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1

Following protein association in vivo with fluorescence fluctuation spectroscopy  

Microsoft Academic Search

The combination of fluorescence correlation spectroscopy and two-photon excitation provides us with a powerful spectroscopic technique. Its submicron resolution and single molecule sensitivity make it an attractive technique for in vivo applications. Experiments have demonstrated that quantitative in vivo fluorescence fluctuation measurements are feasible, despite the presence of autofluorescence and the heterogeneity of the cellular environment. I will demonstrate that

Joachim D. Muller

2003-01-01

2

In-vivo pharmacokinetic study of two fluorescein derivatives by fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

We have already demonstrated the ability of fluorescence spectroscopy and imaging to measure the pH of superficial tissues using pH sensitive fluorescent probes. The purpose of this study was to investigate the in vivo behavior of such fluorescent probes. We report the monitoring of tissue fluorescence after injection of two fluorescein derivatives (carboxyfluorescein and biscarboxyethyl-carboxyfluorescein). The in vivo study was performed on anaesthetized adult Wistar rats. After laparotomy, CF or BCECF solution was injected into the penial vein. Fluorescence spectra were recorded during one hour using an optical multichannel analyzer coupled to a CCD camera. Fiber optic was placed alternatively on the liver area or on the skin. Blood samples were collected and fluorescence was measured in vitro. A clear linear relationship between dose and fluorescence intensity was found in liver for these fluorescent markers. Concerning spectral characteristics, it was found that CF and BCECF spectra show a shift compared to in vivo maximum emission peak and BCECF emission peak was different when recorded in the liver and in the skin. Differences of kinetic profiles are also observed between CF and BCECF. The BCECF derivative displays a fluorescence peak in the liver two minutes after injection, while CF fluorescence peak is observed seven minutes after injection. Clearance of skin fluorescence is slower than the plasmatic one indicating that dye elimination in superficial blood vessels does not follow the same pharmacokinetic behavior. Based on these preliminary findings, fluorescence spectroscopy appears as a tool in pharmacokinetic study in situ and in vivo.

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

1995-12-01

3

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

PubMed

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. PMID:24477382

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

4

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

NASA Astrophysics Data System (ADS)

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.

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

5

Fluorescence Spectroscopy  

NSDL National Science Digital Library

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

Group, Kansas S.; Zollman, Dean A.

2004-03-05

6

Dual-color fluorescence fluctuation spectroscopy in vitro and in vivo  

NASA Astrophysics Data System (ADS)

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

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

2004-06-01

7

[In vivo fluorescence spectroscopy of human skin: principles and application to penetration measurements].  

PubMed

The fluorescence properties of untreated human skin and the penetration of topically applied drugs were measured in vivo using a commercially available fluorescence spectrometer which was adapted to in vivo-measurements on skin by means of optical fibres and a remote viewing head. The intensity and characteristics of the intrinsic fluorescences of human skin and their dependences upon several parameters like pigmentation, water content of the skin or depth of the horny layer are shown. The principles of the penetration measurements based on the fluorescence of the skin or the fluorescence of the drug topically applied to the skin are described and measurements of the penetration of some drugs are reported. PMID:7938148

Harendt, N; Giese, K; Kölmel, K

1994-08-01

8

In Vivo Quantitative Studies of Dynamic Intracellular Processes Using Fluorescence Correlation Spectroscopy  

PubMed Central

It has been a significant challenge to quantitatively study the dynamic intracellular processes in live cells. These studies are essential for a thorough understanding of the underlying mechanisms regulating the signaling pathways and the transitions between cell cycle stages. Our studies of Cdc20, an important mitotic checkpoint protein, throughout the cell cycle demonstrate that fluorescence correlation spectroscopy is a powerful tool for in vivo quantitative studies of dynamic intracellular processes. In this study, Cdc20 is found to be present primarily in a large complex (>1 Mda) during interphase with a diffusion constant of 1.8 ± 0.1 ?m2/s and a concentration of 76 ± 24 nM, consistent with its association with the APC/C. During mitosis, however, a proportion of Cdc20 dissociates from APC/C at a rate of 12 pM/s into a soluble pool with a diffusion constant of 19.5 ± 5.0 ?m2/s, whose size is most consistent with free Cdc20. This free pool accumulates to 50% of total Cdc20 (?40 nM) during chronic activation of the mitotic checkpoint but disappears during mitotic exit at a rate of 31 pM/s. The observed changes in the biochemical assembly states of Cdc20 closely correlate to the known temporal pattern of the activity of APC/CCdc20 in mitosis. Photon counting histograms reveal that both complexes contain only a single molecule of Cdc20. The underlying mechanisms of the activities of APC/CCdc20 throughout the cell cycle are discussed in light of our experimental observations.

Wang, Zifu; Shah, Jagesh V.; Berns, Michael W.; Cleveland, Don W.

2006-01-01

9

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

PubMed Central

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

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

1997-01-01

10

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

PubMed Central

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

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

2012-01-01

11

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

PubMed

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

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

12

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

NASA Astrophysics Data System (ADS)

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

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

13

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

NASA Astrophysics Data System (ADS)

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

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

2013-02-01

14

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

NASA Astrophysics Data System (ADS)

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

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

2007-07-01

15

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

PubMed Central

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.

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

2010-01-01

16

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

NASA Astrophysics Data System (ADS)

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.

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

2005-04-01

17

Fluorescence Correlation Spectroscopy  

NSDL National Science Digital Library

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

Haustein, Elke; Schwille, Petra

2011-06-17

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)

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

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

2012-06-01

19

Optimal Excitation Wavelengths for In Vivo Detection of Oral Neoplasia Using Fluorescence Spectroscopy  

Microsoft Academic Search

There is no satisfactory mechanism to detect premalig- nant lesions in the upper aero-digestive tract. Fluores- cence spectroscopy has potential to bridge the gap be- tween clinical examination and invasive biopsy; however, optimal excitation wavelengths have not yet been deter- mined. The goals of this study were to determine optimal excitation-emission wavelength combinations to discrim- inate normal and precancerous\\/cancerous tissue,

Douglas L. Heintzelman; Urs Utzinger; Holger Fuchs; Andres Zuluaga; Kirk Gossage; Ann M. Gillenwater; Rhonda Jacob; Bonnie Kemp; Rebecca R. Richards-Kortum

2000-01-01

20

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

PubMed Central

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

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

2014-01-01

21

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

PubMed

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

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

2014-05-01

22

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

PubMed Central

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

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

2011-01-01

23

Fluorescence spectroscopy of peptides.  

PubMed

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

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

2014-01-01

24

Probing protein interactions in cells by fluorescence fluctuation spectroscopy  

Microsoft Academic Search

The combination of fluorescence correlation spectroscopy and two-photon excitation provides us with a powerful spectroscopic technique. Its submicron resolution and single molecule sensitivity make it an attractive technique for in vivo applications. Experiments have demonstrated that quantitative in vivo fluorescence fluctuation measurements are feasible, despite the presence of autofluorescence and the heterogeneity of the cellular environment. I will demonstrate that

Joachim Mueller

2003-01-01

25

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

PubMed Central

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

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

2012-01-01

26

Single molecule fluorescence saturation spectroscopy  

NASA Astrophysics Data System (ADS)

Saturation spectroscopy is a powerful method to investigate photophysical parameters of single fluorescent molecules. Nevertheless, the impact of a gradual increase, over a broad range, of the laser excitation on the intramolecular dynamics is not completely understood, particularly concerning their fluorescence emission (the so-called brightness). As we have presented in a previous paper [1], we interpret the evolution of the brightness with the laser power by cascade absorption of two and three photons within a five-level molecular system. This multi-photon consecutive absorption leads us to reconsider the common expression of the saturation curve of fluorescent molecule. Furthermore, this multi-photon absorption process also affects the observation volume of microscope. So, in this paper we propose to interpret the size increase of the confocal observation volume according to simulations based upon two often used expressions of the Point Spread Functions (PSF) in fluorescence microscopy.

