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Sample records for vivo fluorescence spectroscopy

  1. In Vivo Fluorescence Correlation and Cross-Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Mütze, Jörg; Ohrt, Thomas; Petrášek, Zdeněk; Schwille, Petra

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

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

    NASA Astrophysics Data System (ADS)

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

    2009-07-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

  4. 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.

  5. Dual-color fluorescence fluctuation spectroscopy in vitro and in vivo

    NASA Astrophysics Data System (ADS)

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

    2004-06-01

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

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

    PubMed

    Chang, Sung K; Marin, Nena; Follen, Michele; Richards-Kortum, Rebecca

    2006-01-01

    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. PMID:16674198

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

    NASA Astrophysics Data System (ADS)

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

    2012-09-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2003-11-01

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

  9. 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.

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

    PubMed Central

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

    2012-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

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

  12. 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.

  13. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2007-06-01

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

  15. 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

  16. 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.

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

    PubMed Central

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

    2010-01-01

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

  18. 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.; Kaščáková, 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.

  19. 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

  20. Nanosecond fluorescence spectroscopy

    SciTech Connect

    Leskovar, B.

    1985-03-01

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

  1. Protein influence on the plasma membrane dielectric properties: in vivo study utilizing dielectric spectroscopy and fluorescence microscopy.

    PubMed

    Stoneman, M; Chaturvedi, A; Jansma, D B; Kosempa, M; Zeng, C; Raicu, V

    2007-05-01

    We have investigated the origin of the dielectric response of the plasma membrane of living yeast cells (Saccharomyces cerevisiae) by using radiofrequency dielectric spectroscopy. The cells were genetically engineered to overexpress in the membrane of yeast cells a G protein-coupled receptor--the Sterile2-alpha factor receptor protein (Ste2p)--fused to the green fluorescent protein (GFP). Presence of the Ste2-GFP proteins in the plasma membrane was confirmed by exciting the cells at 476 nm and observing with a confocal microscope the emission characteristic of the GFP from individual cells. The dielectric behavior of cells suspended in KCl solution was analyzed over the frequency range 40 Hz-110 MHz and compared to the behavior of control cells that lacked the ability to express Ste2p. A two-shell electrical cell model was used to fit the data starting from known structural parameters and adjustable electrical phase parameters. The best-fit value for the relative permittivity of the plasma membrane showed no significant difference between cells expressing Ste2p (1.63+/-0.11) and the control cells (1.75+/-0.16). This result confirmed earlier predictions that the dielectric properties of the plasma membrane in the radiofrequency range mostly reflect the properties of the hydrophobic layer of the membrane, which is populated by the hydrocarbon tails of the phospholipids and hydrophobic segments of integral membrane proteins. We discuss ways by which dielectric spectroscopy can be improved to be used for tag-free detection of proteins on the membrane. PMID:17350897

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

    NASA Astrophysics Data System (ADS)

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

    2012-11-01

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

  3. 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.

  4. High-Pressure Fluorescence Spectroscopy.

    PubMed

    Maeno, Akihiro; Akasaka, Kazuyuki

    2015-01-01

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

  5. 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

  6. Fluorescence Spectroscopy in a Shoebox

    NASA Astrophysics Data System (ADS)

    Farooq Wahab, M.

    2007-08-01

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

  7. 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 ...

  8. 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.

  9. 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

  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-force spectroscopy at the single molecule level

    NASA Astrophysics Data System (ADS)

    Zhou, Ruobo

    During the past decade, various powerful single-molecule techniques have evolved and helped to address important questions in life sciences. As the single molecule techniques become mature, there is increasingly pressing need to maximize the information content of the analysis in order to be able to study more complex systems that better approximate in-vivo conditions. Here, we develop a fluorescence-force spectroscopy method to combine single-molecule fluorescence spectroscopy with optical tweezers. Optical tweezers are used to manipulate and observe mechanical properties on the nanometer scale and piconewton force range. However, once the force range is in the low piconewton range or less, the spatial resolution of optical tweezers decreases significantly. In combination with fluorescence spectroscopy, like single molecule Forster (or fluorescence) resonance energy transfer (FRET) whose detectable distance range is approximately 3-10 nm, we are able to observe nanometer fluctuations and internal conformational changes in a low-force regime. The possibility to place fluorescent labels at nearly any desired position and a sophisticated design of the experiment increases the amount of information that can be extracted in contrast to pure mechanical or fluorescence experiments. We demonstrate the applications of this method to various biological systems including: 1) to measure the effect of very low forces on the nanometer scale conformational transitions of the DNA four-way (Holliday) junction; 2) to dissect protein diffusion and dissociation mechanisms on single stranded DNA, 3) to calibrate FRET-based in-vivo force sensors and 4) to study mechanical unfolding of single proteins. The results could not have been obtained with fluorescence or force measurement alone, and clearly demonstrates the power and generality of our approach. Finally, we show that self-quenching of two identical fluorophores can be used to detect small conformational dynamics corresponding to sub-nanometer distance changes of single molecules in a FRET-insensitive short range (< 3 nm), extending the detectable distance range of our fluorescence-force spectroscopy method.

  14. 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

  15. 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.

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

    PubMed

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

    2016-03-01

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

  17. 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

  18. 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.

  19. 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.

  20. Fluorescence spectroscopy for wastewater monitoring: A review.

    PubMed

    Carstea, Elfrida M; Bridgeman, John; Baker, Andy; Reynolds, Darren M

    2016-05-15

    Wastewater quality is usually assessed using physical, chemical and microbiological tests, which are not suitable for online monitoring, provide unreliable results, or use hazardous chemicals. Hence, there is an urgent need to find a rapid and effective method for the evaluation of water quality in natural and engineered systems and for providing an early warning of pollution events. Fluorescence spectroscopy has been shown to be a valuable technique to characterize and monitor wastewater in surface waters for tracking sources of pollution, and in treatment works for process control and optimization. This paper reviews the current progress in applying fluorescence to assess wastewater quality. Studies have shown that, in general, wastewater presents higher fluorescence intensity compared to natural waters for the components associated with peak T (living and dead cellular material and their exudates) and peak C (microbially reprocessed organic matter). Furthermore, peak T fluorescence is significantly reduced after the biological treatment process and peak C is almost completely removed after the chlorination and reverse osmosis stages. Thus, simple fluorometers with appropriate wavelength selectivity, particularly for peaks T and C could be used for online monitoring in wastewater treatment works. This review also shows that care should be taken in any attempt to identify wastewater pollution sources due to potential overlapping fluorophores. Correlations between fluorescence intensity and water quality parameters such as biochemical oxygen demand (BOD) and total organic carbon (TOC) have been developed and dilution of samples, typically up to ×10, has been shown to be useful to limit inner filter effect. It has been concluded that the following research gaps need to be filled: lack of studies on the on-line application of fluorescence spectroscopy in wastewater treatment works and lack of data processing tools suitable for rapid correction and extraction of data contained in fluorescence excitation-emission matrices (EEMs) for real-time studies. PMID:26999254

  1. Fluorescence Correlation Spectroscopy: The Case of Subdiffusion

    PubMed Central

    Lubelski, Ariel; Klafter, Joseph

    2009-01-01

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

  2. 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

  3. High-pressure fluorescence correlation spectroscopy.

    PubMed

    Müller, Joachim D; Gratton, Enrico

    2003-10-01

    We demonstrate that a novel high-pressure cell is suitable for fluorescence correlation spectroscopy (FCS). The pressure cell consists of a single fused silica microcapillary. The cylindrical shape of the capillary leads to refraction of the excitation light, which affects the point spread function of the system. We characterize the influence of these beam distortions by FCS and photon-counting histogram (PCH) analysis and identify the optimal position for fluorescence fluctuation experiments in the capillary. At this position within the capillary, FCS and photon-counting histogram experiments are described by the same equations as used in standard FCS experiments. We report the first experimental realization of fluorescence fluctuation spectroscopy under high pressure. A fluorescent dye was used as a model system for evaluating the properties of the capillary under pressure. The autocorrelation function and the photon count distribution were measured in the pressure range from 0 to 300 MPa. The fluctuation amplitude and the diffusion coefficient show a small pressure dependence. The changes of these parameters, which are on the order of 10%, are due to the pressure changes of the viscosity and the density of the aqueous medium. PMID:14507734

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. Two-Photon Fluorescence Correlation Spectroscopy

    NASA Technical Reports Server (NTRS)

    Zimmerli, Gregory A.; Fischer, David G.

    2002-01-01

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

  9. Applications of Fluorescence Correlation Spectroscopy: Polydispersity Measurements.

    PubMed

    Starchev; Buffle; Prez

    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

  10. 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

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

    PubMed

    Lv, Feng; Mao, Lina; Liu, Tianjun

    2014-10-01

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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. Towards a disposable in vivo miniature implantable fluorescence detector

    NASA Astrophysics Data System (ADS)

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

    2006-02-01

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

  20. 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

  1. 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.

  2. Laser Induced Fluorescence Spectroscopy of Soft Tissues of the Oral Cavity

    NASA Astrophysics Data System (ADS)

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

    2011-07-01

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

  3. Imaging cellular dynamics in vivo with multicolor fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Hoffman, Robert M.

    2005-04-01

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

  4. 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, Jrg; 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

  5. 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 μm) 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.

  6. 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%.

  7. 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.

  8. 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.

  9. In vivo study of photosensitizer pharmacokinetics by fluorescence transillumination imaging

    NASA Astrophysics Data System (ADS)

    Shirmanova, Marina; Zagaynova, Elena; Sirotkina, Marina; Snopova, Ludmila; Balalaeva, Irina; Krutova, Irina; Lekanova, Nataliya; Turchin, Ilya; Orlova, Anna; Kleshnin, Michail

    2010-07-01

    The possibility of in vivo investigation of the pharmacokinetics of photosensitizers by means of fluorescence transillumination imaging is demonstrated. An animal is scanned in the transilluminative configuration by a single source and detector pair. Transillumination is chosen as an alternative approach to reflection imaging. In comparison with the traditional back-reflection technique, transillumination is preferable for photosensitizer detection due to its higher sensitivity to deep-seated fluorophores. The experiments are performed on transplantable mouse cervical carcinomas using three drugs: photosens, alasens, and fotoditazin. For quantitative evaluation of the photosensitizer concentration in tumor tissue the fluorescence signal is calibrated using tissue phantoms. We show that the kinetics of photosensitizer tumor uptake obtained by transillumination imaging in vivo agree with data of standard ex vivo methods. The described approach enables rapid and cost-effective study of newly developed photosensitizers in small animals.

  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. 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.

  12. 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.

  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. Widefield multiphoton excited fluorescence microscopy for animal study in vivo

    NASA Astrophysics Data System (ADS)

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

    2010-08-01

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

  16. In vivo layer-resolved characterization of oral dysplasia via nonlinear optical micro-spectroscopy

    PubMed Central

    Edward, Kert; Qiu, Suimin; Resto, Vicente; McCammon, Susan; Vargas, Gracie

    2012-01-01

    Optical spectroscopy has proven to be a powerful technique for studying neoplastic transformation in epithelial tissue. Since specific intra-layer precancerous changes originate in the stratified layers of the oral mucosa, layer-resolved analysis will likely improve both our understanding of the mechanism of premalignant transformation, and clinical diagnostic outcomes. However, the native fluorescence signal in linear spectroscopy typically originates from a multi-layered focal volume. In this study, nonlinear spectroscopy was exploited for in vivo layer-resolved discrimination between normal and dysplastic tissue for the first time. Our results revealed numerous intra-layer specific differences. PMID:22808430

  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. In vivo validation of quantitative frequency domain fluorescence tomography

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

  20. 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.

  1. 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.

  2. 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.

  3. 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

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

    PubMed

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

    2010-01-01

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

  5. 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

  6. 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.

  7. 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.

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

    PubMed

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

    2009-10-01

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

  9. 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.

  10. Pancreatic tissue assessment using fluorescence and reflectance spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2007-07-01

    The ability of multi-modal optical spectroscopy to detect signals from pancreatic tissue was demonstrated by studying human pancreatic cancer xenografts in mice and freshly excised human pancreatic tumor tissue. Measured optical spectra and fluorescence decays were correlated with tissue morphological and biochemical properties. The measured spectral features and decay times correlated well with expected pathological differences in normal, pancreatitis and adenocarcinoma tissue states. The observed differences between the fluorescence and reflectance properties of normal, pancreatitis and adenocarcinoma tissue indicate a possible application of multi-modal optical spectroscopy to differentiating between the three tissue classifications.

