search for the sequences complementary to the primer indicate the existence of additional sites of lower specificity. PMID:7784185

  12. APD detectors for biological fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2006-11-01

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

  13. 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.

  14. Fluorescent protein engineering by in vivo site-directed mutagenesis

    PubMed Central

    Ceballos, Melvys Valledor; Hu, Qinghua; Schiller, Paul; Myers, Richard S.

    2012-01-01

    Summary In vivo site-directed mutagenesis by ssDNA recombineering is a facile method to change the color of fluorescent proteins without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the E. coli genome and egfp carried by a bacterial/mammalian dual expression lentiviral plasmid vector. Fluorescent protein spectra were shifted by subtle modification of the chromophore region and residues interacting with the chromophore of the fluorescent protein. Eight different fluorescent proteins (Violeta, Azure, Aqua, Mar, Celeste, Amarillo, Mostaza and Bronze) were isolated and shown to be useful in multicolor imaging and flow cytometry of bacteria and transgenic human stem cells. To make in vivo site-directed mutagenesis more efficient, the recombineering method was optimized using the fluorescence change as a sensitive quantitative assay for recombination. A set of rules to simplify mutant isolation by recombineering is provided. PMID:22639380

  15. Optical biopsy fiber-based fluorescence spectroscopy instrumentation

    NASA Astrophysics Data System (ADS)

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

    1996-04-01

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

  16. Fluorescence spectroscopy to assess apoptosis in myocardium

    NASA Astrophysics Data System (ADS)

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

    2007-02-01

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

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

    PubMed

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

    2016-01-01

    One-pot green synthesis of fluorescent nitrogen-doped carbon nanodots (CNDs) was developed by hydrothermal treatments of biocompatible polyvinylpyrrolidone (PVP) and glycine. The fluorescent nitrogen-doped CNDs exhibited excellent water solubility, low cytotoxicity, and good salt stability for biological imaging. UV-vis spectroscopy, fluorescence spectroscopy, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) spectroscopy, and Raman spectroscopy were applied to confirm the optical and structural characteristics of the CNDs. Fluorescence of the CNDs was tunable from 417 to 450 nm adjusted by different excitation energy. Fluorescent quantum yield of the CNDs (21.43 %) was significantly increased ~47.59 % in comparison to that of the CNDs (14.52 %) without nitrogen doping by glycine. In the in vivo imaging system (IVIS), fluorescence signal of the nitrogen-doped CNDs was obviously observed in the lungs at 12- and 24-h post-injection. Our work has shown the potential applications of the nitrogen-doped CNDs in fluorescence imaging in vivo. Graphical abstract Synthesis of nitrogen-doped carbon nanodots and its application for vivo bioimaging. PMID:26514673

  18. 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.

  19. 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

  20. Investigating Anomalous Diffusion Using Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Anthony, Neil; Berland, Keith

    2009-11-01

    Models used in fluorescence correlation spectroscopy (FCS) generally assume diffusion of a standard Fickian nature such that the mean square displacement (MSD,<δr^2 >) of the molecular diffusion is linearly proportional to time, i.e. <δr^2 >t. In complex systems the diffusion can be anomalous, which is commonly described via a power law dependence of the MSD, i.e. <δr^2 >t^α. When measuring anomalous dynamics using FCS, the correlation functions are typically measured over a single lengthscale and the anomalous exponent, α, is recovered through curve fitting. The anomalous exponent accurately describes the time dependence of the diffusion over the measurement lengthscale, yet for the majority of experimental systems it has not yet been tested whether the dynamics predicted by the fit are actually observed over different lengthscales -- i.e. whether or not the assumed power law dynamics truly describe the system dynamics. We investigate using scanning FCS methods that simultaneously measure correlation functions over a range of lengthscales in order to determine how accurately the physical models describe the dynamics. We use simulations to test these methods and discuss their application for measuring drug delivery rates in biomedical hydrogels.

  1. 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.

  2. Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms.

    PubMed

    Krieger, Jan W; Singh, Anand P; Bag, Nirmalya; Garbe, Christoph S; Saunders, Timothy E; Langowski, Jörg; Wohland, Thorsten

    2015-12-01

    Single-plane illumination (SPIM) or total internal reflection fluorescence (TIRF) microscopes can be combined with fast and single-molecule-sensitive cameras to allow spatially resolved fluorescence (cross-) correlation spectroscopy (FCS or FCCS, hereafter referred to FCS/FCCS). This creates a powerful quantitative bioimaging tool that can generate spatially resolved mobility and interaction maps with hundreds to thousands of pixels per sample. These massively parallel imaging schemes also cause less photodamage than conventional single-point confocal microscopy-based FCS/FCCS. Here we provide guidelines for imaging FCS/FCCS measurements on commercial and custom-built microscopes (including sample preparation, setup calibration, data acquisition and evaluation), as well as anticipated results for a variety of in vitro and in vivo samples. For a skilled user of an available SPIM or TIRF setup, sample preparation, microscope alignment, data acquisition and data fitting, as described in this protocol, will take ?1 d, depending on the sample and the mode of imaging. PMID:26540588

  3. In vivo Raman spectroscopy of cervix cancers

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

  6. Fluorescent liposomes as contrast agents for in vivo optical imaging of edemas in mice.

    PubMed

    Deissler, Verena; Rüger, Ronny; Frank, Wilhelm; Fahr, Alfred; Kaiser, Werner A; Hilger, Ingrid

    2008-08-01

    This study assesses if specially designed fluorescent liposomes can be used as contrast agent for near-infrared fluorescence (NIRF) optical imaging of cultured macrophages in vitro and for NIRF imaging of inflammatory processes, like edema, in an in vivo mouse model. Fluorescent liposomes are prepared by the film hydration and extrusion method using cholesterol, L-phosphatidylcholine, and the NIR fluorescent dye DY-676-C(18) ester. Photon correlation spectroscopy and flow cytometry reveal that fluorescent liposomes are structurally stable for up to 133 days. Distinct uptake/labeling of cultured murine J774 macrophages is demonstrated by confocal laser scanning microscopy (CLSM), flow cytometry, and macroscopic NIRF imaging system at wavelengths >670 nm. Moreover, CLSM analysis reveals fluorescence signals within intracellular compartments. Ear edema is induced in mice (n = 16) by subcutaneous injection of zymosan A. Whole-body NIRF imaging is performed after intravenous injection (0-24 h) of fluorescent liposomes (55 nmol dye per kg body weight). Distinctly higher fluorescence intensities (1613.6 +/- 61.7 a.u.) are detected at inflamed areas of diseased mice as compared to controls (892.8 +/- 19.4 a.u.). Furthermore, cell isolated from ear lavage reveals the presence of labeled F4/80 positive tissue macrophages. Taken together, the results indicate both that mouse macrophages labeled with fluorescent liposomes can be detected in vitro with fluoro-optical methods and that in vivo optical imaging of inflammatory processes with fluorescent liposomes as contrast agent is feasible. Possibly, early stages of other inflammatory diseases could also be detected by the proposed diagnostic tool in the long term. PMID:18666163

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

    NASA Astrophysics Data System (ADS)

    Katilius, Evaldas; Woodbury, Neal W.

    2004-06-01

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

  8. 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 ...

  9. Native fluorescence spectroscopy of thymus and fat tissues

    NASA Astrophysics Data System (ADS)

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

    1993-08-01

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

  10. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

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

    PubMed

    Winckler, Pascale; Jaffiol, Rodolphe

    2013-05-01

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

  13. 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 α-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

  14. 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

  15. Pigment organization effects on energy transfer and Chl a emission imaged in the diatoms C. meneghiniana and P. tricornutum in vivo: a confocal laser scanning fluorescence (CLSF) microscopy and spectroscopy study.

    PubMed

    Premvardhan, Lavanya; Réfrégiers, Matthieu; Büchel, Claudia

    2013-09-26

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

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

    NASA Astrophysics Data System (ADS)

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

    2010-08-01

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

  17. 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.

  18. 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.

  19. 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

  20. Ex vivo fluorescence molecular tomography of the spine.

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  2. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

  4. NIR Raman and fluorescence spectroscopies diagnose cancer!

    NASA Astrophysics Data System (ADS)

    Liu, C. H.; Das, Bidyut B.; Glassman, Wenling S.; Tang, Gui C.; Zhu, Han-Ru; Akins, Daniel L.; Lubicz, Stephanie; Cleary, Joseph; Prudente, R.; Celmer, Edward J.; Caron, E.; Alfano, Robert R.

    1993-08-01

    NIR Raman scattering and fluorescence were investigated from malignant and normal biomedical media. Raman spectra were obtained from human normal, benign and cancerous tissues of the gynecological (GYN) tracts. Comparing the differences in intensity for the different Raman modes as well as the difference between the number of Raman lines, the normal (GYN) tissues can be distinguished from the malignant tissues. The fluorescence spectra from human breast tissues that were obtained showed that the ratio of fluorescence intensities at 340 nm to 440 nm can be used to distinguish between malignant and non- malignant tissues. Separate studies from normal and malignant breast cell lines show spectral differences assigned to NADH and flavins. These studies show that various optical techniques have the potential to be useful in medical diagnostic applications.

  5. Absorption and fluorescence spectroscopy on a smartphone

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

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

    PubMed

    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. PMID:22894519

  7. Validation of temperature-modulated fluorescence tomography in vivo

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

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

  8. Fluorescence imaging method for in vivo pH monitoring during liposomes uptake in rat liver using a pH-sensitive fluorescent dye.

    PubMed

    Begu, S; Mordon, S; Desmettre, T; Devoisselle, J M

    2005-01-01

    Liposomes are known to be taken up by the liver cells after intravenous injection. Among the few techniques available to follow this process in vivo are perturbed angular correlation spectroscopy, nuclear magnetic resonance spectroscopy, and scintigraphy. The study of the intracellular pathways and liposomal localization in the different liver cells requires sacrifice of the animals, cells separation, and electronic microscopy. In the acidic intracellular compartments, the in situ rate of release of liposomes remains poorly understood. We present a new method to follow the in situ and in vivo uptake of liposomes using a fluorescent pH-sensitive probe 5,6-carboxyfluorescein (5,6-CF). 5,6-CF is encapsulated in liposomes at high concentration (100 mM) to quench its fluorescence. After laparotomy, liposomes are injected into the penile vein of Wistar rats. Fluorescence images of the liver and the skin are recorded during 90 min and the fluorescence intensity ratio is calculated. Ratio kinetics show different profiles depending on the liposomal formulation. The calculated intracellular liver pH values are, respectively, 4.5 to 5.0 and 6.0 to 6.5 for DSPC/chol and DMPC liposomes. After sacrifice and flush with a cold saline solution, the pH of the intracellular site of the liver (ex vivo) is found to be 4.5 to 5.0. This value can be explained by an uptake of liposomes by the liver cells and subsequent localization into the acidic compartment. An intracellular event such as dye release of a drug carrier (liposomes loaded with a fluorescent dye) can be monitored by pH fluorescence imaging and spectroscopy in vivo and in situ. PMID:15910082

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

    NASA Astrophysics Data System (ADS)

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

    2001-05-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

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

    PubMed Central

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

    1994-01-01

    Laser-induced fluorescence at 337-nm excitation was used in vivo to differentiate neoplastic [cervical intraepithelial neoplasia (CIN)], nonneoplastic abnormal (inflammation and human papilloma viral infection), and normal cervical tissues. A colposcope (low-magnification microscope used to view the cervix with reflected light) was used to identify 66 normal and 49 abnormal (5 inflammation, 21 human papilloma virus infection, and 23 CIN) sites on the cervix in 28 patients. These sites were then interrogated spectroscopically. A two-stage algorithm was developed to diagnose CIN. The first stage differentiated histologically abnormal tissues from colposcopically normal tissues with a sensitivity, specificity, and positive predictive value of 92%, 90%, and 88%, respectively. The second stage differentiated preneoplastic and neoplastic tissues from nonneoplastic abnormal tissues with a sensitivity, specificity, and positive predictive value of 87%, 73%, and 74%, respectively. Spectroscopic differences were consistent with a decrease in the absolute contribution of collagen fluorescence, an increase in the absolute contribution of oxyhemoglobin attenuation, and an increase in the relative contribution of reduced nicotinamide dinucleotide phosphate [NAD(P)H] fluorescence as tissue progresses from normal to abnormal in the same patient. These results suggest that in vivo fluorescence spectroscopy of the cervix can be used to diagnose CIN at colposcopy. PMID:7937860

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

    NASA Astrophysics Data System (ADS)

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

    2007-07-01

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

  13. 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.

  14. Investigating Dynamics and Interactions of Biomolecules Using Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Berland, Keith

    2002-10-01

    Fluorescence correlation spectroscopy (FCS) has become a powerful and sensitive research tool for studying molecular dynamics, chemical kinetics, photophysics, and molecular interactions at the single molecule level. This talk will introduce the principle and application of FCS methods in biophysics, and focus on two specific applications. First, we describe the use of two-photon multi-color FCS measurements to quantify molecular interactions between two spectrally distinct fluorescent molecular species. Variations of this method are introduced that allow us to quantify the concentration of specific non-fluorescent DNA sequences with high sensitivity. Second, we describe applications of FCS to study photobleaching and chemical reaction dynamics. In particular, we quantify photobleaching rates and fluorescence flicker in common chemical dyes as well as the green fluorescent protein (GFP) S65T using two-photon excitation.

  15. Nonlinear Laser Fluorescence Spectroscopy of Natural Organic Compounds

    NASA Astrophysics Data System (ADS)

    Fadeev, Victor V.; Shirshin, Evgeny A.

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

  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. Fluorescence lifetime imaging microscopy of nanodiamonds in vivo

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

  18. Optoacoustics, laser-induced fluorescence (LIF), and photometry for investigation of different skin types in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Bartels, Myriam; Meinhardt, Merve; Krebs, Ronald; Petering, Holger; Werfel, Thomas; Anders, Angelika

    2003-10-01

    Precise determination as well as comparison of optical properties of human skin in vivo and in vitro is of great importance to the understanding of effects of UV exposure. Because of that, the absorption properties of epidermal models without and with elanocytes of skin type IV and VI were examined using optical and optoacoustic spectroscopy. The effect of melanin as an important chromophor in human skin was investigated using a photometer, laser induced fluorescence (LIF) and optoacoustics. Moreover, an epidemal model irradiated several times with UVA showed similar absorption characteristics as human skin in vivo. Besides, optoacoustic signals are shown to deliver structural characteristics of different epidermal layers that are about 40 ?m thick. Using laser optoacoustics and laser induced fluorescence, human skin in vivo can be investigated wavelength-resolved. Therefore, optoacoustics is a promising tool for in vivo determination of different skin types, optimization of phototherapy and testing of protective substances like sunscreens in the future.

  19. "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.

  20. Ultrasensitive molecular fluorescence spectroscopy in levitated microdroplets

    SciTech Connect

    Ramsey, J.M.; Whitten, W.B. ); Arnold, S. ); Bronk, B.V. )

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2006-02-01

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

  3. Sucrose Monoester Micelles Size Determined by Fluorescence Correlation Spectroscopy (FCS)

    PubMed Central

    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, Rh. 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

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

    PubMed Central

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

    2006-01-01

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

  5. Fluorescence Imaging In Vivo at Wavelengths beyond 1500?nm.

    PubMed

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

    2015-12-01

    Compared to imaging in the visible and near-infrared regions below 900?nm, imaging in the second near-infrared window (NIR-II, 1000-1700?nm) is a promising method for deep-tissue high-resolution optical imaging in?vivo mainly owing to the reduced scattering of photons traversing through biological tissues. Herein, semiconducting single-walled carbon nanotubes with large diameters were used for in?vivo fluorescence imaging in the long-wavelength NIR region (1500-1700?nm, NIR-IIb). With this imaging agent, 3-4??m wide capillary blood vessels at a depth of about 3?mm could be resolved. Meanwhile, the blood-flow speeds in multiple individual vessels could be mapped simultaneously. Furthermore, NIR-IIb tumor imaging of a live mouse was explored. NIR-IIb imaging can be generalized to a wide range of fluorophores emitting at up to 1700?nm for high-performance in?vivo optical imaging. PMID:26460151

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

    PubMed

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

    2013-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  8. Diffusivity of asphaltene molecules by fluorescence correlation spectroscopy.

    PubMed

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

    2006-07-01

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

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

    SciTech Connect

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

    2011-06-15

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

  10. Multiphoton spectroscopy of human skin in vivo

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  11. Fluorescence spectroscopy of excitation transfer in Photosystem 1

    SciTech Connect

    Mukerji, I.

    1990-12-01

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

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

    NASA Astrophysics Data System (ADS)

    Skala, Melissa Caroline

    2007-12-01

    Cancer morbidity and mortality is greatly reduced when the disease is diagnosed and treated early in its development. Tissue biopsies are the gold standard for cancer diagnosis, and an accurate diagnosis requires a biopsy from the malignant portion of an organ. Light, guided through a fiber optic probe, could be used to inspect regions of interest and provide real-time feedback to determine the optimal tissue site for biopsy. This approach could increase the diagnostic accuracy of current biopsy procedures. The studies in this thesis have characterized changes in tissue optical signals with carcinogenesis, increasing our understanding of the sensitivity of optical techniques for cancer detection. All in vivo studies were conducted on the dimethylbenz[alpha]anthracene treated hamster cheek pouch model of epithelial carcinogenesis. Multiphoton microscopy studies in the near infrared wavelength region quantified changes in tissue morphology and fluorescence with carcinogenesis in vivo. Statistically significant morphological changes with precancer included increased epithelial thickness, loss of stratification in the epithelium, and increased nuclear diameter. Fluorescence changes included a statistically significant decrease in the epithelial fluorescence intensity per voxel at 780 nm excitation, a decrease in the fluorescence lifetime of protein-bound nicotinamide adenine dinucleotide (NADH, an electron donor in oxidative phosphorylation), and an increase in the fluorescence lifetime of protein-bound flavin adenine dinucleotide (FAD, an electron acceptor in oxidative phosphorylation) with precancer. The redox ratio (fluorescence intensity of FAD/NADH, a measure of the cellular oxidation-reduction state) did not significantly change with precancer. Cell culture experiments (MCF10A cells) indicated that the decrease in protein-bound NADH with precancer could be due to increased levels of glycolysis. Point measurements of diffuse reflectance and fluorescence spectra in the ultraviolet to visible wavelength range indicated that the most diagnostic optical signals originate from sub-surface tissue layers. Optical properties extracted from these spectroscopy measurements showed a significant decrease in the hemoglobin saturation, absorption coefficient, reduced scattering coefficient and fluorescence intensity (at 400 nm excitation) in neoplastic compared to normal tissues. The results from these studies indicate that multiphoton microscopy and optical spectroscopy can non-invasively provide information on tissue structure and function in vivo that is related to tissue pathology.

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

    PubMed Central

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

    2010-01-01

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

  14. Dynamic tissue analysis using time- and wavelength-resolved fluorescence spectroscopy for atherosclerosis diagnosis

    PubMed Central

    Sun, Yinghua; Sun, Yang; Stephens, Douglas; Xie, Hongtao; Phipps, Jennifer; Saroufeem, Ramez; Southard, Jeffrey; Elson, Daniel S.; Marcu, Laura

    2011-01-01

    Simultaneous time- and wavelength-resolved fluorescence spectroscopy (STWRFS) was developed and tested for the dynamic characterization of atherosclerotic tissue ex vivo and arterial vessels in vivo. Autofluorescence, induced by a 337 nm, 700 ps pulsed laser, was split to three wavelength sub-bands using dichroic filters, with each sub-band coupled into a different length of optical fiber for temporal separation. STWRFS allows for fast recording/analysis (few microseconds) of time-resolved fluorescence emission in these sub-bands and rapid scanning. Distinct compositions of excised human atherosclerotic aorta were clearly discriminated over scanning lengths of several centimeters based on fluorescence lifetime and the intensity ratio between 390 and 452 nm. Operation of STWRFS blood flow was further validated in pig femoral arteries in vivo using a single-fiber probe integrated with an ultrasound imaging catheter. Current results demonstrate the potential of STWRFS as a tool for real-time optical characterization of arterial tissue composition and for atherosclerosis research and diagnosis. PMID:21369214

  15. Fluorescence spectroscopy using indocyanine green for lymph node mapping

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

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

    NASA Astrophysics Data System (ADS)

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

    1995-03-01

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

  18. 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.

  19. Fluorescence Fluctuation Spectroscopy in the Presence of Immobile Fluorophores

    PubMed Central

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

    2008-01-01

    Fluorescence contributions from immobile sources present a challenge for fluorescence fluctuation spectroscopy (FFS) because the absence of signal fluctuations from stationary fluorophores leads to a biased analysis. This is especially of concern for cellular FFS studies on proteins that interact with immobile structures. Here we present a method that correctly analyzes FFS experiments in the presence of immobile sources by exploiting selective photobleaching of immobile fluorophores. The fluorescence decay due to photobleaching of the immobile species is modeled taking into account the nonuniform illumination volume. The experimentally observed decay curve serves to separate the mobile and immobile fluorescence contribution, which is used to calculate the molecular brightness from the FFS data. We experimentally verify this approach in vitro using the fluorescent protein EGFP as our immobilized species and a diffusing dye of a different color as the mobile one. For this special case, we also use an alternative method of determining the brightness by spectrally resolving the two species. By conducting a dilution study, we show that the correct parameters are obtained using either technique for a wide range of mobile fractions. To demonstrate the application of our technique in living cells, we perform experiments using the histone core protein H2B fused with EGFP expressed in COS-1 cells. We successfully recovered the brightness of the mobile fraction of H2B-EGFP. PMID:18065480

  20. In vivo fluorescence imaging of ?-amyloid plaques with push-pull dimethylaminothiophene derivatives.

    PubMed

    Watanabe, Hiroyuki; Ono, Masahiro; Saji, Hideo

    2015-12-14

    In vivo fluorescence imaging of ?-amyloid (A?) plaques in the brain is expected to be used as a new method for detecting Alzheimer's disease (AD). We synthesized novel push-pull dimethylaminothiophenyl (DTM) derivatives and evaluated their utility as in vivo fluorescence imaging probes targeting A? plaques. As a result, we found that DTM-2 is a promising fluorescent probe for A? plaques in the AD brains. PMID:26455736

  1. 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.

  2. Light emitting diode excitation emission matrix fluorescence spectroscopy.

    PubMed

    Hart, Sean J; JiJi, Renée D

    2002-12-01

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

  3. 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

  4. Optimal algorithm for fluorescence suppression of modulated Raman spectroscopy.

    PubMed

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

    2010-05-24

    Raman spectroscopy permits probing of the molecular and chemical properties of the analyzed sample. However, its applicability has been seriously limited to specific applications by the presence of a strong fluorescence background. In our recent paper [Anal. Chem. 82, 738 (2010)], we reported a new modulation method for separating Raman scattering from fluorescence. By continuously changing the excitation wavelength, we demonstrated that it is possible to continuously shift the Raman peaks while the fluorescence background remains essentially constant. In this way, our method allows separation of the modulated Raman peaks from the static fluorescence background with important advantages when compared to previous work using only two [Appl. Spectrosc. 46, 707 (1992)] or a few shifted excitation wavelengths [Opt. Express 16, 10975 (2008)]. The purpose of the present work is to demonstrate a significant improvement of the efficacy of the modulated method by using different processing algorithms. The merits of each algorithm (Standard Deviation analysis, Fourier Filtering, Least-Squares fitting and Principal Component Analysis) are discussed and the dependence of the modulated Raman signal on several parameters, such as the amplitude and the modulation rate of the Raman excitation wavelength, is analyzed. The results of both simulation and experimental data demonstrate that Principal Component Analysis is the best processing algorithm. It improves the signal-to-noise ratio in the treated Raman spectra, reducing required acquisition times. Additionally, this approach does not require any synchronization procedure, reduces user intervention and renders it suitable for real-time applications. PMID:20588999

  5. 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

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

    PubMed

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

    2015-11-01

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

  7. Frequency-domain fluorescence spectroscopy of human stratum corneum

    NASA Astrophysics Data System (ADS)

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

    1994-08-01

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

  8. 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.

  9. Multiple-fiber probe design for fluorescence spectroscopy in tissue.

    PubMed

    Pfefer, T Joshua; Schomacker, Kevin T; Ediger, Marwood N; Nishioka, Norman S

    2002-08-01

    The fiber-optic probe is an essential component of many quantitative fluorescence spectroscopy systems, enabling delivery of excitation light and collection of remitted fluorescence in a wide variety of clinical and laboratory situations. However, there is little information available on the role of illumination--collection geometry to guide the design of these components. Therefore we used a Monte Carlo model to investigate the effect of multifiber probe design parameters--numerical aperture, fiber diameter, source--collection fiber separation distance, and fiber-tissue spacer thickness--on light propagation and the origin of detected fluorescence. An excitation wavelength of 400 nm and an emission wavelength of 630 nm were simulated. Noteworthy effects included an increase in axial selectivity with decreasing fiber size and a transition with increasing fiber-tissue spacer size from a subsurface peak in fluorophore sensitivity to a nearly monotonic decrease typical of single-fiber probes. We provide theoretical evidence that probe design strongly affects tissue interrogation. Therefore application-specific customization of probe design may lead to improvements in the efficacy of fluorescence-based diagnostic devices. PMID:12153108

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

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

  11. An Analog Filter Approach to Frequency Domain Fluorescence Spectroscopy

    SciTech Connect

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

    2015-04-24

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

  12. An Analog Filter Approach to Frequency Domain Fluorescence Spectroscopy

    DOE PAGESBeta

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

    2015-04-24

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

  13. Spectrally resolved fluorescence correlation spectroscopy based on global analysis.

    PubMed

    Previte, Michael J R; Pelet, Serge; Kim, Ki Hean; Buehler, Christoph; So, Peter T C

    2008-05-01

    Multicolor fluorescence correlation spectroscopy has been recently developed to study chemical interactions of multiple chemical species labeled with spectrally distinct fluorophores. In the presence of spectral overlap, there exists a lower detectability limit for reaction products with multicolor fluorophores. In addition, the ability to separate bound product from reactants allows thermodynamic properties such as dissociation constants to be measured for chemical reactions. In this report, we utilize a spectrally resolved two-photon microscope with single-photon counting sensitivity to acquire spectral and temporal information from multiple chemical species. Further, we have developed a global fitting analysis algorithm that simultaneously analyzes all distinct auto- and cross-correlation functions from 15 independent spectral channels. We have demonstrated that the global analysis approach allows the concentration and diffusion coefficients of fluorescent particles to be resolved despite the presence of overlapping emission spectra. PMID:18351754

  14. Time-resolved confocal fluorescence spectroscopy reveals the structure and metabolic state of epithelial tissue

    NASA Astrophysics Data System (ADS)

    Wu, Yicong; Zheng, Wei; Qu, Jianan Y.