Jaffiol, Rodolphe; Winckler, Pascale

2014-03-01

27

Spatial Distribution and Function of Sterol Regulatory Element-Binding Protein 1a and 2 Homo and Heterodimers by In Vivo Two-Photon Imaging and Spectroscopy Fluorescence Resonance Energy Transfer  

Microsoft Academic Search

Sterol regulatory element-binding proteins (SREBPs) are a subfamily of basic helix-loop-helix-leucine zipper proteins that regulate lipid metabolism. We show novel evidence of the in vivo occurrence and sub- nuclear spatial localization of both exogenously expressed SREBP-1a and -2 homodimers and heterodimers obtained by two-photon imaging and spectroscopy fluorescence resonance energy transfer. SREBP-1a ho- modimers localize diffusely in the nucleus, whereas

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

2005-01-01

28

Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging Microscopy  

Microsoft Academic Search

With few and commercially available add-ons, both confocal and full-field fluorescence microscopes can be adapted to provide more information on the biological sample of interest. In this review we discuss the possibilities offered by two additional functionalities to fluorescence microscopes, fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging mi croscopy (FLIM). FCS measurements at a single point in a sample

Sophia Y. Breusegem; Moshe Levi; Nicholas P. Barry

2006-01-01

29

Fluorescence spectroscopy for colon cancer diagnosis  

NASA Astrophysics Data System (ADS)

Laser-induced fluorescence spectroscopy is a promising technique for detecting colonic dysplasia in vivo but, at present, the biological basis for the success of the method is poorly understood, and little information is provided as to the morphological/molecular origin of tissue fluorescence. We present a methodology for establishing this, taking as a starting point a recently completed prospective laser-induced fluorescence (LIF) clinical study of colonic polyps. The method is based on a morphological model of tissue fluorescence with three components: the intrinsic lineshapes of the fluorophores, the spatial distributions of their intensities, and the optical parameters of tissue. We measure these using fluorescence microspectroscopy, microscopic imaging and tissue optics, respectively. The model predicts the features of the clinical spectra, and quantifies the respective intrinsic and architectural contributions. The results can be inverted to extract biological parameters from the spectra, and used to select optimal excitation wavelength(s) and guide probe design. Implications to detection of nonpolypoid dysplasia are discussed.

Zonios, George I.; Cothren, Robert M.; Arendt, Joseph; Wu, Jun; Crawford, James M.; van Dam, Jacques; Manoharan, Ramasamy; Feld, Michael S.

1995-01-01

30

Calibrated fluorescence imaging of tissue in vivo  

SciTech Connect

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

Qu, Jianan Y.; Hua, Jianwen

2001-06-18

31

Fluorescence spectroscopy for colon cancer diagnosis  

NASA Astrophysics Data System (ADS)

Laser-induced fluorescence spectroscopy is a promising technique for detecting colonic dysplasia in vivo but, at present, the biological basis for the success of the method is poorly understood, and little information is provided as to the morphological/molecular origin of tissue fluorescence. We present a methodology for establishing this, taking as a starting point a recently completed prospective LIF clinical study of colonic polyps. The method is based on a morphological model of tissue fluorescence with three components: the intrinsic lineshapes of the fluorophores, the spatial distributions of their intensities, and the optical parameters of tissue. We measure these using fluorescence microspectroscopy, microscopic imaging and tissue optics, respectively. The model predicts the features of the clinical spectra, and quantifies the respective intrinsic and architectural contributions. The results can be inverted to extract biological parameters from the spectra, and used to select optimal excitation wavelength(s) and guide probe design. Implications for the detection of non-polypoid dysplasia are discussed.

Zonios, George I.; Cothren, Robert M., Jr.; Arendt, Joseph; Wu, Jun; Crawford, James M.; van Dam, Jacques; Manoharan, Ramasamy; Feld, Michael S.

1994-10-01

32

Fluorescence spectroscopy for colon cancer diagnosis  

NASA Astrophysics Data System (ADS)

Laser-induced fluorescence spectroscopy is a promising technique for detecting colonic dysplasia in vivo but, at present, the biological basis for the success of the method is poorly understood, and little information is provided as to the morphological/molecular origin of tissue fluorescence. We present a methodology for establishing this, taking as a starting point a recently completed prospective LIF clinical study of colonic polyps. The method is based on a morphological model of tissue fluorescence with three components: the intrinsic lineshapes of the fluorophores, the spatial distributions of their intensities, and the optical parameters of tissue. We measure these using fluorescence microspectroscopy, microscopic imaging and tissue optics, respectively. The model predicts the features of the clinical spectra, and quantifies the respective intrinsic and architectural contributions. The results can be inverted to extract biological parameters from the spectra, and used to select optimal excitation wavelength(s) and guide probe design. Implications for the detection of non-polypoid dysplasia are discussed.

Zonios, George I.; Cothren, Robert M.; Arendt, Joseph; Wu, Jun; Crawford, James M.; van Dam, Jacques; Manoharan, Ramasamy; Feld, Michael S.

1995-03-01

33

Glucose Recognition in Vitro Using Fluorescent Spectroscopy  

SciTech Connect

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

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

2001-04-25

34

Intraluminal fluorescence spectroscopy catheter with ultrasound guidance  

PubMed Central

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

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

2014-01-01

35

Intraluminal fluorescence spectroscopy catheter with ultrasound guidance  

NASA Astrophysics Data System (ADS)

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

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

2009-05-01

36

Fluorescent proteins as visible in vivo sensors.  

PubMed

Fluorescent proteins have enabled a whole new technology of visible in vivo genetic sensors. Fluorescent proteins have revolutionized biology by enabling what was formerly invisible to be seen clearly. These proteins have allowed us to visualize, in real time, important aspects of cancer in living animals, including tumor cell mobility, invasion, metastasis, and angiogenesis. These multicolored proteins have allowed the color coding of cancer cells growing in vivo and enabled the distinction of host from tumor with single-cell resolution. Whole-body imaging with fluorescent proteins has been shown to be a powerful technology to noninvasively follow the dynamics of metastatic cancer. Whole-body imaging of cancer cells expressing fluorescent proteins has enabled the facile determination of efficacy of candidate antitumor and antimetastatic agents in mouse models. The use of fluorescent proteins to differentially label cancer cells in the nucleus and cytoplasm and high-powered imaging technology have enabled the visualization of the nuclear-cytoplasmic dynamics of cancer cells in vivo, including noninvasive techniques. Fluorescent proteins thus enable both macro- and microimaging technology and thereby provide the basis for the new field of in vivo cell biology. PMID:23244796

Hoffman, Robert M

2013-01-01

37

Modulated Raman spectroscopy technique for real-time fluorescence rejection  

NASA Astrophysics Data System (ADS)

Raman spectroscopy is a label-free and non-invasive method that measures the inelastic scattered light from a sample giving insight into the vibration eigenmodes of the excited molecules. Raman spectroscopy provides a detailed chemical composition of the sample, constituting a sort of its chemical fingerprint. Although Raman spectroscopy is a useful technique to identify and quantify species in a given matrix, it has been severely limited in its applicability by fluorescence. Spectrally, this fluorescence occurs at the same wavelength as the Raman signal and is often several orders of magnitude more intense that the weak chemical transitions probed by Raman spectroscopy. Often, this fluorescence background and its natural variability make biochemical analysis using Raman spectroscopy impractical. In this work, we present the theory and the implementation of an innovative modulated Raman spectroscopy technique to filter out the Raman spectra from the fluorescence background by modulating of the excitation wavelength. The method is based on the continuous wavelength shift of the Raman peaks with the modulation of the laser wavelength while the fluorescence background remains static. Exploiting this physical property allows us to clearly distinguish between the Raman signal and the fluorescence background. Our method is related to wavelength shifting Raman spectroscopy but incorporates two key novel elements: (i) the use of more than two excitation wavelengths and (ii) multi-channel lock-in detection of the Raman signal for suppression of the fluorescence background. Our results establish a direct and practical approach for fluorescence background suppression in 'real-time' Raman spectroscopy for in-vivo biomedical applications.