  11. 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.

  12. 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.

  13. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2002-08-01

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

  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. Uptake of fluorescent gentamicin by vertebrate sensory cells in vivo

    PubMed Central

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

    2008-01-01

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

  19. Near-infrared autofluorescence spectroscopy for in vivo diagnosis of cervical precancer

    NASA Astrophysics Data System (ADS)

    Mo, Jianhua; Zheng, Wei; Low, Jeffrey; Ng, Joseph; Ilancheran, A.; Huang, Zhiwei

    2008-02-01

    The purpose of this study is to explore the feasibility of utilizing near-infrared (NIR) autofluorescence spectroscopy for in vivo diagnosis of precancer (i.e., dysplasia) in the cervix. A rapid NIR spectroscopy system in combination with a fiber-optic probe was developed for the in vivo NIR fluorescence measurements under the 785 nm laser excitation. Multivariate statistical techniques including principal component analysis (PCA) and linear discriminant analysis (LDA) were employed to develop the diagnostic algorithms for spectra classification. Classification result obtained from the PCA-LDA model based on tissue NIR autofluorescence data yielded a diagnostic sensitivity of 84.8% and specificity of 85.1% for discrimination of precancer from normal cervical sites. The results demonstrate that NIR autofluorescence technique has the capacity for the noninvasive, in vivo diagnosis of precancer in the cervix.

  20. "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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2014-01-01

    The sensitivity and resolution of fluorescence-based imaging in vivo is often limited by autofluorescence and other background noise. To overcome these limitations, we have developed a wide-field background-free imaging technique based on magnetic modulation of fluorescent nanodiamond emission. Fluorescent nanodiamonds are bright, photo-stable, biocompatible nanoparticles that are promising probes for a wide range of in vitro and in vivo imaging applications. Our readily applied background-free imaging technique improves the signal-to-background ratio for in vivo imaging up to 100-fold. This technique has the potential to significantly improve and extend fluorescent nanodiamond imaging capabilities on diverse fluorescence imaging platforms. PMID:24761300

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

    PubMed

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

    2013-01-01

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

  9. 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.

  10. 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

  11. 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.

  12. Multiphoton spectroscopy of human skin in vivo

    NASA Astrophysics Data System (ADS)

    Breunig, Hans G.; Weinigel, Martin; Knig, 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.

  13. 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.

  14. 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.

  15. In vivo monitoring of photosensitizer fluorescence during photodynamic therapy

    NASA Astrophysics Data System (ADS)

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

    1995-03-01

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

  16. Spectroscopy analysis of tissues in vivo

    NASA Astrophysics Data System (ADS)

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

    1995-01-01

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

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

    PubMed Central

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

    2011-01-01

    Mechanical tension plays a large role in cell development ranging from morphology to gene expression. On the molecular level, the effects of tension can be seen in the dynamic arrangement of membrane proteins as well as the recruitment and activation of intracellular proteins. Forces applied to biopolymers during in vitro force measurements offer greater understanding of the effects of tension on molecules in live cells, and experimental techniques in test tubes and live cells can often overlap. Indeed, when forces exerted on cellular components can be calibrated ex vivo with force spectroscopy, a powerful tool is available for researchers in probing cellular mechanotransduction on the molecular scale. This review will discuss the techniques used in measuring both cellular traction forces and single-molecule force spectroscopy. Emphasis will be placed on the use of fluorescence reporter systems for the development of in vivo tension sensors that can be used for calibration with single molecule force methods. PMID:21267988

  18. 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.

  19. 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.

  20. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  2. Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope

    NASA Astrophysics Data System (ADS)

    Borejdo, J.; Calander, N.; Gryczynski, Z.; Gryczynski, I.

    2006-08-01

    Study of dynamics of single molecules by Fluorescence Correlation Spectroscopy (FCS) requires that the rate of photon detection per molecule be high, that the background be low, and that there be a large change in fluorescent signal associated with change in a position of a molecule. FCS applied to microscopic Surface Plasmon Coupled Emission (SPCE) suggests a powerful method to meet those requirements. In this method, the observational volume is made shallow by placing a sample on a thin metal film and illuminating it with the laser beam at Surface Plasmon Resonance (SPR) angle through high numerical aperture objective. The illuminating light excites surface plasmons in the metal film that produce an evanescent wave on the aqueous side of the interface. The thickness of the detection volume is a product of evanescent wave penetration depth and distance-dependent fluorescence coupling to surface plasmons. It is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm) to a surface. The fluorescent light is emitted through the metal film only at an SPCE angle. Objective collects emitted light, and a confocal aperture inserted in its conjugate image plane reduces lateral dimensions of the detection volume to a fraction of a micrometer. By using diffusion of fluorescent microspheres, we show that SPCE-FCS is an efficient method to measure molecular diffusion and that on gold surface the height of the detection volume is ~35 nm.

  3. Total internal reflection Raman spectroscopy of barley leaf epicuticular waxes in vivo.

    PubMed

    Greene, Phillip R; Bain, Colin D

    2005-11-10

    The use of total internal reflection (TIR)-Raman spectroscopy to examine cuticular wax layers in vivo at the surface of barley leaves (Hordeum vulgare L. cultivar Pastoral) is demonstrated. The limited penetration depth (40 nm) of the evanescent wave compared to the thickness of the wax layer eliminates problems arising from fluorescence from underlying pigments. Epicuticular wax crystals are observed to be more crystalline than the amorphous wax layer, which is analysed after removal of the wax crystals by cellulose acetate stripping. Carotenoids are detected as trace species in the cuticular waxes. PMID:16198093

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. Cytoskeleton dynamics studied by dispersion-relation fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

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

    PubMed Central

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

    2007-01-01

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

  12. 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.

  13. 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).

  14. New approach on fluorescence spectroscopy for caries detection

    NASA Astrophysics Data System (ADS)

    Hibst, Raimund; Paulus, Robert

    1999-05-01

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

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

    NASA Astrophysics Data System (ADS)

    Makoui, Anali

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

  16. 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

  17. 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.

  18. Quantum process tomography by 2D fluorescence spectroscopy

    SciTech Connect

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

    2015-06-07

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

  19. Optical fiber fluorescence spectroscopy for detecting AFM1 in milk

    NASA Astrophysics Data System (ADS)

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

    2008-04-01

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

  20. 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.

  1. 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

  2. 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.

  3. Accounting for misalignments and thermal fluctuations in fluorescence correlation spectroscopy experiments on membranes.

    PubMed

    Sanguigno, Luigi; Cosenza, Chiara; Causa, Filippo; Netti, Paolo Antonio

    2013-03-21

    Several authors have exploited the ability of the fluorescence correlation spectroscopy to probe motion at the molecular level. In a couple of decades, all their efforts have allowed the application of this technique even to the diffusion measurement of cellular components. Nowadays, the fluorescence correlation spectroscopy is considered a standard tool to measure diffusion in cells both in vivo and in vitro. Unfortunately, while the interpretation and the set-up have been consolidated for 3D diffusion measurements (i.e. diffusion in an aqueous solution), the experiments carried out on flat elements, such as membranes, show unusually high relative errors. Furthermore, long tail correlations are generally detected and ascribed to diffusion anomalies. The 2D fluorescence correlation measurements have been interpreted under certain hypotheses, whereby the membrane is assumed to be perfectly flat, motionless and aligned with the optical axes. Here, we investigated the robustness of these hypotheses, trying to understand, in an elementary but not trivial way, how misalignments and thermal fluctuations affect the temporal correlation of the intensity fluctuation collected during measurements on membranes. PMID:23338952

  4. 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

  5. Identification of active fluorescence stained bacteria by Raman spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2008-04-01

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

  6. 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

  7. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2010-12-01

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

  9. 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.

  10. 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.

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

    PubMed

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

    2014-03-01

    The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 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. PMID:24689603

  12. 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 0.87 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

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

    PubMed

    Werahera, Priya N; Jasion, Edward A; Liu, Yongjun; 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 8×100μm fibers for tissue excitation and a single 200μm 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

  14. 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

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

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

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

  16. Remote excitation fluorescence correlation spectroscopy using silver nanowires

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

    Fluorescence correlation spectroscopy (FCS), a powerful tool to resolve local properties, dynamical process of molecules, rotational and translational diffusion motions, relies on the fluctuations of florescence observables in the observation volume. In the case of rare transition events or small dynamical fluctuations, FCS requires few molecules or even single molecules in the observation volume at a time to minimize the background signals. Metal nanoparticle which possess unique localized surface plasmon resonance (LSPR) have been used to reduce the observation volume down to sub-diffraction limited scale while maintain at high analyst concentration up to tens of micromolar. Nevertheless, the applications of functionalized nanoparticles in living cell are limited due to the continuous diffusion after cell uptake, which makes it difficult to target the region of interests in the cell. In this work, we demonstrate the use of silver nanowires for remote excitation FCS on fluorescent molecules in solution. By using propagation surface plasmon polaritons (SPPs) which supported by the silver nanowire to excite the fluorescence, both illumination and observation volume can be reduced simultaneously. In such a way, less perturbation is induced to the target region, and this will broaden the application scope of silver nanowire as tip in single cell endoscopy.

  17. 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

  18. 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

  19. 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

  20. Comparison of absorption, fluorescence, and polarization spectroscopy of atomic rubidium

    NASA Astrophysics Data System (ADS)

    Ashman, Seth; Stifler, Cayla; Romero, Joaquin

    2015-05-01

    An ongoing spectroscopic investigation of atomic rubidium utilizes a two-photon, single-laser excitation process. Transitions accessible with our tunable laser include 5P1 / 2F' <-- 5S1 / 2 F and 5P3 / 2F' <-- 5S1 / 2 F . The laser is split into a pump and probe beam to allow for Doppler-free measurements of transitions between hyperfine levels. The pump and probe beams are overlapped in a counter-propagating geometry and the laser frequency scans over a transition. Absorption, fluorescence and polarization spectroscopy techniques are applied to this basic experimental setup. The temperature of the vapor cell and the power of the pump and probe beams have been varied to explore line broadening effects and signal-to-noise of each technique. This humble setup will hopefully grow into a more robust experimental arrangement in which double resonance, two-laser excitations are used to explore hyperfine state changing collisions between rubidium atoms and noble gas atoms. Rb-noble gas collisions can transfer population between hyperfine levels, such as 5P3 / 2 (F' = 3) <-- Collision 5P3 / 2 (F ' = 2) , and the probe beam couples 7S1 / 2 (F'' = 2) <-- 5P3 / 2 (F' = 3) . Polarization spectroscopy signal depends on the rate of population transfer due to the collision as well as maintaining the orientation created by the pump laser. Fluorescence spectroscopy relies only on transfer of population due to the collision. Comparison of these techniques yields information regarding the change of the magnetic sublevels, mF, during hyperfine state changing collisions.

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

    PubMed

    Ishii, Kunihiko; Tahara, Tahei

    2013-10-01

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

  2. 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.

  3. 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.

  4. 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.

  5. 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

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

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  7. Biosurfactant templated quantum sized fluorescent gold nanoclusters for in vivo bioimaging in zebrafish embryos.