    2007-02-01

    Autofluorescence spectroscopy has been a widely explored technique for in vivo and noninvasive diagnosis of pre-cancer lesions in epithelium where 90% cancers originate. For extracting more accurate fluorescence information for cancer diagnosis, depth-resolved fluorescence measurements are crucial to assess NADH and FAD in non-keratinized epithelial layer and collagen in stromal layer, respectively. In this study, we achieved the depth-resolved fluorescence spectral measurements of squamous epithelial tissue based on confocal technique. We found that in non-keratinized epithelial layer the fluorescence signals excited at 405 nm were the combination of NADH and FAD fluorescence and could be used for evaluating the redox ratio. Moreover, we found that confocal time-resolved autofluorescence measurements of epithelial tissue with 405 nm excitations could provide the information on the layered tissue structure. All depth-resolved autofluorescence decays were accurately fitted with a dual-exponential function consisting of a short lifetime (0.4 ~ 0.6 ns) and a long lifetime (3 ~ 4 ns) components. The short lifetime component dominated the decay of non-keratinzied epithelial fluorescence while the decay of the signals from keratinized epithelium and stroma were mainly determined by the long lifetime component. The ratio of the amplitudes of two components could be used to differentiate the layered structure of epithelial tissue. In general, the results in this study demonstrated that the combined depth- and timeresolved fluorescence measurements can produce the information on the layered structure and localized biochemistry of epithelial tissue for the diagnosis of tissue pathology.

  15. The photon counting histogram in fluorescence fluctuation spectroscopy.

    PubMed Central

    Chen, Y; Müller, J D; So, P T; Gratton, E

    1999-01-01

    Fluorescence correlation spectroscopy (FCS) is generally used to obtain information about the number of fluorescent particles in a small volume and the diffusion coefficient from the autocorrelation function of the fluorescence signal. Here we demonstrate that photon counting histogram (PCH) analysis constitutes a novel tool for extracting quantities from fluorescence fluctuation data, i.e., the measured photon counts per molecule and the average number of molecules within the observation volume. The photon counting histogram of fluorescence fluctuation experiments, in which few molecules are present in the excitation volume, exhibits a super-Poissonian behavior. The additional broadening of the PCH compared to a Poisson distribution is due to fluorescence intensity fluctuations. For diffusing particles these intensity fluctuations are caused by an inhomogeneous excitation profile and the fluctuations in the number of particles in the observation volume. The quantitative relationship between the detected photon counts and the fluorescence intensity reaching the detector is given by Mandel's formula. Based on this equation and considering the fluorescence intensity distribution in the two-photon excitation volume, a theoretical expression for the PCH as a function of the number of molecules in the excitation volume is derived. For a single molecular species two parameters are sufficient to characterize the histogram completely, namely the average number of molecules within the observation volume and the detected photon counts per molecule per sampling time epsilon. The PCH for multiple molecular species, on the other hand, is generated by successively convoluting the photon counting distribution of each species with the others. The influence of the excitation profile upon the photon counting statistics for two relevant point spread functions (PSFs), the three-dimensional Gaussian PSF conventionally employed in confocal detection and the square of the Gaussian-Lorentzian PSF for two photon excitation, is explicitly treated. Measured photon counting distributions obtained with a two-photon excitation source agree, within experimental error with the theoretical PCHs calculated for the square of a Gaussian-Lorentzian beam profile. We demonstrate and discuss the influence of the average number of particles within the observation volume and the detected photon counts per molecule per sampling interval upon the super-Poissonian character of the photon counting distribution. PMID:10388780

  16. Three-dimensional in vivo fluorescence diffuse optical tomography of breast cancer in humans

    NASA Astrophysics Data System (ADS)

    Corlu, Alper; Choe, Regine; Durduran, Turgut; Rosen, Mark A.; Schweiger, Martin; Arridge, Simon R.; Schnall, Mitchell D.; Yodh, Arjun G.

    2007-05-01

    We present three-dimensional (3D) in vivo images of human breast cancer based on fluorescence diffuse optical tomography (FDOT). To our knowledge, this work represents the first reported 3D fluorescence tomography of human breast cancer in vivo. In our protocol, the fluorophore Indocyanine Green (ICG) is injected intravenously. Fluorescence excitation and detection are accomplished in the soft-compression, parallel-plane, transmission geometry using laser sources at 786 nm and spectrally filtered CCD detection. Phantom and in vivo studies confirm the signals are due to ICG fluorescence, rather than tissue autofluorescence and excitation light leakage. Fluorescence images of breast tumors were in good agreement with those of MRI, and with DOT based on endogenous contrast. Tumorto- normal tissue contrast based on ICG fluorescence was two-to-four-fold higher than contrast based on hemoglobin and scattering parameters. In total the measurements demonstrate that FDOT of breast cancer is feasible and promising.

  17. Three-dimensional in vivo fluorescence diffuse optical tomography of breast cancer in humans.

    PubMed

    Corlu, Alper; Choe, Regine; Durduran, Turgut; Rosen, Mark A; Schweiger, Martin; Arridge, Simon R; Schnall, Mitchell D; Yodh, Arjun G

    2007-05-28

    We present three-dimensional (3D) in vivo images of human breast cancer based on fluorescence diffuse optical tomography (FDOT). To our knowledge, this work represents the first reported 3D fluorescence tomography of human breast cancer in vivo. In our protocol, the fluorophore Indocyanine Green (ICG) is injected intravenously. Fluorescence excitation and detection are accomplished in the soft-compression, parallel-plane, transmission geometry using laser sources at 786 nm and spectrally filtered CCD detection. Phantom and in vivo studies confirm the signals are due to ICG fluorescence, rather than tissue autofluorescence and excitation light leakage. Fluorescence images of breast tumors were in good agreement with those of MRI, and with DOT based on endogenous contrast. Tumorto- normal tissue contrast based on ICG fluorescence was two-to-four-fold higher than contrast based on hemoglobin and scattering parameters. In total the measurements demonstrate that FDOT of breast cancer is feasible and promising. PMID:19546980

  18. Terahertz spectroscopy of pigmentary skin nevi in vivo

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  19. Quantum process tomography by 2D fluorescence spectroscopy

    SciTech Connect

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

    2015-06-07

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

  20. 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

  1. Detectors for single-molecule fluorescence imaging and spectroscopy.

    PubMed

    Michalet, X; Siegmund, O H W; Vallerga, J V; Jelinsky, P; Millaud, J E; Weiss, S

    2007-01-01

    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

  2. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2008-04-01

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

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

    SciTech Connect

    Yankelevich, Diego R.; Department of Biomedical Engineering, University of California, 451 Health Sciences Drive, Davis, California 95616 ; Ma, Dinglong; Liu, Jing; Sun, Yang; Sun, Yinghua; Bec, Julien; Marcu, Laura; Elson, Daniel S.

    2014-03-15

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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

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

    2014-01-01

    The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 ?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

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

    NASA Astrophysics Data System (ADS)

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

    2007-07-01

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

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

    PubMed Central

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

    2011-01-01

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

  9. Human feasibility study of fluorescence spectroscopy guided optical biopsy needle for prostate cancer diagnosis.

    PubMed

    Werahera, Priya N; Jasion, Edward A; Yongjun Liu; Daily, John W; Arangua, Paul; Jones, Clifford; Nash, S Russell; Morrell, Michael; Crawford, E David

    2015-08-01

    Current prostate biopsy cores have a very low diagnostic yield. These biopsies often fail to diagnose prostate cancer since 90% of cores are histopathologically classified as benign. The concentrations of endogenous fluorophores in prostate tissue vary with disease states. Thus, fluorescence spectroscopy could be utilized to quantify these variations for identification of malignant lesions. We investigated clinical feasibility of a 14 gauge (1.98 mm) optical biopsy needle guided by fluorescence spectroscopy for real-time in vivo prostate cancer diagnosis. Built-in optical sensor has 8×100?m fibers for tissue excitation and a single 200?m fiber to collect spectral data. Custom-made fluorometer has 2 light-emitting diodes at 290 and 340 nm and a spectrometer. User interface for fluorometer operation and data collection was developed using LabView software. Each spectral data acquisition required ~2 seconds. The in vivo biopsies were performed during radical retropubic prostatectomy surgery on the exposed prostate with blood flow to the gland intact. A tissue biopsy core was obtained from each biopsy site after acquisition of spectral data. Above procedure was repeated ex vivo after surgical excision of the prostate. Biopsy cores were histopathologically classified as either benign or malignant and correlated with corresponding spectral data. Partial Least Square analysis was performed to determine diagnostically significant principal components as potential classifiers. A linear support vector machine and leave-one-out cross validation method was employed for tissue classification. Thirteen patients were consented to the study. Histopathological analysis found cancer in 29/208 in vivo and 51/224 ex vivo viable biopsy cores. Study results show 72% sensitivity, 66% specificity, and 93% negative predictive value for in vivo and 75%, 80%, and 93%, respectively, for ex vivo malignant versus benign prostatic tissue classification. Optical biopsy needle has a very high negative predictive value to indicate benign tissue while sufficient sensitivity for targeting areas suspicious for cancer within the prostate gland. Hence, the optical biopsy needle can increase the diagnostic yield of prostate biopsies with consequent improvement in patient care. PMID:26737991

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

    PubMed

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

    2016-04-01

    Synchronous fluorescence spectroscopy was used in combination with principal component analysis (PCA) and linear discriminant analysis (LDA) for the differentiation of plum spirits according to their geographical origin. A total of 14 Czech, 12 Hungarian and 18 Slovak plum spirit samples were used. The samples were divided in two categories: colorless (22 samples) and colored (22 samples). Synchronous fluorescence spectra (SFS) obtained at a wavelength difference of 60 nm provided the best results. Considering the PCA-LDA applied to the SFS of all samples, Czech, Hungarian and Slovak colorless samples were properly classified in both the calibration and prediction sets. 100% of correct classification was also obtained for Czech and Hungarian colored samples. However, one group of Slovak colored samples was classified as belonging to the Hungarian group in the calibration set. Thus, the total correct classifications obtained were 94% and 100% for the calibration and prediction steps, respectively. The results were compared with those obtained using near-infrared (NIR) spectroscopy. Applying PCA-LDA to NIR spectra (5500-6000 cm(-1)), the total correct classifications were 91% and 92% for the calibration and prediction steps, respectively, which were slightly lower than those obtained using SFS. PMID:26593555

  11. Fluorescence Correlation Spectroscopy Relates Rafts in Model and Native Membranes

    PubMed Central

    Bacia, Kirsten; Scherfeld, Dag; Kahya, Nicoletta; Schwille, Petra

    2004-01-01

    The lipid raft model has evoked a new perspective on membrane biology. Understanding the structure and dynamics of lipid domains could be a key to many crucial membrane-associated processes in cells. However, one shortcoming in the field is the lack of routinely applicable techniques to measure raft association without perturbation by detergents. We show that both in cell and in domain-exhibiting model membranes, fluorescence correlation spectroscopy (FCS) can easily distinguish a raft marker (cholera toxin B subunit bound to ganglioside (GM1) and a nonraft marker (dialkylcarbocyanine dye diI)) by their decidedly different diffusional mobilities. In contrast, these markers exhibit only slightly different mobilities in a homogeneous artificial membrane. Performing cholesterol depletion with methyl-?-cyclodextrin, which disrupts raft organization, we find an analogous effect of reduced mobility for the nonraft marker in domain-exhibiting artificial membranes and in cell membranes. In contrast, cholesterol depletion has differential effects on the raft marker, cholera toxin B subunit-GM1, rendering it more mobile in artificial domain-exhibiting membranes but leaving it immobile in cell membranes, where cytoskeleton disruption is required to achieve higher mobility. Thus, fluorescence correlation spectroscopy promises to be a valuable tool to elucidate lipid raft associations in native cells and to gain deeper insight into the correspondence between model and natural membranes. PMID:15298908

  12. 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

  13. Fluorescence spectroscopy and chemometric modeling for bioprocess monitoring.

    PubMed

    Faassen, Saskia M; Hitzmann, Bernd

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  15. 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.

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

    PubMed Central

    Wachsmann-Hogiu, Sebastian; Weeks, Tyler

    2009-01-01

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

  18. Assessing Raw and Treated Water Quality Using Fluorescence Spectroscopy

    NASA Astrophysics Data System (ADS)

    Bridgeman, J.; Baker, A.

    2006-12-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  20. Fluorescent N-Doped Carbon Dots as in Vitro and in Vivo Nanothermometer.

    PubMed

    Yang, Yanmei; Kong, Weiqian; Li, Hao; Liu, Juan; Yang, Manman; Huang, Hui; Liu, Yang; Wang, Zhongyang; Wang, Zhiqiang; Sham, Tsun-Kong; Zhong, Jun; Wang, Chao; Liu, Zhuang; Lee, Shuit-Tong; Kang, Zhenhui

    2015-12-16

    The fluorescent N-doped carbon dots (N-CDs) obtained from C3N4 emit strong blue fluorescence, which is stable with different ionic strengths and time. The fluorescence intensity of N-CDs decreases with the temperature increasing, while it can recover to the initial one with the temperature decreasing. It is an accurate linear response of fluorescence intensity to temperature, which may be attributed to the synergistic effect of abundant oxygen-containing functional groups and hydrogen bonds. Further experiments also demonstrate that N-CDs can serve as effective in vitro and in vivo fluorescence-based nanothermometer. PMID:26593857

  1. Near infrared in vivo flow cytometry for tracking fluorescent circulating cells.

    PubMed

    Suo, Yuanzhen; Liu, Tao; Xie, Chengying; Wei, Dan; Tan, Xu; Wu, Liao; Wang, Xiaoling; He, Hao; Shi, Guohua; Wei, Xunbin; Shi, Chunmeng

    2015-09-01

    The in vivo flow cytometry (IVFC) is now a powerful technique in biomedical research, especially for tracking specific cells in circulatory system. The current fluorescence-based IVFC is limited to visible spectrum, while near infrared (NIR) dyes have their advantages, such as deeper penetration, less absorption and less scattering for NIR fluorescence. Here, using an NIR in vivo flow cytometer with a 785 nm laser excitation, the measurement of fluorescent dye IR-780 labeled circulating cells is demonstrated. Representative peaks corresponding to NIR fluorescent circulating cells are detected and quantified. In addition, blood flow information, including the blood flow velocity and flow volume per unit time, is obtained. By simultaneous detection of IR-780 and enhanced green fluorescent protein (EGFP) signals from dual labeled cells, the IR-780 is shown to be a suitable fluorescent dye for multicolor detection by IVFC, including NIR. Thus, the IVFC is extended to the NIR range and shows potential application in biomedical research. PMID:26138257

  2. Fluorescent Molecular Tomography for In Vivo Imaging of Mouse Atherosclerosis.

    PubMed

    Arranz, Alicia; Rudin, Markus; Zaragoza, Carlos; Ripoll, Jorge

    2015-01-01

    Optical imaging technologies such as fluorescence molecular tomography (FMT) are gaining great relevance in cardiovascular research. The main reason is the increased number of available fluorescent agents, especially those termed "activatable probes," which remain quenched under baseline conditions and are fluorescent when a specific enzymatic activity is present. A major characteristic of FMT is the possibility of obtaining quantitative data of fluorescence signal distribution in a noninvasive fashion and using nonionizing radiation, making FMT an invaluable tool for longitudinal studies with biomedical applications. Here, we describe a standard procedure to perform FMT experiments in atherosclerosis mouse models, from the handling of the animals to the reconstruction of the 3D images. PMID:26445804

  3. 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.

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

    PubMed Central

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

    2013-01-01

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

  5. Fluorescence and UV-vis Spectroscopy of Synovial Fluids

    NASA Astrophysics Data System (ADS)

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

    2009-10-01

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

  6. Aqueous solutions of lower alcohols investigated by pyrene fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhao, Li-Jun; Xiao, Han-Shuang

    2012-03-01

    The aqueous solutions of lower alcohols such as methanol, ethanol, 1-propanol and 2-propanol, were studied by fluorescence spectroscopy of pyrene, defining the Py scale for polarity. Sigmoidal curves were used to fit the Py values of aqueous alcohol solutions as a function of the logarithm of water-alcohol mole ratio, i.e., log(WAR). The results from curve fittings were discussed in terms of the structural transitions of aqueous alcohol solutions, as well as the dissociation constants for alcohol- and water-pyrene complexes. The microscopic alcohol and water phases were considered to be saturated with each other, and the structures of dilute aqueous alcohol solutions were found to be more complicated than those of concentrated ones.

  7. Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

    SciTech Connect

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

    2014-08-14

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

    The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for Förster resonance energy transfer (FRET) microscopy and fluorescence fluctuation spectroscopy (FFS) provide important tools for monitoring dynamic protein interactions inside living cells. Fluorescence lifetime imaging microscopy (FLIM) quantitatively maps changes in the spatial distribution of donor FP lifetimes that result from FRET with acceptor FPs. FFS probes dynamic protein associations through its capacity to monitor localized protein diffusion. Here, we use FRET-FLIM combined with FFS in living cells to investigate changes in protein mobility due to protein-protein interactions involving transcription factors and chromatin modifying proteins that function in anterior pituitary gene regulation. The heterochromatin protein 1 alpha (HP1?) plays a key role in the establishment and maintenance of heterochromatin through its interactions with histone methyltransferases. Recent studies, however, also highlight the importance of HP1? as a positive regulator of active transcription in euchromatin. Intriguingly, we observed that the transcription factor CCAAT/enhancer-binding protein alpha (C/EBP?) interacts with HP1? in regions of pericentromeric heterochromatin in mouse pituitary cells. These observations prompted us to investigate the relationship between HP1? dynamic interactions in pituitary specific gene regulation.

  9. Mitochondrial function in vivo: spectroscopy provides window on cellular energetics.

    PubMed

    Amara, Catherine E; Marcinek, David J; Shankland, Eric G; Schenkman, Kenneth A; Arakaki, Lorilee S L; Conley, Kevin E

    2008-12-01

    Mitochondria integrate the key metabolic fluxes in the cell. This role places this organelle at the center of cellular energetics and, hence, mitochondrial dysfunction underlies a growing number of human disorders and age-related degenerative diseases. Here we present novel analytical and technical methods for evaluating mitochondrial metabolism and (dys)function in human muscle in vivo. Three innovations involving advances in optical spectroscopy (OS) and magnetic resonance spectroscopy (MRS) permit quantifying key compounds in energy metabolism to yield mitochondrial oxidation and phosphorylation fluxes. The first of these uses analytical methods applied to optical spectra to measure hemoglobin (Hb) and myoglobin (Mb) oxygenation states and relative contents ([Hb]/[Mb]) to determine mitochondrial respiration (O2 uptake) in vivo. The second uses MRS methods to quantify key high-energy compounds (creatine phosphate, PCr, and adenosine triphosphate, ATP) to determine mitochondrial phosphorylation (ATP flux) in vivo. The third involves a functional test that combines these spectroscopic approaches to determine mitochondrial energy coupling (ATP/O2), phosphorylation capacity (ATP(max)) and oxidative capacity (O2max) of muscle. These new developments in optical and MR tools allow us to determine the function and capacity of mitochondria noninvasively in order to identify specific defects in vivo that are associated with disease in human and animal muscle. The clinical implication of this unique diagnostic probe is the insight into the nature and extent of dysfunction in metabolic and degenerative disorders, as well as the ability to follow the impact of interventions designed to reverse these disorders. PMID:18930151

  10. [Outlier Detection of Time Series Three-Dimensional Fluorescence Spectroscopy].

    PubMed

    Yu, Shao-hui; Zhang, Yu-jun; Zhao, Nan-jing

    2015-06-01

    The qualitative and quantitative analysis are often interfered by the outliers in time series three-dimensional fluorescence spectroscopy. In this work, an efficient outlier detection method is proposed by taking advantage of the characteristics in time dimension and the spectral dimension. Firstly, the wavelength points that are mostly the outliers are extracted by the variance in time dimension. Secondly, by the analysis of the existence styles of outliers and similarity score of any two samples, the cumulative similarity is introduced in spectral dimension. At last, fluorescence intensity at each wavelength of all samples is modified by the correction matrix in time dimension and the outlier detection is completed according the to cumulative similarity scores. The application of the correction matrix in time dimension not only improves the validity of the method but also reduces the computation by the choice of characteristics region in correction matrix. Numerical experiments show that the outliers can still be detected by the 50 percent of all points in spectral dimension. PMID:26601379

  11. 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.

  12. Vibrational fluorescence spectroscopy of single conjugated polymer molecules

    NASA Astrophysics Data System (ADS)

    Müller, J. G.; Anni, M.; Scherf, U.; Lupton, J. M.; Feldmann, J.

    2004-07-01

    Fluorescence spectroscopy of conjugated polymers at the single molecule level provides unique insight into the nature of the emitting state of these organic semiconductors. We are able to verify the picture that molecular excitations form the primary photoexcitations in conjugated polymers by identifying individual chromophore units on rigid rod-like chains of a ladder-type polymer. The observation of a well-defined substructure in the vibronic progression as well as the presence of sum-frequency vibrational modes in the higher order vibrational bands demonstrate the sensitivity of the method. We find that conjugated polymers are excellent materials for single molecule experiments, exhibiting narrow transition lines accompanied only by a limited number of discrete vibrational modes offset by hundreds of cm-1 . We conclude that the high level of structural rigidity of the molecule as well as the presence of shielding sidegroups on the polymer chain reduces vibrational coupling both to the amorphous matrix as well as limiting the number of internal vibrational modes, in contrast to the case for small dye molecules. By studying the fluorescence from different single molecules we are able to image intramolecular and intermolecular disorder directly. We observe a distribution in energy of the electronic transitions due to the characteristic energetic disorder. The intensity of the vibronic side bands is also found to vary from molecule to molecule, which we propose to be related to conformational influence on the strength of coupling between the electronic excitation and vibrational modes. Structural relaxation and intramolecular energy transfer are studied by single molecule site-selective fluorescence. Our results suggest that even in rigid polymer molecules structural relaxation leads to a small Stokes shift of <70cm-1 upon electronic excitation of a single chromophore on a polymer chain at low temperatures. The influence of vibrational and structural relaxation on intramolecular energy transfer in these multichromophoric systems is also discussed.

  13. Detection of atheroma using Photofrin IIr and laser-induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    1991-06-01

    The goal of this study was to investigate laser induced fluorescence spectroscopy (LIFS) as a method of localization of atherosclerotic lesions not visible by angiography using Photofrin IIr enhanced fluorescence. Twenty-four New Zealand White rabbits divided into six groups varying in type of arterial wall lesion and Photofrin IIr administration time (i.v.) were used. Aortic wall fluorescence signals were acquired from the aortic arch to iliac bifurcation. The output of a He-Cd laser (442 nm, 17 mW) was directed at the arterial wall through a 400 micron fiber. The fluorescence signal created in the arterial wall was collected via the same fiber and analyzed by an optical multi-channel analyzer (OMA). The ratio of fluorescence intensities at 630 nm (Photofrin IIr) and 540 nm (autofluorescence of artery wall) was analyzed (I630nm/I540nm). Intensity ratio values 24 hours after administration of Photofrin IIr were found to be as follows: in normal artery wall of 0.30 +/- 0.14 (n equals 3), in mechanically damaged wall of 0.91 +/- 0.65 (n equals 2) and, in atheromatous tissue, 0.88 +/- 0.54 (n equals 4). The intensity ratio of atheromatous tissue without Photofrin IIr was 0.23 +/- 0.04 (n equals 7). These results suggest that the use of Photofrin IIr allows in vivo atheroma detection by LIFS because of its ability to accumulate in atheroma. In addition, accumulation of Photofrin IIr was found in artery walls traumatized by balloon catheter intervention. Using this method, a catheter-based LIFS system may be developed for atheroma detection.

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

    NASA Astrophysics Data System (ADS)

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

    2005-04-01

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

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

    PubMed Central

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

    2013-01-01

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

  16. 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

  17. Methods of single-molecule fluorescence spectroscopy and microscopy

    NASA Astrophysics Data System (ADS)

    Moerner, W. E.; Fromm, David P.

    2003-08-01

    Optical spectroscopy at the ultimate limit of a single molecule has grown over the past dozen years into a powerful technique for exploring the individual nanoscale behavior of molecules in complex local environments. Observing a single molecule removes the usual ensemble average, allowing the exploration of hidden heterogeneity in complex condensed phases as well as direct observation of dynamical state changes arising from photophysics and photochemistry, without synchronization. This article reviews the experimental techniques of single-molecule fluorescence spectroscopy and microscopy with emphasis on studies at room temperature where the same single molecule is studied for an extended period. Key to successful single-molecule detection is the need to optimize signal-to-noise ratio, and the physical parameters affecting both signal and noise are described in detail. Four successful microscopic methods including the wide-field techniques of epifluorescence and total internal reflection, as well as confocal and near-field optical scanning microscopies are described. In order to extract the maximum amount of information from an experiment, a wide array of properties of the emission can be recorded, such as polarization, spectrum, degree of energy transfer, and spatial position. Whatever variable is measured, the time dependence of the parameter can yield information about excited state lifetimes, photochemistry, local environmental fluctuations, enzymatic activity, quantum optics, and many other dynamical effects. Due to the breadth of applications now appearing, single-molecule spectroscopy and microscopy may be viewed as useful new tools for the study of dynamics in complex systems, especially where ensemble averaging or lack of synchronization may obscure the details of the process under study.

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

    NASA Astrophysics Data System (ADS)

    Peters, Sven; Hammer, Martin; Schweitzer, Dietrich

    2011-07-01

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

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

    PubMed

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

    2015-12-01

    CP43 is a chlorophyll-binding protein, which acts as a conduit for the excitation energy transfer. The thermal stability of apo-CP43 was studied by intrinsic fluorescence, exogenous ANS fluorescence, and circular dichroism spectroscopy. Under heat treatment, the structure of apo-CP43 changed and existed transition state occurred between 56 and 62 °C by the intrinsic, exogenous ANS fluorescence and the analysis of hydrophobicity. Besides, the isosbestic point of the sigmoidal curve was 58.10 ± 1.02 °C by calculating ?-helix transition and the Tm was 56.45 ± 0.52 and 55.59 ± 0.68 °C by calculating the unfolded fraction of tryptophan and tyrosine fluorescence, respectively. During the process of unfolding, the hydrophobic structure of C-terminal segment firstly started to expose at 40 °C, and then the hydrophobic cluster adjacent to the N-terminal segment also gradually exposed to hydrophilic environment with increasing temperature. Our results indicated that heat treatment, especially above 40 °C, has an important impact on the structural stability of apo-CP43. PMID:26071019

  20. Frequently asked questions about in vivo chlorophyll fluorescence: practical issues.

    PubMed

    Kalaji, Hazem M; Schansker, Gert; Ladle, Richard J; Goltsev, Vasilij; Bosa, Karolina; Allakhverdiev, Suleyman I; Brestic, Marian; Bussotti, Filippo; Calatayud, Angeles; Dąbrowski, Piotr; Elsheery, Nabil I; Ferroni, Lorenzo; Guidi, Lucia; Hogewoning, Sander W; Jajoo, Anjana; Misra, Amarendra N; Nebauer, Sergio G; Pancaldi, Simonetta; Penella, Consuelo; Poli, DorothyBelle; Pollastrini, Martina; Romanowska-Duda, Zdzislawa B; Rutkowska, Beata; Serôdio, João; Suresh, Kancherla; Szulc, Wiesław; Tambussi, Eduardo; Yanniccari, Marcos; Zivcak, Marek

    2014-11-01

    The aim of this educational review is to provide practical information on the hardware, methodology, and the hands on application of chlorophyll (Chl) a fluorescence technology. We present the paper in a question and answer format like frequently asked questions. Although nearly all information on the application of Chl a fluorescence can be found in the literature, it is not always easily accessible. This paper is primarily aimed at scientists who have some experience with the application of Chl a fluorescence but are still in the process of discovering what it all means and how it can be used. Topics discussed are (among other things) the kind of information that can be obtained using different fluorescence techniques, the interpretation of Chl a fluorescence signals, specific applications of these techniques, and practical advice on different subjects, such as on the length of dark adaptation before measurement of the Chl a fluorescence transient. The paper also provides the physiological background for some of the applied procedures. It also serves as a source of reference for experienced scientists. PMID:25119687

  1. Longitudinal in vivo two-photon fluorescence imaging

    PubMed Central

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

    2014-01-01

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

  2. 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.