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

2010-02-01

38

Laser-induced fluorescence spectroscopy in tissue local necrosis detection  

NASA Astrophysics Data System (ADS)

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

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

2014-03-01

39

Fluorescence-force spectroscopy at the single molecule level  

NASA Astrophysics Data System (ADS)

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.

Zhou, Ruobo

40

Fluorescence spectroscopy of rhodopsins: insights and approaches.  

PubMed

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

Alexiev, Ulrike; Farrens, David L

2014-05-01

41

Colon cancer diagnosis using fluorescence spectroscopy and fluorescence imaging technique  

NASA Astrophysics Data System (ADS)

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.

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

1997-12-01

42

Extraction of masked fluorescence peaks through synchronous fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

2012-02-01

43

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

Microsoft Academic Search

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

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

1998-01-01

44

Ultraviolet, Visible, and Fluorescence Spectroscopy  

Microsoft Academic Search

\\u000a Spectroscopy in the ultraviolet–visible (UV–Vis) range is one of the most commonly encountered laboratory techniques in food\\u000a analysis. Diverse examples, such as the quantification of macrocomponents (total carbohydrate by the phenol-sulfuric acid\\u000a method), quantification of microcomponents, (thiamin by the thiochrome fluorometric procedure), estimates of rancidity (lipid\\u000a oxidation status by the thiobarbituric acid test), and surveillance testing (enzyme-linked immunoassays), are presented

Michael H. Penner

45

Fluorescence Spectroscopy Investigations of Cutaneous Tissues  

Microsoft Academic Search

Fluorescence Spectroscopy of the human skin is very prominent for early diagnosis and differentiation of cutaneous diseases. Selection of proper excitation sources and sensitive detectors gives wide range of possibilities related to real-time determination of existing pathological conditions. A problem with using laser as an excitation source is the high expenses associated with the operation of these types of installations.

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

2007-01-01

46

Fluorescence spectroscopy of complex aromatic mixtures.  

PubMed

The contribution of two- to seven-ring polycyclic aromatic hydrocarbons (PAH) and of larger aromatic structures contained in complex PAH-laden mixtures collected in flames was evaluated by fluorescence spectroscopy. A composition procedure of the fluorescence spectra of individual PAHs, analyzed by gas chromatography/mass spectrometry (GC/MS) was applied for the evaluation of their contribution to the fluorescence spectra of PAH-laden mixtures. In this way, it was possible to put in evidence the contribution to the total fluorescence spectrum of high molecular weight aromatic species present in the PAH-laden mixtures and not detectable by GC/MS. Qualitative and quantitative interpretation of synchronous and conventional fluorescence spectra of PAH-laden mixtures formed in combustion processes was proposed. The composition procedure was showed to be reliable in the UV-visible region for samples dissolved in cyclohexane solutions, but failed in the UV region when the solvent contained heavy atoms, as in the case of dichloromethane. However, the heavy-atom solvent effect was not sufficient to explain the depression of the UV fluorescence signal. Energy transfer interaction between fluorene and other fluorescing PAHs was suggested to be also responsible for this effect on the basis of fluorescence studies performed on single PAHs and their mixtures in cyclohexane, methanol, and dichloromethane. PMID:15053681

Apicella, Barbara; Ciajolo, Anna; Tregrossi, Antonio

2004-04-01

47

Fluorescence Correlation Spectroscopy: The Case of Subdiffusion  

PubMed Central

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.

Lubelski, Ariel; Klafter, Joseph

2009-01-01

48

Fluorescence correlation spectroscopy: the case of subdiffusion.  

PubMed

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

Lubelski, Ariel; Klafter, Joseph

2009-03-18

49

Total Internal Reflection with Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

\\u000a The method in which total internal reflection illumination is combined with fluorescence correlation spectroscopy (TIR-FCS)\\u000a is reviewed. Included are descriptions of the conceptual basis and experimental design, as well as a variety of applications\\u000a and the theoretical models associated with these measurements. Emerging, more complex versions of TIR-FCS, related techniques,\\u000a and possible future directions for TIR-FCS are also outlined.

Nancy L. Thompson; Punya Navaratnarajah; Xiang Wang

50

High-Pressure Fluorescence Correlation Spectroscopy  

PubMed Central

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.

Muller, Joachim D.; Gratton, Enrico

2003-01-01

51

In vivo multiphoton fluorescence microscopy of epithelial precancer  

NASA Astrophysics Data System (ADS)

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

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

2011-02-01

52

Plasmon-controlled fluorescence: a new paradigm in fluorescence spectroscopy  

PubMed Central

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

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

2009-01-01

53

Theory and applications of fluorescence spectroscopy in food research  

Microsoft Academic Search

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

Gale M. Strasburg; Richard D. Ludescher

1995-01-01

54

Cross Talk Free Fluorescence Cross Correlation Spectroscopy in Live Cells  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) is now a widely used technique to measure small ensembles of labeled biomolecules with single molecule detection sensitivity (e.g., low endogenous concentrations). Fluorescence cross correlation spectroscopy (FCCS) is a derivative of this technique that detects the synchronous movement of two biomolecules with different fluorescence labels. Both methods can be applied to live cells and, therefore, can

Elmar Thews; Margarita Gerken; Reiner Eckert; Johannes Zäpfel; Carsten Tietz; Jörg Wrachtrup

2005-01-01

55

Two-Photon Fluorescence Correlation Spectroscopy  

NASA Technical Reports Server (NTRS)

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

Zimmerli, Gregory A.; Fischer, David G.

2002-01-01

56

Noninvasive skin fluorescence spectroscopy for diabetes screening.  

PubMed

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

Stirban, Alin

2013-07-01

57

The Photon Counting Histogram in Fluorescence Fluctuation Spectroscopy  

Microsoft Academic Search

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

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

1999-01-01

58

Polarization spectroscopy of single fluorescent molecules  

SciTech Connect

Polarization spectroscopy of single fluorescent molecules is used to probe their rotational dynamics. When a molecule is immobilized on a dry surface, its in-plane dipole orientation is precisely determined by utilizing its transition dipole moment. An angular offset between the absorption and the emission dipoles was detected from a single fluorophore revealing its binding geometry to the surface. In an aqueous environment, DNA-tethered fluorophores display dynamics that are well-described by a hindered rotational diffusion model. A detailed description of the model is given, including calculations to estimate depolarization effects resulting from the high numerical aperture objective used to collect fluorescence photons. Protein-conjugated fluorophores display very distinct dynamics with continuous evolution of the rotational profile, possibly reflecting fluctuations in the polypeptide chain. When protein-conjugated fluorophores are allowed to freely diffuse in solution, it is possible to determine the fluorescence polarization anisotropy of each molecule that traverses the laser beam. The anisotropy values could, in principle, be used to identify conformational states of single molecules without the potential artifacts associated with surface immobilization.

Ha, T.; Weiss, S. [Lawrence Berkeley National Lab., CA (United States)] [Lawrence Berkeley National Lab., CA (United States); Laurence, T.A.; Chemla, D.S. [Lawrence Berkeley National Lab., CA (United States). Material Sciences Div.] [Lawrence Berkeley National Lab., CA (United States). Material Sciences Div.; [Univ. of California, Berkeley, CA (United States). Physics Dept.