    PubMed

    S, Chandirasekar; C, Chandrasekaran; T, Muthukumarasamyvel; G, Sudhandiran; N, Rajendiran

    2016-07-01

    We report the biosurfactant (sodium cholate) templated bright bluish-green emitting gold nanoclusters (AuNCs) by green chemical approach. Optical properties of the AuNCs were studied using UV-vis and luminescence spectroscopy. Lifetime of the fluorescent AuNCs was measured using time correlated single photon counting technique (TCSPC). High-resolution transmission electron microscopy (HR-TEM) and dynamic light scattering (DLS) were used to measure the sizes of the clusters. In-vivo toxicity and bioimaging studies of sodium cholate (NaC) templated AuNCs were carried out at different developmental stages of zebrafish embryos. The survival rate, hatching rate, heart rate, malformation and apoptotic gene expression experiments shows no significant toxicity in developing embryos up to 100μL/mL of AuNCs concentration and the AuNCs stained embryos exhibited green fluorescence with high intensity over the period from 4 to 96hpf (hours post fertilization) which shows that AuNCs were stable in living organisms. PMID:27037785

  8. Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Smith, Elizabeth Myhra

    The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

  9. 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

  10. 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

  11. Silica-porphyrin hybrid nanotubes for in vivo cell tracking by near-infrared fluorescence imaging.

    PubMed

    Hayashi, Koichiro; Nakamura, Michihiro; Ishimura, Kazunori

    2012-04-21

    Near-infrared fluorescent silica-porphyrin hybrid nanotubes (HNTs) were successfully synthesized by π-π stacking, electrostatic interaction and a sol-gel reaction. The HNTs-labeled macrophages were detected in vivo, and the minimum detectable number of cells was 200. Furthermore, the biodistribution of HNTs-labeled macrophages was tracked by fluorescence imaging. PMID:22437325

  12. 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

  13. Time-resolved fluorescence spectroscopy and ultrasound backscatter microscopy for nondestructive evaluation of vascular grafts

    PubMed Central

    Fatakdawala, Hussain; Griffiths, Leigh G.; Humphrey, Sterling; Marcu, Laura

    2014-01-01

    Abstract. Quantitative and qualitative evaluations of structure and composition are important in monitoring development of engineered vascular tissue both in vitro and in vivo. Destructive techniques are an obstacle for performing time-lapse analyses from a single sample or animal. This study demonstrates the ability of time-resolved fluorescence spectroscopy (TRFS) and ultrasound backscatter microscopy (UBM), as nondestructive and synergistic techniques, for compositional and morphological analyses of tissue grafts, respectively. UBM images and integrated backscatter coefficients demonstrate the ability to visualize and quantify postimplantation changes in vascular graft biomaterials such as loss of the external elastic lamina and intimal/medial thickening over the grafted region as well as graft integration with the surrounding tissue. TRFS results show significant changes in spectra, average lifetime, and fluorescence decay parameters owing to changes in collagen, elastin, and cellular content between normal and grafted tissue regions. These results lay the foundation for the application of a catheter-based technique for in vivo evaluation of vascular grafts using TRFS and UBM. PMID:25147960

  14. 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.

  15. FM spectroscopy in fluorescence in laser-cooled rubidium

    NASA Astrophysics Data System (ADS)

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

    1998-07-01

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

  16. 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.

  17. 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.

  18. 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.

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

    SciTech Connect

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

    1982-07-01

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

  20. [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

  1. Continuous Fluorescence Microphotolysis and Correlation Spectroscopy Using 4Pi Microscopy

    PubMed Central

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

    2007-01-01

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

  2. Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2007-11-01

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

  3. 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.

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

    PubMed Central

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

    2013-01-01

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

  5. Microneedles rollers as a potential device to increase ALA diffusion and PpIX production: evaluations by wide-field fluorescence imaging and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Gracielli Sousa, R. Phamilla; de Menezes, Priscila F. C.; Fujita, Alessandra K. L.; Requena, Michelle B.; Govone, Angelo Biassi; Escobar, André; de Nardi, Andrigo B.; Kurachi, Cristina; Bagnato, Vanderlei Salvador

    2014-03-01

    One of the limitations of topical photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) is the poor ability to penetrate biological barriers of skin and the recurrence rates in treatments. This study aimed to identify possible signs of increased diffusion of ALA-induced PpIX by fluorescence images and fluorescence spectroscopy. The research was done using in vivo porcine skin model. Before the cream application, microholes was performed with microneedles rollers in only one direction, afterward the ALA cream was applied at a 2.5cm2 area in triplicate and an occlusive dressing was placed. PpIX production was monitored using fluorescence spectroscopy collected at skin surface after 70, 100, 140, and 180 minutes of ALA incubation. About 100 fluorescence spectra of each treatment were collected, distributed by about five points for each site. Wide-field fluorescence imaging was made after 70, 90, and 170 minutes after treatment. The results obtained by imaging analysis indicated increase of the PpIX diffusion in the skin surface using the microneedles rollers (MNs) before ALA application. Circular regions of red fluorescence around the microholes were observed. In addition, the fluorescence spectra showed a greater intensity (2 times as many) in groups microneedles rollers associated. In conclusion, our data shown greater homogeneity and PpIX production in the groups pre-treated with microneedles indicating that the technique can be used to greater uniformity of PpIX production throughout the area to be treated reducing the chances of recurrent tumor as well as has potential for decreasing the time of therapy. (FUNDING SUPPORT:CAPES, CNPq and FAPESP)

  6. Transcutaneous Raman spectroscopy of murine bone in vivo.

    PubMed

    Schulmerich, Matthew V; Cole, Jacqueline H; Kreider, Jaclynn M; Esmonde-White, Francis; Dooley, Kathryn A; Goldstein, Steven A; Morris, Michael D

    2009-03-01

    Raman spectroscopy can provide valuable information about bone tissue composition in studies of bone development, biomechanics, and health. In order to study the Raman spectra of bone in vivo, instrumentation that enhances the recovery of subsurface spectra must be developed and validated. Five fiber-optic probe configurations were considered for transcutaneous bone Raman spectroscopy of small animals. Measurements were obtained from the tibia of sacrificed mice, and the bone Raman signal was recovered for each probe configuration. The configuration with the optimal combination of bone signal intensity, signal variance, and power distribution was then evaluated under in vivo conditions. Multiple in vivo transcutaneous measurements were obtained from the left tibia of 32 anesthetized mice. After collecting the transcutaneous Raman signal, exposed bone measurements were collected and used as a validation reference. Multivariate analysis was used to recover bone spectra from transcutaneous measurements. To assess the validity of the transcutaneous bone measurements cross-correlations were calculated between standardized spectra from the recovered bone signal and the exposed bone measurements. Additionally, the carbonate-to-phosphate height ratios of the recovered bone signals were compared to the reference exposed bone measurements. The mean cross-correlation coefficient between the recovered and exposed measurements was 0.96, and the carbonate-to-phosphate ratios did not differ significantly between the two sets of spectra (p > 0.05). During these first systematic in vivo Raman measurements, we discovered that probe alignment and animal coat color influenced the results and thus should be considered in future probe and study designs. Nevertheless, our noninvasive Raman spectroscopic probe accurately assessed bone tissue composition through the skin in live mice. PMID:19281644

  7. 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

  8. 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.

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

    PubMed

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

    2016-05-01

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

  10. A Rapid and Convenient Method for in Vivo Fluorescent Imaging of Protoscolices of Echinococcus multilocularis

    PubMed Central

    Yang, Tao; Wang, Sibo; Zhang, Xuyong; Xia, Jie; Guo, Jun; Hou, Jixue; Zhang, Hongwei; Chen, Xueling; Wu, Xiangwei

    2016-01-01

    Human and animal alveolar echinococcosis (AE) are important helminth infections endemic in wide areas of the Northern hemisphere. Monitoring Echinococcus multilocularis viability and spread using real-time fluorescent imaging in vivo provides a fast method to evaluate the load of parasite. Here, we generated a kind of fluorescent protoscolices in vivo imaging model and utilized this model to assess the activity against E. multilocularis protoscolices of metformin (Met). Results indicated that JC-1 tagged E. multilocularis can be reliably and confidently used to monitor protoscolices in vitro and in vivo. The availability of this transient in vivo fluorescent imaging of E. multilocularis protoscolices constitutes an important step toward the long term bio-imaging research of the AE-infected mouse models. In addition, this will be of great interest for further research on infection strategies and development of drugs and vaccines against E. multilocularis and other cestodes. PMID:27180584

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

    NASA Astrophysics Data System (ADS)

    Glassman, Wenling Sha

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

  12. 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.

  13. 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 62C by the intrinsic, exogenous ANS fluorescence and the analysis of hydrophobicity. Besides, the isosbestic point of the sigmoidal curve was 58.101.02C by calculating ?-helix transition and the Tm was 56.450.52 and 55.590.68C 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 40C, 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 40C, has an important impact on the structural stability of apo-CP43. PMID:26071019

  14. 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).

  15. Fluorescence Spectroscopy: An Adjunct Diagnostic Tool to Image-Guided Core Needle Biopsy of the Breast

    PubMed Central

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

    2009-01-01

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

  16. Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo.

    PubMed

    Sawa, Masaaki; Hsu, Tsui-Ling; Itoh, Takeshi; Sugiyama, Masakazu; Hanson, Sarah R; Vogt, Peter K; Wong, Chi-Huey

    2006-08-15

    Glycomics is emerging as a new field for the biology of complex glycoproteins and glycoconjugates. The lack of versatile glycan-labeling methods has presented a major obstacle to visualizing at the cellular level and studying glycoconjugates. To address this issue, we developed a fluorescent labeling technique based on the Cu(I)-catalyzed [3 + 2] cycloaddition, or click chemistry, which allows rapid, versatile, and specific covalent labeling of cellular glycans bearing azide groups. The method entails generating a fluorescent probe from a nonfluorescent precursor, 4-ethynyl-N-ethyl-1,8-naphthalimide, by clicking the fluorescent trigger, the alkyne at the 4 position, with an azido-modified sugar. Using this click-activated fluorescent probe, we demonstrate incorporation of an azido-containing fucose analog into glycoproteins via the fucose salvage pathway. Distinct fluorescent signals were observed by flow cytometry when cells treated with 6-azidofucose were labeled with the click-activated fluorogenic probe or biotinylated alkyne. The intracellular localization of fucosylated glycoconjugates was visualized by using fluorescence microscopy. This technique will allow dynamic imaging of cellular fucosylation and facilitate studies of fucosylated glycoproteins and glycolipids. PMID:16895981

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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

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

    PubMed Central

    Sevenich, F W; Langowski, J; Weiss, V; Rippe, K

    1998-01-01

    Fluorescence anisotropy and fluorescence correlation spectroscopy measurements of rhodamine-labeled DNA oligonucleotide duplexes have been used to determine equilibrium binding constants for DNA binding of the prokaryotic transcription activator protein NtrC. Measurements were made with wild-type NtrC from Escherichia coli and the constitutively active mutant NtrCS160Ffrom Salmonella using DNA duplexes with one or two binding sites. The following results were obtained: (i) the dissociation constant K d for binding of one NtrC dimer to a single binding site was the same for the wild-type and mutant proteins within the error of measurement. (ii) The value of K d decreased from 1.4 +/- 0.7 x 10(-11) M at 15 mM K acetate to 5.8 +/- 2.6 x 10(-9) M at 600 mM K acetate. From the salt dependence of the dissociation constant we calculated that two ion pairs form upon binding of one dimeric protein to the DNA. (iii) Binding of two NtrC dimers to the DNA duplex with two binding sites occured with essentially no cooperativity. Titration curves of NtrCS160Fbinding to the same duplex demonstrated that more than two protein dimers of the mutant protein could bind to the DNA. PMID:9490780

  4. 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

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

    PubMed

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

    2014-04-01

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

  6. 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

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

    NASA Astrophysics Data System (ADS)

    Tsai, Tsuimin; Chen, Chin-Tin; Wang, Chih-Yu; Chiang, Chung-Ping; Kuo, Yu-Luan

    2001-10-01

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

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

    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 (ɛnu), an important parameter of nucleoplasmic rheology. Our FCS analyses show that ɛnu 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 °C. ɛnu was reduced by 31 to 36% upon hypotonic exposure and increased by 28 to 52% from 37 to 24 °C. In addition, we found that ɛnu of HeLa cells reached the lowest value in the S phase and that there was no significant difference of ɛnu 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 ɛnu in living cells synchronized in the G1, S, and G2 phases.

  17. 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.

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

    PubMed

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

    2013-11-11

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

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

    PubMed Central

    Mo, Weirong; Rohrbach, Daniel

    2012-01-01

    Abstract. We report the tomographic imaging of a photodynamic therapy (PDT) photosensitizer, 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH) in vivo with time-domain fluorescence diffuse optical tomography (TD-FDOT). Simultaneous reconstruction of fluorescence yield and lifetime of HPPH was performed before and after PDT. The methodology was validated in phantom experiments, and depth-resolved in vivo imaging was achieved through simultaneous three-dimensional (3-D) mappings of fluorescence yield and lifetime contrasts. The tomographic images of a human head-and-neck xenograft in a mouse confirmed the preferential uptake and retention of HPPH by the tumor 24-h post-injection. HPPH-mediated PDT induced significant changes in fluorescence yield and lifetime. This pilot study demonstrates that TD-FDOT may be a good imaging modality for assessing photosensitizer distributions in deep tissue during PDT monitoring. PMID:22894467

  20. Analyses of the Dynamic Properties of Nuclear Lamins by Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS).

    PubMed

    Takeshi, Shimi; Pack, Chan-Gi; Goldman, Robert D

    2016-01-01

    The major structural components of the nuclear lamina are the A- and B-type nuclear lamin proteins which are also present in the nucleoplasm. Studies of molecular movements of the lamins in both the lamina and nucleoplasm of living cell nuclei have provided insights into their roles in maintaining nuclear architecture. In this chapter, we present protocols for quantitatively measuring the mobilities of lamin proteins by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) in mammalian cell nuclei. PMID:27147036

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

    PubMed

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

    2014-03-01

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

  2. 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.