  3. In vivo two-dimensional NMR correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Kraft, Robert A.

    1999-10-01

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

  4. Interaction of fluorescent dyes with DNA and spermine using fluorescence spectroscopy.

    PubMed

    Gracie, K; Smith, W E; Yip, P; Sutter, J U; Birch, D J S; Graham, D; Faulds, K

    2014-08-01

    Oligonucleotides labelled with fluorescent dyes are widely used as probes for the identification of DNA sequences in detection methods using optical spectroscopies such as fluorescence and surface enhanced Raman scattering (SERS). Spermine is widely used in surface enhanced based assays as a charge reduction and aggregating agent as it interacts strongly with the phosphate backbone and has shown to enhance the signal of a labelled oligonucleotide. The fluorescence intensity of two commonly used labels, FAM and TAMRA, were compared when spermine was added under different experimental conditions. There was a marked difference upon conjugating the free dye to an oligonucleotide, when FAM was conjugated to an oligonucleotide there was around a six fold decrease in emission, compared to a six fold increase when TAMRA was conjugated to an oligonucleotide. Dye labelled single and double stranded DNA also behaved differently with double stranded DNA labelled with FAM being a much more efficient emitter in the mid pH range, however TAMRA becomes increasingly less efficient as the pH rises. Upon addition of the base spermine, signal enhancement from the FAM labelled oligonucleotide is observed. Increasing probe concentrations of TAMRA oligonucleotide above 0.5 μM led to signal reduction most likely through quenching, either by an interaction with guanine, or through self-quenching. By using different bases for comparison, spermine and triethylamine (TEA), different affects were observed in the measured fluorescence signals. When TEA was added to FAM, a reduction in the pH dependence of fluorescence was observed, which may be useful for mid pH range assays. With the drive to increase information content and decrease time and complexity of DNA assays it is likely that more assays will be carried out in complex media such as extracted DNA fragments and PCR product. This model study indicates that dye DNA and dye spermine interactions are dye specific and that extreme care with conditions is necessary particularly if it is intended to determine the concentrations of multiple analytes using probes labelled with different dyes. PMID:24915043

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

    PubMed Central

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

    2015-01-01

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

  6. 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

  7. 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.

  8. Laboratory studies of in vivo fluorescence of phytoplankton

    NASA Technical Reports Server (NTRS)

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

    1978-01-01

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

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

    PubMed

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

    2013-09-01

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

  10. In vivo and in situ imaging of experimental invasive pulmonary aspergillosis using fibered confocal fluorescence microscopy.

    PubMed

    Morisse, Hélène; Heyman, Loraine; Salaün, Mathieu; Favennec, Loïc; Picquenot, Jean Michel; Bohn, Pierre; Thiberville, Luc

    2012-05-01

    Invasive pulmonary aspergillosis (IPA) is a highly fatal disease in immunosuppressed patients. In this study, we assessed fibered confocal fluorescence microscopy (FCFM), a new endoscopic technique that enables in vivo microscopic imaging of the distal lung, as a tool for in vivo imaging of IPA. IPA was induced in immunosuppressed rats using a wild strain of Aspergillus fumigatus (n = 6) or a fluorescent transformed TAG-RFP A. fumigatus strain (n = 10). Subpleural areas of pulmonary infection were imaged in vivo using FCFM employing a transthoracic approach. Results were compared to three immunosuppressed control groups, i.e., non-inoculated rats (n = 4), rats inoculated with sterile Phospate-buffer saline (PBS; n = 5), and rats inoculated with Geosmithia argillacea (n = 6). Only hyphae of TAG-RFP A. fumigatus were detectable both in vitro and in vivo by FCFM. In vivo, a local infiltration of fluorescent alveolar macrophages was observed with FCFM in IPA areas in all fungal infections groups, but also in focal inflammatory areas in the immunosuppressed PBS group. A specific fibrillar fluorescence was observed in IPA areas with the TAG-RFP A. fumigatus group, with a 83% sensitivity, a 100% specificity, a 100% positive predictive value and 94% negative predictive value. FCFM provides a new tool to study host-aspergillus interactions in vivo. PMID:22004362

  11. Simple and rapid determination of homozygous transgenic mice via in vivo fluorescence imaging

    PubMed Central

    Li, Wei; Xiao, Gaofang; Li, Yanqing; Xie, Raoying; Huang, Hailu; Zhong, Lin; Wu, Qinghong; Wang, Wanshan; Huang, Wenhua; Yao, Kaitai; Xiao, Dong; Sun, Yan

    2015-01-01

    Setting up breeding programs for transgenic mouse strains require to distinguish homozygous from the heterozygous transgenic animals. The combinational use of the fluorescence reporter transgene and small animal in-vivo imaging system might allow us to rapidly and visually determine the transgenic mice homozygous for transgene(s) by the in vivo fluorescence imaging. RLG, RCLG or Rm17LG transgenic mice ubiquitously express red fluorescent protein (RFP). To identify homozygous RLG transgenic mice, whole-body fluorescence imaging for all of newborn F2-generation littermates produced by mating of RFP-positive heterozygous transgenic mice (F1-generation) derived from the same transgenic founder was performed. Subsequently, the immediate data analysis of the in vivo fluorescence imaging was carried out, which greatly facilitated us to rapidly and readily distinguish RLG transgenic individual(s) with strong fluorescence from the rest of F2-generation littermates, followed by further determining this/these RLG individual(s) showing strong fluorescence to be homozygous, as strongly confirmed by mouse mating. Additionally, homozygous RCLG or Rm17LG transgenic mice were also rapidly and precisely distinguished by the above-mentioned optical approach. This approach allowed us within the shortest time period to obtain 10, 8 and 2 transgenic mice homozygous for RLG, RCLG and Rm17LG transgene, respectively, as verified by mouse mating, indicating the practicality and reliability of this optical method. Taken together, our findings fully demonstrate that the in vivo fluorescence imaging offers a visual, rapid and reliable alternative method to the traditional approaches (i.e., mouse mating and real-time quantitative PCR) in identifying homozygous transgenic mice harboring fluorescence reporter transgene under the control of a ubiquitous promoter in the situation mentioned in this study. PMID:26472024

  12. Simple and rapid determination of homozygous transgenic mice via in vivo fluorescence imaging.

    PubMed

    Lin, Xiaolin; Jia, Junshuang; Qin, Yujuan; Lin, Xia; Li, Wei; Xiao, Gaofang; Li, Yanqing; Xie, Raoying; Huang, Hailu; Zhong, Lin; Wu, Qinghong; Wang, Wanshan; Huang, Wenhua; Yao, Kaitai; Xiao, Dong; Sun, Yan

    2015-11-17

    Setting up breeding programs for transgenic mouse strains require to distinguish homozygous from the heterozygous transgenic animals. The combinational use of the fluorescence reporter transgene and small animal in-vivo imaging system might allow us to rapidly and visually determine the transgenic mice homozygous for transgene(s) by the in vivo fluorescence imaging. RLG, RCLG or Rm17LG transgenic mice ubiquitously express red fluorescent protein (RFP). To identify homozygous RLG transgenic mice, whole-body fluorescence imaging for all of newborn F2-generation littermates produced by mating of RFP-positive heterozygous transgenic mice (F1-generation) derived from the same transgenic founder was performed. Subsequently, the immediate data analysis of the in vivo fluorescence imaging was carried out, which greatly facilitated us to rapidly and readily distinguish RLG transgenic individual(s) with strong fluorescence from the rest of F2-generation littermates, followed by further determining this/these RLG individual(s) showing strong fluorescence to be homozygous, as strongly confirmed by mouse mating. Additionally, homozygous RCLG or Rm17LG transgenic mice were also rapidly and precisely distinguished by the above-mentioned optical approach. This approach allowed us within the shortest time period to obtain 10, 8 and 2 transgenic mice homozygous for RLG, RCLG and Rm17LG transgene, respectively, as verified by mouse mating, indicating the practicality and reliability of this optical method. Taken together, our findings fully demonstrate that the in vivo fluorescence imaging offers a visual, rapid and reliable alternative method to the traditional approaches (i.e., mouse mating and real-time quantitative PCR) in identifying homozygous transgenic mice harboring fluorescence reporter transgene under the control of a ubiquitous promoter in the situation mentioned in this study. PMID:26472024

  13. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2007-02-01

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

  16. Dispersed Fluorescence Spectroscopy of Jet-Cooled Methylcyclohexoxy Radicals

    NASA Astrophysics Data System (ADS)

    Alam, Jahangir; Reza, Md Asmaul; Mason, Amy; Liu, Jinjun

    2015-06-01

    Vibrational structures of the nearly degenerate tilde X and tilde A states of all four positional isomers of the methylcyclohexoxy (MCHO) radicals were studied by jet-cooled dispersed fluorescence (DF) spectroscopy, which unravels the effect of methyl substitution at different positions on the six-membered ring. Experimentally observed vibronic transitions in the DF spectra were assigned based on vibrational frequencies from quantum chemical calculations and predicted Franck-Condon factors that take into account the Duschinsky rotation. DF spectra of 2-, 3-, and 4-MCHO radicals are dominated by CO-stretch progressions or the progressions of CO-stretch modes in combination with the excited vibrational modes. DF spectra of two lowest-energy conformers of the tertiary 1-MCHO radical, chair-axial and chair equatorial, are significantly different from each other and from those of the other three positional isomers. Strong C-CH_3 stretch progressions as well as progressions of its combination bands with the CO stretch modes or the excited modes were observed. Such differences between the isomers and the conformers can be explained by variation of geometry and symmetry of the electronic states of cyclohexoxy upon methyl substitution at different positions. DF study of MCHO provides direct measurement of the energy separation between the tilde A and tilde X states that are subject to the pseudo-Jahn-Teller effect.

  17. 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.

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

    PubMed

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

    2009-01-01

    Noninvasive and reliable quantification of rheological characteristics in the nucleus is extremely useful for fundamental research and practical applications in medicine and biology. This study examines the use of fluorescence correlation spectroscopy (FCS) to noninvasively determine nucleoplasmic viscosity (eta(nu)), an important parameter of nucleoplasmic rheology. Our FCS analyses show that eta(nu) of lung adenocarcinoma (ASTC-a-1) and HeLa cells are 1.77+/-0.42 cP and 1.40+/-0.27 cP, respectively, about three to four times larger than the water viscosity at 37 degrees C. eta(nu) was reduced by 31 to 36% upon hypotonic exposure and increased by 28 to 52% from 37 to 24 degrees C. In addition, we found that eta(nu) of HeLa cells reached the lowest value in the S phase and that there was no significant difference of eta(nu) between in the G1 and G2 phases. Last, nucleoplasmic viscosity was found to be larger than cytoplasmic viscosity in both HeLa and ASTC-a-1 cells. These results indicate that FCS can be used as a noninvasive tool to investigate the microenvironment of living cells. This is the first report on the measurement of eta(nu) in living cells synchronized in the G1, S, and G2 phases. PMID:19405743

  19. Inference of protein diffusion probed via fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Tsekouras, Konstantinos

    2015-03-01

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

  20. DOM transformations in stream biofilms shown by fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2012-04-01

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

  1. Intraoperative metastases detection by laser-induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    1991-06-01

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

  2. Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

    DOE PAGESBeta

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

    2014-08-14

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-06-01

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

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

    NASA Astrophysics Data System (ADS)

    Salomatina, Elena V.; Pravdin, Alexander B.

    2003-10-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-09-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2008-01-01

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

  8. In vivo Raman spectroscopy for oral cancers diagnosis

    NASA Astrophysics Data System (ADS)

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

    2012-01-01

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

  9. 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

  10. Biocompatible fluorescent nanoparticles for in vivo stem cell tracking

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

  11. 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.

  12. 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.

  13. 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

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

    NASA Astrophysics Data System (ADS)

    Zhou, Chao

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

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

    NASA Astrophysics Data System (ADS)

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

    2007-07-01

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

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

    SciTech Connect

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

    1990-09-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  18. Frequency-domain fluorescent diffusion tomography of turbid media and in-vivo tissues

    NASA Astrophysics Data System (ADS)

    Yang, Ye; Iftimia, Nicusor; Xu, Yong; Jiang, Huabei

    2001-06-01

    The reconstruction of fluorescence lifetime distributions in heterogeneous turbid media and tumor-bearing animals are experimentally demonstrated by frequency-domain measurements. A set of coupled diffusion equations are used to describe the propagation of excitation and fluorescent emission light in multiply scattering media. A finite element based reconstruction algorithm combined with Marquardt and Tikhonov regularization methods are used to obtain the fluorescence images. The experimental set-up is an automatic multi-channel frequency-domain system. 16 sources and 16 detectors are used. Experiments are performed using indocyanine green (ICG) and 3,3'-diethylthiatricarbocyanine iodide (DTTCI) in tissue-like phantoms of both single- and multi-target configurations with considerations of perfect and imperfect uptake of fluorescence dyes in the scattering media. ICG are used in tumor-bearing animal studies. Our results show that the fluorescence lifetime image of the heterogeneities within a circular surrounding medium and in-vivo tissue can be reconstructed successfully.

  19. 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.

  20. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

  2. Fluorescence lifetime spectroscopy for breast cancer margins assessment

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-09-01

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

  5. 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

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

    PubMed

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

    2009-01-01

    We interrogate the ability of free-space fluorescence tomography to image small animals in vivo using charge-coupled device (CCD) camera measurements over 360-deg noncontact projections. We demonstrate the performance of normalized dual-wavelength measurements that are essential for in-vivo use, as they account for the heterogeneous distribution of photons in tissue. In-vivo imaging is then showcased on mouse lung and brain tumors cross-validated by x-ray microcomputed tomography and histology. PMID:19566290

  7. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  9. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2009-09-01

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

  12. Development of a time-gated fluorescence lifetime microscope for in vivo corneal metabolic imaging

    NASA Astrophysics Data System (ADS)

    Silva, Susana F.; Batista, Ana; Castejón, Olga C.; Quadrado, Maria João.; Domingues, José Paulo; Morgado, Miguel

    2015-07-01

    Metabolic imaging can be a valuable tool in the early diagnosis of corneal diseases. Cell metabolic changes can be assessed through non-invasive optical methods due to the autofluorescence of metabolic co-factors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). Both molecules exhibit double exponential fluorescence decays, with well-separated short and long lifetime components, which are related to their protein-bound and free states. Corneal metabolism can be monitored by measuring the relative contribution of these two components. Here we report on the development of a fluorescence lifetime imaging microscope for in vivo measurement of FAD fluorescence lifetimes in corneal cells. The microscope is based on one-photon fluorescence excitation, through a pulsed blue diode laser. Fluorescence lifetime imaging is achieved using the Time-Gated technique. Structured illumination is used to improve the low axial resolution of wide-field time-gated FLIM. A Digital Micromirror Device (DMD) is used to produce the sinusoidal patterns required by structural illumination. The DMD control is integrated with the acquisition software of the imaging system which is based on an ultra-high speed gated image intensifier coupled to a CCD camera. We present preliminary results concerning optical and timing performance of the fluorescence lifetime microscope. Preliminary tests with ex-vivo bovine corneas are also described.

  13. Mini review of ultrafast fluorescence polarization spectroscopy [invited].

    PubMed

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

    2013-02-10

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

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

    PubMed Central

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

    2014-01-01

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

  15. 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.

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

    SciTech Connect

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

    2014-10-15

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

  17. A fluorescence spectroscopy study of traditional Chinese medicine Angelica

    NASA Astrophysics Data System (ADS)

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

    2013-10-01

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

  18. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2007-02-01

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

  20. Fluorescence modeling for optimized-binary compressive detection Raman spectroscopy.

    PubMed

    Rehrauer, Owen G; Mankani, Bharat R; Buzzard, Gregery T; Lucier, Bradley J; Ben-Amotz, Dor

    2015-09-01

    The recently-developed optimized binary compressive detection (OB-CD) strategy has been shown to be capable of using Raman spectral signatures to rapidly classify and quantify liquid samples and to image solid samples. Here we demonstrate that OB-CD can also be used to quantitatively separate Raman and fluorescence features, and thus facilitate Raman-based chemical analyses in the presence of fluorescence background. More specifically, we describe a general strategy for fitting and suppressing fluorescence background using OB-CD filters trained on third-degree Bernstein polynomials. We present results that demonstrate the utility of this strategy by comparing classification and quantitation results obtained from liquids and powdered mixtures, both with and without fluorescence. Our results demonstrate high-speed Raman-based quantitation in the presence of moderate fluorescence. Moreover, we show that this OB-CD based method is effective in suppressing fluorescence of variable shape, as well as fluorescence that changes during the measurement process, as a result of photobleaching. PMID:26368484

  1. Fluorescence spectroscopy: A promising tool for carbonate petrology

    SciTech Connect

    Vice, M.A.; Bensley, D.F.; Utgaard, J.E. . 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.

  2. 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.

  3. 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.

  4. Non-invasive in vivo tracking of fibrin degradation by fluorescence imaging.

    PubMed

    Wolbank, Susanne; Pichler, Valentin; Ferguson, James Crawford; Meinl, Alexandra; van Griensven, Martijn; Goppelt, Andreas; Redl, Heinz

    2015-08-01

    Fibrin-based sealants consist of natural coagulation factors involved in the final phase of blood coagulation, during which fibrinogen is enzymatically converted by thrombin to form a solid-phase fibrin clot. For applications in tissue regeneration, a controlled process of matrix degradation within a certain period of time is essential for optimal wound healing. Hence, it is desirable to follow the kinetics of fibrinolysis at the application site. Non-invasive molecular imaging systems enable real-time tracking of processes in the living animal. In this study, a non-invasive fluorescence based imaging system was applied to follow and quantify site-specific degradation of fibrin sealant. To enable non-invasive tracking of fibrin in vivo, fibrin-matrix was labelled by incorporation of a fluorophore-conjugated fibrinogen component. Protein degradation and release of fluorescence were, in a first step, correlated in vitro. In vivo, fluorophore-labelled fibrin was subcutaneously implanted in mice and followed throughout the experiment using a multispectral imaging system. For the fluorescent fibrin, degradation correlated with the release of fluorescence from the clots in vitro. In vivo it was possible to follow and quantify implanted fibrin clots throughout the experiment, demonstrating degradation kinetics of approximately 16?days in the subcutaneous compartment, which was further confirmed by histological evaluation of the application site. PMID:25044309

  5. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2012-08-01

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

  7. 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

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

    PubMed Central

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

    2010-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Markovic, Stacey; Li, Siyuan; Niedre, Mark

    2015-03-01

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

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

    PubMed

    Xu, Heng; Rice, Brad W

    2009-01-01

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

  11. 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

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

    NASA Astrophysics Data System (ADS)

    Bartlett, Matthew Allen

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

  13. 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

  14. Prediction of myocardial damage depth induced by extracellular photosensitization reaction using fluorescence measurement in vivo

    NASA Astrophysics Data System (ADS)

    Takahashi, M.; Ogawa, E.; Nakamura, T.; Kawakami, H.; Machida, N.; Yajima, M.; Kurotsu, M.; Ito, A.; Kimura, T.; Arai, T.

    2014-03-01

    We experimentally studied the correlation between myocardial damage depth due to the extracellular photosensitization reaction (PR) using talaporfin sodium and fluorescence-fall amount (FA), which is calculated from the measured backscattering fluorescence intensity via a manipulatable 7 Fr. laser catheter during the PR operation in vivo to establish treatment depth predictor for a non-thermal tachyarrhythmia treatment. The PR was performed to left and/or right ventricle in the open-chest canine heart. The laser irradiation of 663+/-2 nm in wavelength via the laser catheter was operated 15 min after the intravenous administration of talaporfin sodium with concentration of 36.2+/-8.0 μg/ml in plasma. The irradiation was operated with irradiance of 5, 10, 20 W/cm2, and duration of 5, 10, 20 s. Backscattering fluorescence of 710+/-2 nm in wavelength was measured via the laser catheter during the PR. The FA was calculated multiplying the irradiation duration by the fluorescence-fall, which is subtraction of the fluorescence intensity at the kickoff and end of the irradiation. The canine heart was extracted 1 week after the PR and HE stained specimen was histologically evaluated. The correlation of the myocardial damage depth and FA was investigated. We found that FA obtained a logarithmic relation to the myocardial damage depth. We think that the FA might be available to predict the PR induced myocardial damage depth for the application of tachyarrhythmia treatment under catheterization in vivo.

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed

    Ntziachristos, Vasilis; Bremer, Christoph; Weissleder, Ralph

    2003-01-01

    A recent development in biomedical imaging is the non-invasive mapping of molecular events in intact tissues using fluorescence. Underpinning to this development is the discovery of bio-compatible, specific fluorescent probes and proteins and the development of highly sensitive imaging technologies for in vivo fluorescent detection. Of particular interest are fluorochromes that emit in the near infrared (NIR), a spectral window, whereas hemoglobin and water absorb minimally so as to allow photons to penetrate for several centimetres in tissue. In this review article we concentrate on optical imaging technologies used for non-invasive imaging of the distribution of such probes. We illuminate the advantages and limitations of simple photographic methods and turn our attention to fluorescence-mediated molecular tomography (FMT), a technique that can three-dimensionally image gene expression by resolving fluorescence activation in deep tissues. We describe theoretical specifics, and we provide insight into its in vivo capacity and the sensitivity achieved. Finally, we discuss its clinical feasibility. PMID:12541130

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

    PubMed Central

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

    2013-01-01

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

  18. Mesenchymal stem cells: isolation, characterisation and in vivo fluorescent dye tracking.

    PubMed

    Weir, Christopher; Morel-Kopp, Marie-Christine; Gill, Anthony; Tinworth, Kellie; Ladd, Leigh; Hunyor, Stephen N; Ward, Christopher

    2008-10-01

    Cell therapies have been used to regenerate the heart by direct myocardial delivery, by coronary infusion and by surface attached scaffolds. Multipotent mesenchymal stem cells (MSC) with capacity to differentiate into cardiomyocytes and other cell lines have been predominantly trialled in rodents. However, large animal models are increasingly needed to translate basic research into new, safe regenerative therapies. Understanding the mode of action of cell therapies in the mammalian heart has been limited by cell tracking capability. This study examined the ability to track the fate of allogeneic MSC in sheep using various fluorescent dyes. MSC isolated from sheep bone marrow were grown in culture following extraction and flow cytometric characterisation. After labelling with fluorescent tracking dyes (e.g. CFSE and DiI) cells were tested for in vitro and in vivo signal up to six weeks. Labelling effect on cell division and differentiation was studied. Several dyes lost fluorescence and slowed cell division. However, the thiol reactive dye CM-DiI showed detectable in vivo fluorescence in labelled MSC six weeks after injection into sheep skeletal muscle and two weeks after implantation of an MSC coated biomaterial scaffold. CM-DiI labelled MSC differentiated in vitro showed label retention over four weeks. The fluorescent membrane dye CM-DiI tracks implanted sheep MSC and provides an alternative to traditional cell markers such as gene modified GFP. PMID:18396458

  19. 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.

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

    PubMed Central

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

    2016-01-01

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

  1. 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 (FAPα) is a cell surface glycoprotein which is selectively expressed by tumor-associated fibroblasts in malignant tumors but rarely on normal tissues. FAPα 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 FAPα inhibitors has led to the discovery of many FAPα-targeting inhibitors including a drug candidate in a phase II clinical trial, the development of imaging probes to monitor the expression and activity of FAPα in vivo has largely lagged behind. Herein we report an activatable near infrared (NIR) fluorescent probe (ANPFAP) for in vivo optical imaging of FAPα. 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 FAPα 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 FAPα both in vitro and in vivo. In vitro assay on ANPFAP indicated the specificity of the probe to FAPα. The in vivo optical imaging using ANPFAP showed fast tumor uptake as well as high tumor to background contrast on U87MG tumor models with FAPα expression, while much lower signal and tumor contrast were observed in the C6 tumor without FAPα 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 FAPα expressing tumors. PMID:22812530

  2. In-vivo fluorescence lifetime imaging for monitoring the efficacy of the cancer treatment

    PubMed Central

    Ardeshirpour, Yasaman; Chernomordik, Victor; Hassan, Moinuddin; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir

    2015-01-01

    Purpose Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in-vivo fluorescence lifetime imaging with HER2 targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. Experimental Design HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice, bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pre-treatment size. Results Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (~0.13ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment) the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03ns. Conclusions The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in-vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients. PMID:24671949

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

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

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

    SciTech Connect

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

    1986-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

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

  7. Unravelling molecular mechanisms in the fluorescence spectra of doxorubicin in aqueous solution by femtosecond fluorescence spectroscopy.

    PubMed

    Changenet-Barret, Pascale; Gustavsson, Thomas; Markovitsi, Dimitra; Manet, Ilse; Monti, Sandra

    2013-02-28

    Doxorubicin (DOX) is a potent anti-tumoral agent widely used for cancer therapy. Despite numerous studies, the fluorescence properties of DOX, usually exploited for the characterization of the interaction with biological media, have until now led to controversial interpretations, mainly due to self-association of the drug in aqueous solution. We present here the first femtosecond study of DOX based on measurements with the fluorescence up-conversion technique in combination with time-correlated single photon counting using the same laser source. We provide evidence that fluorescence signals of DOX stem from monomers and dimers. DOX dimerization induces a dramatic decrease in the fluorescence quantum yield from 3.9 × 10(-2) to 10(-5) associated with the red shift of the fluorescence spectrum by ~25 nm. While the fluorescence lifetime of the monomer is 1 ns, the dimer fluorescence is found to decay with a lifetime of about 2 ps. In contrast to monomers, the fluorescence anisotropy of dimers is found to be negative. These experimental observations are consistent with an ultrafast internal conversion (<200 fs) between two exciton states, possibly followed by a charge separation process. PMID:23340955

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

  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.