1999-08-19

59

Total internal reflection fluorescence microscopy imaging-guided confocal single-molecule fluorescence spectroscopy  

PubMed Central

We have developed an integrated spectroscopy system combining total internal reflection fluorescence microscopy imaging with confocal single-molecule fluorescence spectroscopy for two-dimensional interfaces. This spectroscopy approach is capable of both multiple molecules simultaneously sampling and in situ confocal fluorescence dynamics analyses of individual molecules of interest. We have demonstrated the calibration with fluorescent microspheres, and carried out single-molecule spectroscopy measurements. This integrated single-molecule spectroscopy is powerful in studies of single molecule dynamics at interfaces of biological and chemical systems.

Zheng, Desheng; Kaldaras, Leonora; Lu, H. Peter

2012-01-01

60

In vivo identification of colonic dysplasia using fluorescence endoscopic imaging  

Microsoft Academic Search

Background: 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 the collection of fluorescence images of adenomatous polyps in vivo. Methods: Fluorescence images were collected during colonoscopy in 30 patients. A total of 12 adenomatous and 6 hyperplastic polyps were identified. A

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

1999-01-01

61

Optical biopsy fiber-based fluorescence spectroscopy instrumentation  

NASA Astrophysics Data System (ADS)

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

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

1996-04-01

62

Fluorescence Lifetime Cross Correlation Spectroscopy Resolves EGFR and Antagonist Interaction in Live Cells  

Microsoft Academic Search

Fluorescence correlation or cross-correlation spectroscopy (FCS or FCCS), a single molecule technique, has the ability to provide highly sensitive information on interaction and dynamics of biomolecules both in vitro and in vivo. However, the inherent drawback of FCS is that species with similar molecular weight could not be differentiated. Although FCCS could resolve this through cross-correlation, it suffers from nonideal

Jiji Chen; Joseph Irudayaraj

2010-01-01

63

Fluorescence spectroscopy to assess apoptosis in myocardium  

NASA Astrophysics Data System (ADS)

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.

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

2007-03-01

64

Detecting Nanodomains in Living Cell Membrane by Fluorescence Correlation Spectroscopy  

NASA Astrophysics Data System (ADS)

Cell membranes actively participate in numerous cellular functions. Inasmuch as bioactivities of cell membranes are known to depend crucially on their lateral organization, much effort has been focused on deciphering this organization on different length scales. Within this context, the concept of lipid rafts has been intensively discussed over recent years. In line with its ability to measure diffusion parameters with great precision, fluorescence correlation spectroscopy (FCS) measurements have been made in association with innovative experimental strategies to monitor modes of molecular lateral diffusion within the plasma membrane of living cells. These investigations have allowed significant progress in the characterization of the cell membrane lateral organization at the suboptical level and have provided compelling evidence for the in vivo existence of raft nanodomains. We review these FCS-based studies and the characteristic structural features of raft nanodomains. We also discuss the findings in regards to the current view of lipid rafts as a general membrane-organizing principle.

He, Hai-Tao; Marguet, Didier

2011-05-01

65

Fluorescence Spectroscopy of Neoplastic and Non-Neoplastic Tissues  

PubMed Central

Abstract Fast and non-invasive, diagnostic techniques based on fluorescence spectroscopy have the potential to link the biochemical and morphologic properties of tissues to individual patient care. One of the most widely explored applications of fluorescence spectroscopy is the detection of endoscopically invisible, early neoplastic growth in epithelial tissue sites. Currently, there are no effective diagnostic techniques for these early tissue transformations. If fluorescence spectroscopy can be applied successfully as a diagnostic technique in this clinical context, it may increase the potential for curative treatment, and thus, reduce complications and health care costs. Steady-state, fluorescence measurements from small tissue regions as well as relatively large tissue fields have been performed. To a much lesser extent, time-resolved, fluorescence measurements have also been explored for tissue characterization. Furthermore, sources of both intrinsic (endogenous fluorophores) and extrinsic fluorescence (exogenous fluorophores) have been considered. The goal of the current report is to provide a comprehensive review on steady-state and time-resolved, fluorescence measurements of neoplastic and non-neoplastic, biologic systems of varying degrees of complexity. First, the principles and methodology of fluorescence spectroscopy are discussed. Next, the endogenous fluorescence properties of cells, frozen tissue sections and excised and intact bulk tissues are presented; fluorescence measurements from both animal and human tissue models are discussed. This is concluded with future perspectives.

Ramanujam, Nirmala

2000-01-01

66

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

Microsoft Academic Search

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

Yoshikazu Suzuki; Tomomi Tani; Kazuo Sutoh; Shinji Kamimura

2002-01-01

67

In vivo near-infrared fluorescence imaging  

Microsoft Academic Search

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

John V Frangioni

2003-01-01

68

Luciferase intramolecular dynamics studied by time-resolved fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

Earlier a comprehensive study of reaction substrate, product and their complexes with the enzyme has been carried out by means of stationary fluorescence spectroscopy. A hypothesis has been suggested according to which protein changes its conformation during the reaction. In this respect it is quite interesting to study both static and dynamic properties of the enzyme molecule. Luckily, according to genetic engineering data, luciferase molecule contains only one tryptophan residue, which makes it a very convenient object for the studies by means of time-resolved fluorescence spectroscopy. The scope of this paper is the study of luciferase tryptophan fluorescence. The methods applied were fluorescence spectrochronography and anisotropy decay analysis.

Brovko, Lubov Y.; Chikishev, Andrey Y.; Gandelman, Olga A.; Shkurinov, Aleksandr P.

1993-06-01

69

Handheld multispectral fluorescence lifetime imaging system for in vivo applications  

PubMed Central

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.

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

70

Handheld multispectral fluorescence lifetime imaging system for in vivo applications.  

PubMed

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

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

2014-03-01

71

Structured illumination fluorescence correlation spectroscopy for velocimetry in Zebrafish embryos  

NASA Astrophysics Data System (ADS)

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

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

2013-02-01

72

Imaging cellular dynamics in vivo with multicolor fluorescent proteins  

NASA Astrophysics Data System (ADS)

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

Hoffman, Robert M.

2005-04-01

73

Quantitative Determination of DNA-Ligand Binding Using Fluorescence Spectroscopy  

ERIC Educational Resources Information Center

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

Healy, Eamonn F.

2007-01-01

74

Fluorescence correlation spectroscopy: the technique and its applications  

Microsoft Academic Search

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

Oleg Krichevsky; Grégoire Bonnet

2002-01-01

75

Fluorescence Correlation Spectroscopy of Biomolecules: Kinetic Studies Using Microfluidic Devices  

Microsoft Academic Search

Fluorescence correlation spectroscopy (fcs) uses the autocorrelation of the fluctuation of the fluorescence intensity resulted by single fluorophores traversing a confined excitation volume. Information about motion and interactions can be gathered on the single molecule level. Usually either diffusion or convection is dominant on the timescales a molecule travels through the focal volume of a laser beam. In some conditions

Peter Galajda

2005-01-01

76

TOPICAL REVIEW: Single-molecule fluorescence spectroscopy of biomolecular folding  

Microsoft Academic Search

Single-molecule fluorescence spectroscopy is emerging as an important tool for studying biomolecular folding dynamics. Its usefulness stems from its ability to directly map heterogeneities in folding pathways and to provide information about the energy landscape of proteins and ribonucleic acid (RNA) molecules. Single-molecule fluorescence techniques relevant for folding studies, including methods for trapping and immobilizing molecules, are described and compared