  3. 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.

  4. 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.

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

    PubMed

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

    2009-09-01

    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

  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. 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.

  8. 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.

  9. 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.

  10. 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

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

    NASA Astrophysics Data System (ADS)

    Rujihan, Suparat; Damrongsak, Badin; Kittidachachan, Pattareeya

    2013-06-01

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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-10-01

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

  18. Near-Infrared Squaraine Dye Encapsulated Micelles for in Vivo Fluorescence and Photoacoustic Bimodal Imaging.

    PubMed

    Sreejith, Sivaramapanicker; Joseph, James; Lin, Manjing; Menon, Nishanth Venugopal; Borah, Parijat; Ng, Hao Jun; Loong, Yun Xian; Kang, Yuejun; Yu, Sidney Wing-Kwong; Zhao, Yanli

    2015-06-23

    Combined near-infrared (NIR) fluorescence and photoacoustic imaging techniques present promising capabilities for noninvasive visualization of biological structures. Development of bimodal noninvasive optical imaging approaches by combining NIR fluorescence and photoacoustic tomography demands suitable NIR-active exogenous contrast agents. If the aggregation and photobleaching are prevented, squaraine dyes are ideal candidates for fluorescence and photoacoustic imaging. Herein, we report rational selection, preparation, and micelle encapsulation of an NIR-absorbing squaraine dye (D1) for in vivo fluorescence and photoacoustic bimodal imaging. D1 was encapsulated inside micelles constructed from a biocompatible nonionic surfactant (Pluoronic F-127) to obtain D1-encapsulated micelles (D1(micelle)) in aqueous conditions. The micelle encapsulation retains both the photophysical features and chemical stability of D1. D1(micelle) exhibits high photostability and low cytotoxicity in biological conditions. Unique properties of D1(micelle) in the NIR window of 800-900 nm enable the development of a squaraine-based exogenous contrast agent for fluorescence and photoacoustic bimodal imaging above 820 nm. In vivo imaging using D1(micelle), as demonstrated by fluorescence and photoacoustic tomography experiments in live mice, shows contrast-enhanced deep tissue imaging capability. The usage of D1(micelle) proven by preclinical experiments in rodents reveals its excellent applicability for NIR fluorescence and photoacoustic bimodal imaging. PMID:26022724

  19. 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.

  20. 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.

  1. 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.

  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. New Homogeneous Standards by Atomic Layer Deposition for Synchrotron X-Ray Fluorescence and Absorption Spectroscopies

    NASA Astrophysics Data System (ADS)

    Butterworth, A.; Becker, N.; Gainsforth, Z.; Lanzirotti, A.; Newville, M.; Proslier, T.; Stodolna, J.; Sutton, S.; Tyliszczak, T.; Westphal, A. J.; Zasadzinski, J.

    2012-03-01

    New homogeneous multi-layer film standards synthesized using Atomic Layer Deposition and characterized by multiple analytical methods, including ellipsometry, RBS, TEM, and synchrotron x-ray fluorescence and absorption spectroscopies.

  4. 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...

  5. 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.

  6. 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

  7. Fluorescence Spectroscopy in Rapid Analysis of Scallop Adductor Muscle

    NASA Astrophysics Data System (ADS)

    Sugawara, Tomoaki; Nomura, Yasutomo; Kato, Sanae; Yoshioka, Takeya; Kinoshita, Yasunori; Oda, Isao

    Autofluorescence of refrigerated raw scallop adductor muscle was investigated. Fluorescence of reduced nicotinamide adenine dinucleotide (NADH) with peak wavelength at 458 nm was observed in fresh scallops. The fluorescence emission intensity of NADH decreased until 48 hours in the storage time at time. As for the fluorescence excitation spectra, the peak wavelength became long from 361 to 369 nm as the storage time became long.

  8. 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 μM. 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

  9. In vivo fluorescence lifetime detection of an activatable probe in infarcted myocardium

    NASA Astrophysics Data System (ADS)

    Goergen, Craig J.; Chen, Howard H.; Bogdanov, Alexei; Sosnovik, David E.; Kumar, Anand T. N.

    2012-05-01

    Activatable fluorescent molecular probes are predominantly nonfluorescent in their inactivated state due to intramolecular quenching, but increase fluorescence yield significantly after enzyme-mediated hydrolysis of peptides. Continuous wave in vivo detection of these protease-activatable fluorophores in the heart, however, is limited by the inability to differentiate between activated and nonactivated fractions of the probe and is frequently complicated by large background signal from probe accumulation in the liver. Using a cathepsin-activatable near-infrared probe (PGC-800), we demonstrate here that fluorescence lifetime (FL) significantly increases in infarcted murine myocardial tissue (0.67 ns) when compared with healthy myocardium (0.59 ns) after 24 h. Furthermore, we show that lifetime contrast can be used to distinguish in vivo cardiac fluorescence from background nonspecific liver signal. The results of this study show that lifetime contrast is a helpful addition to preclinical imaging of activatable fluorophores in the myocardium by reporting molecular activity in vivo due to changes in intramolecular quenching. This characterization of FL from activatable molecular probes will be helpful for advancing in vivo imaging of enzyme activity.

  10. 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.

  11. In vivo imaging of orthotopic prostate cancer with far-red gene reporter fluorescence tomography and in vivo and ex vivo validation

    PubMed Central

    Darne, Chinmay D.; Tan, I-Chih; Wu, Grace; Wilganowski, Nathaniel; Robinson, Holly; Azhdarinia, Ali; Zhu, Banghe; Rasmussen, John C.; Sevick-Muraca, Eva M.

    2013-01-01

    Abstract. Fluorescence gene reporters have recently become available for excitation at far-red wavelengths, enabling opportunities for small animal in vivo gene reporter fluorescence tomography (GRFT). We employed multiple projections of the far-red fluorescence gene reporters IFP1.4 and iRFP, excited by a point source in transillumination geometry in order to reconstruct the location of orthotopically implanted human prostate cancer (PC3), which stably expresses the reporter. Reconstruction was performed using a linear radiative-transfer-based regularization-free tomographic method. Positron emission tomography (PET) imaging of a radiolabeled antibody-based agent that targeted epithelial cell adhesion molecule overexpressed on PC3 cells was used to confirm in vivo GRFT results. Validation of GRFT results was also conducted from ex vivo fluorescence imaging of resected prostate tumor. In addition, in mice with large primary prostate tumors, a combination of GRFT and PET showed that the radiolabeled antibody did not penetrate the tumor, consistent with known tumor transport limitations of large (∼150  kDa) molecules. These results represent the first tomography of a living animal using far-red gene reporters. PMID:23797877

  12. 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.

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

    PubMed Central

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

    2015-01-01

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

  14. Elastic-scattering spectroscopy for quantitative measurement of chemotherapy and PDT drug concentrations in vivo

    NASA Astrophysics Data System (ADS)

    Bigio, Irving J.; Mourant, Judith R.; Los, Gerrit

    1999-01-01

    We have applied elastic-scattering spectroscopy (ESS) for noninvasive, real-time in vivo measurement of the concentrations of certain drugs in tissue, utilizing a simple fiber-optic-probe spectroscopic system. The system uses a broadband light source, enabling the detection of compounds with absorption bands in most regions of the visible, as well as the NIR to 1700 nm. Subcutaneous tumors were grown in 4 Nude mice; the mice were treated with one of two chemotherapy agents, and the ESS system was used to perform pharmacokinetics measurements on the tumors following drug administration. Time histories of the drug concentrations in the tumors agreed with the known pharmacokinetics of the two drugs, and HPLC assays following sacrifice showed good correlation with the ESS values. Most photodynamic therapy agents and many chemotherapy drugs, including any that are not fluorescent, are ideal candidates for the ESS system. The measurement can be calibrated absolutely, and is not susceptible to problems associated with fluorescence assay methods.

  15. 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.

  16. Real-time in-vivo endoscopic imaging of fluorescence from human colonic adenomas

    NASA Astrophysics Data System (ADS)

    Wang, Thomas D.; Van Dam, Jacques; Crawford, James M.; Wang, Yang; Itzkan, Irving; Feld, Michael S.

    1998-04-01

    Previous in vitro studies showed that autofluorescence images of colonic mucosa collected endoscopically can be used to detect dysplasia with high sensitivity. This method is extended to collection of fluorescence images of adenomatous polyps in vivo. Fluorescence images were collected during colonoscopy in 30 patients. A total of 12 adenomatous and 6 hyperplastic polyps were identified. An optical fiber excitation probe, located in the instrument channel of the colonoscope, delivered 300 mW of near- ultraviolet light at (lambda) ex equals 351 and 364 nm. Mucosal fluorescence in the spectral bandwidth between 400 and 700 nm was imaged, processed, and displayed with various likelihoods of associated dysplasia. Adenomatous polyps exhibited decreased fluorescence intensity compared to adjacent mucosa with normal appearance. With the fluorescence threshold set to 80% of the average intensity of normal mucosa, a sensitivity of 83% for dysplasia detection was achieved. All hyperplastic polyps were correctly identified as being non-dysplastic. Optimal identification of dysplastic regions was obtained with the colonoscope oriented at near-normal incidence to the polyps. At higher angles of incidence, artifacts due to illumination shadows were introduced. The dysplasia associated with adenomatous polyps can be detected in vivo on fluorescence imaging with high sensitivity, thus demonstrating the potential to guide endoscopic biopsy.

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

    PubMed Central

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

    2015-01-01

    Chronic inflammation is considered as a critical cause of a host of disorders, such as cancer, rheumatoid arthritis, atherosclerosis, and neurodegenerative diseases, although the exact mechanism is yet to be explored. Imaging tools that can specifically target inflammation are therefore important to help reveal the role of inflammation in disease progression, and allows for developing new therapeutic strategies to ultimately improve patient care. The purpose of this study was to develop a new in vivo inflammation imaging approach by targeting the cannabinoid receptor type 2 (CB2R), an emerging inflammation biomarker, using a unique near infrared (NIR) fluorescent probe. Herein, we report the first in vivo CB2R-targeted NIR inflammation imaging study using a synthetic fluorescent probe developed in our laboratory, NIR760-mbc94. In vitro binding assay and fluorescence microscopy study indicate NIR760-mbc94 specifically binds towards CB2R in mouse RAW264.7 macrophage cells. Furthermore, in vivo imaging was performed using a Complete Freund’s Adjuvant (CFA)-induced inflammation mouse model. NIR760-mbc94 successfully identified inflamed tissues and the probe uptake was blocked by a CB2R ligand, SR144528. Additionally, immunofluorescence staining in cryosectioned tissues validated the NIR760-mbc94 uptake in inflamed tissues. In conclusion, this study reports the first in vivo CB2R-targeted inflammation imaging using an NIR fluorescent probe. Specific targeting of NIR760-mbc94 has been demonstrated in macrophage cells, as well as a CFA-induced inflammation mouse model. The combined evidence indicates that NIR760-mbc94 is a promising inflammation imaging probe. Moreover, in vivo CB2R-targeted fluorescence imaging may have potential in the study of inflammation-related diseases. PMID:26069858

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

    PubMed

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

    2015-01-01

    Chronic inflammation is considered as a critical cause of a host of disorders, such as cancer, rheumatoid arthritis, atherosclerosis, and neurodegenerative diseases, although the exact mechanism is yet to be explored. Imaging tools that can specifically target inflammation are therefore important to help reveal the role of inflammation in disease progression, and allows for developing new therapeutic strategies to ultimately improve patient care. The purpose of this study was to develop a new in vivo inflammation imaging approach by targeting the cannabinoid receptor type 2 (CB2R), an emerging inflammation biomarker, using a unique near infrared (NIR) fluorescent probe. Herein, we report the first in vivo CB2R-targeted NIR inflammation imaging study using a synthetic fluorescent probe developed in our laboratory, NIR760-mbc94. In vitro binding assay and fluorescence microscopy study indicate NIR760-mbc94 specifically binds towards CB2R in mouse RAW264.7 macrophage cells. Furthermore, in vivo imaging was performed using a Complete Freund's Adjuvant (CFA)-induced inflammation mouse model. NIR760-mbc94 successfully identified inflamed tissues and the probe uptake was blocked by a CB2R ligand, SR144528. Additionally, immunofluorescence staining in cryosectioned tissues validated the NIR760-mbc94 uptake in inflamed tissues. In conclusion, this study reports the first in vivo CB2R-targeted inflammation imaging using an NIR fluorescent probe. Specific targeting of NIR760-mbc94 has been demonstrated in macrophage cells, as well as a CFA-induced inflammation mouse model. The combined evidence indicates that NIR760-mbc94 is a promising inflammation imaging probe. Moreover, in vivo CB2R-targeted fluorescence imaging may have potential in the study of inflammation-related diseases. PMID:26069858