    2015-07-01

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

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

    PubMed

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

    2014-09-01

    Here, we aimed to assess the feasibility of anti-ESAT-6 monoclonal antibody (mAb) coupling with IR783 and rhodamine fluorescent probe in the detection of ESAT-6 expression in tuberculosis tissue of mice using near-infrared fluorescence imaging. IR783 and rhodamine were conjugated to the anti-ESAT-6 mAb or IgG. Mice in the experimental group were injected with fluorescence-labeled mAb probe, and mice in the control group were injected with fluorescence-labeled non-specific IgG antibody. Twenty-four hours later, the lung tissue of mice was examined using ex vivo near-infrared fluorescence imaging. In addition, the contrast-to-noise ratio (CNR) was calculated by measuring the signal intensities of the pulmonary lesions, normal lung tissue and background noise. The frozen lung tissue section was examined under fluorescence microscopy and compared with hemoxylin and eosin (HE) staining. The ex vivo near-infrared fluorescence imaging showed that the fluorescence signal in the lung tuberculosis lesions in the experimental group was significantly enhanced, whereas there was only a weak fluorescence signal or even no fluorescence signal in the control group. CNR values were 64.40 ± 7.02 (n = 6) and 8.75 ± 3.87 (n = 6), respectively (t = 17.01, p < 0.001). The fluorescence accumulation distribution detected under fluorescence microscopy was consistent with HE staining of the tuberculosis region. In conclusion, anti-ESAT-6 mAb fluorescent probe could target and be applied in specific ex vivo imaging of mice tuberculosis, and may be of further use in tuberculosis in living mice. PMID:24170605

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

    PubMed Central

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

    2015-01-01

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

  12. In vivo photoacoustic and fluorescence cystography using clinically relevant dual modal indocyanine green.

    PubMed

    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

  13. 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

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

    PubMed

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

    2016-01-01

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

  15. 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

  16. Long wavelength fluorescence based biosensors for in vivo continuous monitoring of metabolites

    NASA Astrophysics Data System (ADS)

    Thomas, Joseph; Ambroise, Arounaguiry; Birchfield, Kara; Cai, Wensheng; Sandmann, Christian; Singh, Sarabjit; Weidemaier, Kristin; Pitner, J. Bruce

    2006-02-01

    The early stage development studies of novel implantable continuous metabolite sensor systems for glucose, lactate and fatty acids are discussed. These sensors utilize non-enzymatic "reagentless" sensor systems based on NIR fluorophore-labeled binding proteins. For in vivo applications, NIR fluorescence based systems (beyond 600 nm) have the added benefit of reduced interference from background scattering, tissue and serum absorption and cell auto-fluorescence. The long wavelength emission facilitates implanted sensor disks to transmit fluorescence to an external reader through wireless connections and the resulting fluorescence signals can be correlated to metabolite concentrations. We have developed a prototype optical system that uses a bifurcated optical fiber to transmit excitation and read emission at the surface of the skin. With this system, fluorescence signals were read over time through animal skin. The changes in glucose concentration were studied using immobilized sensor proteins and were compared to non-immobilized sensors in solution. For sensors in solution, no response delay was observed. For immobilized systems, the fluorescence response showed a delay corresponding to the diffusion time for the metabolite to equilibrate within the sensor.

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

    NASA Astrophysics Data System (ADS)

    Markovic, Stacey

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

  18. 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).

  19. Soft X-ray spectroscopy of metalloproteins using fluorescence detection

    NASA Astrophysics Data System (ADS)

    Cramer, S. P.; Chen, J.; George, S. J.; van Elp, J.; Moore, J.; Tensch, O.; Colaresi, J.; Yocum, M.; Mullins, O. C.; Chen, C. T.

    1992-08-01

    Fluorescence detection methods have been developed for measuring the L 2.3 X-ray absorption spectra of first transition series metalloprotiens. Samples are prepared as thin films on silicon supports, and mounted on a liquid helium cooled cold finger in a UHV chamber. A windowless Ge array detector discriminates metal L fluorescence from oxygen K a background. The high resolution, strong sensitivity to chemical environment and amenability to quantitative spectral shape analysis indicate that L-edges of the first transition series metals are a useful probe for bioinorganic studies.

  20. Laser-induced fluorescent spectroscopy of steroid hormones

    NASA Astrophysics Data System (ADS)

    Samoilova, Elena S.; Fedorov, Vyacheslav I.; Cherkasova, Olga P.; Meshalkin, Yuri P.

    2002-07-01

    The laser-induced fluorescence spectra of steroid hormones - estradiol, estriol, estrone, androstenedione - are obtained at excitation of the fourth harmonic of Nd:YAG laser radiation. The quantum yields of fluorescence of these substances were rated by means of the relative method. They are 1.11 X 10-1, 5.20 X 10-3, 8.47 X 10-5. The water solution of tryptophan was used as a standard. The set-up sensitivity for high and average quantum yields substances has been defined.

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  2. Conventional and confocal epi-reflection and fluorescence microscopy of the rat kidney in vivo.

    PubMed

    Boyde, A; Capasso, G; Unwin, R J

    1998-01-01

    To visualize superficial and accessible renal tubule cells functioning in situ and to relate what we can 'see' to what we know of their function from more invasive in vivo or less direct in vitro studies means applying and adapting recent advances in epifluorescence and confocal microscopy to improve image resolution and to combine this with the use of fluorescent labels to monitor the handling of specific molecules by the proximal and distal renal tubule cells in vivo. Doing this in living tissue is novel, especially in the kidney. Application of confocal microscopy to the imaging of living tissue, as opposed to isolated cells, has not been widely reported. The kidney surface has been imaged before using the confocal microscope and in preliminary studies we have extended this by using a different confocal system with and without fluorescence. While the studies published up to now have been morphological, comparing standard renal (structural) histology of surface glomeruli and renal tubules with the corresponding in vivo confocal images, more dynamic, real-time studies have been limited. Individual red blood cells can be seen flowing around the peritubule capillary network and nucleated white blood cells can also be distinguished. Tubule cells, endothelial cells, the proximal tubule cell brush border and cell mitochondria can be visualized. Filtration and secretion can be observed, and the early and late parts of the proximal tubule distinguished, and the distal tubule recognized. Localization of fluorescently labeled insulin to the luminal brush border and progressive uptake of label and distribution within proximal tubule cells toward the basolateral (blood side) membrane can be demonstrated. The possibility of monitoring hemodynamic changes and tracking the filtration, uptake, secretion and absorption of fluorescently tagged molecules, as well as intracellular fluorescence, e.g. calcium or pH, is an exciting prospect and is ripe for detailed exploration. PMID:9730655

  3. 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

  4. 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.

  5. Quantitative analysis of essential oils of Thymus daenensis using laser-induced fluorescence and Raman spectroscopy.

    PubMed

    Khoshroo, H; Khadem, H; Bahreini, M; Tavassoli, S H; Hadian, J

    2015-11-10

    Laser-induced fluorescence and Raman spectroscopy are used for the investigation of different genotypes of Thymus daenensis native to the Ilam province of Iran. Different genotypes of T. daenensis essential oils, labeled T1 through T7, possess slight differences with regard to the composition of the thymol. The gas chromatography-mass spectrometry (GC-MS) method is performed to determine the concentration of each constituent as a reference method. The Raman spectra of different concentrations of pure thymol dissolved in hexane as standard samples are obtained via a laboratory prototype Raman spectroscopy setup for the calculation of the calibration curve. The regression coefficient and limit of detection are calculated. The possibility of the differentiation of different genotypes of T. daenensis is also examined by laser-induced fluorescence spectroscopy, although we do not know the exact amounts of their components. All the fluorescence spectral information is used jointly by cluster analysis to differentiate between 7 genotypes. Our results demonstrate the acceptable precision of Raman spectroscopy with GC-MS and corroborate the capacity of Raman spectroscopy in applications in the quantitative analysis field. Furthermore, the cluster analysis results show that laser-induced fluorescence spectroscopy is an acceptable technique for the rapid classification of different genotypes of T. daenensis without having any previous information of their exact amount of constituents. So, the ability to rapidly and nondestructively differentiate between genotypes makes it possible to efficiently select high-quality herbs from many samples. PMID:26560783

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  8. Au:CdHgTe quantum dots for in vivo tumor-targeted multispectral fluorescence imaging.

    PubMed

    Han, Sihai; Mu, Ying; Zhu, Qiangyuan; Gao, Yibo; Li, Zuhong; Jin, Qinhan; Jin, Wei

    2012-05-01

    Near-infrared gold-doped CdHgTe quantum dots (QDs) with improved photoluminescence and biocompatibility were developed using an aqueous solution route with L-glutathione and L-cysteine as stabilizers. As-prepared Au:CdHgTe QDs were covalently linked to arginine-glycine-aspartic acid (RGD) peptide, anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb), and anti- carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) MAb separately. Three Au:CdHgTe QD bioconjugates (QD800-RGD, QD820-anti-CEACAM1, and QD840-anti-EGFR) were successfully used as probes for in vivo tumor-targeted multispectral fluorescence imaging of xenografts. Fluorescence signals from the QD bioconjugates used to detect three tumor markers were spectrally unmixed, and their co-localization was analyzed. The results indicate that multiple tumor markers could be simultaneously detected by multispectral fluorescence imaging in vivo using QD bioconjugates as probes. This approach has excellent potential as an imaging method for the noninvasive exploration and detection of multiple tumor markers in vivo, thereby substantially aiding the diagnosis of cancer. PMID:22447216

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    SciTech Connect

    Mayasich, S.A.; Zygmont, N.J. CDM Federal Programs Corp., South Plainfield, NJ )

    1993-06-01

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

  12. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2007-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

  16. Fluorescence spectroscopy of the retina from scrapie-infected mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently, we have proposed that the fluorescence spectra of sheep retina can be well correlated to the presence or absence of scrapie. Scrapie is the most widespread TSE (transmissible spongiform encephalopathy) affecting sheep and goats worldwide. Mice eyes have been previously reported as a model ...

  17. 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

  18. Variation of fluorescence spectroscopy during the menstrual cycle

    NASA Astrophysics Data System (ADS)

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

    2002-06-01

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

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

    PubMed

    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

  20. 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.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-04-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-08-01

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

  5. Ultrasensitive near-infrared fluorescence-enhanced probe for in vivo nitroreductase imaging.

    PubMed

    Li, Yuhao; Sun, Yun; Li, Jiachang; Su, Qianqian; Yuan, Wei; Dai, Yu; Han, Chunmiao; Wang, Qiuhong; Feng, Wei; Li, Fuyou

    2015-05-20

    Nitroreductase (NTR) can be overexpressed in hypoxic tumors, thus the selective and efficient detection of NTR is of great importance. To date, although a few optical methods have been reported for the detection of NTR in solution, an effective optical probe for NTR monitoring in vivo is still lacking. Therefore, it is necessary to develop a near-infrared (NIR) fluorescent detection probe for NTR. In this study, five NIR cyanine dyes with fluorescence reporting structure decorated with different nitro aromatic groups, Cy7-1-5, have been designed and explored for possible rapid detection of NTR. Our experimental results presented that only a para-nitro benzoate group modified cyanine probe (Cy7-1) could serve as a rapid NIR fluorescence-enhanced probe for monitoring and bioimaging of NTR. The structure-function relationship has been revealed by theoretical study. The linker connecting the detecting and fluorescence reporting groups and the nitro group position is a key factor for the formation of hydrogen bonds and spatial structure match, inducing the NTR catalytic ability enhancement. The in vitro response and mechanism of the enzyme-catalyzed reduction of Cy7-1 have been investigated through kinetic optical studies and other methods. The results have indicated that an electro-withdrawing group induced electron-transfer process becomes blocked when Cy7-1 is catalytically reduced to Cy7-NH2 by NTR, which is manifested in enhanced fluorescence intensity during the detection process. Confocal fluorescence imaging of hypoxic A549 cells has confirmed the NTR detection ability of Cy7-1 at the cellular level. Importantly, Cy7-1 can detect tumor hypoxia in a murine hypoxic tumor model, showing a rapid and significant enhancement of its NIR fluorescence characteristics suitable for fluorescence bioimaging. This method may potentially be used for tumor hypoxia diagnosis. PMID:25923361

  6. Potential of fluorescence spectroscopy to predict fatty acid composition of beef.

    PubMed

    Aït-Kaddour, A; Thomas, A; Mardon, J; Jacquot, S; Ferlay, A; Gruffat, D

    2016-03-01

    The present study aimed to evaluate and compare the ability of front face (FFFS) and synchronous fluorescence spectroscopy (SFS) to predict total fat and FA composition of beef LT muscles coming from 36 animals of 3 breeds (Angus, Limousin and Blond d'Aquitaine). The regression models were performed by using Partial Least Square (PLS) method. In spite of the low number of samples used, the results of this preliminary study demonstrated the ability of fluorescence spectroscopy to predict meat lipids. Nonetheless, the results suggested that the fluorescence spectroscopy is more suited to measure SFA (R(2)p?0.66; RPD?2.29) and MUFA (R(2)p?0.48; RPD?1.49) than PUFA (R(2)p?0.48; RPD?1.63). Moreover, R(2) and RPD factors obtained with FFFS were greater compared to the ones obtained with SFS suggesting that FFFS is more adapted to measure lipid composition of beef meat. PMID:26656871

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

    PubMed

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

    2014-12-01

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

  8. Remote filament-induced fluorescence spectroscopy from thin clouds of smoke

    NASA Astrophysics Data System (ADS)

    Daigle, J.-F.; Kamali, Y.; Roy, G.; Chin, S. L.

    2008-12-01

    Remote filament-induced fluorescence spectroscopy is used to probe a cloud of smoke, produced from burning mosquito coils, located at a distance of 25 m from the laser source and LIDAR detector. CN, CH and C2 molecular fragments were identified in the sample. We demonstrate that temporally gated measurement is an efficient technique to easily suppress spectral contaminations, such as white light and atmospheric N2 fluorescence.

  9. High-speed multispectral fluorescence lifetime imaging implementation for in vivo applications

    PubMed Central

    Shrestha, Sebina; Applegate, Brian E.; Park, Jesung; Xiao, Xudong; Pande, Paritosh; Jo, Javier A.

    2016-01-01

    Fluorescence lifetime imaging microscopy (FLIM) offers a noninvasive approach for characterizing the biochemical composition of biological tissue. In recent years, there has been an increasing interest in the application of multispectral FLIM for medical diagnosis. Central to the clinical translation of FLIM technology is the development of robust, fast, and cost-effective FLIM instrumentation suitable for in vivo tissue imaging. Unfortunately, the predominant multispectral FLIM approaches suffer from limitations that impede the development of high-speed instruments for in vivo applications. We present a cost-effective scanning multispectral FLIM implementation capable of achieving pixel rates on the order of tens of kilohertz, which will facilitate the evaluation of FLIM for in vivo applications. PMID:20680057

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

    PubMed

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

    2013-04-01

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

  11. MoI density measurements by laser induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    1999-01-01

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

  12. The laser-induced fluorescence spectroscopy of yttrium monosulfide

    NASA Astrophysics Data System (ADS)

    Zang, Jianzheng; Zhang, Qun; Zhang, Deping; Qin, Chengbing; Zhang, Qiang; Chen, Yang

    2015-07-01

    We have investigated the laser-induced fluorescence (LIF) excitation spectra and dispersed fluorescence (DF) spectra of yttrium monosulfide (YS) in the energy range of 17 860-20 700 cm-1. Rotational analyses indicated that almost all of the intense vibronic bands can be attributed to the new [ 19.38 ]2?+ (??) - X2?+ (??) transition system. The missing (1, 2), (2, 1) and (3, 3) bands are caused by their very small Franck-Condon factors, as confirmed by our calculations. The new 2?+ state has been suggested to arise from the electronic configuration of (core)1?22?21?33?2?, featuring a charge-transfer nature. Moreover, the spin-rotation parameter ? for the newly observed 2?+ state has been determined to be 0.0206 cm-1, the magnitude of which is larger than the known ? (X2?+) (0.001427 cm-1) but smaller than ? (B2?+) (-0.1515 cm-1).

  13. Time-resolved fluorescence spectroscopy for chemical sensors

    NASA Astrophysics Data System (ADS)

    Draxler, Sonja; Lippitsch, Max E.

    1996-07-01

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

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

    NASA Astrophysics Data System (ADS)

    Cerussi, Albert Edward

    1999-09-01

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

  15. Quantification of DNA and RNA with absorption and fluorescence spectroscopy.

    PubMed

    Gallagher, S R

    2001-05-01

    Reliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers in cell and molecular biology. In addition to the traditional absorbance measurements at 260 nm, two more sensitive fluorescence techniques-Hoescht 33258 dye binding, ethidium bromide binding are described. These three procedures cover a range from 5 to 10 ng/ml DNA to 50 mg/ml DNA. PMID:18228286

  16. 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

  17. 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

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

    NASA Astrophysics Data System (ADS)

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

    2010-09-01

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

  19. Sensitive ?-galactosidase-targeting fluorescence probe for visualizing small peritoneal metastatic tumours in vivo

    PubMed Central

    Asanuma, Daisuke; Sakabe, Masayo; Kamiya, Mako; Yamamoto, Kyoko; Hiratake, Jun; Ogawa, Mikako; Kosaka, Nobuyuki; Choyke, Peter L.; Nagano, Tetsuo; Kobayashi, Hisataka; Urano, Yasuteru

    2015-01-01

    Fluorescence-guided diagnostics is one of the most promising approaches for facile detection of cancer in situ. Here we focus on ?-galactosidase, which is overexpressed in primary ovarian cancers, as a molecular target for visualizing peritoneal metastases from ovarian cancers. As existing fluorescence probes are unsuitable, we have designed membrane-permeable HMRef-?Gal, in which the optimized intramolecular spirocyclic function affords >1,400-fold fluorescence enhancement on activation. We confirm that HMRef-?Gal sensitively detects intracellular ?-galactosidase activity in several ovarian cancer lines. In vivo, this probe visualizes metastases as small as <1?mm in diameter in seven mouse models of disseminated human peritoneal ovarian cancer (SHIN3, SKOV3, OVK18, OVCAR3, OVCAR4, OVCAR5 and OVCAR8). Because of its high brightness, real-time detection of metastases with the naked eye is possible. Endoscopic fluorescence detection of metastases is also demonstrated. The results clearly indicate preclinical potential value of the probe for fluorescence-guided diagnosis of peritoneal metastases from ovarian cancers. PMID:25765713

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

    NASA Astrophysics Data System (ADS)

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

    2013-08-01

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

  1. Evaluation of tea quality by two-dimensional fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Lu, Hao; Dong, Yongjiang; Yan, Chunsheng

    2015-02-01

    In this paper, light-emitting diode (LED) based two-dimensional fluorescence correlation spectroscopy was used to discriminate tea leaves with different grades. The distance between LED and tea samples was changed as an external variable. As the fluorescence spectral data collected through the experiment was large, principal component regression (PCR) was used to extract the important information and analyze the spectral data. The final two-dimensional fluorescence correlation spectra contour maps showed obvious difference between different tea leaves and the predictive results based on the leave-one-out method. It showed the strong ability of this spectral method for tea classification.

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

    NASA Astrophysics Data System (ADS)

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

    2011-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

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

    NASA Astrophysics Data System (ADS)

    Carstea, Elfrida M.; Bridgeman, John

    2015-04-01

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

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

    NASA Astrophysics Data System (ADS)

    Neuweiler, Hannes; Scheffler, Silvia; Sauer, Markus

    2005-08-01

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

  6. 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.

  7. 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.

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

    PubMed Central

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

    2014-01-01

    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

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  10. 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

  11. 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.

  12. Classification evaluation of tobaccos using LED-induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

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

  13. 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

  14. Near-infrared fluorescent peptide probes for imaging of tumor in vivo and their biotoxicity evaluation.

    PubMed

    Liu, Liwei; Lin, Guimiao; Yin, Feng; Law, Wing-Cheung; Yong, Ken-Tye

    2016-04-01

    Optical imaging techniques are becoming increasingly urgent for the early detection and monitoring the progression of tumor development. However, tumor vasculature imaging has so far been largely unexplored because of the lack of suitable optical probes. In this study, we demonstrated the preparation of near-infrared (NIR) florescent RGD peptide probes for noninvasive imaging of tumor vasculature during tumor angiogenesis. The peptide optical probes combined the advantages of NIR emission and RGD peptide, which possesses minimal biological absorption and specially targets the integrin, which highly expressed on activated tumor endothelial cells. In vivo optical imaging of nude mice bearing pancreatic tumor showed that systemically delivered NIR probes enabled us to visualize the tumors at 24 hours post-injection. In addition, we have performed in vivo toxicity study on the prepared fluorescent RGD peptide probes formulation. The blood test results and histological analysis demonstrated that no obvious toxicity was found for the mice treated with RGD peptide probes for two weeks. These studies suggest that the NIR fluorescent peptide probes can be further designed and employed for ultrasensitive fluorescence imaging of angiogenic tumor vasculature, as well as imaging of other pathophysiological processes accompanied by activation of endothelial cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 910-916, 2016. PMID:26691353

  15. 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

  16. Fluorescence spectroscopy of small peptides interacting with microheterogeneous micelles.

    PubMed

    Romani, Ana Paula; Marquezin, Cassia Alessandra; Ito, Amando Siuiti

    2010-01-01

    Many peptides containing tryptophan have therapeutic uses and can be studied by their fluorescent properties. The biological activity of these peptides involves interactions with many cellular components and micelles can function as carriers inside organisms. We report results from the interaction of small peptides containing tryptophan with several microheterogeneous systems: sodium dodecyl sulphate (SDS) micelles; sodium dodecyl sulphate-poly(ethylene oxide) (SDS-PEO) aggregates; and neutral polymeric micelles. We observed that specific parameters, such as wavelength of maximum emission and fluorescence anisotropy, could be used to ascertain the occurrence of interactions. Affinity constants were determined from changes in the intensity of emission while structural modifications in rotameric conformations were verified from time-resolved measurements. Information about the location and diffusion of peptides in the microheterogeneous systems were obtained from tryptophan emission quenching experiments using N-alkylpyridinium ions. The results show the importance of electrostatic and hydrophobic effects, and of the ionization state of charged residues, in the presence of anionic and amphiphilic SDS in the microheterogeneous systems. Conformational stability of peptides is best preserved in the interaction with the neutral polymeric micelles. PMID:19761821

  17. Steady state fluorescence spectroscopy of the photosystem II core complex.

    PubMed

    Cai, Xia; Wang, Shui-Cai; He, Jun-Fang; Liu, Xiao; Peng, Ju-Fang; Kuang, Ting-Yun

    2006-04-01

    Spectroscopic properties within the core complex of photosystem II were investigated by studying the influence of the wavelength of excitation on the fluorescence emission spectrum. At two temperatures, when the core complex of PSII isolated from spinach was excited at six different excitation wavelengths ranging from 436 nm to 520 nm, there is no difference in the maxima of the emission spectra of the core complex, and when the core complex was excited at 480, 489, 495 and 507 nm respectively, fluorescence intensities of maxima decrease with increasing of the absorbance of the beta-carotene molecules at the four excitation wavelengths. The extent of change of the shoulder of the spectra beyond 700 nm depends on the kind of pigment molecule excited. The excitation wavelength can influence the way of energy transfer in the core complex of photosystem II. By Gaussian deconvolution analysis, at least seven groups of chlorophyll a molecules were discovered. They are Chl a(660), Chl a(670), Chl a(680), Chl a(682), Chl a(684), Chl a(687) and Chl a(690). PMID:16622315

  18. 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

  19. Near-field fluorescence cross-correlation spectroscopy on planar membranes.

    PubMed

    Kelly, Christopher V; Wakefield, Devin L; Holowka, David A; Craighead, Harold G; Baird, Barbara A

    2014-07-22

    The organization and dynamics of plasma membrane components at the nanometer scale are essential for biological functions such as transmembrane signaling and endocytosis. Planarized nanoscale apertures in a metallic film are demonstrated as a means of confining the excitation light for multicolor fluorescence spectroscopy to a 55 ± 10 nm beam waist. This technique provides simultaneous two-color, subdiffraction-limited fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy on planar membranes. The fabrication and implementation of this technique are demonstrated for both model membranes and live cells. Membrane-bound proteins were observed to cluster upon the addition of a multivalent cross-linker: On supported lipid bilayers, clusters of cholera toxin subunit B were formed upon cross-linking by an antibody specific for this protein; on living cells, immunoglobulin E bound to its receptor (Fc?RI) on the plasma membranes of RBL mast cells was observed to form clusters upon exposure to a trivalent antigen. The formation of membrane clusters was quantified via fluorescence intensity vs time and changes in the temporal auto- and cross-correlations above a single nanoscale aperture. The illumination profile from a single aperture is analyzed experimentally and computationally with a rim-dominated illumination profile, yielding no change in the autocorrelation dwell time with changes in aperture diameter from 60 to 250 nm. This near-field fluorescence cross-correlation methodology provides access to nanoscale details of dynamic membrane interactions and motivates further development of near-field optical methods. PMID:25004429

  20. 2D fluorescence spectroscopy for monitoring ion-exchange membrane based technologies - Reverse electrodialysis (RED).