Gilad Haran

2003-01-01

77

Coupling evaporation-based, microfluidic concentration and confocal fluorescence spectroscopy  

Microsoft Academic Search

Detection limits in confocal fluorescence spectroscopy (CFS) have traditionally been restrained by the low molecular detection efficiencies associated with femtoliter probe volumes. In this report, we address this issue by designing a microfluidic evaporator capable of accepting large sample volumes and concentrating biomolecules to a nanoliter-sized, interrogation chamber. Single molecule fluorescence detection within this chamber is enhanced through microfluidic recirculation,

C. M. Puleo; H. C. Yeh; K. J. Liu; T. Rane; T. H. Wang

2008-01-01

78

Principles and Applications of Fluorescence Correlation Spectroscopy (FCS)  

Microsoft Academic Search

\\u000a \\u000a Fluorescence correlation spectroscopy (FCS) is one of the various modern optical techniques that allow access to single fluorescently labeled biomolecules in aqueous\\u000a solution. In contrast to other fluorescence measurements, however, the parameter of primary interest is not the emission intensity\\u000a itself, but rather spontaneous intensity fluctuations caused by the minute deviations of a small ensemble of molecules from\\u000a thermal equilibrium.

Petra Schwille; Jonas Ries

79

Applications of Dual-Color Confocal Fluorescence Spectroscopy in Biotechnology  

Microsoft Academic Search

\\u000a Classical applications of fluorescence spectroscopy detect emission which is collected from comparatively large ensembles\\u000a of fluorescent particles, i.e. the signal is averaged over space and time. In contrast to this, confocal fluorescence methods\\u000a restrict the probe volume to a tiny spot of less than one femtoliter, which is the size of a typical bacterial cell. The high\\u000a spatial resolution can

A. Koltermann; U. Kettling; J. Stephan; M. Rarbach; T. Winkler; M. Eigen

80

Fluorescent protein engineering by in vivo site-directed mutagenesis.  

PubMed

In vivo site-directed mutagenesis by single-stranded deoxyribonucleic acid recombineering is a facile method to change the color of fluorescent proteins (FPs) without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the Escherichia 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 FP. Eight different FPs (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

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

2012-08-01

81

In vivo Raman spectroscopy of cervix cancers  

NASA Astrophysics Data System (ADS)

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

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

2014-03-01

82

In vivo study of photosensitizer pharmacokinetics by fluorescence transillumination imaging  

NASA Astrophysics Data System (ADS)

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.

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

2010-07-01

83

Statistical filtering in fluorescence microscopy and fluorescence correlation spectroscopy.  

PubMed

We review the principles and applications of statistical filtering in multichannel fluorescence microscopy. This alternative approach to separation of signals from individual fluorophore populations has many important advantages, especially when spectral and/or temporal overlap, or the complicated nature of those signals, makes their discrimination or sorting impossible by means of hardware. This situation is typically encountered for biological samples. This review of well established statistical filtering techniques and of emerging, very promising new methods of analysis reveals remarkable progress in bioanalytical applications of fluorescence microscopy. PMID:24908406

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

2014-08-01

84

Some Recent Advances in Fluorescence Spectroscopy  

Microsoft Academic Search

Fluorescence analysis methods continue to increase rapidly in importance in an ever?wider range of application areas. This growth highlights the need for the development and use of better standard materials and methods to ensure interlaboratory compatibility of results. Further impetus to the use of fluorescence methods has been provided by combining them with modern chemometric approaches that extract more information

James N. Miller

2006-01-01

85

Fluorescence Correlation Spectroscopy: A Review of Biochemical and Microfluidic Applications  

PubMed Central

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

Tian, Yu; Martinez, Michelle M.

2011-01-01

86

Clinical applications of in vivo fluorescence confocal laser scanning microscopy  

NASA Astrophysics Data System (ADS)

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 1.0?m, field of view 200?m x 100?m (lateral resolution , 0.3?m). 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.

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

2008-03-01

87

Fluorescence spectroscopy as a tool to investigate protein interactions.  

PubMed

Recent advances in the use of fluorescence spectroscopy to study protein interactions have primarily involved combinations of classic fluorescence techniques, novel probe and coupling chemistries, and advances in laser excitation and detection capabilities. For example, new coupling strategies for fluorescent probes have allowed the first determination of the DeltaG° describing the insertion of a protein into a membrane. Fluorescently labeled oligonucleotides with specific protein-binding sequences have been used to study both protein-DNA associations and oligonucleotide hybridization using anisotropy changes. The first kinetic data describing a DNA-protein binding event was collected with stopped-flow fluorescence instrumentation. Combining scanning fluctuation correlation spectroscopy with a two-photon excitation source improved this technique so that it may now be used to study protein self-associations. PMID:9013650

Brown; Royer

1997-02-01

88

Fluorescent lectins for local in vivo visualization of peripheral nerves.  

PubMed

Damage to peripheral nerves caused during a surgical intervention often results in function loss. Fluorescence imaging has the potential to improve intraoperative identification and preservation of these structures. However, only very few nerve targeting agents are available. This study describes the in vivo nerve staining capabilities of locally administered fluorescent lectin-analogues. To this end WGA, PNA, PHA-L and LEL were functionalized with Cy5 (?ex max 640 nm; ?em max 680 nm). Transfer of these imaging agents along the sciatic nerve was evaluated in Thy1-YFP mice (n = 12) after intramuscular injection. Migration from the injection site was assessed in vivo using a laboratory fluorescence scanner and ex vivo via fluorescence confocal microscopy. All four lectins showed retrograde movement and staining of the epineurium with a signal-to-muscle ratio of around two. On average, the longest transfer distance was obtained with WGA-Cy5 (0.95 cm). Since WGA also gave minimal uptake in the lymphatic system, this lectin type revealed the highest potential as a migration imaging agent to visualize nerves. PMID:25006792

KleinJan, Gijs Hendrik; Buckle, Tessa; van Willigen, Danny Michel; Oosterom, Matthias Nathanaël van; Spa, Silvia Johara; Kloosterboer, Harmen Egbert; van Leeuwen, Fijs Willem Bernhard

2014-01-01

89

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

Microsoft Academic Search

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

Longjiang Zheng; Yuanting Hu

2009-01-01

90

Multiparameter fluorescence image spectroscopy to study molecular interactions.  

PubMed

Multiparameter Fluorescence Image Spectroscopy (MFIS) is used to monitor simultaneously a variety of fluorescence parameters in confocal fluorescence microscopy. As the photons are registered one by one, MFIS allows for fully parallel recording of Fluorescence Correlation/Cross Correlation Spectroscopy (FCS/FCCS), fluorescence lifetime and pixel/image information over time periods of hours with picosecond accuracy. The analysis of the pixel fluorescence information in higher-dimensional histograms maximizes the selectivity of fluorescence microscopic methods. Moreover it facilitates a statistically-relevant data analysis of the pixel information which makes an efficient detection of heterogeneities possible. The reliability of MFIS has been demonstrated for molecular interaction studies in different complex environments: (I) detecting the heterogeneity of diffusion properties of the dye Rhodamine 110 in a sepharose bead, (II) Förster Resonance Energy Transfer (FRET) studies in mammalian HEK293 cells, and (III) FRET study of the homodimerisation of the transcription factor BIM1 in plant cells. The multidimensional analysis of correlated changes of several parameters measured by FRET, FCS, fluorescence lifetime and anisotropy increases the robustness of the analysis significantly. The economic use of photon information allows one to keep the expression levels of fluorescent protein-fusion proteins as low as possible (down to the single-molecule level). PMID:19337660

Weidtkamp-Peters, Stefanie; Felekyan, Suren; Bleckmann, Andrea; Simon, Rüdiger; Becker, Wolfgang; Kühnemuth, Ralf; Seidel, Claus A M