  19. 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

  20. 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

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

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

    SciTech Connect

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

    2014-10-15

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

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

    NASA Astrophysics Data System (ADS)

    Punj, Deep; Rigneault, Hervé; Wenger, Jérôme

    2014-05-01

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

  4. In vivo diffuse correlation spectroscopy investigation of the ocular fundus

    NASA Astrophysics Data System (ADS)

    Cattini, Stefano; Staurenghi, Giovanni; Gatti, Antonietta; Rovati, Luigi

    2013-05-01

    Diffuse correlation spectroscopy (DCS) measurements in vivo recorded from rabbits' ocular fundus are presented. Despite the complexity of these ocular tissues, we provide a clear and simple demonstration of the DCS abilities to analyze variations in physiological quantities of clinical interest. Indeed, the reported experimental activities demonstrate that DCS can reveal both choroidal-flow and temperature variations and detect nano- and micro-aggregates in ocular fundus circulation. Such abilities can be of great interest both in fundamental research and practical clinical applications. The proposed measuring system can be useful in: (a) monitoring choroidal blood flow variations, (b) determining the end-point for photo-dynamic therapy and transpupillary thermo therapy and, (c) managing the dye injection and determining an end-point for dye-enhanced photothrombosis. Moreover, it could allow both diagnoses when the presence of nano- and micro-aggregates is related to specific diseases and verifying the effects of nanoparticle injection in nanomedicine. Even though the reported results demonstrate the applicability of DCS to investigate ocular fundus, a detailed and accurate investigation of the limits of detection is beyond the scope of this article.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

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

  12. 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

  13. 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

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

    PubMed Central

    Aouani, Heykel; Schn, Peter; Brasselet, Sophie; Rigneault, Herv; Wenger, Jrme

    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 m 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

  15. Fast two-dimensional fluorescence correlation spectroscopy technique for tea quality detection.

    PubMed

    Dong, Yongjiang; Lu, Hao; Yong, Zhengdong; Yan, Chunsheng; He, Sailing

    2015-08-10

    A fast two-dimensional fluorescence correlation spectroscopy technique based on light emitting diodes is developed, which uses light intensity and excitation wavelength as quickly changeable and easily controllable external perturbations. A compact and automatic system is set up to detect tea quality. A partial least square regression method is used to create predictive models for tea grades. Compared to the traditional fluorescence spectroscopy method, this convenient two-dimensional correlation spectroscopy technique is more accurate according to our experimental results and is promising for practical applications. PMID:26368372

  16. 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.

  17. 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

  18. 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

  19. Laser-induced fluorescence spectroscopy for combustion diagnostics

    NASA Technical Reports Server (NTRS)

    Crosley, D. R.; Smith, G. P.

    1983-01-01

    The types of spectroscopic and collisional measurements that are needed to develop laser-induced fluorescence as a diagnostic technique are discussed, with emphasis placed on combustion measurements. The spectroscopic measurements under collision-free conditions include production of radicals, excitation scan studies, lifetime measurements, and fluorescence scans. The collisional studies discussed here are quenching, energy transfer, and polarization phenomena. The results of recent laboratory experiments are presented.

  20. Fluorescence spectroscopy of gastrointestinal tumors using δ-ALA

    NASA Astrophysics Data System (ADS)

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

    2007-03-01

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

  1. 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.

  2. 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

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

    PubMed Central

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

    2011-01-01

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

  4. Fluorescence spectroscopy of anisole at elevated temperatures and pressures

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

  5. In Vivo Lighted Fluorescence via Fenton Reaction: Approach for Imaging of Hydrogen Peroxide in Living Systems.

    PubMed

    Liu, Changhui; Chen, Weiju; Qing, Zhihe; Zheng, Jing; Xiao, Yue; Yang, Sheng; Wang, Lili; Li, Yinhui; Yang, Ronghua

    2016-04-01

    By virtue of its high sensitivity and rapidity, Fenton reaction has been demonstrated as a powerful tool for in vitro biochemical analysis; however, in vivo applications of Fenton reaction still remain to be exploited. Herein, we report, for the first time, the design, formation and testing of Fenton reaction for in vivo fluorescence imaging of hydrogen peroxide (H2O2). To realize in vivo fluorescence imaging of H2O2 via Fenton reaction, a functional nanosphere, Fc@MSN-FDNA/PTAD, is fabricated from mesoporous silica nanoparticle (MSN), a Fenton reagent of ferrocene (Fc), ROX-labeled DNA (FDNA), and a cationic perylene derivative (PTAD). The ferrocene molecules are locked in the pore entrances of MSN, and exterior of MSN is covalently immobilized with FDNA. As a key part, PTAD acts as not only the gatekeeper of MSN but also the efficient quencher of ROX. H2O2 can permeate into the nanosphere and react with ferrocene to product hydroxyl radical (·OH) via Fenton reaction, which cleaves FDNA to detach ROX from PTAD, thus in turn, lights the ROX fluorescence. Under physiological condition, H2O2 can be determined from 5.0 nM to 1.0 μM with a detection limit of 2.4 nM. Because of the rapid kinetics of Fenton reaction and high specificity for H2O2, the proposed method meets the requirement for real applications. The feasibility of Fc@MSN-FDNA/PTAD for in vivo applications is demonstrated for fluorescence imaging of exogenous and endogenous H2O2 in cells and mice. We expect that this work will not only contribute to the H2O2-releated studies but also open up a new way to exploit in vivo Fenton reaction for biochemical research. PMID:26948406

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

    PubMed Central

    2015-01-01

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

  7. 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.

  8. 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

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

    PubMed Central

    Keese, Daniel; Korkusuz, Hdayi; 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

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. Non-destructive identification of varnishes by UV fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Thoury, Mathieu; Elias, Mady; Frigerio, Jean Marc; Barthou, Carlos

    2005-06-01

    Qualitative UV-fluorescence of varnishes is commonly used to locate repaints on paintings or to specify the homogeneousness of a varnish layer. Photographers can now use flash UV-lamps coupled with a CCD camera to obtain colour images of the fluorescence of paintings, unveiling thus both interest and difficulty to interpret these colours. Starting from this point of view, UV-fluorescence spectra appear to be a potential technique to characterize the nature of varnishes and, if possible, their state of degradation. This identification will be non-invasive, without contact, obtained in real time and workable in situ, as the identification of pigments or dyes by reflectance spectrometry which is already done in our group. The last goal will be to realize both identifications with the same device. Emission fluorescence spectra are implemented with the Jobin-Yvon Fluorolog-3, providing an incident wavelength laying between 200 and 850 nm. The emission spectra are implemented with an optical fiber linked to a Jobin-Yvon spectrometer HR460 and a multi-channel CCD detector. In a first step, popular, fresh, raw resins used between the XVI th and the XIX th century, as mastic, dammar and sandarac, have been used to prepare varnishes films with different solvents. The fluorescence spectra of these films have been carried out at different excitation wavelengths to build databases. After having tested the coherence, the limits and the accuracy of the method, we suggest different applications of our method. A synthesis of the results will be presented to characterize each varnish by their fluorescence spectra.

  20. 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).

  1. 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.

  2. 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

  3. 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

  4. 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

  5. The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors.

    PubMed

    Kilpatrick, Laura E; Hill, Stephen J

    2016-04-15

    The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand-receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties. PMID:27068980

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. Comparative analysis of infrared fluorescence generation in multiphoton spectroscopy

    NASA Astrophysics Data System (ADS)

    Legros, Philippe; Choquet, Daniel; Mottay, Eric P.; Deguil, Nelly; Salin, Francois

    2004-06-01

    We have applied a new, 1030 nm wavelength, infrared diode-pumped femtosecond laser source to multiphoton microscopy, and present comparative results on the efficiency of fluorescence generation versus wavelength for several fluorophores. It is shown that an emission wavelength of 1030 nm is optimal both for GFP and DsRed excitation.

  13. 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 ...

  14. 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.

  15. 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

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

    PubMed

    Wegner, K David; Hildebrandt, Niko

    2015-07-21

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

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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).

  13. 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

  14. 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.

  15. 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.

  16. 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.

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

    SciTech Connect

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

    2003-11-15

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

  18. Tissue autofluorescence spectroscopy: in-vivo alterations may reflect cellular proliferation

    NASA Astrophysics Data System (ADS)

    Savage, Howard E.; Kolli, Venkateswara; Zhang, Jian C.; Alfano, Robert R.; Sacks, Peter G.; Schantz, Stimson P.

    1994-05-01

    We report on the in vivo fluorescence spectroscopy of ten oral tongue cancers in previously untreated patients. Spectral profiles of the tongue tumor in each patient were compared to those of the corresponding normal contralateral oral tongue mucosa. Spectral characteristics were generated using a xenon lamp as a light source and the results described herein were restricted to one excitation scan obtained using a Mediscience-CD Scanner. The ratio of the main peak at 320-350 nm to the secondary peak at 373-376 nm and the area under the curve between 300 and 400 nm in the excitation scan were used as measurements to calculate differences between the normal versus oral tongue cancer. Significance was determined using the paired t-st. The ratio of the main peak to the secondary peak was higher in the tumor scans when compared to the corresponding contralateral mucosa. When the area under the curves was analyzed, the tumor tissue had reproducible lower values as compared to the contralateral normal sites.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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

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

    PubMed Central

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

    2013-01-01

    Previously, assays for sister chromatid segregation patterns relied on incorporation of BrdU and indirect methods to infer segregation patterns after two cell divisions. Here we describe a method to differentially label sister chromatids of murine cells and directly assay sister chromatid segregation patterns following one cell division in vitro and in vivo by adaptation of the well-established CO-FISH (chromosome orientation fluorescent in situ hybridization) technique. 5-bromo-2′-deoxyuridine (BrdU) is incorporated into newly-formed DNA strands, followed by photolysis and exonuclease digestion to create single-stranded sister chromatids containing parental template DNA only. Such single-stranded sister chromatids are differentially labeled using unidirectional probes to major satellite sequences coupled to fluorescent markers. Differentially-labeled sister chromatids in post-mitotic cells are visualized using fluorescence microscopy and sister chromatid segregation patterns can be directly assayed after one cell division. This procedure requires four days for in vivo mouse tissues, and two days for in vitro cultured cells. PMID:20595964

  6. 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

  7. Photoinhibition of photosynthesis in vivo results in singlet oxygen production detection via nitroxide-induced fluorescence quenching in broad bean leaves.