    PubMed

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

    2016-01-01

    Reverse electrodialysis (RED) is one of the emerging, membrane-based technologies for harvesting salinity gradient energy. In RED process, fouling is an undesirable operation constraint since it leads to a decrease of the obtainable net power density due to increasing stack electric resistance and pressure drop. Therefore, early fouling detection is one of the main challenges for successful RED technology implementation. In the present study, two-dimensional (2D) fluorescence spectroscopy was used, for the first time, as a tool for fouling monitoring in RED. Fluorescence excitation-emission matrices (EEMs) of ion-exchange membrane surfaces and of natural aqueous streams were acquired during one month of a RED stack operation. Fouling evolvement on the ion-exchange membrane surfaces was successfully followed by 2D fluorescence spectroscopy and quantified using principal components analysis (PCA). Additionally, the efficiency of cleaning strategy was assessed by measuring the membrane fluorescence emission intensity before and after cleaning. The anion-exchange membrane (AEM) surface in contact with river water showed to be significantly affected due to fouling by humic compounds, which were found to cross through the membrane from the lower salinity (river water) to higher salinity (sea water) stream. The results obtained show that the combined approach of using 2D fluorescence spectroscopy and PCA has a high potential for studying fouling development and membrane cleaning efficiency in ion exchange membrane processes. PMID:26497936

  1. Near-Field Fluorescence Cross-Correlation Spectroscopy on Planar Membranes

    PubMed Central

    2015-01-01

    The organization and dynamics of plasma membrane components at the nanometer scale are essential for biological functions such as transmembrane signaling and endocytosis. Planarized nanoscale apertures in a metallic film are demonstrated as a means of confining the excitation light for multicolor fluorescence spectroscopy to a 55 ± 10 nm beam waist. This technique provides simultaneous two-color, subdiffraction-limited fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy on planar membranes. The fabrication and implementation of this technique are demonstrated for both model membranes and live cells. Membrane-bound proteins were observed to cluster upon the addition of a multivalent cross-linker: On supported lipid bilayers, clusters of cholera toxin subunit B were formed upon cross-linking by an antibody specific for this protein; on living cells, immunoglobulin E bound to its receptor (Fc?RI) on the plasma membranes of RBL mast cells was observed to form clusters upon exposure to a trivalent antigen. The formation of membrane clusters was quantified via fluorescence intensity vs time and changes in the temporal auto- and cross-correlations above a single nanoscale aperture. The illumination profile from a single aperture is analyzed experimentally and computationally with a rim-dominated illumination profile, yielding no change in the autocorrelation dwell time with changes in aperture diameter from 60 to 250 nm. This near-field fluorescence cross-correlation methodology provides access to nanoscale details of dynamic membrane interactions and motivates further development of near-field optical methods. PMID:25004429

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

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  3. Single-molecule dynamics of phytochrome-bound fluorophores probed by fluorescence correlation spectroscopy

    PubMed Central

    Miller, Abigail E.; Fischer, Amanda J.; Laurence, Ted; Hollars, Christopher W.; Saykally, Richard J.; Lagarias, J. Clark; Huser, Thomas

    2006-01-01

    Fluorescence correlation spectroscopy (FCS) was used to investigate the hydrodynamic and photophysical properties of PR1 (phytofluor red 1), an intensely red fluorescent biliprotein variant of the truncated cyanobacterial phytochrome 1 (Cph1?, which consists of the N-terminal 514 amino acids). Single-molecule diffusion measurements showed that PR1 has excellent fluorescence properties at the single-molecule level, making it an interesting candidate for red fluorescent protein fusions. FCS measurements for probing dimer formation in solution over a range of protein concentrations were enabled by addition of Cph1? apoprotein (apoCph1?) to nanomolar solutions of PR1. FCS brightness analysis showed that heterodimerization of PR1 with apoCph1? altered the chemical environment of the PR1 chromophore to further enhance its fluorescence emission. Fluorescence correlation measurements also revealed interactions between apoCph1? and the red fluorescent dyes Cy5.18 and Atto 655 but not Alexa Fluor 660. The concentration dependence of protein:dye complex formation indicated that Atto 655 interacted with, or influenced the formation of, the apoCph1 dimer. These studies presage the utility of phytofluor tags for probing single-molecule dynamics in living cells in which the fluorescence signal can be controlled by the addition of various chromophores that have different structures and photophysical properties, thereby imparting different types of information, such as dimer formation or the presence of open binding faces on a protein. PMID:16844775

  4. In vivo fluorescence and photodynamic activity of zinc phthalocyanine administered in liposomes.

    PubMed

    van Leengoed, H L; Cuomo, V; Versteeg, A A; van der Veen, N; Jori, G; Star, W M

    1994-05-01

    Zinc(II) phthalocyanine, a hydrophobic photosensitiser, was incorporated in unilamellar liposomes and studied in vivo for fluorescence kinetics and photodynamic activity. An observation chamber mounted in a dorsal skinfold of female WAG/Rij rats was used as a model system. In the chamber, an isogeneic mammary carcinoma was transplanted in the subcutaneous tissue. Phthalocyanine fluorescence was excited at 610 nm with a power density of 0.25 mW cm-2 and was detected above 665 nm through a high-pass filter using a two-stage image intensifier coupled to a charge-coupled device (CCD) camera. Following i.v. administration of 0.14 mg kg-1 of the drug, the fluorescence pharmacokinetics of the dye in vasculature, normal tissue and tumour tissue was determined as a function of time. Tumour fluorescence increased slowly to a maximum about 3 h post injection (p.i.), and remained well above the normal tissue fluorescence till 24 h p.i. Fluorescence in the circulation was always stronger than in the tissues. A treatment light dose at a wavelength of 675 nm was delivered 24 h p.i. One group of six animals received a total light dose of 150 J cm-2 (100 mW cm-2). A second group of six animals received a total light dose of 450 J cm-2 at the same dose rate. Vascular damage resulting from treatment was observed only at the final stages of the irradiation, despite the relatively high levels of fluorescence in the circulation. Immediate post-treatment (re)transplantation of the content of the chamber into the flank always resulted in tumour regrowth, confirming the presence of viable tumour cells following photodynamic therapy (PDT). When the chamber was left intact, the light dose of 450 J cm-2 yielded complete tissue necrosis. The role of the dye-carrier complex in shielding the vascular surrounding from photoproducts was studied in a third group of animals. The presence of peroxides was demonstrated in the serum of these animals after PDT with zinc phthalocyanine in liposomes (ZnPc-lip) using a total light dose of 450 J cm-2. This ex vivo observation supports the previously reported observations in vitro that the carrier complex is able to quench the photoproducts resulting from photoactivation of the photosensitiser which is present in the circulation. PMID:8180012

  5. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Application of Fluorescence Spectroscopy for Rapid Detection of Pathogens in Food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The potential of fluorescence spectroscopy was investigated for the detection food bone pathogens. E coli, Salmonella and Campylobactor, the most commonly present in food, were selectively identified. Each pathogen, grown in agar plate, was diluted in saline and prepared in different concentrations....

  8. In vivo macroscopic HPD fluorescence reflectance imaging on small animals bearing surface ARO/NPA tumor

    NASA Astrophysics Data System (ADS)

    Autiero, Maddalena; Celentano, Luigi; Laccetti, Paolo; Marotta, Marcello; Mettivier, Giovanni; Montesi, Maria C.; Riccio, Patrizia; Russo, Paolo; Roberti, Giuseppe

    2005-08-01

    Recently multimodal imaging systems have been devised because the combination of different imaging modalities results in the complementarity and integration of the techniques and in a consequent improvement of the diagnostic capabilities of the multimodal system with respect to each separate imaging modality. We developed a simple and reliable HematoPorphyrin (HP) mediated Fluorescence Reflectance Imaging (FRI) system that allows for in vivo real time imaging of surface tumors with a large field of view. The tumor cells are anaplastic human thyroid carcinoma-derived ARO cells, or human papillary thyroid carcinoma-derived NPA cells. Our measurements show that the optical contrast of the tumor region image is increased by a simple digital subtraction of the background fluorescence and that HP fluorescence emissivity of ARO tumors is about 2 times greater than that of NPA tumors, and about 4 times greater than that of healthy tissues. This is also confirmed by spectroscopic measurements on histological sections of tumor and healthy tissues. It was shown also the capability of this system to distinguish the tumor type on the basis of the different intensity of the fluorescence emission, probably related to the malignancy degree. The features of this system are complementary with those ones of a pixel radionuclide detection system, which allows for relatively time expensive, narrow field of view measurements, and applicability to tumors also deeply imbedded in tissues. The fluorescence detection could be used as a large scale and quick analysis tool and could be followed by narrow field, higher resolution radionuclide measurements on previously determined highly fluorescent regions.

  9. 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.

  10. Nonlinear Theory of Anomalous Diffusion and Application to Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Boon, Jean Pierre; Lutsko, James F.

    2015-07-01

    The nonlinear theory of anomalous diffusion is based on particle interactions giving an explicit microscopic description of diffusive processes leading to sub-, normal, or super-diffusion as a result of competitive effects between attractive and repulsive interactions. We present the explicit analytical solution to the nonlinear diffusion equation which we then use to compute the correlation function which is experimentally measured by correlation spectroscopy. The theoretical results are applicable in particular to the analysis of fluorescence correlation spectroscopy of marked molecules in biological systems. More specifically we consider the cases of fluorescently labeled lipids in the plasma membrane and of fluorescent apoferritin (a spherically shaped oligomer) in a crowded dextran solution and we find that the nonlinear correlation spectra reproduce very well the experimental data indicating sub-diffusive molecular motion.

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

    PubMed Central

    Romantsov, Tatyana; Fishov, Itzhak; Krichevsky, Oleg

    2007-01-01

    The morphology and dynamics of DNA in a bacterial nucleoid affects the kinetics of such major processes as DNA replication, gene expression. and chromosome segregation. In this work, we have applied fluorescence correlation spectroscopy to assess the structure and internal dynamics of isolated Escherichia coli nucleoids. We show that structural information can be extracted from the amplitude of fluorescence correlation spectroscopy correlation functions of randomly labeled nucleoids. Based on the developed formalism we estimate the characteristic size of nucleoid structural units for native, relaxed, and positively supercoiled nucleoids. The degree of supercoiling was varied using the intercalating agent chloroquine and evaluated from fluorescence microscopy images. The relaxation of superhelicity was accompanied by 15-fold decrease in the length of nucleoid units (from ?50 kbp to ?3 kbp). PMID:17259281

  12. [Development and evaluation of a spectroscopy system for classification of laser-induced arterial fluorescence spectra].

    PubMed

    Morguet, A J; Andreas, S; Gabriel, R E; Nyga, R; Kreuzer, H

    1997-06-01

    The present study evaluated the potential of fluorescence guidance of laser angioplasty without using a second laser for fluorescence excitation. A prototype spectroscopy system with a grating spectrograph, microchannel plate, CCD array and digital image processor on a personal computer was developed and coupled to a clinical XeCl excimer laser. Using multifibre catheters, specimens of human aorta were ablated in physiological saline and blood. The spectra thus generated were recorded and validated histologically. Five types of spectra could be differentiated. Based on a training set, classification algorithms were developed using multiple linear regression and linear discriminant analysis with intensity ratios as predictor variables. Discriminant analysis yielded prospective classification of the remaining validation spectra with high sensitivity and specificity for each type. These data demonstrate that fluorescence spectroscopy during excimer laser ablation at 308 nm does not require a diagnostic laser. Principal types of atherosclerotic lesions and the media can be differentiated spectroscopically in physiological saline and blood. PMID:9312308

  13. Nonlinear Theory of Anomalous Diffusion and Application to Fluorescence Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Boon, Jean Pierre; Lutsko, James F.

    2015-12-01

    The nonlinear theory of anomalous diffusion is based on particle interactions giving an explicit microscopic description of diffusive processes leading to sub-, normal, or super-diffusion as a result of competitive effects between attractive and repulsive interactions. We present the explicit analytical solution to the nonlinear diffusion equation which we then use to compute the correlation function which is experimentally measured by correlation spectroscopy. The theoretical results are applicable in particular to the analysis of fluorescence correlation spectroscopy of marked molecules in biological systems. More specifically we consider the cases of fluorescently labeled lipids in the plasma membrane and of fluorescent apoferritin (a spherically shaped oligomer) in a crowded dextran solution and we find that the nonlinear correlation spectra reproduce very well the experimental data indicating sub-diffusive molecular motion.

  14. Characterising organic matter in recirculating aquaculture systems with fluorescence EEM spectroscopy.

    PubMed

    Hambly, A C; Arvin, E; Pedersen, L-F; Pedersen, P B; Seredy?ska-Sobecka, B; Stedmon, C A

    2015-10-15

    The potential of recirculating aquaculture systems (RAS) in the aquaculture industry is increasingly being acknowledged. Along with intensified application, the need to better characterise and understand the accumulated dissolved organic matter (DOM) within these systems increases. Mature RASs, stocked with rainbow trout and operated at steady state at four feed loadings, were analysed by dissolved organic carbon (DOC) analysis and fluorescence excitation-emission matrix (EEM) spectroscopy. The fluorescence dataset was then decomposed by PARAFAC analysis using the drEEM toolbox. This revealed that the fluorescence character of the RAS water could be represented by five components, of which four have previously been identified in fresh water, coastal marine water, wetlands and drinking water. The fluorescence components as well as the DOC showed positive correlations with feed loading, however there was considerable variation between the five fluorescence components with respect to the degree of accumulation with feed loading. The five components were found to originate from three sources: the feed; the influent tap water (groundwater); and processes related to the fish and the water treatment system. This paper details the first application of fluorescence EEM spectroscopy to assess DOM in RAS, and highlights the potential applications of this technique within future RAS management strategies. PMID:26141427

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

    NASA Astrophysics Data System (ADS)

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

    1990-08-01

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

  16. In vivo optimizing of intracellular production of heterologous protein in Pichia pastoris by fluorescent scanning.

    PubMed

    Zhang, Yaodong; Yang, Bolun

    2006-10-15

    Specific monitoring of recombinant protein titer in DNA recombinant biotechnology traditionally has relied on SDS-PAGE, Western blotting, or bioactivity-based assays, but these are labor-intensive, time-consuming, and destructive and are not a good choice for the optimization of recombinant protein production. We describe a study in which enhanced green fluorescence protein (EGFP) was fused to the C terminus of a model protein glutathione S-transferase (GST) to optimize the chimeric protein production in Pichia pastoris by measurements of fluorescence of living cells in a 96-well microtiter plate using simple fluorescent scanning. Several common factors (e.g., time course of expression, effect of methanol concentration, frequency of methanol addition, medium pH) were tested using this strategy. Western blotting assay showed that the correct full-length GST-EGFP chimeric protein was expressed intracellularly in P. pastoris. The fluorescence intensity and GST bioactivity of cell extract yielded a direct correlation. The results show that the reported method provides an attractive platform for the optimization of recombinant protein production in vivo in real time as well as handling at least 96 samples in parallel. PMID:16930521

  17. 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

  18. Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo

    NASA Astrophysics Data System (ADS)

    Razansky, Daniel; Distel, Martin; Vinegoni, Claudio; Ma, Rui; Perrimon, Norbert; Köster, Reinhard W.; Ntziachristos, Vasilis

    2009-07-01

    Fluorescent proteins have become essential reporter molecules for studying life at the cellular and sub-cellular level, re-defining the ways in which we investigate biology. However, because of intense light scattering, most organisms and tissues remain inaccessible to current fluorescence microscopy techniques at depths beyond several hundred micrometres. We describe a multispectral opto-acoustic tomography technique capable of high-resolution visualization of fluorescent proteins deep within highly light-scattering living organisms. The method uses multiwavelength illumination over multiple projections combined with selective-plane opto-acoustic detection for artifact-free data collection. Accurate image reconstruction is enabled by making use of wavelength-dependent light propagation models in tissue. By performing whole-body imaging of two biologically important and optically diffuse model organisms, Drosophila melanogaster pupae and adult zebrafish, we demonstrate the facility to resolve tissue-specific expression of eGFP and mCherrry fluorescent proteins for precise morphological and functional observations in vivo.

  19. Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

    NASA Astrophysics Data System (ADS)

    Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

    2014-10-01

    Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.

  20. Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

    DOE PAGESBeta

    Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

    2014-10-31

    Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip windowmore » surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.« less

  1. Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

    SciTech Connect

    Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

    2014-10-31

    Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.

  2. 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.

  3. Fiber based in-vivo imaging of epithelial FAD fluorescence: experiments and simulations

    NASA Astrophysics Data System (ADS)

    Kanakaraj, Bala Nivetha; Narayanan Unni, Sujatha

    2015-03-01

    Fluorescence from endogenous fluorophores has been emerging as a promising biomarker for tissue discrimination resulting a noninvasive screening methodology to understand the biochemical and morphological variations in tissues associated with cancer development. We have developed a scan based fiber optic probe system to image increased flavin adenine dinucleotide (FAD) fluorescence from epithelial tissues under conditions mimicking dysplasia surrounded by normal tissues. Experiments were conducted on optical phantoms mimicking epithelial tissues excited by 450nm LED source. The spectral emission from the sample is collected via optical fibers and the imaging is performed by scanning the sample using a translation stage at desired resolution. Monte Carlo simulations were also performed by devising an optical model corresponding to epithelial tissue and the results were correlated with experimental fluorescence measurements. This whole field imaging approach could be useful for in vivo assessment of tissue pathologies based on auto fluorescence and can give a better quantitative approach for estimation of tissue properties by correlating the experimental and simulated data.

  4. Correlative electron and fluorescence microscopy of magnetotactic bacteria in liquid: toward in vivo imaging.

    PubMed

    Woehl, Taylor J; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A; Prozorov, Tanya

    2014-01-01

    Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria. PMID:25358460

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

    NASA Astrophysics Data System (ADS)

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

    2012-07-01

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

  6. Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

    PubMed Central

    Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

    2014-01-01

    Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria. PMID:25358460

  7. Compact low-cost detector for in vivo assessment of microphytobenthos using laser induced fluorescence

    NASA Astrophysics Data System (ADS)

    Utkin, A. B.; Vieira, S.; Marques da Silva, J.; Lavrov, A.; Leite, E.; Cartaxana, P.

    2013-03-01

    The development of a compact low-cost detector for non-destructive assessment of microphytobenthos using laser induced fluorescence was described. The detector was built from a specially modified commercial miniature fiber optic spectrometer (Ocean Optics USB4000). Its usefulness is experimentally verified by the study of diatom-dominated biofilms inhabiting the upper layers of intertidal sediments of the Tagus Estuary, Portugal. It is demonstrated that, operating with a laser emitter producing 30 mJ pulses at the wavelength of 532 nm, the detector is capable to record fluorescence signals with sufficient intensity for the quantitative biomass characterization of the motile epipelic microphytobenthic communities and to monitor their migratory activity. This paves the way for building an entire emitter-detector LIF system for microphytobenthos monitoring, which will enable microalgae communities occupying hardly accessible intertidal flats to be monitored in vivo at affordable cost.

  8. 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

  9. In Vivo Optical Imaging of Acute Cell Death Using a Near-Infrared Fluorescent Zinc-Dipicolylamine Probe

    PubMed Central

    Smith, Bryan A.; Gammon, Seth T.; Xiao, Shuzhang; Wang, Wei; Chapman, Sarah; McDermott, Ryan; Suckow, Mark A.; Johnson, James R.; Piwnica-Worms, David; Gokel, George W.; Smith, Bradley D.; Leevy, W. Matthew

    2013-01-01

    Cell death is a fundamental biological process that is present in numerous disease pathologies. Fluorescent probes that detect cell death have been developed for a myriad of research applications ranging from microscopy to in vivo imaging. Here we describe a synthetic near infrared conjugate of zinc(II)-dipicolylamine (Zn2+-DPA) for in vivo imaging of cell death. Chemically induced in vivo models of myopathy were established using an ionphore, ethanol, or ketamine as chemical cytotoxins. The Zn2+-DPA fluorescent probe or corresponding control was subsequently injected and whole animal fluorescence imaging demonstrated probe uptake at the site of muscle damage, which was confirmed by ex vivo and histological analyses. Further, a comparative study with a near-infrared fluorescent conjugate Annexin V showed less intense uptake at the site of muscle damage and high accumulation in the bladder. The results indicate that the fluorescent Zn2+-DPA conjugate is an effective probe for in vivo cell death detection and in some cases may be an appropriate alternative to fluorescent Annexin V conjugates. PMID:21323375

  10. In vivo glucose measurement by surface-enhanced Raman spectroscopy.

    PubMed

    Stuart, Douglas A; Yuen, Jonathan M; Shah, Nilam; Lyandres, Olga; Yonzon, Chanda R; Glucksberg, Matthew R; Walsh, Joseph T; Van Duyne, Richard P

    2006-10-15

    This paper presents the first in vivo application of surface-enhanced Raman scattering (SERS). SERS was used to obtain quantitative in vivo glucose measurements from an animal model. Silver film over nanosphere surfaces were functionalized with a two-component self-assembled monolayer, and subcutaneously implanted in a Sprague-Dawley rat such that the glucose concentration of the interstitial fluid could be measured by spectroscopically addressing the sensor through an optical window. The sensor had relatively low error (RMSEC = 7.46 mg/dL (0.41 mM) and RMSEP = 53.42 mg/dL (2.97 mM). PMID:17037923

  11. Multimodal in vivo imaging of oral cancer using fluorescence lifetime, photoacoustic and ultrasound techniques

    PubMed Central

    Fatakdawala, Hussain; Poti, Shannon; Zhou, Feifei; Sun, Yang; Bec, Julien; Liu, Jing; Yankelevich, Diego R.; Tinling, Steven P.; Gandour-Edwards, Regina F.; Farwell, D. Gregory; Marcu, Laura

    2013-01-01

    This work reports a multimodal system for label-free tissue diagnosis combining fluorescence lifetime imaging (FLIm), ultrasound backscatter microscopy (UBM), and photoacoustic imaging (PAI). This system provides complementary biochemical, structural and functional features allowing for enhanced in vivo detection of oral carcinoma. Results from a hamster oral carcinoma model (normal, precancer and carcinoma) are presented demonstrating the ability of FLIm to delineate biochemical composition at the tissue surface, UBM and related radiofrequency parameters to identify disruptions in the tissue microarchitecture and PAI to map optical absorption associated with specific tissue morphology and physiology. PMID:24049693

  12. In vivo near-infrared fluorescence imaging of CD105 expression during tumor angiogenesis

    PubMed Central

    Yang, Yunan; Zhang, Yin; Hong, Hao; Liu, Glenn; Leigh, Bryan R.; Cai, Weibo

    2011-01-01

    Objectives Angiogenesis is an indispensable process during tumor development. The currently accepted standard method for quantifying tumor angiogenesis is to assess microvessel density (MVD) based on CD105 staining, which is an independent prognostic factor for survival in patients of most solid tumor types. The goal of this study is to evaluate tumor angiogenesis in a mouse model by near-infrared fluorescence (NIRF) imaging of CD105 expression. Methods TRC105, a human/murine chimeric anti-CD105 monoclonal antibody, was conjugated to a NIRF dye (IRDye 800CW; Ex: 778 nm; Em: 806 nm). FACS analysis and microscopy studies were performed to compare the CD105 binding affinity of TRC105 and 800CW-TRC105. In vivo/ex vivo NIRF imaging, blocking studies, and ex vivo histology were performed on 4T1 murine breast tumor-bearing mice to evaluate the ability of 800CW-TRC105 to target tumor angiogenesis. Another chimeric antibody, Cetuximab, was used as an isotype-matched control. Results FACS analysis of HUVECs revealed no difference in CD105 binding affinity between TRC105 and 800CW-TRC105, which was further validated by fluorescence microscopy. 800CW conjugation of TRC105 was achieved in excellent yield (> 85%), with an average of 0.4 800CW molecules per TRC105. Serial NIRF imaging after intravenous injection of 800CW-TRC105 revealed that the 4T1 tumor could be clearly visualized as early as 30 minutes post-injection. Quantitative region-of-interest (ROI) analysis showed that the tumor uptake peaked at about 16 h post-injection. Based on ex vivo NIRF imaging at 48 h post-injection, tumor-uptake of 800CW-TRC105 was higher than most organs thus providing excellent tumor contrast. Blocking experiments, control studies with 800CW-Cetuximab and 800CW, as well as ex vivo histology all confirmed the in vivo target specificity of 800CW-TRC105. Conclusions This is the first successful NIRF imaging study of CD105 expression in vivo. Fast, prominent, persistent, and CD105-specific uptake of the probe during tumor angiogenesis was observed in mouse models. 800CW-TRC105 may be used in the clinic for imaging tumor angiogenesis within the lesions close to the skin surface, tissues accessible by endoscopy, or during image-guided surgery. PMID:21814852

  13. 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.

  14. Tracking structural transitions of bovine serum albumin in surfactant solutions by fluorescence correlation spectroscopy and fluorescence lifetime analysis.

    PubMed

    Zhang, Xuzhu; Poniewierski, Andrzej; Hou, Sen; Soza?ski, Krzysztof; Wisniewska, Agnieszka; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Sun, Lili; Ho?yst, Robert

    2015-03-28

    The structural dynamics of proteins is crucial to their biological functions. A precise and convenient method to determine the structural changes of a protein is still urgently needed. Herein, we employ fluorescence correlation spectroscopy (FCS) to track the structural transition of bovine serum albumin (BSA) in low concentrated cationic (cetyltrimethylammonium chloride, CTAC), anionic (sodium dodecyl sulfate, SDS), and nonionic (pentaethylene glycol monododecyl ether, C12E5 and octaethylene glycol monododecyl ether, C12E8) surfactant solutions. BSA is labelled with the fluorescence dye called ATTO-488 (ATTO-BSA) to obtain steady fluorescence signals for measurements. We find that the diffusion coefficient of BSA decreases abruptly with the surfactant concentration in ionic surfactant solutions at concentrations below the critical micelle concentration (CMC), while it is constant in nonionic surfactant solutions. According to the Stokes-Sutherland-Einstein equation, the hydrodynamic radius of BSA in ionic surfactant solutions amounts to ?6.5 nm, which is 1.7 times larger than in pure water or in nonionic surfactant solutions (3.9 nm). The interaction between BSA and ionic surfactant monomers is believed to cause the structural transition of BSA. We confirm this proposal by observing a sudden shift of the fluorescence lifetime of ATTO-BSA, from 2.3 ns to ?3.0 ns, in ionic surfactant solutions at the concentration below CMC. No change in the fluorescence lifetime is detected in nonionic surfactant solutions. Moreover, by using FCS we are also able to identify whether the structural change of protein results from its self-aggregation or unfolding. PMID:25682837

  15. Analysis of in vivo ROP GTPase activity at the subcellular level by fluorescence resonance energy transfer microscopy.

    PubMed

    Zhu, Lei; Fu, Ying

    2012-01-01

    Proteins generally interact with some other proteins to achieve their cellular functions. Fluorescence resonance energy transfer (FRET) microscopy provides a powerful technique to elucidate such interactions in vivo. FRET occurs when two properly chosen fluorophores are sufficiently close (less than 10 nm). Aided by multiple colored fluorescent proteins (FPs), FRET microscopy has been widely used in live cells for detection of protein-protein interaction and in some cases protein activity in a real-time in vivo manner, which contributes to the understanding of the mechanisms for the regulation of many cellular activities, such as signal transduction pathways. Here, we describe a convenient and fast FRET imaging microscopy involving transiently expressed proteins fused with an FRET pair of fluorescent proteins (e.g., cyn fluorescent protein and yellow fluorescent protein). We describe an example of the FRET-based assay used to analyze ROP GTPase activity in live plant cells. PMID:22576092

  16. In vivo monitoring of toxic metals: assessment of neutron activation and x-ray fluorescence techniques

    SciTech Connect

    Ellis, K.J.

    1986-01-01

    To date, cadmium, lead, aluminum, and mercury have been measured in vivo in humans. The possibilities of monitoring other toxic metals have also been demonstrated, but no human studies have been performed. Neutron activation analysis appears to be most suitable for Cd and Al measurements, while x-ray fluorescence is ideally suited for measurement of lead in superficial bone. Filtered neutron beams and polarized x-ray sources are being developed which will improve in vivo detection limits. Even so, several of the current facilities are already suitable for use in epidemiological studies of selected populations with suspected long-term low-level ''environmental'' exposures. Evaluation and diagnosis of patients presenting with general clinical symptoms attributable to possible toxic metal exposure may be assisted by in vivo examination. Continued in vivo monitoring of industrial workers, especially follow-up measurements, will provide the first direct assessment of changes in body burden and a direct measure of the biological life-times of these metals in humans. 50 refs., 4 figs., 2 tabs.