2009-04-01

91

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

NASA Astrophysics Data System (ADS)

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

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

2012-12-01

92

In vivo lipidomics using single-cell Raman spectroscopy  

PubMed Central

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

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

2011-01-01

93

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

PubMed

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

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

2012-07-01

94

Ex Vivo Fluorescence Molecular Tomography of the Spine  

PubMed Central

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

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

2012-01-01

95

Method for X-ray fluorescence spectroscopy  

Microsoft Academic Search

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

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

1985-01-01

96

Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy  

Microsoft Academic Search

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

Lifang Liang; Da Xing; Tongshen Chen; Yihui Pei

2007-01-01

97

Measurement of diffusion in articular cartilage using fluorescence correlation spectroscopy  

Microsoft Academic Search

Background  Fluorescence correlation spectroscopy (FCS) provides information about translational diffusion of fluorescent molecules in\\u000a tiny detection volumes at the single-molecule level. In normal states, cartilage tissue lacks vascularity, so chondrocyte\\u000a metabolism depends on diffusion for molecular exchanges. The abundant extracellular matrix (ECM) of cartilage is maintained\\u000a by a limited number of chondrocytes. ECM plays an important role in the regulation of

Jeong Ik Lee; Masato Sato; Kiminori Ushida; Joji Mochida

2011-01-01

98

Fluorescence correlation spectroscopy and its potential for intracellular applications  

Microsoft Academic Search

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

Petra Schwille

2001-01-01

99

Pancreatic tissue assessment using fluorescence and reflectance spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

2007-07-01

100

Fluorescent Lifetime Spectroscopy in Random Media  

NASA Astrophysics Data System (ADS)

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

Hutchinson, Christina Laura

101

Intramolecular Fluorescence Correlation Spectroscopy in a Feedback Tracking Microscope  

PubMed Central

Abstract We derive the statistics of the signals generated by shape fluctuations of large molecules studied by feedback tracking microscopy. We account for the influence of intramolecular dynamics on the response of the tracking system and derive a general expression for the fluorescence autocorrelation function that applies when those dynamics are linear. We show that in comparison to traditional fluorescence correlation spectroscopy, tracking provides enhanced sensitivity to translational diffusion, molecular size, heterogeneity, and long-timescale decays. We demonstrate our approach using a three-dimensional tracking microscope to study genomic ?-phage DNA molecules with various fluorescence label configurations.

McHale, Kevin; Mabuchi, Hideo

2010-01-01

102

Validation of temperature-modulated fluorescence tomography in vivo  

NASA Astrophysics Data System (ADS)

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

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

2014-02-01

103

Nonlinear Laser Fluorescence Spectroscopy of Natural Organic Compounds  

NASA Astrophysics Data System (ADS)

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

Fadeev, Victor V.; Shirshin, Evgeny A.

104

Fluorescence spectroscopy of Foscan induced feline tissues  

Microsoft Academic Search

Laser-induced fluorescence from feline tissue injected with meta-tetra(hydroxyphenyl) chlorin (mTHPC, FoscanR) was measured to determine the relative drug concentration in the five tissues (kidney, liver, lung, nasal planum and thyroid). Seventeen animals were administered differing initial dosages (0 mg\\/kg, 0.15 mg\\/kg, 0.03 mg\\/kg, 0.60 mg\\/kg), and were sacrificed at varying time intervals (24, 48, 72, 96, 336 hours). The tissues

Samuel J. Rhoades; James P. Wicksted; A. Y. Hamad; Avigdor M. Ronn; Greg A. Campbell; C. S. Arnold; Kenneth E. Bartels

2000-01-01

105

Fluorescence spectroscopy of Foscan induced feline tissues  

NASA Astrophysics Data System (ADS)

Laser-induced fluorescence from feline tissue injected with meta-tetra(hydroxyphenyl) chlorin (mTHPC, FoscanR) was measured to determine the relative drug concentration in the five tissues (kidney, liver, lung, nasal planum and thyroid). Seventeen animals were administered differing initial dosages (0 mg/kg, 0.15 mg/kg, 0.03 mg/kg, 0.60 mg/kg), and were sacrificed at varying time intervals (24, 48, 72, 96, 336 hours). The tissues obtained by necropsy were analyzed using a laser-induced backscattered fluorescence method that measured the emission spectra from the various tissue samples. It was shown that the animals injected with the largest initial dosage (0.60 mg/kg) of mTHPC almost always had the largest fluorescent intensities. It was also noted that highly vascular tissue (kidney, liver, lung) had more distinguishable peaks than those tissues farther from the venous system. Using this method, the optimal times to find the largest final concentration were 24 hours for lung and liver, 48 hours for kidney and thyroid and 72 hours for nasal planum.

Rhoades, Samuel J.; Wicksted, James P.; Hamad, A. Y.; Ronn, Avigdor M.; Campbell, Greg A.; Arnold, C. S.; Bartels, Kenneth E.

2000-05-01

106

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

NASA Astrophysics Data System (ADS)

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

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

2008-03-01

107

In vivo Diagnosis of Cervical Intraepithelial Neoplasia Using 337-nm- Excited Laser-Induced Fluorescence  

NASA Astrophysics Data System (ADS)

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

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

1994-10-01

108

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

NASA Astrophysics Data System (ADS)

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.

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

2007-08-01

109

Fluorescence spectroscopy of fulvic acids from fen peatlands  

Microsoft Academic Search

Intensive cultivation and agricultural use of peatlands lead to the degradation and mineralization of peat. Fulvic acids (FA) as the most mobile part of peat organic matter can be considered as an early indicator of its changes. One of the most sensitive and simple methods for studying the structural chemistry of humic substances is fluorescence spectroscopy. The objective of this

Victoria Maryganova; Lech Wojciech Szajdak

2010-01-01

110

Endogenous Fluorescence Spectroscopy of Cell Suspensions for Chemopreventive Drug Monitoring¶  

Microsoft Academic Search

Cancer chemopreventive agents such as N-4-(hydroxyphenyl)- retinamide (4HPR) are thought to prevent cancers by suppress- ing growth or inducing apoptosis in precancerous cells. Mechanisms by which these drugs affect cells are often not known, and the means to monitor their effects is not available. In this study endogenous fluorescence spectroscopy was used to measure metabolic changes in response to treatment

Nathaniel D. Kirkpatrick; Changping Zou; Molly A. Brewer; William R. Brands; Rebekah A. Drezek; Urs Utzinger

2005-01-01

111

Investigating Dynamics and Interactions of Biomolecules Using Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

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

Keith Berland

2002-01-01

112

Protein–Protein Interactions Determined by Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) is an emerging technique where the interaction between biomolecules is detected through their correlated motion. It offers the advantage of high (single-molecule) sensitivity; independence of molecular orientation or distance; and simultaneous measurement of molecular interactions, concentrations, and mobilities. Here we introduce the principle of the technique and review some recent examples from the literature where FCS

J. Langowski

2008-01-01

113

Fibre optic fluorescence spectroscopy for monitoring fish freshness  

NASA Astrophysics Data System (ADS)

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

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

2012-02-01

114

EPR Spectroscopy of Function In Vivo  

Microsoft Academic Search

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

Harold M. Swartz; Nadeem Khan

115

Deep tissue fluorescence imaging and in vivo biological applications  

PubMed Central

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

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

2012-01-01

116

Deep tissue fluorescence imaging and in vivo biological applications  

NASA Astrophysics Data System (ADS)

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

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

2012-11-01

117

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

NASA Astrophysics Data System (ADS)

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.

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

2006-03-01

118

Solid-phase fluorescence spectroscopy to characterize organic wastes.  