    PubMed

    Hideg, E; Kálai, T; Hideg, K; Vass, I

    1998-08-18

    In plants experiencing environmental stress, the formation of reactive oxygen is often presumed. In this study, singlet oxygen was detected in broad bean (Vicia faba) leaves that were photoinhibited in vivo. Detection was based on the reaction of singlet oxygen with DanePy (dansyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole) yielding a nitroxide radical (DanePyO) which is EPR active and also features lower fluorescence compared to DanePy. The two (fluorescent and spin) sensor fuctions of DanePy are commensurate, which makes detecting singlet oxygen possible with a spectrofluorimeter in samples hard to measure with EPR spectroscopy [Kálai, T., Hideg, E., Vass, I., and Hideg, K. (1998) Free Radical Biol. Med. 24, 649-652]. We found that in leaves saturated with DanePy, the fluorescence of this double sensor was decreased when the leaves were photoinhibited by 1500 micromol m-2 s-1 photosynthetically active radiation. This fluorescence quenching is the first direct experimental evidence that photoinhibition of photosynthesis in vivo is accompanied by 1O2 production and is, at least partly, governed by the process characterized as acceptor side-induced photoinhibition in vitro. PMID:9708975

  8. Near-infrared fluorescence imaging platform for quantifying in vivo nanoparticle diffusion from drug loaded implants

    PubMed Central

    Markovic, Stacey; Belz, Jodi; Kumar, Rajiv; Cormack, Robert A; Sridhar, Srinivas; Niedre, Mark

    2016-01-01

    Drug loaded implants are a new, versatile technology platform to deliver a localized payload of drugs for various disease models. One example is the implantable nanoplatform for chemo-radiation therapy where inert brachytherapy spacers are replaced by spacers doped with nanoparticles (NPs) loaded with chemotherapeutics and placed directly at the disease site for long-term localized drug delivery. However, it is difficult to directly validate and optimize the diffusion of these doped NPs in in vivo systems. To better study this drug release and diffusion, we developed a custom macroscopic fluorescence imaging system to visualize and quantify fluorescent NP diffusion from spacers in vivo. To validate the platform, we studied the release of free fluorophores, and 30 nm and 200 nm NPs conjugated with the same fluorophores as a model drug, in agar gel phantoms in vitro and in mice in vivo. Our data verified that the diffusion volume was NP size-dependent in all cases. Our near-infrared imaging system provides a method by which NP diffusion from implantable nanoplatform for chemo-radiation therapy spacers can be systematically optimized (eg, particle size or charge) thereby improving treatment efficacy of the platform. PMID:27069363

  9. 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

  10. In vivo imaging of choroidal angiogenesis using fluorescence-labeled cationic liposomes

    PubMed Central

    Hua, Jing; Gross, Nikolai; Schulze, Brita; Michaelis, Uwe; Bohnenkamp, Hermann; Guenzi, Eric; Hansen, Lutz L.; Martin, Gottfried

    2012-01-01

    Purpose Precise monitoring of active angiogenesis in neovascular eye diseases such as age-related macular degeneration (AMD) enables sensitive use of antiangiogenic drugs and reduces adverse side effects. So far, no in vivo imaging methods are available to specifically label active angiogenesis. Here, we report such a technique using fluorophore-labeled cationic liposomes (CL) detected with a standard clinical in vivo scanning laser ophthalmoscope (SLO). Methods C57Bl/6 mice underwent laser coagulations at day 0 (d0) to induce choroidal neovascularization (CNV). Liposomes labeled with Oregon green, rhodamine (Rh), or indocyanine green (ICG) were injected into the tail vein at various time points after laser coagulation, and their fluorescence was observed in vivo 60 min later using an SLO, or afterwards in choroidal flatmounts or cryosections. Results SLO detected accumulated fluorescence only in active CNV lesions with insignificant background noise. The best signal was obtained with CL-ICG. Choroidal flatmounts and cryosections of the eye confirmed the location of retained CL in CNV lesions. Neutral liposomes, in contrast, showed no accumulation. Conclusions These results establish fluorophore-labeled CL as high affinity markers to selectively stain active CNV. This novel, non-invasive SLO imaging technique could improve risk assessment and indication for current intraocular antiangiogenic drugs in neovascular eye diseases, as well as monitor therapeutic outcomes. Labeling of angiogenic vessels using CL can be of interest not only for functional imaging in ophthalmology but also for other conditions where localization of active angiogenesis is desirable. PMID:22605917

  11. Near-infrared fluorescence imaging platform for quantifying in vivo nanoparticle diffusion from drug loaded implants.

    PubMed

    Markovic, Stacey; Belz, Jodi; Kumar, Rajiv; Cormack, Robert A; Sridhar, Srinivas; Niedre, Mark

    2016-01-01

    Drug loaded implants are a new, versatile technology platform to deliver a localized payload of drugs for various disease models. One example is the implantable nanoplatform for chemo-radiation therapy where inert brachytherapy spacers are replaced by spacers doped with nanoparticles (NPs) loaded with chemotherapeutics and placed directly at the disease site for long-term localized drug delivery. However, it is difficult to directly validate and optimize the diffusion of these doped NPs in in vivo systems. To better study this drug release and diffusion, we developed a custom macroscopic fluorescence imaging system to visualize and quantify fluorescent NP diffusion from spacers in vivo. To validate the platform, we studied the release of free fluorophores, and 30 nm and 200 nm NPs conjugated with the same fluorophores as a model drug, in agar gel phantoms in vitro and in mice in vivo. Our data verified that the diffusion volume was NP size-dependent in all cases. Our near-infrared imaging system provides a method by which NP diffusion from implantable nanoplatform for chemo-radiation therapy spacers can be systematically optimized (eg, particle size or charge) thereby improving treatment efficacy of the platform. PMID:27069363

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

    PubMed Central

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

    2014-01-01

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

  13. 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

  14. 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 (FcεRI) 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

  15. 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.

  16. 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...

  17. 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.

  18. 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....

  19. 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.

  20. 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.

  1. Fluorescence-free biochemical characterization of cells using modulated Raman spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

    The use of Raman spectroscopy for biomedical applications requires overcoming the obstacle of the broad fluorescence background that is generally generated in biological samples. Recently, we have developed a new modulation method for separating the weak Raman peaks from the strong fluorescence background. The novel method is based on the periodical modulation of the excitation wavelength and uses the principle of multi-channel lock-in detection. By continuously modulating the excitation wavelength it is possible to shift the Raman peaks while the fluorescence background remains essentially constant. The powerful capabilities of this novel method are demonstrated by acquiring spectra from different location (nucleus, cytoplasm and membrane) inside a CHO cell. In fact, we show that our modulated Raman spectroscopy provides, with higher efficiency than the standard one, Raman spectra of different locations within a single cell, suggesting that this minimally invasive optical technology could be applied for bio-medical diagnosis and imaging.

  2. 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.

  3. Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution.

    PubMed Central

    Schwille, P; Meyer-Almes, F J; Rigler, R

    1997-01-01

    The present paper describes a new experimental scheme for following diffusion and chemical reaction systems of fluorescently labeled molecules in the nanomolar concentration range by fluorescence correlation analysis. In the dual-color fluorescence cross-correlation spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as their reaction product can be followed in parallel. By using two differently labeled reaction partners, the selectivity to investigate the temporal evolution of reaction product is significantly increased compared to ordinary one-color fluorescence autocorrelation systems. Here we develop the theoretical and experimental basis for carrying out measurements in a confocal dual-beam fluorescence correlation spectroscopy setup and discuss conditions that are favorable for cross-correlation analysis. The measurement principle is explained for carrying out DNA-DNA renaturation kinetics with two differently labeled complementary strands. The concentration of the reaction product can be directly determined from the cross-correlation amplitude. Images FIGURE 2 FIGURE 3 PMID:9083691

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

    PubMed

    Hambly, A C; Arvin, E; Pedersen, L-F; Pedersen, P B; Seredyńska-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

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. In vivo multiphoton NADH fluorescence reveals depth-dependent keratinocyte metabolism in human skin.

    PubMed

    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 μmoles/10(6) cells/h. PMID:23332078

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. Bright and stable near infra-red fluorescent protein for in vivo imaging

    PubMed Central

    Filonov, Grigory S.; Piatkevich, Kiryl D.; Ting, Li-Min; Zhang, Jinghang; Kim, Kami; Verkhusha, Vladislav V.

    2011-01-01

    The ability of non-invasive monitoring of deep-tissue developmental, metabolic, and pathogenic processes will advance modern biotechnology. Imaging of live mammals using fluorescent probes is more feasible within a “near-infrared optical window” (NIRW)1. Here we report a phytochrome-based near infra-red fluorescent protein (iRFP) with the excitation/emission maxima at 690/713 nm. Bright fluorescence in a living mouse proved iRFP to be a superior probe for non-invasive imaging of internal mammalian tissues. Its high intracellular stability, low cytotoxicity, and lack of the requirement to add external biliverdin-chromophore makes iRFP as easy to use as conventional GFP-like proteins. Compared to earlier phytochrome-derived fluorescent probes, the iRFP protein has better in vitro characteristics and performs well in cells and in vivo, having greater effective brightness and photostability. Compared to the far-red GFP-like proteins, iRFP has substantially higher signal to background ratio in a mouse model owing to its infra-red shifted spectra. PMID:21765402

  16. 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.

  17. InVivo Multiphoton NADH Fluorescence Reveals Depth-Dependent Keratinocyte Metabolism in Human Skin

    PubMed Central

    Balu, Mihaela; Mazhar, Amaan; Hayakawa, CaroleK.; Mittal, Richa; Krasieva, TatianaB.; Knig, Karsten; Venugopalan, Vasan; Tromberg, BruceJ.

    2013-01-01

    We employ a clinical multiphoton microscope to monitor invivo 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. 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

  19. Asymmetric Rhodamine-Based Fluorescent Probe for Multicolour In Vivo Imaging.

    PubMed

    Iwatate, Ryu J; Kamiya, Mako; Urano, Yasuteru

    2016-01-01

    To achieve rapid and sensitive detection of cancer, activatable fluorescent probes targeting proteases that are overexpressed in various types of cancer have been developed, based on the hydroxymethyl rhodamine green (HMRG) scaffold. However, to visualize altered activities of multiple enzymes in cancer sites, other scaffolds with distinct fluorescence properties from those of HMRG are needed. A novel asymmetrically modified rhodamine with suitable absorption/emission, brightness and equilibrium constant of intramolecular spirocyclization, working in the yellow/orange region, is introduced. As a proof of concept, a probe targeting γ-glutamyl transpeptidase (gGlu-HMJCR) was developed on the basis of the new scaffold. Simultaneous visualization and discrimination of tumours expressing γ-glutamyl transpeptidase (with gGlu-HMJCR) and cathepsins (with Z-Phe-Arg-HMRG) by colour were achieved in a mouse model in vivo. PMID:26744125

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

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

  1. Micro-endoscope for in vivo widefield high spatial resolution fluorescent imaging

    PubMed Central

    Saunter, C D; Semprini, S.; Buckley, C.; Mullins, J; Girkin, J M

    2012-01-01

    In this paper we report the design, testing and use of a scannerless probe specifically for minimally invasive imaging of deep tissue in vivo with an epi-fluorescence modality. The probe images a 500 μm diameter field of view through a 710 μm outer diameter probe with a maximum tissue penetration depth of 15 mm specifically configured for eGFP imaging. Example results are given from imaging the pituitary gland of rats and zebrafish hearts with lateral resolution of 2.5 μm. PMID:22741074

  2. 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.

  3. 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

  4. Sensitive and high resolution subcutaneous fluorescence in vivo imaging using upconversion nanoparticles and microarrays.

    PubMed

    Li, Xin; Li, Zhuoqi; Gan, Wupeng; Wang, Tongzhou; Zhao, Songmin; Lu, Ying; Cheng, Jing; Huang, Guoliang

    2013-07-01

    A sensitive and high resolution small animal in vivo imaging system using upconversion nanoparticles (UNPs) and microarrays was developed. The fluorescence tomography using UNPs could achieve higher precision than that using ordinary fluorophores, which was theoretically explained by the finite element method (FEM). Given the autofluorescence-insensitive property of UNPs, a high subcutaneous detection sensitivity of 0.93 × 10(-4) wt% could be achieved with a UNP volume of ∼10 μL in tissue phantoms. Furthermore, UNP fluorophore microarrays (25, 50 and 100 μm arrays) embedded under mouse skin were prepared for subcutaneous in vivo detection. An optical clearing method was applied to enhance the skin transparency and improve the spatial resolution. The results demonstrated that the optimized system could achieve a spatial resolution of 50 μm for in vivo detection of subcutaneous UNP microarrays. Taken together, we conclude that the proposed system and UNP microarrays could achieve sensitive, high resolution subcutaneous in vivo detection, and have great potential for high throughput detection of tumors and other diseases. PMID:23687650

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

    PubMed Central

    Vinegoni, Claudio; Razansky, Daniel; Pitsouli, Chrysoula; Perrimon, Norbert; Ntziachristos, Vasilis; Weissleder, Ralph

    2009-01-01

    Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (>1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery. We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism. The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size. We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours. PMID:19696720

  13. Mesoscopic fluorescence tomography for in-vivo imaging of developing Drosophila.

    PubMed

    Vinegoni, Claudio; Razansky, Daniel; Pitsouli, Chrysoula; Perrimon, Norbert; Ntziachristos, Vasilis; Weissleder, Ralph

    2009-01-01

    Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (>1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery. We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism. The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size. We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours. PMID:19696720

  14. Precise quantification of cellular uptake of cell-penetrating peptides using fluorescence-activated cell sorting and fluorescence correlation spectroscopy.