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

    PubMed

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

    2009-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-10-01

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

  19. Interactions of Indocyanine Green and Lipid in Enhancing Near-Infrared Fluorescence Properties: The Basis for Near-Infrared Imaging in Vivo

    PubMed Central

    2015-01-01

    Indocyanine green (ICG) is a near-infrared (NIR) contrast agent commonly used for in vivo cardiovascular and eye imaging. For medical diagnosis, ICG is limited by its aqueous instability, concentration-dependent aggregation, and rapid degradation. To overcome these limitations, scientists have formulated ICG in various liposomes, which are spherical lipid membrane vesicles with an aqueous core. Some encapsulate ICG, while others mix it with liposomes. There is no clear understanding of lipid–ICG interactions. Therefore, we investigated lipid–ICG interactions by fluorescence and photon correlation spectroscopy. These data were used to design stable and maximally fluorescent liposomal ICG nanoparticles for NIR optical imaging of the lymphatic system. We found that ICG binds to and is incorporated completely and stably into the lipid membrane. At a lipid:ICG molar ratio of 250:1, the maximal fluorescence intensity was detected. ICG incorporated into liposomes enhanced the fluorescence intensity that could be detected across 1.5 cm of muscle tissue, while free ICG only allowed 0.5 cm detection. When administered subcutaneously in mice, lipid-bound ICG in liposomes exhibited a higher intensity, NIR image resolution, and enhanced lymph node and lymphatic vessel visualization. It also reduced the level of fluorescence quenching due to light exposure and degradation in storage. Lipid-bound ICG could provide additional medical diagnostic value with NIR optical imaging for early intervention in cases of lymphatic abnormalities. PMID:24512123

  20. Prediction of cell culture media performance using fluorescence spectroscopy.

    PubMed

    Ryan, Paul W; Li, Boyan; Shanahan, Michael; Leister, Kirk J; Ryder, Alan G

    2010-02-15

    Cell culture media used in industrial mammalian cell culture are complex aqueous solutions that are inherently difficult to analyze comprehensively. The analysis of media quality and variance is of utmost importance in efficient manufacturing. We are exploring the use of rapid "holistic" analytical methods that can be used for routine screening of cell culture media used in industrial biotechnology. The application of rapid fluorescence spectroscopic techniques to the routine analysis of cell culture media (Chinese hamster ovary cell-based manufacture) was investigated. We have developed robust methods which can be used to identify compositional changes and ultimately predict the efficacy of individual fed batch media in terms of downstream protein product yield with an accuracy of +/-0.13 g/L. This is achieved through the implementation of chemometric methods such as multiway robust principal component analysis (MROBPCA), and n-way partial least-squares-discriminant analysis and regression (NPLS-DA and NPLS). This ability to observe compositional changes and predict product yield before media use has enormous potential and should permit the effective elimination of one of the major process variables leading to more consistent product quality and improved yield. These robust and reliable methods have the potential to become an important part of upstream biopharmaceutical quality control and analysis. PMID:20088547

  1. Rapid screening test for porphyria diagnosis using fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Lang, A.; Stepp, H.; Homann, C.; Hennig, G.; Brittenham, G. M.; Vogeser, M.

    2015-07-01

    Porphyrias are rare genetic metabolic disorders, which result from deficiencies of enzymes in the heme biosynthesis pathway. Depending on the enzyme defect, different types of porphyrins and heme precursors accumulate for the different porphyria diseases in erythrocytes, liver, blood plasma, urine and stool. Patients with acute hepatic porphyrias can suffer from acute neuropathic attacks, which can lead to death when undiagnosed, but show only unspecific clinical symptoms such as abdominal pain. Therefore, in addition to chromatographic methods, a rapid screening test is required to allow for immediate identification and treatment of these patients. In this study, fluorescence spectroscopic measurements were conducted on blood plasma and phantom material, mimicking the composition of blood plasma of porphyria patients. Hydrochloric acid was used to differentiate the occurring porphyrins (uroporphyrin-III and coproporphyrin-III) spectroscopically despite their initially overlapping excitation spectra. Plasma phantom mixtures were measured using dual wavelength excitation and the corresponding concentrations of uroporphyrin-III and coproporphyrin-III were determined. Additionally, three plasma samples of porphyria patients were examined and traces of coproporphyrin-III and uroporphyrin-III were identified. This study may therefore help to establish a rapid screening test method with spectroscopic differentiation of the occurring porphyrins, which consequently allows for the distinction of different porphyrias. This may be a valuable tool for clinical porphyria diagnosis and rapid or immediate treatment.

  2. Probing Ternary Complex Equilibria of Crown Ether Ligands by Time-Resolved Fluorescence Spectroscopy

    PubMed Central

    2015-01-01

    Ternary complex formation with solvent molecules and other adventitious ligands may compromise the performance of metal-ion-selective fluorescent probes. As Ca(II) can accommodate more than 6 donors in the first coordination sphere, commonly used crown ether ligands are prone to ternary complex formation with this cation. The steric strain imposed by auxiliary ligands, however, may result in an ensemble of rapidly equilibrating coordination species with varying degrees of interaction between the cation and the specific donor atoms mediating the fluorescence response, thus diminishing the change in fluorescence properties upon Ca(II) binding. To explore the influence of ligand architecture on these equilibria, we tethered two structurally distinct aza-15-crown-5 ligands to pyrazoline fluorophores as reporters. Due to ultrafast photoinduced electron-transfer (PET) quenching of the fluorophore by the ligand moiety, the fluorescence decay profile directly reflects the species composition in the ground state. By adjusting the PET driving force through electronic tuning of the pyrazoline fluorophores, we were able to differentiate between species with only subtle variations in PET donor abilities. Concluding from a global analysis of the corresponding fluorescence decay profiles, the coordination species composition was indeed strongly dependent on the ligand architecture. Altogether, the combination of time-resolved fluorescence spectroscopy with selective tuning of the PET driving force represents an effective analytical tool to study dynamic coordination equilibria and thus to optimize ligand architectures for the design of high-contrast cation-responsive fluorescence switches. PMID:25313708

  3. Probing ternary complex equilibria of crown ether ligands by time-resolved fluorescence spectroscopy.

    PubMed

    Morgan, M Thomas; Sumalekshmy, S; Sarwar, Mysha; Beck, Hillary; Crooke, Stephen; Fahrni, Christoph J

    2014-12-11

    Ternary complex formation with solvent molecules and other adventitious ligands may compromise the performance of metal-ion-selective fluorescent probes. As Ca(II) can accommodate more than 6 donors in the first coordination sphere, commonly used crown ether ligands are prone to ternary complex formation with this cation. The steric strain imposed by auxiliary ligands, however, may result in an ensemble of rapidly equilibrating coordination species with varying degrees of interaction between the cation and the specific donor atoms mediating the fluorescence response, thus diminishing the change in fluorescence properties upon Ca(II) binding. To explore the influence of ligand architecture on these equilibria, we tethered two structurally distinct aza-15-crown-5 ligands to pyrazoline fluorophores as reporters. Due to ultrafast photoinduced electron-transfer (PET) quenching of the fluorophore by the ligand moiety, the fluorescence decay profile directly reflects the species composition in the ground state. By adjusting the PET driving force through electronic tuning of the pyrazoline fluorophores, we were able to differentiate between species with only subtle variations in PET donor abilities. Concluding from a global analysis of the corresponding fluorescence decay profiles, the coordination species composition was indeed strongly dependent on the ligand architecture. Altogether, the combination of time-resolved fluorescence spectroscopy with selective tuning of the PET driving force represents an effective analytical tool to study dynamic coordination equilibria and thus to optimize ligand architectures for the design of high-contrast cation-responsive fluorescence switches. PMID:25313708

  4. Fullerol in human lens and retinal pigment epithelial cells: time domain fluorescence spectroscopy and imaging.

    PubMed

    Taroni, Paola; D'Andrea, Cosimo; Valentini, Gianluca; Cubeddu, Rinaldo; Hu, Dan-Ning; Roberts, Joan E

    2011-06-01

    Fullerol is a fullerene derivative that is extensively hydroxylated [nano-C(60)(OH)(24)] and this makes it water-soluble. These fullerene derivatives have shown promise as drug carriers that bypass ocular barriers but fullerols are also potentially phototoxic to human lens and retinal tissues. Fluorescence imaging is a powerful and non-invasive means of probing nanoparticles in biological systems. However, fullerol nanoparticles have a very low level of fluorescence and have not as yet been imaged in vitro and in vivo. Using specialized measurements including time-correlated single photon counting (TCSPC), fullerol fluorescence was determined in aqueous solutions and detected in both human lens and retinal pigment epithelial cells. Time-resolved fluorescence of fullerol (5-200 ?M) was characterized in aqueous environment, where the fluorescence decay is best fitted with three lifetimes (3 ns, 0.7-0.9 ns and 0.2 ns). Time-resolved microspectrofluorimetry and time-gated fluorescence imaging were performed on both human lens and retinal pigment epithelial cells incubated with increasing fullerol doses (5-500 ?M and 5-50 ?M, respectively). Upon increasing concentration, we observe some shortening of the lifetimes, a reduction in the relative amplitude of the shortest-living component and a corresponding increase in the weight of the intermediate-living species. Time-gated imaging of fullerol fluorescence provided information on its intracellular distribution that correlates with progressive cell damage. Therefore time-gated imaging may potentially be used as a means to investigate fullerol distribution and toxicity in the human lens and retina in vivo. PMID:21298184

  5. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds

    NASA Astrophysics Data System (ADS)

    Holt, David; Parthasarathy, Ashwin B.; Okusanya, Olugbenga; Keating, Jane; Venegas, Ollin; Deshpande, Charuhas; Karakousis, Giorgos; Madajewski, Brian; Durham, Amy; Nie, Shuming; Yodh, Arjun G.; Singhal, Sunil

    2015-07-01

    Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ˜16. Ten animals showed no residual tumor cells in the wound bed (mean SBR<2, P<0.001). None had a local recurrence at >1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

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

    PubMed

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

    2014-05-01

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

  7. Fluorescence lifetime spectroscopy in multiple-scattering environments: an application to biotechnology

    NASA Astrophysics Data System (ADS)

    Cerussi, Albert E.; Gratton, Enrico; Fantini, Sergio

    1999-07-01

    Over the past few years, there has been significant research activity devoted to the application of fluorescence spectroscopy to strongly scattering media, where photons propagate diffusely. Much of this activity focused on fluorescence as a source of contrast enhancement in optical tomography. Our efforts have emphasized the quantitative recovery of fluorescence parameters for spectroscopy. Using a frequency-domain diffusion-based model, we have successfully recovered the lifetime, the absolute quantum yield, the fluorophore concentration, and the emission spectrum of the fluorophore, as well as the absorption and the reduced scattering coefficients at the emission wavelength of the medium in different measurements. In this contribution, we present a sensitive monitor of the binding between ethidium bromide and bovine cells in fresh milk. The spectroscopic contrast was the approximately tenfold increase in the ethidium bromide lifetime upon binding to DNA. The measurement clearly demonstrated that we could quantitatively measure the density of cells in the milk, which is an application vital to the tremendous economic burden of bovine subclinical mastitis detection. Furthermore, we may in principle use the spirit of this technique as a quantitative monitor of the binding of fluorescent drugs inside tissues. This is a first step towards lifetime spectroscopy in tissues.

  8. Assessing the blinking state of fluorescent quantum dots in free solution by combining fluorescence correlation spectroscopy with ensemble spectroscopic methods.

    PubMed

    Dong, Chaoqing; Liu, Heng; Ren, Jicun

    2014-11-01

    The current method for investigating the blinking behavior is to immobilize quantum dots (QDs) in the matrix and then apply a fluorescent technique to monitor the fluorescent trajectories of individual QDs. So far, no method can be used to directly assess the blinking state of ensemble QDs in free solution. In this study, a new method was described to characterize the blinking state of the QDs in free solution by combining single molecule fluorescence correlation spectroscopy (FCS) with ensemble spectroscopic methods. Its principle is based on the observation that the apparent concentration of bright QDs obtained by FCS is less than its actual concentration measured by ensemble spectroscopic method due to the QDs blinking. We proposed a blinking index (Kblink) for characterizing the blinking state of QDs, and Kblink is defined as the ratio of the actual concentration (Cb,actual) measured by the ensemble spectroscopic method to the apparent concentration (Cb,app) of QDs obtained by FCS. The effects of certain factors such as laser intensity, growth process, and ligands on blinking of QDs were investigated. The Kblink data of QDs obtained were successfully used to characterize the blinking state of QDs and explain certain experimental results. PMID:25290853

  9. Center for Fluorescence Spectroscopy: advanced studies of fluorescence dynamics, lifetime imaging, clinical sensing, two-photon excitation, and light quenching

    NASA Astrophysics Data System (ADS)

    Lakowicz, Joseph R.; Malak, Henryk M.; Gryczynski, Ignacy; Szmacinski, Henryk; Kusba, Jozef; Akkaya, Engin; Terpetschnig, Ewald A.; Johnson, Michael L.

    1994-08-01

    The Center for Fluorescence Spectroscopy (CFS) is a multi-user facility providing state of the art time-resolved fluorescence instrumentation and software for scientists, whose research can be enhanced by such experimental data. The CFS is a national center, supported by the National Center for Research Resources Division of the National Institutes of Health, and in part by the National Science Foundation. Both time-domain (TD) and frequency- domain (FD) measurements (10 MHz to 10 Ghz) are available, with a wide range of excitation and emission wavelengths (UV to NIR). The data can be used to recover distances and site-to-site diffusion in protein, interactions between macromolecules, accessibility of fluorophores to quenchers, and the dynamic properties of proteins, membranes and nucleic acids. Current software provides for analysis of multi-exponential intensity and anisotropy decays, lifetime distribution, distance distributions for independent observation of fluorescence donors and acceptors, transient effects in collisional quenching, phase-modulation spectra and time-resolved emission spectra. Most programs provide for global analysis of multiple data sets obtained under similar experimental conditions. Data can be analyzed on-site by connection with the CFS computers through the internet. During six years of operation we have established scientific collaborations with over 30 academic and industrial groups in the United States. These collaborations have resulted in 63 scientific papers.

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

    PubMed Central

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

    2013-01-01

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

  11. 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

  12. 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

  13. Fluorescence spectroscopy as a tool for determination of coumarins by multivariate calibration.

    PubMed

    Polá?ek, Roman; Májek, Pavel; Hrobo?ová, Katarína; Sádecká, Jana

    2015-03-01

    At present, it is necessary to check the quality of many food products in which the content of coumarins is limited. Since a rapid and simple method for the determination of coumarin (COU), 4-hydroxycoumarin (4HC) and dicoumarol (DC) in tea samples was needed, we developed an alternative option to chromatography, i.e., fluorescence spectroscopy with multivariate calibration. The synchronous fluorescence spectra were recorded at constant wavelength differences 70, 80 and 90 nm from 200 to 400 nm. The different experimental parameters affecting the synchronous fluorescence intensities of the analytes were carefully studied and optimized. Partial least squares (PLS) method and multi linear regression (MLR) were compared on determining the concentrations. The best results were obtained by the PLS method on synchronous fluorescence spectra at ???=?90 nm. The results from the analysis of herbal tea Melilotus officinalis by synchronous fluorescence spectroscopy with PLS model are equivalent with the results from HPLC. Fisher F-test and Student's t-test confirmed this finding. PMID:25595059

  14. Optical fluorescence spectroscopy to detect hepatic necrosis after normothermic ischemia: animal model

    NASA Astrophysics Data System (ADS)

    Romano, Renan A.; Vollet-Filho, Jose D.; Pratavieira, Sebastião.; Fernandez, Jorge L.; Kurachi, Cristina; Bagnato, Vanderlei S.; Castro-e-Silva, Orlando; Sankarankutty, Ajith K.

    2015-06-01

    Liver transplantation is a well-established treatment for liver failure. However, the success of the transplantation procedure depends on liver graft conditions. The tissue function evaluation during the several transplantation stages is relevant, in particular during the organ harvesting, when a decision is made concerning the viability of the graft. Optical fluorescence spectroscopy is a good option because it is a noninvasive and fast technique. A partial normothermic hepatic ischemia was performed in rat livers, with a vascular occlusion of both median and left lateral lobes, allowing circulation only for the right lateral lobe and the caudate lobe. Fluorescence spectra under excitation at 532 nm (doubled frequency Nd:YAG laser) were collected using a portable spectrometer (USB2000, Ocean Optics, USA). The fluorescence emission was collected before vascular occlusion, after ischemia, and 24 hours after reperfusion. A morphometric histology analysis was performed as the gold standard evaluation - liver samples were analyzed, and the percentage of necrotic tissue was obtained. The results showed that changes in the fluorescence emission after ischemia can be correlated with the amount of necrosis evaluated by a morphometric analysis, the Pearson correlation coefficient of the generated model was 0.90 and the root mean square error was around 20%. In this context, the laser-induced fluorescence spectroscopy technique after normothermic ischemia showed to be a fast and efficient method to differentiate ischemic injury from viable tissues.

  15. Oblique-incidence illumination and collection for depth-selective fluorescence spectroscopy.

    PubMed

    Pfefer, T Joshua; Agrawal, Anant; Drezek, Rebekah A

    2005-01-01

    Optimization of device-tissue interface parameters may lead to an improvement in the efficacy of fluorescence spectroscopy for minimally invasive disease detection. Although illumination-collection geometry has been shown to have a strong influence on the spatial origin of detected fluorescence, devices that deliver and/or collect light at oblique incidence are not well understood. Simulations are performed using a Monte Carlo model of light propagation in homogeneous tissue to characterize general trends in the intensity and spatial origin of fluorescence detected by angled geometries. Specifically, the influence of illumination angle, collection angle, and illumination-collection spot separation distance are investigated for low and high attenuation tissue cases. Results indicate that oblique-incidence geometries have the potential to enhance the selective interrogation of superficial or subsurface fluorophores at user-selectable depths up to about 0.5 mm. Detected fluorescence intensity is shown to increase significantly with illumination and collection angle. Improved selectivity and signal intensity over normal-incidence geometries result from the overlap of illumination and collection cones within the tissue. Cases involving highly attenuating tissue produce a moderate reduction in the depth of signal origin. While Monte Carlo modeling indicates that oblique-incidence designs can facilitate depth-selective fluorescence spectroscopy, optimization of device performance will require application-specific consideration of optical and biological parameters. PMID:16178649

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

    NASA Astrophysics Data System (ADS)

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

    2011-09-01

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

  17. 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

  18. Near-infrared fluorescent probe for imaging mitochondrial hydrogen polysulfides in living cells and in vivo.

    PubMed

    Gao, Min; Yu, Fabiao; Chen, Hao; Chen, Lingxin

    2015-04-01

    Hydrogen polysulfides (H2Sn, n > 1), derived from hydrogen sulfide (H2S), have attracted increasing attention in biochemical research, which may perform as the actual signaling molecules during cell signaling processes. Because of the closed biological and chemical relationship between H2S and H2Sn, it is of great value to develop sensitive and specific techniques to distinguish the intracellular level of H2Sn. To improve the understanding of the physiological and pathological roles played by H2Sn, we now develop a specific fluorescent probe Mito-ss for capturing H2Sn in cells and in vivo. When triggered by H2Sn, Mito-ss replies a turn-on fluorescence signal and exhibits a higher selectivity toward H2Sn than other abundant competing biothiols, such as glutathione, cysteine and H2S. The probe Mito-ss can also be applied to visual H2Sn in living cells, as well as in vivo, providing a potentially powerful approach for probing H2Sn in biological systems. PMID:25751615

  19. Reaction-based epoxide fluorescent probe for in vivo visualization of hydrogen sulfide.

    PubMed

    Sathyadevi, Palanisamy; Chen, Yu-Jen; Wu, Shou-Cheng; Chen, Yen-Hao; Wang, Yun-Ming

    2015-06-15

    Hydrogen sulfide (H2S) has emerged as the most important biosynthetic gasotransmitters along with nitric oxide (NO) and carbon monoxide (CO). In this study, we report the design and the synthesis of a new epoxide fluorescent probe 7-glycidyloxy-9-(2-glycidyloxycarbonylphenyl)-2-xanthone (FEPO) for use in in vivo visualization of hydrogen sulfide. The probe employs a fluorescein as a fluorophore, and is equipped with an operating epoxide unit. FEPO functions via epoxide ring opening upon nucleophilic attack of H2S. This ring opening strategy may open a new avenue for the development of various H2S fluorescent sensors. FEPO showed high selectivity and high sensitivity for H2S. FEPO's cytotoxicity was tested using MTT (2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide) assay. Furthermore, the use of confocal imaging of H2S and in vivo imaging in live zebra fish demonstrated FEPO's potential biological applications. We anticipate that, owing to their ideal properties, probes of this type will find great uses in exploring the role of H2S in biology. PMID:25660659

  20. In vivo tracking of bone marrow fibroblasts with fluorescent carbocyanine dye.

    PubMed

    Ferrari, A; Hannouche, D; Oudina, K; Bourguignon, M; Meunier, A; Sedel, L; Petite, H

    2001-09-01

    Recent advances in the field of tissue engineering have culminated in new tissue substitutes that combine a biomaterial and precursor cells. The effectiveness of these materials is generally assessed in animals, but few studies explore the fate of the transplanted cells in vivo, despite its paramount importance for understanding the function of the engineered tissues. Current methods that use reporter genes or chimeric animals are not always well suited to solving tissue-engineering problems. We therefore developed a new method for irreversible labeling of cells to track their fate in vivo. We used a fluorescent lipophilic probe, CM-Dil, that avidly binds to the cell membrane. Human bone marrow stromal fibroblasts could be labeled with 20 microM CM-Dil in 30 min. The CM-Dil was not cytotoxic and did not affect cell proliferation in vitro. Cells could be monitored for up to 30 days when placed in a coral scaffold and implanted intramuscularly or in a bony site. However, the fluorescence intensity decreased roughly in parallel with the number of cell divisions. This fact needs to be taken into account during the design and interpretation of experiments. We believe that this technique is also of interest for other cell types. PMID:11372053

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

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

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

  2. Can the Indo-1 fluorescence approach measure brain intracellular calcium in vivo? A multiparametric study of cerebrocortical anoxia and ischemia.

    PubMed

    Ligeti, L; Mayevsky, A; Ruttner, Z; Kovach, A G; McLaughlin, A C

    1997-02-01

    Indo-1 fluorescence was used to monitor intracellular calcium levels in the cat brain in vivo, using the approach proposed by Uematsu et al. [Uematsu D., Greenberg J. H., Reivich M., Karp A. In vivo measurement of cytosolic free calcium during cerebral ischemia and reperfusion. Ann Neurol 1988; 24: 420-428]. In addition, extracellular calcium and potassium levels, NADH redox state, electrocorticogram (ECoG), DC potential and relative cerebral blood flow were monitored simultaneously. Changes in the Indo-1 fluorescence ratio F400/F506 were monitored during anoxia, reversible ischemia and irreversible ischemia. Although these perturbations resulted in the expected changes in extracellular calcium and potassium levels, NADH redox state, ECoG and other physiological parameters, they did not result in significant increases in the F400/F506 ratio. The apparent insensitivity of the in vivo Indo-1 approach is due to the difficulty in obtaining accurate fluorescence signals from Indo-1 in the brain. Two reasons for this difficulty appear to be problems in loading Indo-1 into the brain, and problems in correcting Indo-1 fluorescence signals for changes in NADH fluorescence and changes in absorption of intrinsic chromophores. Under the conditions of our in vivo cat experiments, Indo-1 fluorescence is not a viable approach for measuring changes in cerebral intracellular calcium levels. PMID:9132294

  3. Functional surface engineering of C-dots for fluorescent biosensing and in vivo bioimaging.

    PubMed

    Ding, Changqin; Zhu, Anwei; Tian, Yang

    2014-01-21

    Nanoparticles are promising scaffolds for applications such as imaging, chemical sensors and biosensors, diagnostics, drug delivery, catalysis, energy, photonics, medicine, and more. Surface functionalization of nanoparticles introduces an additional dimension in controlling nanoparticle interfacial properties and provides an effective bridge to connect nanoparticles to biological systems. With fascinating photoluminescence properties, carbon dots (C-dots), carbon-containing nanoparticles that are attracting considerable attention as a new type of quantum dot, are becoming both an important class of imaging probes and a versatile platform for engineering multifunctional nanosensors. In order to transfer C-dots from proof-of-concept studies toward real world applications such as in vivo bioimaging and biosensing, careful design and engineering of C-dot probes is becoming increasingly important. A comprehensive knowledge of how C-dot surfaces with various properties behave is essential for engineering C-dots with useful imaging properties such as high quantum yield, stability, and low toxicity, and with desirable biosensing properties such as high selectivity, sensitivity, and accuracy. Several reviews in recent years have reported preparation methods and properties of C-dots and described their application in biosensors, catalysis, photovoltatic cells, and more. However, no one has yet systematically summarized the surface engineering of C-dots, nor the use of C-dots as fluorescent nanosensors or probes for in vivo imaging in cells, tissues, and living organisms. In this Account, we discuss the major design principles and criteria for engineering the surface functionality of C-dots for biological applications. These criteria include brightness, long-term stability, and good biocompatibility. We review recent developments in designing C-dot surfaces with various functionalities for use as nanosensors or as fluorescent probes with fascinating analytical performance, and we emphasize applications in bioimaging and biosensing in live cells, tissues, and animals. In addition, we highlight our work on the design and synthesis of a C-dot ratiometric biosensor for intracellular Cu(2+) detection, and a twophoton fluorescent probe for pH measurement in live cells and tissues. We conclude this Account by outlining future directions in engineering the functional surface of C-dots for a variety of in vivo imaging applications, including dots with combined targeting, imaging and therapeutic-delivery capabilities, or high-resolution multiplexed vascular imaging. With each application C-dots should open new horizons of multiplexed quantitative detection, high-resolution fluorescence imaging, and long-term, real-time monitoring of their target. PMID:23911118

  4. 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.

  5. Rapid detection of authenticity and adulteration of walnut oil by FTIR and fluorescence spectroscopy: a comparative study.