PubMed

The production of solid organic waste (SOW) such as sewage sludge (SS) or municipal solid waste (MSW) has been continuously increasing in Europe since the beginning of the 1990'. Today, the European Union encourages the stabilization of these wastes using biologic processes such as anaerobic digestion and/or composting to produce bio-energy and organic fertilizers. However, the design and management of such biologic processes require knowledge about the quantity and quality of the organic matter (OM) contained in the SOW. The current methods to characterize SOW are tedious, time-consuming and often insufficiently informative. In this paper, we assess the potential of solid-phase fluorescence (SPF) spectroscopy to quickly provide a relevant characterization of SOW. First, we tested well known model compounds (tryptophan, bovine serum albumin, lignin and humic acid) and biologic matrix (Escherichia coli) in three dimensional solid-phase fluorescence (3D-SPF) spectroscopy. We recorded fluorescence spectra from proteinaceous samples but we could not record the fluorescence emitted by lignin and humic acid powders. For SOW samples, fluorescence spectra were successfully recorded for MSW and most of its sub-components (foods, cardboard) but impossible for SS, sludge compost (SC) and ligno-cellulosic wastes. Based on visual observations and additional assays, we concluded that the presence of highly light-absorptive chemical structures in such dark-colored samples was responsible for this limitation. For such samples, i.e. lignin, humic acid, SS, SC and ligno-cellulosic wastes, we show that laser induced fluorescence (LIF) spectroscopy enables the acquisition of 2D fluorescence spectra. PMID:21696938

Muller, Mathieu; Milori, Débora Marcondes Bastos Pereira; Déléris, Stéphane; Steyer, Jean-Philippe; Dudal, Yves

2011-01-01

119

Ultrasensitive molecular fluorescence spectroscopy in levitated microdroplets  

SciTech Connect

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

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

1990-01-01

120

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

PubMed Central

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.

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

2011-01-01

121

Single-molecule fluorescence spectroscopy in (bio)catalysis  

PubMed Central

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

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

2007-01-01

122

Diagnosis of corneal pathology by laser fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

2012-09-01

123

Quantitative Prediction of Synthetic Food Colors by Fluorescence Spectroscopy and Radial Basis Function Neural Networks  

Microsoft Academic Search

In this paper, quantitative prediction of synthetic food colors by fluorescence spectroscopy and radial basis function neural networks is introduced. Taking Amaranth as an example, for sample solution of Amaranth with different concentrations, the fluorescence spectroscopy excited by the light with the wavelength of 400 nm is measured. The peak wavelength of Amaranth solution fluorescence spectroscopy is about 640 nm,

Chen Guo-qing; Su Zhou-ping; Wu Ya-min; Wang Jun; Wei Bai-lin; Zhu Tuo

2009-01-01

124

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

NASA Astrophysics Data System (ADS)

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

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

2012-02-01

125

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

NASA Astrophysics Data System (ADS)

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.

Skala, Melissa Caroline

126

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

PubMed

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

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

2014-04-01

127

Fluorescence spectroscopy of excitation transfer in Photosystem 1  

SciTech Connect

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

Mukerji, I.

1990-12-01

128

Emerging applications of fluorescence spectroscopy in medical microbiology field  

PubMed Central

There are many diagnostic techniques and methods available for diagnosis of medically important microorganisms like bacteria, viruses, fungi and parasites. But, almost all these techniques and methods have some limitations or inconvenience. Most of these techniques are laborious, time consuming and with chances of false positive or false negative results. It warrants the need of a diagnostic technique which can overcome these limitations and problems. At present, there is emerging trend to use Fluorescence spectroscopy as a diagnostic as well as research tool in many fields of medical sciences. Here, we will critically discuss research studies which propose that Fluorescence spectroscopy may be an excellent diagnostic as well as excellent research tool in medical microbiology field with high sensitivity and specificity.

2009-01-01

129

The power and prospects of fluorescence microscopies and spectroscopies.  

PubMed

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

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

2003-01-01

130

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

PubMed Central

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

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

2010-01-01

131

Fluorescence correlation spectroscopy of molecular motions and kinetics  

Microsoft Academic Search

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

Michael Gösch; Rudolf Rigler

2005-01-01

132

TOPICAL REVIEW: Single-molecule fluorescence spectroscopy of biomolecular folding  

NASA Astrophysics Data System (ADS)

Single-molecule fluorescence spectroscopy is emerging as an important tool for studying biomolecular folding dynamics. Its usefulness stems from its ability to directly map heterogeneities in folding pathways and to provide information about the energy landscape of proteins and ribonucleic acid (RNA) molecules. Single-molecule fluorescence techniques relevant for folding studies, including methods for trapping and immobilizing molecules, are described and compared in this review. Some emphasis is placed on fluorescence resonance energy transfer, which is particularly useful for studying conformational dynamics of biomolecules. Studies on protein and RNA folding using this methodology are reviewed and set in the more general context of folding science. Finally, some of the interesting future prospects in this field are delineated.

Haran, Gilad

2003-08-01

133

Fluorescence spectroscopy using indocyanine green for lymph node mapping  

NASA Astrophysics Data System (ADS)

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

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

2014-02-01

134

Multiphoton spectroscopy of human skin in vivo  

NASA Astrophysics Data System (ADS)

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

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

2012-02-01

135

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

PubMed Central

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

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

2011-01-01

136

Ultrasensitive fluorescence correlation spectroscopy of highly parallelized microfluidic devices  

NASA Astrophysics Data System (ADS)

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

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

2012-02-01

137

Measurement of diffusion in articular cartilage using fluorescence correlation spectroscopy  

PubMed Central

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

2011-01-01

138

Lifetime fluorescence spectroscopy for in situ investigation of osteogenic differentiation  

NASA Astrophysics Data System (ADS)

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

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

2003-07-01

139

Fluorescence Spectroscopy of Gas-phase Polycyclic Aromatic Hydrocarbons  

NASA Astrophysics Data System (ADS)

The purpose of this investigation was to produce fluorescence spectra of polycyclic aromatic hydrocarbon (PAH) molecules in the gas-phase for comparison with blue luminescence (BL) emission observed in astrophysical sources te{ref2,ref3,ref4}. The BL occurs roughly from 350 to 450 nm, with a sharp peak near 380 nm. PAHs with three to four rings, e.g. anthracene and pyrene, were found to produce luminescence in the appropriate spectral region, based on existing studies. Relatively few studies of the gas-phase fluorescence of PAHs exist; those that do exist have dealt primarily with the same samples commonly available for purchase such as pyrene and anthracene. In an attempt to understand the chemistry of the nebular environment we also obtained several nitrogen substituted PAHs from our colleagues at NASA Ames. In order to simulate the astrophysical environment we also took spectra by heating the PAHs in a flame. The flame environment counteracts the formation of eximers and permits the spectroscopy of free-flying neutral molecules. Experiments with coal tar demonstrate that fluorescence spectroscopy reveals primarily the presence of the smallest molecules, which are most abundant and which possess the highest fluorescence efficiencies. One gas-phase PAH that seems to fit the BL spectrum most closely is phenanthridine. In view of the results from the spectroscopy of coal tar, a compound containing a mixture of PAHs ranging from small to very large PAH molecules, we can not preclude the presence of larger PAHs in interstellar sources exhibiting BL.

Thomas, J. D.; Witt, A. N.