    PubMed

    Rezgui, Rachid; Blumer, Katy; Yeoh-Tan, Gilbert; Trexler, Adam J; Magzoub, Mazin

    2016-07-01

    Cell-penetrating peptides (CPPs) have emerged as a potentially powerful tool for drug delivery due to their ability to efficiently transport a whole host of biologically active cargoes into cells. Although concerted efforts have shed some light on the cellular internalization pathways of CPPs, quantification of CPP uptake has proved problematic. Here we describe an experimental approach that combines two powerful biophysical techniques, fluorescence-activated cell sorting (FACS) and fluorescence correlation spectroscopy (FCS), to directly, accurately and precisely measure the cellular uptake of fluorescently-labeled molecules. This rapid and technically simple approach is highly versatile and can readily be applied to characterize all major CPP properties that normally require multiple assays, including amount taken up by cells (in moles/cell), uptake efficiency, internalization pathways, intracellular distribution, intracellular degradation and toxicity threshold. The FACS-FCS approach provides a means for quantifying any intracellular biochemical entity, whether expressed in the cell or introduced exogenously and transported across the plasma membrane. PMID:27033412

  15. 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.

  16. 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.

  17. 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

  18. 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

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

    PubMed Central

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

    2014-01-01

    Abstract 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 km2 and ∼3 million inhabitants. Samples were collect at six monitoring sites covering 107 km of the main river. BOD, COD, nitrogen, and phosphorus concentration indicates a high input of sewage to the river. Specific absorbance at 254 and 285 nm (SUVA254 and A285/COD) did not show significant variation between sites monitored, indicating the presence of both dissolved compounds found in domestic effluents and humic and fulvic compounds derived from allochthonous organic matter. Correlations between BOD and tryptophan-like fluorescence peak (peak T2, r=0.7560, and peak T1, r=0.6949) and tyrosine-like fluorescence peak (peak B, r=0.7321) indicated the presence of labile organic matter and thus confirmed the presence of sewage in the river. Results showed that fluorescence and absorbance spectroscopy provide useful information on pollution in rivers from critical watersheds and together are a robust method that is simpler and more rapid than traditional methods employed by regulatory agencies. PMID:25469076

  20. Biomarkers of in vivo fluorescence imaging in allergic airway inflammation.

    PubMed

    Wang, Fa-Ping; Fan, Ying-Qi; Li, Su-Yun; Mao, Hui

    2016-04-01

    Airway inflammation is a central component of the manifestation of asthma but is relatively inaccessible to study. Current imaging techniques such as X-ray CT, MRI, and PET, have advanced noninvasive research on pulmonary diseases. However, these techniques mainly facilitate the anatomical or structural assessment of the diseased lung and/or typically use radioactive agents. In vivo fluorescence imaging is a novel method for noninvasive, real-time, and specific monitoring of lung airway inflammation, which is particularly important to gain a further understanding asthma. Compared to conventional techniques, fluorescent imaging has the advantages of rapid feedback, as well as high sensitivity and resolution. Recently, there has been an increase in the identification of biomarkers, including matrix metalloproteinases, cathepsins, selectins, folate receptor-beta, nanoparticles, as well as sialic acid-binding immunoglobulin-like lectin-F to assess the level of airway inflammation in asthma. Recent advances in our understanding of these biomarkers as molecular probes for in vivo imaging are discussed in this review. PMID:26902991

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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.

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

    PubMed

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

    2006-07-28

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

  9. 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.

  10. 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

  11. Laser-induced fluorescence spectroscopy of benign and malignant cutaneous lesions

    NASA Astrophysics Data System (ADS)

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

    2005-04-01

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

  12. 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

  13. 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.

  14. 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.

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

    PubMed Central

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

    1986-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2009-05-01

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

  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. 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.

  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. Imaging fluorescence correlation spectroscopy: nonuniform IgE distributions on planar membranes.

    PubMed Central

    Huang, Z; Thompson, N L

    1996-01-01

    Fluorescence correlation spectroscopy is useful for detecting and characterizing molecular clusters that are smaller than or approximately equal to optical resolution in size. Here, we report the development of an approach in which the pixel-to-pixel fluorescence fluctuations from a single fluorescence image are spatially autocorrelated. In these measurements, tetramethylrhodamine-labeled, anti-trinitrophenyl IgE antibodies were specifically bound to substrate-supported planar membranes composed of trinitrophenyl-aminocaproyldipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine. The antibody-coated membranes were illuminated with the evanescent field from a totally internally reflected laser beam, and the fluorescence arising from the IgE-coated membranes was recorded with a cooled CCD camera. The image was corrected for the elliptical Gaussian shape of the evanescent illumination after background subtraction. The spatial autocorrelation functions of the resulting images generated two useful parameters: the extrapolated initial values, which were related to the average cluster intensity and density; and the correlation distances, which were related to the average cluster size. These parameters varied with the IgE density, and unlabeled polyclonal anti-IgE enhanced the nonuniform IgE distributions. The autocorrelation functions calculated from images of planar membranes containing fluorescently labeled lipids rather than bound, labeled IgE demonstrated that the spatial nonuniformities were prominent only in the presence of IgE. Fluorescent beads were used to demonstrate the principles and the methods. Images FIGURE 3 PMID:8785359

  1. Laser-induced fluorescence spectroscopy of colonic dysplasia: prospects for optical histological analysis

    NASA Astrophysics Data System (ADS)

    Manoharan, Ramasamy; Zonios, George I.; Cothren, Robert M., Jr.; Arendt, Joseph; Van Dam, Jacques; Feld, Michael S.

    1995-05-01

    Several groups have shown that laser-induced fluorescence spectroscopy can detect dysplastic changes in human colon tissues. We present an approach based on analysis of the underlying tissue microstructure for extracting histological information from such spectral signals. The method employs fluorescence microscopy and tissue optics to model the `bulk' fluorescence collected with an optical fiber probe in a clinical setting. For both colonic normal and adenoma, we measured the intrinsic fluorescence lineshapes, the spatial distributions of the fluorophores, and optical parameters of tissue. Numerical and analytical solutions to the radiative transfer equation were then used to compute fluorescence spectra. The results of the model were in excellent agreement with clinical spectra collected during colonoscopy, using 370 nm excitation. Four factors were found to be responsible for the spectral differences between normal tissue and adenoma: fluorescence of mucosal collagen, dysplastic cell, and submucosa, and hemoglobin attenuation. Preliminary results indicate that these parameters can be extracted from individual clinical spectra by reversing the modeling procedure.

  2. 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

  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. 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 μm) by means of a Twyman-Green interferometer and repeatedly scanned on the sample, allowing acquisition of information on flows at 4 ms-3 μm 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.

  6. 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 μm) by means of a Twyman-Green interferometer and repeatedly scanned on the sample, allowing acquisition of information on flows at 4 ms-3 μm 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

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

    PubMed Central

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

    2011-01-01

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

  8. 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

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

    NASA Astrophysics Data System (ADS)

    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.

  10. Analysis of RNA Folding and Ribonucleoprotein Assembly by Single-Molecule Fluorescence Spectroscopy

    PubMed Central

    Pljevaljčić, Goran; Robertson-Anderson, Rae; van der Schans, Edwin; Millar, David

    2013-01-01

    Summary To execute their diverse range of biological functions, RNA molecules must fold into specific tertiary structures and/or associate with one or more proteins to form ribonucleoprotein (RNP) complexes. Single-molecule fluorescence spectroscopy is a powerful tool for the study of RNA folding and RNP assembly processes, directly revealing different conformational subpopulations that are hidden in conventional ensemble measurements. Moreover, kinetic processes can be observed without the need to synchronize a population of molecules. In this chapter, we describe the fluorescence spectroscopic methods used for single-molecule measurements of freely diffusing or immobilized RNA molecules or RNA-protein complexes. We also provide practical protocols to prepare the fluorescently labeled RNA and protein molecules required for such studies. Finally, we provide two examples of how these various preparative and spectroscopic methods are employed in the study of RNA folding and RNP assembly processes. PMID:22573447

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. Amyloid-β 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-β (Aβ) 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 Aβ 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 Aβ 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 Aβ 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 Aβ plaques in the brain. PMID:26717442

  20. 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.

  1. 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

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

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

  3. 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.

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

    USGS Publications Warehouse

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

    2012-01-01

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

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. Fluorescence imaging and spectroscopy of ALA-induced protoporphyrin IX preferentially accumulated in tumor tissue

    NASA Astrophysics Data System (ADS)

    Stepp, Herbert G.; Baumgartner, Reinhold; Beyer, Wolfgang; Knuechel, Ruth; Koerner, T. O.; Kriegmair, M.; Rick, Kai; Steinbach, Pia; Hofstetter, Alfons G.

    1995-12-01

    In a clinical pilot study performed on 104 patients suffering from bladder cancer it could be shown that intravesical instillation of a solution of 5-aminolevulinic acid (5-ALA) induces a tumorselective accumulation of Protoporphyrin IX (PPIX). Malignant lesions could be detected with a sensitivity of 97% and a specificity of 67%. The Kr+-laser as excitation light source could successfully be replaced by a filtered short arc Xe-lamp. Its emission wavelength band (375 nm - 440 nm) leads to an efficiency of 58% for PPIX- excitation compared to the laser. Two-hundred-sixty mW of output power at the distal end of a slightly modified cystoscope could be obtained. This is sufficient for recording fluorescence images with a target integrating color CCD-camera. Red fluorescence and blue remitted light are displayed simultaneously. Standard white light observation is possible with the same instrumentation. Pharmacokinetic measurements were performed on 18 patients after different routes of 5-ALA application (oral, inhalation and intravesical instillation). PPIX-fluorescence measurements were made on the skin and on the blood plasma. Pharmacokinetic of 5-ALA could be performed on blood plasma. Endoscopical florescence spectroscopy showed the high fluorescence contrast between tumor and normal tissue with a mean value of 10.7. Forthcoming clinical multicenter studies require an objective measure of the fluorescence intensity. Monte Carlo computer simulations showed that artifacts due to observation geometry and varying absorption can largely be reduced by ratioing fluorescence (red channel of camera) to remission (blue channel). Real time image ratioing provides false color images with a reliable fluorescence information.

  10. 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.

  11. 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

  12. 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

  13. Identification of a Novel Indoline Derivative for in Vivo Fluorescent Imaging of Blood-Brain Barrier Disruption in Animal Models

    PubMed Central

    2013-01-01

    Disruption of the blood-brain barrier (BBB) can occur in various pathophysiological conditions. Administration of extraneous tracers that can pass the disrupted, but not the intact, BBB and detection of the extravasation have been widely used to assess BBB disruption in animal models. Although several fluorescent tracers have been successfully used, the administration of these tracers basically requires intravascular injection, which can be laborious when using small animals such as zebrafish. To identify fluorescent tracers that could be easily administered into various animal models and visualize the BBB disruption in vivo, we prepared nine structurally related indoline derivatives (IDs) as a minimum set of diverse fluorescent compounds. We found that one ID, ZMB741, had the highest affinity for serum albumin and emitted the strongest fluorescence in the presence of serum albumin of the nine IDs tested. The affinity to serum albumin and the fluorescence intensity was superior to those of Evans blue and indocyanine green that have been conventionally used to assess the BBB disruption. We showed that ZMB741 could be administered into zebrafish by static immersion or mice by intraperitoneal injection and visualizes the active disruption of their BBB. These results suggest that ZMB741 can be a convenient and versatile tool for in vivo fluorescent imaging of BBB disruption in various animal models. The strategy used in this study can also be applied to diversity-oriented libraries to identify novel fluorescent tracers that may be superior to ZMB741. PMID:23668665

  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. 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.

  16. Dual-beam laser illuminator of fluorescence microscope for in vivo microcirculation studies.

    PubMed

    Shibata, M; Ichioka, S; Kamiya, A

    1999-07-01

    A new fluorescence intravital microscope of long working distance (39 mm) has been developed for the observation of microcirculation in a wide visual field by designing a simple epi-illumination technique with dual laser beams. Cross-illumination, in which a pair of laser beams is symmetrically placed on either side of the objective such that they intersect at the focal plane of the objective, was employed to produce uniform distribution of the incident light in the object plane. In vitro experiments using a fluorescein isothiocyanate dextran (FITC-dextran; molecular weight = 70,000) solution of known concentration confirmed uniform tracer excitation in a wide visual field (approximately 30 mm2), and a linear correlation between fluorescence intensity and tracer concentration (r = 0.999), ranging between 5 mumol l-1 and 25 mumol l-1. In vivo observations in the microcirculation of a hamster cheek pouch indicated that the present technique had the advantage of high contrast compared with the image obtained by bright-field transillumination. This microscope illuminator may prove useful for the evaluation of vascular permeability under physiological and inflammatory conditions, with sufficient quantitative reliability to determine tracer concentrations in all parts of the microvascular network. Furthermore, a long working distance in this technique could have considerable advantages for the application to nail-fold capillaroscopy in humans. PMID:10696696

  17. 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.