    PubMed

    Li, Bingning; Wang, Haixia; Zhao, Qiaojiao; Ouyang, Jie; Wu, Yanwen

    2015-08-15

    Fourier transform infrared (FTIR) and fluorescence spectroscopy combined with soft independent modeling of class analogies (SIMCA) and partial least square (PLS) were used to detect the authenticity of walnut oil and adulteration amount of soybean oil in walnut oil. A SIMCA model of FTIR spectra could differentiate walnut oil and other oils into separate categories; the classification limit of soybean oil in walnut oil was 10%. Fluorescence spectroscopy could differentiate oil composition by the peak position and intensity of emission spectrum without multivariate analysis. The classification limit of soybean oil adulterated in walnut oil by fluorescence spectroscopy was below 5%. The deviation of the prediction model for fluorescence spectra was lower than that for FTIR spectra. Fluorescence spectroscopy was more applicable than FTIR in the adulteration detection of walnut oil, both from the determination limit and prediction deviation. PMID:25794716

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

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

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

  7. X-Ray Fluorescence Correlation Spectroscopy: A Method for Studying Particle Dynamics in Condensed Matter

    NASA Astrophysics Data System (ADS)

    Wang, Jin; Sood, Ajay K.; Satyam, Parlapalli V.; Feng, Yiping; Wu, Xiao-Zhong; Cai, Zhonghou; Yun, Wenbing; Sinha, Sunil K.

    1998-02-01

    We have demonstrated that x-ray fluorescence correlation spectroscopy, in conjunction with microfocused synchrotron x-ray beams, can be used for elucidating particle dynamics. The dynamics of gold and ferromagnetic colloidal particles and aggregates undergoing both diffusion and sedimentation in water has been studied by measuring the time autocorrelation of the x-ray fluorescence intensity from a small illuminated volume. The dynamical parameters obtained are in excellent agreement with theoretical estimates and other measurements. Potential applications of the technique are discussed.

  8. Silver Nanostructures for Fluorescence Correlation Spectroscopy: Reduced Volumes and Increased Signal Intensities

    PubMed Central

    Choudhury, Sharmistha Dutta; Ray, Krishanu; Lakowicz, Joseph R.

    2016-01-01

    Fluorescence correlation spectroscopy (FCS) is a widely used technique to investigate the interactions and dynamics of molecules, below micromolar concentrations. Silver nanostructure (AgNS) substrates can extend the applicability of FCS to higher concentrations, which is useful for many biologically relevant reactions. Additionally, these substrates can improve detection efficiency by increasing fluorescence signal intensities. The ease of preparation of the AgNS substrates in comparison to previously investigated materials prepared by top-down nanofabrication is expected to make them readily available and suitable for various FCS applications.

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

    NASA Technical Reports Server (NTRS)

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

    1988-01-01

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

  10. Host-Guest Complexation Studied by Fluorescence Correlation Spectroscopy: Adamantane–Cyclodextrin Inclusion

    PubMed Central

    Granadero, Daniel; Bordello, Jorge; Pérez-Alvite, Maria Jesus; Novo, Mercedes; Al-Soufi, Wajih

    2010-01-01

    The host-guest complexation between an Alexa 488 labelled adamantane derivative and ?-cyclodextrin is studied by Fluorescence Correlation Spectroscopy (FCS). A 1:1 complex stoichiometry and a high association equilibrium constant of K = 5.2 × 104 M?1 are obtained in aqueous solution at 25 °C and pH = 6. The necessary experimental conditions are discussed. FCS proves to be an excellent method for the determination of stoichiometry and association equilibrium constant of this type of complexes, where both host and guest are nonfluorescent and which are therefore not easily amenable to standard fluorescence spectroscopic methods. PMID:20162009

  11. 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.

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

    NASA Astrophysics Data System (ADS)

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

    1995-05-01

    The detection and quantification of the concentration of exogenous chromophores in-vivo by their fluorescence is complicated by many physical and geometrical parameters. Measurement of such signals is advantageous in determining the pharmacokinetics of photosensitizers such as those used in photodynamic therapy (PDT) or to assist in the diagnosis of tissue histological state. To overcome these difficulties a ratio based fiber optic contact fluorometer has been developed. This fluorescence detection system (FDS) uses the ratio of the fluorescence emission peak of the exogenous chromophore to that of endogenous chromophores, i.e. autofluorescence, to correct for a variety of parameters affecting the magnitude of the measured signals. By doing so it also minimizes the range of baseline measurements prior to exogenous drug injection, for various tissue types. Design of the FDS and results of its testing in animals and patients using the second generation photosensitizer Tin ethyletiopurpurin (SnET2) are presented. These results support the feasibility and usefulness of the Ratio FDS system.

  13. Development of in-vivo fluorescence imaging with the Matrix-Free method

    NASA Astrophysics Data System (ADS)

    Zacharopoulos, Athanasios; Garofalakis, Anikitos; Ripoll, Jorge; Arridge, Simon

    2010-11-01

    Non-contact Fluorescence Molecular Tomography is an emerging technique for imaging of fluorescent probes or proteins in live animals. One of the main characteristics of the non-contact acquisition systems in comparison to the usual fibre-based systems is the much denser boundary data sets that are created. When model-based reconstruction methods are used that rely on the inversion of a derivative operator, the large number of measurements poses a challenge since the explicit formulation and storage of the Jacobian matrix could be in general not feasible. In this paper we test a matrix-free method that addresses the problems of large data sets and reduces the computational cost and memory requirements for the reconstruction. More specifically we challenged the Matrix-Free method with in-vivo measurements from mice where fluorescence tubes of different but controlled concentrations are inserted, to assess the quantification performance of the method. We extended the test with simulations, using realistic geometries extracted from a mouse-atlas and including prior known information about the optical properties of tissue into the forward model.

  14. Surface chemistry architecture of silica nanoparticles determine the efficiency of in vivo fluorescence lymph node mapping.

    PubMed

    Helle, Marion; Rampazzo, Enrico; Monchanin, Morgane; Marchal, Frédéric; Guillemin, François; Bonacchi, Sara; Salis, Francesca; Prodi, Luca; Bezdetnaya, Lina

    2013-10-22

    Near-infrared (NIR) imaging of the lymphatic system offers a sensitive, versatile, and accurate lymph node mapping to locate the first, potentially metastatic, draining nodes in the operating room. Many luminescent nanoprobes have received great attention in this field, and the design of nontoxic and bright nanosystems is of crucial importance. Fluorescent NIR-emitting dye doped silica nanoparticles represent valuable platforms to fulfill these scopes, providing sufficient brightness, resistance to photobleaching, and hydrophilic nontoxic materials. Here, we synthesized these highly stable core-shell nanoparticles with a programmable surface charge positioning and determined the effect of these physicochemical properties on their in vivo behavior. In addition, we characterized their fluorescence kinetic profile in the right axillary lymph node (RALN) mapping. We found that nanoparticles with negative charges hidden by a PEG shell are more appropriate than those with external negative charges in the mapping of lymph nodes. We also demonstrated the efficient excretion of these nanostructures by the hepatobiliary route and their nontoxicity in mice up to 3 months postinjection. These results indicate the potential future development of these fluorescent nanosystems for LN mapping. PMID:24070236

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

    PubMed Central

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

    2009-01-01

    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. PMID:19587837

  16. Effect of indocyanin green formulation on blood clearance and in vivo fluorescence kinetic profile of skin

    NASA Astrophysics Data System (ADS)

    Devoisselle, Jean-Marie; Soulie-Begu, Sylvie; Mordon, Serge R.; Mestres, G.; Desmettre, Thomas; Maillols, H.

    1995-12-01

    Indocyanine green has been used to measure cardiac and liver functions. More recently, it has been proposed as a contrast agent in ophthalmic angiography, tumor imaging and as an infrared absorbing dye in the context of laser-induced thermal damage of blood vessels. The aim of the study is to overcome the disadvantage of a very short blood half-time and to participate to a better confinement in blood vessels. Indocyanine green was administered intravenously to Wistar rats at a 7.5 mg/kg dose. Formulations consist in indocyanine green aqueous solution and o/w emulsion. Blood samples were collected and analyzed by spectrophotometry. Fluorescence was recorded in vivo by spectrofluorometry using an optic fiber coupled to an optical multichannel analyzer. The fiber optic was placed at a 4 mm distance from the skin surface. Results show that aqueous solution of indocyanine green leads to a rapid blood clearance. To the administration of ICG emulsion belongs the advantage of increasing the half-time and the residence time of indocyanine green in skin. It may be noted that however the formulation is, the observed blood clearance profiles are quite different from the tissue fluorescence kinetic profiles. The dye could have a longer residence time (20 - 60 min. plateau phase). Moreover, a shift of the maximum emission peak is noted after i.v. administration. The study of ICG fluorescence in the presence of model membranes shows that ICG is able to interact with phospholipid bilayers. These findings may be interesting for therapeutic applications of indocyanine green requiring a high level of dye in tissues for a great period of time and participate to the knowledge of ICG behavior in vivo.

  17. 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

  18. 31P NMR spectroscopy of in vivo tumors

    NASA Astrophysics Data System (ADS)

    Ng, T. C.; Evanochko, W. T.; Hiramoto, R. N.; Ghanta, V. K.; Lilly, M. B.; Lawson, A. J.; Corbett, T. H.; Durant, J. R.; Glickson, J. D.

    A probe, suitable for any wide-bore NMR spectrometer, was constructed for monitoring high-resolution spectra of in vivo subcutaneously implanted tumors in mice. Preliminary studies of a variety of murine tumors (MOPC 104E myeloma, Dunn osteosarcoma, colon-26, ovarian M5, and mammary adenocarcinoma as well as human colon, mammary, and lung tumors in athymic mice) indicate that the 31P NMR spectrum is a sensitive monitor of progressive metabolic changes that occur during untreated tumor growth and an early indicator of tumor response to chemotherapy, hyperthermia, and X radiation. Response to each of these therapeutic modalities is accompanied by distinctly different spectral changes.

  19. Clinical approved fluorescent dyes coupled to endomicroscopy for in vivo diagnostic of peritoneal carcinomatosis

    NASA Astrophysics Data System (ADS)

    Abbaci, Muriel; Dartigues, Peggy; Soufan, Ranya; De Leeuw, Frederic; Fabre, Monique; Laplace-Builhé, Corinne

    2015-03-01

    Peritoneal carcinomatosis is metastatic stage aggravating digestive, gynecological or bladder cancer dissemination and the preoperative evaluation of lesions remains difficult. There is therefore a need for minimal invasive innovative techniques to establish a precise preoperative assessment of cancer peritoneal cavity. Probe-based confocal laser endomicroscopy (pCLE) provides dynamic images of the microarchitecture of tissues during an endoscopy. The PERSEE project proposes new developments in robotics and pCLE for the exploration of the peritoneal cavity during laparoscopy. Two fluorescent dyes, Patent blue V and Indocyanine green have been evaluated on human ex vivo samples to improve the contrast of pCLE images. For a future implementation in clinical study, two topically staining protocols operable in vivo have been validated on 70 specimens from 25 patients with a peritoneal carcinomatosis. The specimens were then imaged by pCLE with an optical probe designed for the application. A histo-morphological correlative study was performed on 350 pCLE images and 70 standard histological preparations. All images were interpreted in a random way by two pathologists. Differential histological diagnostics such as normal peritoneum or pseudomyxoma could be recognized on fluorescence images. The statistical analysis of the correlative study is underway. These dyes already approved for human use are interesting for pCLE imaging because some micromorphological criteria look like to conventional histology and are readable by pathologist. Thus pCLE images using both dyes do not require a specific semiology unlike to what is described in the literature, for pCLE associated with fluorescein for the in vivo imaging of pancreatic cysts.

  20. New surface plasmons approach to single molecule detection (SMD) and fluorescence correlation spectroscopy (FCS)

    NASA Astrophysics Data System (ADS)

    Gryczynski, Z.; Gryczynski, I.; Matveeva, E. G.; Calander, N.; Grygorczyk, R.; Akopova, I.; Bharill, S.; Muthu, P.; Klidgar, S.; Borejdo, J.

    2007-02-01

    We report new approach to Fluorescence Correlation Spectroscopy (FCS) and Single Molecule Detection (SMD) based on Surface Plasmon-Coupled Emission (SPCE) technology. The use of SPCE offers significant reduction of fluorescence volume (detection volume) reduction decreasing background contribution. Fluorophore interaction with surface plasmons increases the rate of photon detection and makes fluorescence very sensitive to change in a position of emitting molecule. The effective thickness of the fluorescence volume in SPCE experiments depends on two factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. The excitation with the laser beam at Surface Plasmon Resonance (SPR) angle (Kretschmann configuration) through the high numerical aperture objective makes observation volume very shallow below 100 nm. The layer thickness is further reduced by the metal quenching of excited fluorophores immediately close to the interface (~10 nm). The fluorescence light is emitted through the metal film only at the SPCE angle. Any fluorescence occurring at the distances greater than the coupling distance is effectively reflected (~92%) by the metal film and not transmitted to the objective. The thickness of the detected volume can be 20-50 nm, depending on the prism dielectric constants and orientation of the excited dipoles. In addition the signal is very sensitive to the change in fluorophore position and orientation. Such strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles opens new possibilities to study conformational changes of macromolecules in real time.

  1. Electronic excited states of guanine-cytosine hairpins and duplexes studied by fluorescence spectroscopy.

    PubMed

    Brazard, Johanna; Thazhathveetil, Arun K; Vayá, Ignacio; Lewis, Frederick D; Gustavsson, Thomas; Markovitsi, Dimitra

    2013-08-01

    Guanine-cytosine hairpins, containing a hexaethylene glycol bridge, are studied by steady-state fluorescence spectroscopy and time-correlated single photon counting; their properties are compared to those of duplexes with the same sequence. It is shown that, both in hairpins and in duplexes, base pairing induces quenching of the ππ* fluorescence, the quantum yield decreasing by at least two orders of magnitude. When the size of the systems increases from two to ten base pairs, a fluorescent component decaying on the nanosecond time-scale appears at energy higher than that stemming from the bright states of non-interacting mono-nucleotides (ca. 330 nm). For ten base pairs, this new fluorescence forms a well-defined band peaking at 305 nm. Its intensity is about 20% higher for the hairpin compared to the duplex. Its position (red-shifted by 1600 cm(-1)) and width (broader by 1800 cm(-1) FWHM) differ from those observed for large duplexes containing 1000 base pairs, suggesting the involvement of electronic coupling. Fluorescence anisotropy reveals that the excited states responsible for high energy emission are not populated directly upon photon absorption but are reached during a relaxation process. They are assigned to charge transfer states. According to the emerging picture, the amplitude of conformational motions determines whether instantaneous deactivation to the ground state or emission from charge transfer states will take place, while ππ* fluorescence is associated to imperfect base-pairing. PMID:23736116

  2. 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

  3. 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

  4. 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.

  5. 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

  6. Auto-fluorescence lifetime and light reflectance spectroscopy for breast cancer diagnosis: potential tools for intraoperative margin detection

    PubMed Central

    Sharma, Vikrant; Shivalingaiah, Shivaranjani; Peng, Yan; Euhus, David; Gryczynski, Zygmunt; Liu, Hanli

    2012-01-01

    This study investigates the use of two spectroscopic techniques, auto-fluorescence lifetime measurement (AFLM) and light reflectance spectroscopy (LRS), for detecting invasive ductal carcinoma (IDC) in human ex vivo breast specimens. AFLM used excitation at 447 nm with multiple emission wavelengths (532, 562, 632, and 644 nm), at which auto-fluorescence lifetimes and their weight factors were analyzed using a double exponent model. LRS measured reflectance spectra in the range of 500-840 nm and analyzed the spectral slopes empirically at several distinct spectral regions. Our preliminary results based on 93 measured locations (i.e., 34 IDC, 31 benign fibrous, 28 adipose) from 6 specimens show significant differences in 5 AFLM-derived parameters and 9 LRS-based spectral slopes between benign and malignant breast samples. Multinomial logistic regression with a 10-fold cross validation approach was implemented with selected features to classify IDC from benign fibrous and adipose tissues for the two techniques independently as well as for the combined dual-modality approach. The accuracy for classifying IDC was found to be 96.4 ± 0.8%, 92.3 ± 0.8% and 96 ± 1.3% for LRS, AFLM, and dual-modality, respectively. PMID:22876347

  7. 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

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Quantum dots in bioanalysis: a review of applications across various platforms for fluorescence spectroscopy and imaging.

    PubMed

    Petryayeva, Eleonora; Algar, W Russ; Medintz, Igor L

    2013-03-01

    Semiconductor quantum dots (QDs) are brightly luminescent nanoparticles that have found numerous applications in bioanalysis and bioimaging. In this review, we highlight recent developments in these areas in the context of specific methods for fluorescence spectroscopy and imaging. Following a primer on the structure, properties, and biofunctionalization of QDs, we describe select examples of how QDs have been used in combination with steady-state or time-resolved spectroscopic techniques to develop a variety of assays, bioprobes, and biosensors that function via changes in QD photoluminescence intensity, polarization, or lifetime. Some special attention is paid to the use of Förster resonance energy transfer-type methods in bioanalysis, including those based on bioluminescence and chemiluminescence. Direct chemiluminescence, electrochemiluminescence, and charge transfer quenching are similarly discussed. We further describe the combination of QDs and flow cytometry, including traditional cellular analyses and spectrally encoded barcode-based assay technologies, before turning our attention to enhanced fluorescence techniques based on photonic crystals or plasmon coupling. Finally, we survey the use of QDs across different platforms for biological fluorescence imaging, including epifluorescence, confocal, and two-photon excitation microscopy; single particle tracking and fluorescence correlation spectroscopy; super-resolution imaging; near-field scanning optical microscopy; and fluorescence lifetime imaging microscopy. In each of the above-mentioned platforms, QDs provide the brightness needed for highly sensitive detection, the photostability needed for tracking dynamic processes, or the multiplexing capacity needed to elucidate complex systems. There is a clear synergy between advances in QD materials and spectroscopy and imaging techniques, as both must be applied in concert to achieve their full potential. PMID:23452487

  10. Revealing the photophysics of gold-nanobeacons via time-resolved fluorescence spectroscopy.

    PubMed

    Wei, Guoke; Simionesie, Dorin; Sefcik, Jan; Sutter, Jens U; Xue, Qingjiang; Yu, Jun; Wang, Jinliang; Birch, David J S; Chen, Yu

    2015-12-15

    We demonstrate that time-resolved fluorescence spectroscopy is a powerful tool to investigate the conformation states of hairpin DNA on the surface of gold nanoparticles (AuNPs) and energy transfer processes in Au-nanobeacons. Long-range fluorescence quenching of Cy5 by AuNPs has been found to be in good agreement with electrodynamics modeling. Moreover, time-correlated single-photon counting (TCSPC) is shown to be promising for real-time monitoring of the hybridization kinetics of Au-nanobeacons, with up to 60% increase in decay time component and 300% increase in component fluorescence fraction observed. Our results also indicate the importance of the stem and spacer designs for the performance of Au-nanobeacons. PMID:26670500

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

    NASA Astrophysics Data System (ADS)

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

    2004-04-01

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

  12. Detection of mercuric bromide in a gas phase flow cell by laser photofragment fluorescence spectroscopy

    SciTech Connect

    Tong, X.; Barat, R.B.; Poulos, A.T.

    1999-09-15

    Photofragment fluorescence (PFF) spectroscopy offers real-time monitoring capability with high-analytical sensitivity and selectivity for volatile mercury compounds found in process gas streams, such as incinerator stacks. In this work, low concentrations (6 ppb to 30 ppm) of mercuric bromide (HgBr{sub 2}) vapor were introduced into an atmospheric pressure flow cell. The PFF technique used 222 nm laser radiation to photolyze HgBr{sub 2} and excite fluorescence from the resulting Hg atoms at 253.7 nm. The fluorescence intensity was linear with laser fluence over the range of 45--180 mJ/cm{sup 2}. Extrapolated detection limits by this method below 1 ppb of HgBr{sub 2} in the absence of air are estimated. A linear dynamic detection range up to 0.7 ppm is reported.

  13. 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

  14. Site-specific interaction of thrombin and inhibitors observed by fluorescence correlation spectroscopy.

    PubMed Central

    Klingler, J; Friedrich, T

    1997-01-01

    We report on the application of fluorescence correlation spectroscopy (FCS) to observe the interaction between thrombin and thrombin inhibitors. Two site-specific fluorescent labels were used to distinguish between inhibitors directed to the active site, the exosite, or both binding sites of thrombin. For several well-known inhibitors of thrombin, the binding sites observed by FCS correspond to previous studies. The interaction of the recently discovered thrombin inhibitor ornithodorin from the tick Ornithodorus moubata with thrombin was investigated. It was found that this inhibitor, like hirudin and rhodniin, binds to both the active site and exosite of thrombin simultaneously. This study shows the feasibility of FCS as a sensitive and selective method for observing protein-ligand interactions. As an additional technique, simultaneous labeling with both fluorescent labels was successfully demonstrated. Images FIGURE 1 PMID:9336216

  15. Fluorescence spectroscopy for assessment of liver transplantation grafts concerning graft viability and patient survival

    NASA Astrophysics Data System (ADS)

    Vollet Filho, José D.; da Silveira, Marina R.; Castro-e-Silva, Orlando; Bagnato, Vanderlei S.; Kurachi, Cristina

    2015-06-01

    Evaluating transplantation grafts at harvest is essential for its success. Laser-induced fluorescence spectroscopy (LIFS) can help monitoring changes in metabolic/structural conditions of tissue during transplantation. The aim of the present study is to correlate LIFSobtained spectra of human hepatic grafts during liver transplantation with post-operative patients' mortality rate and biochemical parameters, establishing a method to exclude nonviable grafts before implantation. Orthotopic liver transplantation, piggyback technique was performed in 15 patients. LIFS was performed under 408nm excitation. Collection was performed immediately after opening donor's abdominal cavity, after cold perfusion, end of back-table period, and 5 min and 1 h after warm perfusion at recipient. Fluorescence information was compared to lactate, creatinine, bilirubin and INR levels and to survival status. LIFS was sensitive to liver changes during transplantation stages. Study-in-progress; initial results indicate correlation between fluorescence and life/death status of patients.

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

    SciTech Connect

    McGown, L.B.

    1989-01-01

    During the first project period, we have explored several different aspects of phase-resolved fluorescence spectroscopy (PRFS) for the fingerprinting of complex samples. It should be noted that our goal is not only fingerprinting'' per se, but also includes the characterization of complex samples with respect to dynamic interactions of luminescent molecules with each other and with sample matrix constituents. Each area of investigation is discussed in the following sections.

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

    PubMed Central

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

    2009-01-01

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

  18. Tea quality and classification evaluation using multi-wavelength light-emitting diodes induced fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Feng, Chao; Liu, Xuan; Yan, Chunsheng; He, Sailing; Mei, Liang

    2013-03-01

    In this paper, we demonstrated a detective system to evaluate the quality and classification of different tea samples based on multi-wavelength LED-induced fluorescence spectroscopy. By utilizing multiple excitation wavelengths, we obtained much more physical and chemical information from the detected samples than single excitation wavelength. By utilizing principal component analysis (PCA), we extracted the dominant features of the samples to classify and characterize the tea samples.

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  20. In vivo tomographic imaging of lung colonization of tumour in mouse with simultaneous fluorescence and X-ray CT.

    PubMed

    Zhang, Bin; Gao, Fuping; Wang, Mengjiao; Cao, Xu; Liu, Fei; Wang, Xin; Luo, Jianwen; Wang, Guangzhi; Bai, Jing

    2014-01-01

    Non-invasive in vivo imaging of diffuse and wide-spread colonization within the lungs, rather than distinct solid primary tumors, is still a challenging work. In this work, a lung colonization mouse model bearing A549 human lung tumor was simultaneously scanned by a dual-modality fluorescence molecular tomography (FMT) and X-ray computed tomography (CT) system in vivo. A two steps method which incorporates CT structural information into the FMT reconstruction procedure is employed to provide concurrent anatomical and functional information. By using the target-specific fluorescence agent, the fluorescence tomographic results show elevated fluorescence intensity deep within the lungs which is colonized with diffuse and wide-spread tumors. The results were confirmed with ex vivo fluorescence reflectance imaging and histological examination of the lung tissues. With FMT reconstruction combined with the CT information, the dual-modality FMT/micro-CT system is expected to offer sensitive and noninvasive imaging of diffuse tumor colonization within the lungs in vivo. PMID:23696158

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

    NASA Astrophysics Data System (ADS)

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

    2012-11-01

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

  2. 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.

  3. 256 × 2 SPAD line sensor for time resolved fluorescence spectroscopy.

    PubMed

    Krstajić, Nikola; Levitt, James; Poland, Simon; Ameer-Beg, Simon; Henderson, Robert

    2015-03-01

    We present a CMOS chip 256 × 2 single photon avalanche diode (SPAD) line sensor, 23.78 µm pitch, 43.7% fill factor, custom designed for time resolved emission spectroscopy (TRES). Integrating time-to-digital converters (TDCs) implement on-chip mono-exponential fluorescence lifetime pre-calculation allowing timing of 65k photons/pixel at 200 Hz line rate at 40 ps resolution using centre-of-mass method (CMM). Per pixel time-correlated single-photon counting (TCSPC) histograms can also be generated with 320 ps bin resolution. We characterize performance in terms of dark count rate, instrument response function and lifetime uniformity for a set of fluorophores with lifetimes ranging from 4 ns to 6 ns. Lastly, we present fluorescence lifetime spectra of multicolor microspheres and skin autofluorescence acquired using a custom built spectrometer. In TCSPC mode, time-resolved spectra are acquired within 5 minutes whilst in CMM mode spectral lifetime signatures are acquired within 2 ms for fluorophore in cuvette and 200 ms for skin autofluorescence. We demonstrate CMOS line sensors to be a versatile tool for time-resolved fluorescence spectroscopy by providing parallelized and flexible spectral detection of fluorescence decay. PMID:25836796

  4. Advances in the in Vivo Raman Spectroscopy of Malignant Skin Tumors Using Portable Instrumentation

    PubMed Central

    Kourkoumelis, Nikolaos; Balatsoukas, Ioannis; Moulia, Violetta; Elka, Aspasia; Gaitanis, Georgios; Bassukas, Ioannis D.

    2015-01-01

    Raman spectroscopy has emerged as a promising tool for real-time clinical diagnosis of malignant skin tumors offering a number of potential advantages: it is non-intrusive, it requires no sample preparation, and it features high chemical specificity with minimal water interference. However, in vivo tissue evaluation and accurate histopathological classification remain a challenging task for the successful transition from laboratory prototypes to clinical devices. In the literature, there are numerous reports on the applications of Raman spectroscopy to biomedical research and cancer diagnostics. Nevertheless, cases where real-time, portable instrumentations have been employed for the in vivo evaluation of skin lesions are scarce, despite their advantages in use as medical devices in the clinical setting. This paper reviews the advances in real-time Raman spectroscopy for the in vivo characterization of common skin lesions. The translational momentum of Raman spectroscopy towards the clinical practice is revealed by (i) assembling the technical specifications of portable systems and (ii) analyzing the spectral characteristics of in vivo measurements. PMID:26132563

  5. Advances in the in Vivo Raman Spectroscopy of Malignant Skin Tumors Using Portable Instrumentation.

    PubMed

    Kourkoumelis, Nikolaos; Balatsoukas, Ioannis; Moulia, Violetta; Elka, Aspasia; Gaitanis, Georgios; Bassukas, Ioannis D

    2015-01-01

    Raman spectroscopy has emerged as a promising tool for real-time clinical diagnosis of malignant skin tumors offering a number of potential advantages: it is non-intrusive, it requires no sample preparation, and it features high chemical specificity with minimal water interference. However, in vivo tissue evaluation and accurate histopathological classification remain a challenging task for the successful transition from laboratory prototypes to clinical devices. In the literature, there are numerous reports on the applications of Raman spectroscopy to biomedical research and cancer diagnostics. Nevertheless, cases where real-time, portable instrumentations have been employed for the in vivo evaluation of skin lesions are scarce, despite their advantages in use as medical devices in the clinical setting. This paper reviews the advances in real-time Raman spectroscopy for the in vivo characterization of common skin lesions. The translational momentum of Raman spectroscopy towards the clinical practice is revealed by (i) assembling the technical specifications of portable systems and (ii) analyzing the spectral characteristics of in vivo measurements. PMID:26132563

  6. 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

  7. 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

  8. X-ray fluorescence correlation spectroscopy for studying particle dynamics in condensed matter

    NASA Astrophysics Data System (ADS)

    Wang, Jin; Sood, Ajay K.; Satyam, Parlapalli; Feng, Yiping; Wu, Xiao-zhong; Cai, Zhonghou; Yun, Wenbing; Sinha, Sunil K.

    1997-07-01

    Photon correlation spectroscopy probing fluctuations in scattered or fluorescent intensity to study particle dynamics in fluids is by now well established in the visible light regime. With the advent of high-brilliance synchrotron radiation sources, correlation spectroscopy utilizing scattered radiation has recently been extended to the x-ray wavelength regime by using spatially coherent x-rays to study the time fluctuations of the corresponding speckle patterns. In this presentation, we report the development of a new technique, x-ray fluorescence correlation spectroscopy (XFCS) for elucidating the dynamics of particles. This technique does not require coherent beams but relies on intense microfocused x-ray beams. Further, it is element specific. As a demonstration of this method, the dynamics of gold colloidal particles and aggregates undergoing diffusion and sedimentation in water was studied by measuring the time autocorrelation of the gold fluorescence intensity from a small illuminated volume. The values of the translational diffusion constants and sedimentation velocities obtained are in excellent agreement with theoretical estimates and other measurements. Further potential applications of the technique are discussed.

  9. 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

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

    PubMed

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

    2013-11-01

    A prerequisite for in vivo fluorescence imaging is the safety of fluorescent probes. Among all fluorescent probes, fluorescent proteins (FPs) might be the safest ones, which have been widely used in biological sciences at the gene level. But FPs have not been used in vivo in the purified form yet due to the instability of proteins. Here, we individually coat near-infrared (NIR) FPs (NIRFPs) with a silica nanoshell, resulting in NIRFP@silica, one of the safest and brightest NIR fluorescent nanoprobes with a quantum yield of 0.33 for in vivo imaging. The silica shell not only protects NIRFPs from denaturation and metabolic digestion, but also enhances the quantum yield and photostability of the coated NIRFPs. When injected via the tail vein, NIRFP@silica NPs can distribute all over the mouse body, and then can be efficiently eliminated through urine in 24 h, demonstrating its potential applications as a safe and robust NIR fluorescence probe for whole body imaging. PMID:24056841

  11. Amyloid-? Deposits Target Efficient Near-Infrared Fluorescent Probes: Synthesis, in Vitro Evaluation, and in Vivo Imaging.

    PubMed

    Fu, Hualong; Tu, Peiyu; Zhao, Liu; Dai, Jiapei; Liu, Boli; Cui, Mengchao

    2016-02-01

    The formation of extracellular amyloid-? (A?) plaques is a common molecular change that underlies several debilitating human conditions, including Alzheimer's disease (AD); however, the existing near-infrared (NIR) fluorescent probes for the in vivo detection of A? plaques are limited by undesirable fluorescent properties and poor brain kinetics. In this work, we designed, synthesized, and evaluated a new family of efficient NIR probes that target A? plaques by incorporating hydroxyethyl groups into the ligand structure. Among these probes, DANIR 8c showed excellent fluorescent properties with an emission maximum above 670 nm upon binding to A? aggregates and also displayed a high sensitivity (a 629-fold increase in fluorescence intensity) and affinity (Kd = 14.5 nM). Because of the improved hydrophilicity that was induced by hydroxyls, 8c displayed increased initial brain uptake and a fast washout from the brain, as well as an acceptable biostability in the brain. In vivo NIR fluorescent imaging revealed that 8c could efficiently distinguish between AD transgenic model mice and normal controls. Overall, 8c is an efficient and veritable NIR fluorescent probe for the in vivo detection of A? plaques in the brain. PMID:26717442

  12. In-vivo two-dimensional NMR spectroscopy: inventory and perspectives

    NASA Astrophysics Data System (ADS)

    Ziegler, A.; Décorps, M.

    1998-02-01

    Two-dimensional spectroscopy, routinely used in high-resolution NMR, may present a number of advantages fo in-vivo applications (resonance assignments, follow-up of metabolism...). The main features of experiments on living systems (animal models and clinical applications) are reviewed: short T2* decay times, low field magnets, water suppression, localization, motion artefacts... The need for adapting 2D techniques is analyzed. Recent applications and developments are reviewed (J-resolved, homonuclear correlation spectroscopy. La spectroscopie à deux dimensions, couramment utilisée en RMN haute résolution, pourrait présenter de nombreux avantages dans les applications in vivo. Les caractéristiques de l'expérimentation sur des systèmes vivants (en clinique ou sur des modèles animaux) sont détaillées : T2* courts, “faibles" champ B0, suppression de l'eau et des signaux indésirables, localisation du signal, artefacts de mouvement... Les conséquences pour l'utilisation in vivo des techniques RMN 2D sont passées en revue. Les applications et développements récents sont mentionnés (spectroscopie J-résolu, spectroscopie de corrélation homonucléaire).

  13. Disassembly of structurally modified viral nanoparticles: characterization by fluorescence correlation spectroscopy.

    PubMed

    Toivola, Jouni; Gilbert, Leona; Michel, Patrik; White, Daniel; Vuento, Matti; Oker-Blom, Christian

    2005-12-01

    Analysis of the breakdown products of engineered viral particles can give useful information on the particle structure. We used various methods to breakdown both a recombinant enveloped virus and virus-like particles (VLPs) from two non-enveloped viruses and analysed the resulting subunits by fluorescence correlation spectroscopy (FCS). Analysis of the enveloped baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), displaying the green fluorescent protein (GFP) fused to its envelope protein gp64 was performed in the presence and absence of 5 mM SDS and 25 mM DTT. Without treatment, the viral particle showed a diffusion time of 3.3 ms. In the presence of SDS, fluorescent subunits with diffusion times of 0.2 ms were observed. Additional treatment with DTT caused a drop in the diffusion time to 0.1 ms. Changes in the amplitude of the autocorrelation function suggested a 3-fold increase in fluorescent particle number when viral particles were treated with SDS, and a further 1.5-fold increase with additional treatment with DTT. Thus, the data showed that an average of 4.5 molecules of gp64-GFP was incorporated in the membrane of the modified baculovirus. Further, this suggests that each fluorescent gp64 trimer carries on average 1.5 fluorescent units. Similar experiments were carried out with two non-enveloped fluorescent virus-like particles (fVLPs) that displayed enhanced green fluorescent protein (EGFP). These, fVLPs of canine and human B19 parvoviruses were treated with 6 M urea and 5 mM SDS, respectively. Correspondingly, the original hydrodynamic radii of 17 and 14 nm were reduced to 9 and 5 nm after treatment. Here, the change in the amplitude of the autocorrelation curve suggested a 10-fold increase in particle number when viral particles of CPV were treated with 6 M urea at 50 degrees C for 10 min. For EGFP-B19, there was a decrease in the amplitude, accompanied by a 9-fold increase in the number of fluorescent units with SDS treatment. The results showed that approximately 10 and 9 fluorescent units were associated with the corresponding CPV and B19 VLPs. In summary, we were able to estimate the number of fluorescent subunits in a baculovirus containing a GFP-fusion with its gp64 envelope protein and in two different parvo-VLPs containing EGFP-fused with their VP2 capsid proteins. PMID:16314283

  14. In vivo spatial frequency domain spectroscopy of two layer media

    NASA Astrophysics Data System (ADS)

    Yudovsky, Dmitry; Nguyen, John Quan M.; Durkin, Anthony J.

    2012-10-01

    Monitoring of tissue blood volume and local oxygen saturation can inform the assessment of tissue health, healing, and dysfunction. These quantities can be estimated from the contribution of oxyhemoglobin and deoxyhemoglobin to the absorption spectrum of the dermis. However, estimation of blood related absorption in skin can be confounded by the strong absorption of melanin in the epidermis and epidermal thickness and pigmentation varies with anatomic location, race, gender, and degree of disease progression. Therefore, a method is desired that decouples the effect of melanin absorption in the epidermis from blood absorption in the dermis for a large range of skin types and thicknesses. A previously developed inverse method based on a neural network forward model was applied to simulated spatial frequency domain reflectance of skin for multiple wavelengths in the near infrared. It is demonstrated that the optical thickness of the epidermis and absorption and reduced scattering coefficients of the dermis can be determined independently and with minimal coupling. Then, the same inverse method was applied to reflectance measurements from a tissue simulating phantom and in vivo human skin. Oxygen saturation and total hemoglobin concentrations were estimated from the volar forearms of weakly and strongly pigmented subjects using a standard homogeneous model and the present two layer model.

  15. Equipment design issues for the in vivo X-ray fluorescence analysis of bone lead.

    PubMed Central

    Thomas, B J

    1991-01-01

    Several groups have reported the development of systems, based on the principle of X-ray fluorescence, for the in vivo measurement of bone lead concentrations. These systems have used the detection of either the characteristic L or K X-rays resulting from excitation by a suitable photon source. This paper examines design issues related to the development of these systems. These design issues are, in most instances, a result of consideration of the physical principles involved, and hence there are many features common to the systems developed by the individual groups. Design issues discussed in this paper include the selection of the site for measurement, source-sample-detector configuration, and collimation. Specific examples from published work are used to demonstrate the relevant features. PMID:2040249

  16. Differences in in vivo fluorescence yield between three phytoplankton size classes

    USGS Publications Warehouse

    Alpine, Andrea E.; Cloern, James E.

    1985-01-01

    The size-dependent relationship between in vivo fluorescence (IVF) and chlorophyll a was determined for monthly phytoplankton samples from the San Francisco Bay estuary. Chlorophyll a and IVF were both measured on netplankton (>22 μm), nanoplankton (5–22 μm), and ultraplankton (<5 μm) samples that were separated with screens. IVF and chlorophyll a were linearly related for each size class, but the IVF per unit chlorophyll a (R) was significantly different between these three size classes. The ultraplankton R was twice that of the nanoplankton which was in turn twice the netplankton R. Hence, accurate size fractionation of phytoplankton biomass from measures of IVF requires correction for size-dependent variations in R.

  17. In vivo measurement of hepatic iodine concentration using fluorescent excitation analysis.

    PubMed

    Koehler, R E; Kaufman, L; Brito, A; Nelson, J A

    1976-01-01

    Hepatic iodine concentration was measured in the live dog by external use of fluorescent excitation analysis. The number of characteristic photons produced by interaction of exciting radiation from an americium-241 source with iodine within the tissue is proportional to the tissue iodine concentrations. A correction is made for absorption of radiation by the abdominal wall and other tissues lying between the volume of liver being assayed and the detector collimator. The technique is applicable to the in vivo measurement of iodine concentrations from 0.5 to 40 mg/g. Accuracy of the technique is approximately +/- 10%, which is within the range of variation in iodine concentration at various sites within the liver. Radiation dose is low, and radiolabeled tracer compounds need not be used. PMID:1262179

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

    NASA Astrophysics Data System (ADS)

    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

    2015-06-01

    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.

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

    SciTech Connect

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

    2007-01-19

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

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

    PubMed Central

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

    2015-01-01

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

  1. 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.

  2. Time-resolved fluorescence polarization spectroscopy of visible and near infrared dyes in picosecond dynamics

    NASA Astrophysics Data System (ADS)

    Pu, Yang; Alfano, Robert R.

    2015-03-01

    Near-infrared (NIR) dyes absorb and emit light within the range from 700 to 900 nm have several benefits in biological studies for one- and/or two-photon excitation for deeper penetration of tissues. These molecules undergo vibrational and rotational motion in the relaxation of the excited electronic states, Due to the less than ideal anisotropy behavior of NIR dyes stemming from the fluorophores elongated structures and short fluorescence lifetime in picosecond range, no significant efforts have been made to recognize the theory of these dyes in time-resolved polarization dynamics. In this study, the depolarization of the fluorescence due to emission from rotational deactivation in solution will be measured with the excitation of a linearly polarized femtosecond laser pulse and a streak camera. The theory, experiment and application of the ultrafast fluorescence polarization dynamics and anisotropy are illustrated with examples of two of the most important medical based dyes. One is NIR dye, namely Indocyanine Green (ICG) and is compared with Fluorescein which is in visible range with much longer lifetime. A set of first-order linear differential equations was developed to model fluorescence polarization dynamics of NIR dye in picosecond range. Using this model, the important parameters of ultrafast polarization spectroscopy were identified: risetime, initial time, fluorescence lifetime, and rotation times.

  3. 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

  4. 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.

  5. Interaction Studies of Greenly Synthesized Gold Nanoparticles with Bovine Serum Albumin (BSA) Using Fluorescence Spectroscopy.

    PubMed

    Ravikumar, Sambandam; Sreekanth, T V M; Eom, In-Yong

    2015-12-01

    In the present study, gold nanoparticles (AuNPs) with an average particle size of -41.23 nm were synthesized using eco-friendly reducing material (i.e., aqueous Nelumbo nucifera root extract). Rapid reduction results in the formation of polydispersed nanoparticles. The formation of AuNPs was characterized by surface plasmon resonance (SPR) which was determined by UV-Vis spectra (band at 544 nm), FTIR, SEM-EDX, TEM, HR-TEM, and XRD. This study aims to investigate the interaction between AuNPs and Bovine Serum Albumin (BSA) using fluorescence spectroscopy. The analysis of fluorescence spectra and intensity at physiological pH in an aqueous solution indicates that AuNPs have a potent ability to quench the BSA fluorescence by both quenching mechanisms. Resonance light scattering spectra indicated the formation of BSA-AuNPs complex. The number of binding sites and binding constants were determined based on fluorescence quenching at different temperatures. The thermodynamic parameters were also calculated at various temperatures that indicate that hydrophobic forces are abundant in the AuNPs-BSA complex. Negative ΔG degrees values suggest that the binding process is spontaneous. Synchronous fluorescence spectra showed a blue shift and CD spectra showed an increase in a-helicity content which is an indication of increasing hydrophobicity. PMID:26682387

  6. 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

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

    PubMed

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

    2014-01-01

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

  8. Detection of non-melanoma skin cancer by in vivo fluorescence imaging with fluorocoxib A.

    PubMed

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

    2015-02-01

    Non-melanoma skin cancer (NMSC) is the most common form of cancer in the US and its incidence is increasing. The current standard of care is visual inspection by physicians and/or dermatologists, followed by skin biopsy and pathologic confirmation. We have investigated the use of in vivo fluorescence imaging using fluorocoxib A as a molecular probe for early detection and assessment of skin tumors in mouse models of NMSC. Fluorocoxib A targets the cyclooxygenase-2 (COX-2) enzyme that is preferentially expressed by inflamed and tumor tissue, and therefore has potential to be an effective broadly active molecular biomarker for cancer detection. We tested the sensitivity of fluorocoxib A in a BCC allograft SCID hairless mouse model using a wide-field fluorescence imaging system. Subcutaneous allografts comprised of 1000 BCC cells were detectable above background. These BCC allograft mice were imaged over time and a linear correlation (R(2) = 0.8) between tumor volume and fluorocoxib A signal levels was observed. We also tested fluorocoxib A in a genetically engineered spontaneous BCC mouse model (Ptch1(+/-) K14-Cre-ER2 p53(fl/fl)), where sequential imaging of the same animals over time demonstrated that early, microscopic lesions (100 ?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

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

    SciTech Connect

    Bergmann, Uwe; Cramer, Stephen P.

    2001-08-02

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

  10. In Vivo Imaging of Flavoprotein Fluorescence During Hypoxia Reveals the Importance of Direct Arterial Oxygen Supply to Cerebral Cortex Tissue.

    PubMed

    Chisholm, K I; Ida, K K; Davies, A L; Papkovsky, D B; Singer, M; Dyson, A; Tachtsidis, I; Duchen, M R; Smith, K J

    2016-01-01

    Live imaging of mitochondrial function is crucial to understand the important role played by these organelles in a wide range of diseases. The mitochondrial redox potential is a particularly informative measure of mitochondrial function, and can be monitored using the endogenous green fluorescence of oxidized mitochondrial flavoproteins. Here, we have observed flavoprotein fluorescence in the exposed murine cerebral cortex in vivo using confocal imaging; the mitochondrial origin of the signal was confirmed using agents known to manipulate mitochondrial redox potential. The effects of cerebral oxygenation on flavoprotein fluorescence were determined by manipulating the inspired oxygen concentration. We report that flavoprotein fluorescence is sensitive to reductions in cortical oxygenation, such that reductions in inspired oxygen resulted in loss of flavoprotein fluorescence with the exception of a preserved 'halo' of signal in periarterial regions. The findings are consistent with reports that arteries play an important role in supplying oxygen directly to tissue in the cerebral cortex, maintaining mitochondrial function. PMID:26782217

  11. Near-infrared spectroscopy of renal tissue in vivo

    NASA Astrophysics Data System (ADS)

    Grosenick, Dirk; Steinkellner, Oliver; Wabnitz, Heidrun; Macdonald, Rainer; Niendorf, Thoralf; Cantow, Kathleen; Flemming, Bert; Seeliger, Erdmann

    2013-03-01

    We have developed a method to quantify hemoglobin concentration and oxygen saturation within the renal cortex by near-infrared spectroscopy. A fiber optic probe was used to transmit the radiation of three semiconductor lasers at 690 nm, 800 nm and 830 nm to the tissue, and to collect diffusely remitted light at source-detector separations from 1 mm to 4 mm. To derive tissue hemoglobin concentration and oxygen saturation of hemoglobin the spatial dependence of the measured cw intensities was fitted by a Monte Carlo model. In this model the tissue was assumed to be homogeneous. The scaling factors between measured intensities and simulated photon flux were obtained by applying the same setup to a homogeneous semi-infinite phantom with known optical properties and by performing Monte Carlo simulations for this phantom. To accelerate the fit of the tissue optical properties a look-up table of the simulated reflected intensities was generated for the needed range of absorption and scattering coefficients. The intensities at the three wavelengths were fitted simultaneously using hemoglobin concentration, oxygen saturation, the reduced scattering coefficient at 800 nm and the scatter power coefficient as fit parameters. The method was employed to study the temporal changes of renal hemoglobin concentration and blood oxygenation on an anesthetized rat during a short period of renal ischemia induced by aortic occlusion and during subsequent reperfusion.

  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. Structural Changes of Yellow Cameleon Domains Observed by Quantitative FRET Analysis and Polarized Fluorescence Correlation Spectroscopy

    PubMed Central

    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.

    2008-01-01

    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

  14. Probing local conformation and dynamics of molecular complexes using phase-selective fluorescence correlation and coherence spectroscopy

    NASA Astrophysics Data System (ADS)

    Lott, Geoffrey Adam

    When two or more fluorescent chromophores are closely spaced in a macromolecular complex, dipolar coupling leads to delocalization of the excited states, forming excitons. The relative transition frequencies and magnitudes are sensitive to conformation, which can then be studied with optical spectroscopy. Non-invasive fluorescence spectroscopy techniques are useful tools for the study of dilute concentrations of such naturally fluorescent or fluorescently labeled biological systems. This dissertation presents two phase-selective fluorescence spectroscopy techniques for the study of dynamical processes in bio-molecular systems across a wide range of timescales. Polarization-modulated Fourier imaging correlation spectroscopy (PM-FICS) is a novel phase-selective fluorescence spectroscopy for simultaneous study of translational and conformational dynamics. We utilize modulated polarization and intensity gratings with phase-sensitive signal collection to monitor the collective fluctuations of an ensemble of fluorescent molecules. The translational and conformational dynamics can be separated and analyzed separately to generate 2D spectral densities and joint probability distributions. We present results of PM-FICS experiments on DsRed, a fluorescent protein complex. Detailed information on thermally driven dipole-coupled optical switching pathways is found, for which we propose a conformation transition mechanism. 2D phase-modulation electronic coherence spectroscopy is a third-order nonlinear spectroscopy that uses collinear pulse geometry and acousto-optic phase modulation to isolate rephasing and nonrephasing contributions to the collected fluorescence signal. We generate 2D spectra, from which we are able to determine relative dipole orientations, and therefore structural conformation, in addition to detailed coupling information. We present results of experiments on magnesium tetraphenylporphyrin dimers in lipid vesicle bilayers. The 2D spectra show clearly resolved diagonal and off-diagonal features, evidence of exciton behavior. The amplitudes of the distinct spectral features change on a femtosecond timescale, revealing information on time-dependent energy transfer dynamics. This dissertation includes co-authored and previously published material.

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

    SciTech Connect

    Brack, W.; Rottler, H.; Frank, H.

    1998-10-01

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

  16. In vivo simulation environment for fluorescence molecular tomography using Monte Carlo method

    NASA Astrophysics Data System (ADS)

    Zhang, Yizhai; Xu, Qiong; Li, Jin; Tang, Shaojie; Zhang, Xin

    2008-12-01

    Optical sensing of specific molecular target using near-infrared light has been recognized to be the crucial technology, have changing human's future. The imaging of Fluorescence Molecular Tomography is the most novel technology in optical sensing. It uses near-infrared light(600-900nm) as instrument and utilize fluorochrome as probe to take noncontact three-dimensional imaging for live molecular targets and to exhibit molecular process in vivo. In order to solve the problem of forward simulation in FMT, this paper mainly introduces a new simulation modeling. The modeling utilizes Monte Carlo method and is implemented in C++ programming language. Ultimately its accuracy has been testified by comparing with analytic solutions and MOSE from University of Iowa and Chinese Academy of Science. The main characters of the modeling are that it can simulate both of bioluminescent imaging and FMT and take analytic calculation and support more than one source and CCD detector simultaneously. It can generate sufficient and proper data and pre-preparation for the study of fluorescence molecular tomography.

  17. Confocal in vivo microscopy and confocal laser-scanning fluorescence microscopy in keratoconus.

    PubMed

    Somodi, S; Hahnel, C; Slowik, C; Richter, A; Weiss, D G; Guthoff, R

    1996-11-01

    The purpose of this study was the determination of morphological changes in the corneal epithelium and the keratocyte network in keratoconus. In all, 33 eyes of 19 patients were examined in vivo using the confocal slit-scanning microscope Microphthal. After penetrating keratoplasty, recipients' trephanates were stained with the Live/Dead kit and examined using the confocal laser-scanning fluorescence microscope Diaphot 300/Odyssey. The fluorescence images were reconstructed three-dimensionally. All findings were compared with data from healthy corneas. Morphological alterations were found only in the area of the corneal apex; obviously elongated superficial epithelial cells arranged in a whorl-like fashion were found. Near Bowman's membrane, highly reflective changes and fold-like structures were visible. The anterior stroma also showed an increased reflectivity. In the posterior stroma, typical findings were Vogt's striae and keratocytes with extremely long processes arranged nearly in parallel. In scarred stroma the keratocytes were spindle-shaped and arranged irregularly. The spatial organization of the living keratocyte network could be demonstrated through three-dimensional reconstructions. PMID:9479549

  18. Identification of Listeria monocytogenes In Vivo-Induced Genes by Fluorescence-Activated Cell Sorting

    PubMed Central

    Wilson, Rebecca L.; Tvinnereim, A. R.; Jones, Bradley D.; Harty, John T.

    2001-01-01

    Listeria monocytogenes is a gram-positive, intracellular, food-borne pathogen capable of causing severe infections in immunocompromised or pregnant individuals, as well as numerous animal species. Genetic analysis of Listeria pathogenesis has identified several genes which are crucial for virulence. The transcription of most of these genes has been shown to be induced upon entry of Listeria into the host cell. To identify additional genes that are induced in vivo and may be required for L. monocytogenes pathogenesis, a fluorescence-activated cell-sorting technique was initiated. Random fragments of the L. monocytogenes chromosome were cloned into a plasmid carrying a promoterless green fluorescent protein (GFP) gene, and the plasmids were transformed into the L. monocytogenes actA mutant DP-L1942. Fluorescence-activated cell sorting (FACS) was used to isolate L. monocytogenes clones that exhibited increased GFP expression within macrophage-like J774 cells but had relatively low levels of GFP expression when the bacteria were extracellular. Using this strategy, several genes were identified, including actA, that exhibited such an expression profile. In-frame deletions of two of these genes, one encoding the putative L. monocytogenes uracil DNA glycosylase (ung) and one encoding a protein with homology to the Bacillus subtilis YhdP hemolysin-like protein, were constructed and introduced into the chromosome of wild-type L. monocytogenes 10403s. The L. monocytogenes 10403s ung deletion mutant was not attenuated for virulence in mice, while the yhdP mutant exhibited a three- to sevenfold reduction in virulence. PMID:11447181

  19. Fluorescence and UV/VIS absorption spectroscopy studies on polymer blend films for photovoltaics

    NASA Astrophysics Data System (ADS)

    van Stam, Jan; Lindqvist, Camilla; Hansson, Rickard; Ericsson, Leif; Moons, Ellen

    2015-08-01

    The quinoxaline-based polymer TQ1 (poly[2,3-bis-(3-octyloxyphenyl)quinoxaline-5,8-diyl-alt-thiophene-2,5- diyl]) is a promising candidate as electron donor in organic solar cells. In combination with the electron acceptor [6,6]- phenyl-C71- butyric acid methyl ester (PC70BM), TQ1 has resulted in solar cells with power conversion efficiencies of 7 %. We have studied TQ1 films, with and without PC70BM, spin-casted from different solvents, by fluorescence spectroscopy and UV/VIS absorption spectroscopy. We used chloroform (CF), chlorobenzene (CB), and odichlorobenzene (o-DCB) as solvents for the coating solutions and 1-chloronaphthalene (CN) as solvent additive. CN addition has been shown to enhance photo-conversion efficiency of these solar cells. Phase-separation causes lateral domain formation in the films and the domain size depends on the solvent . These morphological differences coincide with changes in the spectroscopic patterns of the films. From a spectroscopic point of view, TQ1 acts as fluorescent probe and PC70BM as quencher. The degree of fluorescence