  18. 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 quenching is coupled to the morphology through the distance between TQ1 and PC70BM. Furthermore, if using a bad solvent for PC70BM, morphological regions rich in the fullerene yield emission characteristic for aggregated PC70BM. Clear differences were found, comparing the TQ1:PC70BM blend films casted from different solvents and at different ratios between the donor and acceptor. The morphology also influences the UV/VIS absorption spectra, yielding further information on the composition. The results show that fluorescence and UV/VIS absorption spectroscopy can be used to detect aggregation in blended films and that these methods extend the morphological information beyond the scale accessible with microscopy.

  19. Laser-Assisted Cryosurgery in ex vivo Mice Hepatic Tissue: Viability Assays Using Green Fluorescent Protein

    PubMed Central

    Duperray, B.; Godinez, F.; Guillén, G.; Slade, A.; Aguilar, G.

    2010-01-01

    An experimental investigation is carried out to develop a novel approach to cryosurgery, where laser heating counteracts tissue freezing to better confine damage to the targeted cancerous tissue within a lethal low-temperature isothermal boundary—an approach we refer to as laser-assisted cryosurgery (LAC). The advantage of this procedure relative to conventional cryosurgery assisted with urethral warmers or cryoheaters is that laser heating provides volumetric rather than superficial heating, which leads to deeper penetration, more homogeneous tissue protection and better demarcation of the destructive freezing effect to a well-defined targeted volume. Tissue viability assays are performed using green fluorescence protein (GFP) as a viability marker and correlated with temperature history after performing LAC procedures on ex vivo mice hepatic tissue. The limit for cell denaturation at the irradiated surface predicted by GFP analysis is further confirmed using reverse transcription polymerase chain reaction (RT-PCR). In addition, the correlation between GFP fluorescence and cell viability and loss of GFP fluorescence in non-viable cells has been tested and validated by histological analysis using a standard cell viability measuring method (hematoxylin and eosin staining). Analysis of our experimental measurements show that reproducible thermal gradients (of 236 °C/cm) and predictable tissue necrosis can be reliably produced by LAC without exceeding temperature thresholds for cell denaturation (of Tsurf ≈ 48 °C) beyond preset tissue boundaries (with resolution of 0.1 °C/mm). The results have shown the feasibility of controlling temperatures at specified tissue locations to prevent hyperthermal or freezing damage. PMID:20963494

  20. In vivo high-resolution fluorescence microendoscopy for ovarian cancer detection and treatment monitoring

    PubMed Central

    Zhong, W; Celli, J P; Rizvi, I; Mai, Z; Spring, B Q; Yun, S H; Hasan, T

    2009-01-01

    Background: In patients with advanced ovarian cancer (OvCa), microscopic residual tumour nodules that remain after surgical debulking frequently escape detection by current treatment assessment methods and lead to disease recurrence. The aim of this study was to evaluate the use of high-resolution fibre-optic fluorescence imaging of the clinically approved photodynamic therapy (PDT) agent benzoporphyin-derivative monoacid ring A (BPD-MA) for detection of microscopic OvCa and for monitoring treatment response. Methods: Our fluorescence microendoscope consists of a flexible imaging fibre coupled to a custom epi-fluorescence system optimised for imaging BPD-MA, which, after a single administration, serves as both an imaging agent and a light-activated therapeutic agent. After characterisation in an in vitro OvCa 3D model, we used the flexible imaging fibre to minimally invasively image the peritoneal cavity of a disseminated OvCa murine model using BPD-MA administered intraperitoneally (i.p.). To evaluate longitudinal changes in response to treatment, we compared sets of images obtained before and after PDT with those from untreated mice imaged at the same time points. Results: By comparison with histopathology, we report an 86% sensitivity for tumour detection in vivo using the microendoscope. Using a custom routine to batch process-image data in the monitoring study, treated mice exhibited an average decrease of 58.8% in tumour volumes compared with an increase of 59.3% in untreated controls (P<0.05). Conclusions: Our findings indicate the potential of this approach as a reporter of treatment outcome that could aid in the rational design of strategies to mitigate recurrent OvCa. PMID:19920823

  1. 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

  2. A simple preparation of Ag@graphene nanocomposites for surface-enhanced Raman spectroscopy of fluorescent anticancer drug

    NASA Astrophysics Data System (ADS)

    Meng, Ying; Yan, Xueying; Wang, Yi

    2016-05-01

    A simple method was developed to synthesize Ag@graphene nanocomposites with rough Ag nanoparticles (AgNPs) conjugated with graphene nanosheets, and the nanocomposites could be used as substrates for effective surface-enhanced Raman spectroscopy (SERS) of fluorescent anticancer drug (Dox) since they could not only enhance the Raman signals but also suppress the fluorescent signals.

  3. Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways

    NASA Astrophysics Data System (ADS)

    Shirshin, Evgeny; Cherkasova, Olga; Tikhonova, Tatiana; Berlovskaya, Elena; Priezzhev, Alexander; Fadeev, Victor

    2015-05-01

    We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation.

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

    PubMed

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

    2010-02-01

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

  5. Fluorescent and bioluminescent nanoprobes for in vitro and in vivo detection of matrix metalloproteinase activity

    PubMed Central

    Lee, Hawon; Kim, Young-Pil

    2015-01-01

    Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade the extracellular matrix (ECM) and regulate the extracellular microenvironment. Despite the significant role that MMP activity plays in cell-cell and cell-ECM interactions, migration, and differentiation, analyses of MMPs in vitro and in vivo have relied upon their abundance using conventional immunoassays, rather than their enzymatic activities. To resolve this issue, diverse nanoprobes have emerged and proven useful as effective activity-based detection tools. Here, we review the recent advances in luminescent nanoprobes and their applications in in vitro diagnosis and in vivo imaging of MMP activity. Nanoprobes with the purpose of sensing MMP activity consist of recognition and detection units, which include MMP-specific substrates and luminescent (fluorescent or bioluminescent) nanoparticles, respectively. With further research into improvement of the optical performance, it is anticipated that luminescent nanoprobes will have great potential for the study of the functional roles of proteases in cancer biology and nanomedicine. [BMB Reports 2015; 48(6): 313-318] PMID:25817215

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

    Recently we described a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. This technique was applied to in vivo imaging of murine small intestine and colon. Individuals with Inflammatory Bowel Disease (IBD), commonly presenting as Crohn's disease or Ulcerative Colitis, are at increased risk for developing colorectal cancer. We have developed a Giα2 gene knock out mouse IBD model that develops colitis and colon cancer. The challenge is to study the disease in the whole animal, while maintaining high resolution imaging at millimeter depth. In the Giα2-/- mice, we have been successful in imaging Lgr5-GFP positive stem cell reporters that are found in crypts of niche structures, as well as deeper structures, in the small intestine and colon at depths greater than 1mm. In parallel with these in vivo deep tissue imaging experiments, we have also pursued autofluorescence FLIM imaging of the colon and small intestine-at more shallow depths (roughly 160μm)- on commercial two photon microscopes with excellent structural correlation (in overlapping tissue regions) between the different technologies.

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

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

  8. In vivo imaging of small animals with optical tomography and near-infrared fluorescent probes

    NASA Astrophysics Data System (ADS)

    Palmer, Matthew R.; Shibata, Yasushi; Kruskal, Jonathan B.; Lenkinski, Robert E.

    2002-06-01

    A developmental optical tomography has been designed for imaging small animals in vivo using near IR fluorophores. The system employs epi-illumination via a 450 W Xe arc lamp, filtered and collimated to illuminate a 10 cm square movable stage. Emission light is filtered then collected by a high- resolution, high quantum efficiency, cooled CCD camera. Stage movement and image acquisition are under the control of a personal computer running system integration and automation software. During an experiment, the anesthetized animal is secured to the stage and up to 200 projections can be acquired over 180 degrees rotation. Angular sampling of the light distribution at a point on the surface is used to determine relative contributions form ballistic and diffuse photons. We have employed the system to investigate a number of applications of in-vivo fluorescent imaging. In dynamic studies, hepatic function has been visualized in nude mice following intravenous injection of indocyanine green (ICG) and cerebrospinal fluid flow as been measured by injection of ICG-lipoprotein conjugate in the subarachnoid space of the lumbar spine followed by dynamic imaging of the brain. Further applications in physiological imaging, cancer detection, and molecular imaging are under investigation in our laboratory.

  9. Fluorescence-based laser capture microscopy technology facilitates identification of critical in vivo cytomegalovirus transcriptional programs.

    PubMed

    Kreklywich, Craig N; Smith, Patricia P; Jones, Carmen Baca; Cornea, Anda; Orloff, Susan L; Streblow, Daniel N

    2014-01-01

    Cytomegalovirus gene expression in highly permissive, cultured fibroblasts occurs in three kinetic classes known as immediate early, early, and late. Infection of these cells results in a predictable transcriptional program leading to high levels of virus production. Infection of other, so-called, nonpermissive cell types results in a transcriptional program that either fails to produce virus particles or production is substantially reduced compared to fibroblasts. We have found that CMV gene expression profiles in tissues from infected hosts differ greatly from those observed in infected tissue culture cells. The number of viral genes expressed in tissues is much more limited, and the number of highly active genes does not correlate with viral DNA load. Additionally, viral gene expression in vivo is tissue selective with no two tissues expressing the exact same viral gene profile. Thus, in vivo CMV gene expression appears to be governed by mechanisms that are still uncharacterized. Cytomegalovirus remains in a persistent phase for the lifetime of the host. During this phase only a limited number of host cells are infected, and it is very difficult to detect CMV gene expression in whole tissues without sub-fractionating infected vs. uninfected cells. Herein, we describe the development of a fluorescence-based laser capture microscopy technique coupled with small sample size microarray analysis to determine the viral gene expression in 50-100 infected cells isolated from frozen RCMV-infected tissue sections. PMID:24639226

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

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

    PubMed Central

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

    2013-01-01

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

  12. Retrograde fluorescent labeling allows for targeted extracellular single-unit recording from identified neurons in vivo.

    PubMed

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

    2013-01-01

    The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular(1), juxtacellular(2) or loose-patch(3) iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology(4-6). However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons(7), but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult(8-11). The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS. The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals(12). We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes(13). However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods(11, 14). Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation. PMID:23928906

  13. Fluorescence spectroscopy of soil pellets : The use of CP/PARAFAC.

    NASA Astrophysics Data System (ADS)

    Mounier, Stéphane; Nicolodeli, Gustavo; Redon, Roland; Hacherouf, Kalhed; Milori, Debora M. B. P.

    2014-05-01

    Fluorescence spectroscopy is one of the most sensitive techniques available for analytical purposes. It is relatively easy to implement, phenomenologically straightforward and well investigated. Largely non-invasive and fast, so that it can be useful for environmental applications. Fluorescence phenomenon is highly probable in molecular systems containing atoms with lone pairs of electrons such as C=O, aromatic, phenolic, quinone and more rigid unsaturated conjugated systems. These functional groups are present in humic substances (HS) from soils (Senesi, 1990; N. Senesi et al., 1991) and represent the main fluorophors of Soil Organic Matter (SOM). The extension of the conjugated electronic system, the level of heteroatom substitution and type and number of substituting groups under the aromatic rings strongly affect the intensity and wavelength of molecular fluorescence. However, to analyse the SOM it is generally done a chemical extraction that allows measuring the fluorescence response of the liquid extract. To avoid this fractionation of the SOM, Milori et al. (2006) proposed the application of laser induced fluorescence spectroscopy (LIFS) in whole soil. This work intends to assess the technical feasibility of 3D fluorescence spectroscopy using lamp for excitation to analyse solids opaque samples prepared with different substances. Seventy four (74) solid samples were prepared from different mixtures of boric acid (BA), humic substance acid and tryptophan (TRP) powder. The compounds were mixture and a pellet was done by using pressure (8 ton). The pellets were measured using a spectrofluorimeter HITACHI F4500, and a 3D fluorescence tensor was done from emission spectra (200-600 nm) with excitation range from 200 to 500 nm. The acquisition parameters were: step at 5 nm, scan speed at 2400 nm.min-1, response time at 0.1 s, excitation and emission slits at 5 nm and photomultiplier voltage at 700 V. Further