140

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

NASA Astrophysics Data System (ADS)

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

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

1995-03-01

141

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

NASA Astrophysics Data System (ADS)

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

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

1994-10-01

142

Long-Term Retention of Fluorescent Quantum Dots In Vivo  

NASA Astrophysics Data System (ADS)

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

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

143

Characterization of fluorescence-labeled DNA by time-resolved fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

A strategy for DNA characterization was developed, which exploits the normally unwanted interactions of the dye with its environment. With a single suitable dye, acting as a sensitive probe, one can characterize the neighboring DNA-base by time-resolved fluorescence spectroscopy due to specific dynamic quenching reactions affecting the fluorescence lifetime. By specific coupling of the dye to a linker-group at the 3' terminus of DNA a one- label/one-lane concept for DNA sequencing seems to be possible and all problems with multiple dyes can be avoided. To study the feasibility of this concept, fluorescence lifetimes of various model phosphorothioate oligodeoxyribonucleotides, different in length and labelled with a specially synthesized coumarin derivative, were measured in different aqueous solutions with a DFDL-UV picosecond fluorescence spectrometer. The influence of nucleic acid-dye interactions on the lifetime and quantum yield of the coupled dye is investigated. In mononucleotides fluorescence lifetimes range between 5.3 and 1.4 ns. The order in the quenching efficiency of the bases is adenine (A) equals 0, cytosine (C) < thymine (T) < guanine (G). In labelled di- and oligonucleotides the order of quenching changes slightly: A equals 0, C < G fluorescence lifetime is small and depends on the diastereomer, sequence and solvent. Comparison of quantum yields and lifetimes shows an additional static quenching due to complex formation especially in oligonucleotides.

Seidel, Claus; Rittinger, K.; Cortes, J.; Goody, R. S.; Koellner, Malte; Wolfrum, Juergen M.; Greulich, Karl O.

1991-06-01

144

In vivo Repeatability and Reproducibility of the Quantitative Light-Induced Fluorescence Method  

Microsoft Academic Search

In vivo repeatability and reproducibility of the quantitative light-induced fluorescence (QLF) method were tested with respect to three variables: lesion area, and average and maximum changes in lesion fluorescence. To test the image-capturing stages, three analysts each captured images of 15 incipient smooth surface lesions in vivo, and the images were analysed by one of the analysts. To test the

Sofia Tranæus; Xie-Qi Shi; Lars-Erik Lindgren; Karin Trollsås; Birgit Angmar-Månsson

2002-01-01

145

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

NASA Astrophysics Data System (ADS)

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

Sullivan, Kelley Diane

146

In-vivo optical imaging and spectroscopy of cerebral hemodynamics  

Microsoft Academic Search

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

Chao Zhou

2007-01-01

147

Hybrid phosphorescence and fluorescence native spectroscopy for breast cancer detection.  

PubMed

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

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

2007-01-01

148

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

Microsoft Academic Search

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

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

1999-01-01

149

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

Microsoft Academic Search

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

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

2007-01-01

150

Brain cancer probed by native fluorescence and stokes shift spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

2012-12-01

151

Investigation of asphaltene association by front-face fluorescence spectroscopy.  

PubMed

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

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

2003-07-01

152

Multiple-fiber probe design for fluorescence spectroscopy in tissue.  

PubMed

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

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

2002-08-01

153

Characterizing specific phage-protein interactions by fluorescence correlation spectroscopy  

PubMed Central

The interactions of several affinity reagent displayed T7 and M13 phage particles with their corresponding target molecules were examined using Fluorescence Correlation Spectroscopy (FCS). Diffusion times, relative fractions of each component in the recognition reactions at the equilibrium state, and ultimately the dissociation constants were deduced from analyzing the fluorescence autocorrelation curves. Although the sample preparation and FCS characterization of icosahedral T7-related systems were relatively straight forward, procedures with filamentous M13-related systems were complicated by the physical size of M13 and its aggregate formation. Methods that accommodate the FCS measurement of the M13 phage via changing confocal optics, fitting procedures, and aggregate discrimination are presented and discussed.

Bahns, John T.; Liu, Chin-Mei; Chen, Liaohai

2004-01-01

154

Oxidation monitoring by fluorescence spectroscopy reveals the age of fingermarks.  

PubMed

No forensic method exists that can reliably estimate the age of fingermarks found at a crime scene. Information on time passed since fingermark deposition is desired as it can be used to distinguish between crime related and unrelated fingermarks and to support or refute statements made by the fingermark donors. We introduce a non-contact method that can estimate the age of fingermarks. Fingermarks were approached as protein-lipid mixtures and an age-estimation model was build based on the expected protein and lipid oxidation reactions. Two measures of oxidation are required from the fingermark to estimate its age: 1)?the relative amount of fluorescent oxidation products 2)?the rate at which these products are formed. Fluorescence spectroscopy was used to obtain these measures. We tested the method on 44?fingermarks and were able to estimate the age of 55?% of the male fingermarks, up to three weeks old with an uncertainty of 1.9?days. PMID:24847728

van Dam, Annemieke; Schwarz, Janina C V; de Vos, Judith; Siebes, Maria; Sijen, Titia; van Leeuwen, Ton G; Aalders, Maurice C G; Lambrechts, Saskia A G

2014-06-10

155

Detectors for single-molecule fluorescence imaging and spectroscopy  

PubMed Central

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

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

2010-01-01

156

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

NASA Astrophysics Data System (ADS)

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

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

2014-03-01

157

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

PubMed

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

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

2014-03-01

158

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

NASA Astrophysics Data System (ADS)

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

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

2011-01-01

159

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

NASA Astrophysics Data System (ADS)

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

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

2007-07-01

160

Laser-induced fluorescence and dispersed fluorescence spectroscopy of jet-cooled 1-phenylpropargyl radical  

NASA Astrophysics Data System (ADS)

The D1(2A'')-D0(2A'') electronic transition of the resonance-stabilized 1-phenylpropargyl radicalooled discharge of 3-phenyl-1-propyne, has been investigated in detail by laser-induced fluorescence excitation and dispersed single vibronic level fluorescence (SVLF) spectroscopy. The transition is dominated by the origin band at 21 007 cm-1, with weaker Franck-Condon activity observed in a' fundamentals and even overtones and combinations of a'' symmetry. Ab initio and density functional theory calculations of the D0 and D1 geometries and frequencies were performed to support and guide the experimental assignments throughout. Analysis of SVLF spectra from 16 D1 vibronic levels has led to the assignment of 15 fundamental frequencies in the excited state and 19 fundamental frequencies in the ground state; assignments for many more normal modes not probed directly by fluorescence spectroscopy are also suggested. Duschinsky mixing, in which the excited state normal modes are rotated with respect to the ground state modes, is prevalent throughout, in vibrations of both a' and a'' symmetry.

Reilly, Neil J.; Nakajima, Masakazu; Gibson, Bligh A.; Schmidt, Timothy W.; Kable, Scott H.

2009-04-01

161

A universal model of restricted diffusion for fluorescence correlation spectroscopy.  

PubMed

Fluorescence correlation spectroscopy (FCS) is frequently used to study the processes of restricted diffusion. The most important quantity to determine is the size of the structures that hinder the Brownian motion of the molecules. We study three qualitatively different models of restricted diffusion, widely applied in biophysics and material science: Diffusion constrained by elastic force (i), walking confined diffusion (ii), and hop diffusion (iii). They cover the diversity of statistical behaviors, from purely Gaussian (i) to sharply non-Gaussian on intermediate time scales (ii) and, additionally, discrete (iii). We test whether one can use the Gaussian approximation of the FCS autocorrelation function to interpret the non-Gaussian data. We show that (i-iii) have approximately the same mean square displacements. Using simulations, we show that the FCS data suspected of restricted diffusion can be reliably interpreted using one archetypal model (i). Even if the underlying mechanism of the restriction is different or unknown, the accuracy of fitting the confinement size is excellent, and diffusion coefficients are also estimated with a good accuracy. This study gives a physical insight into the statistical behavior of different types of restricted diffusion and into the ability of fluorescence correlation spectroscopy to distinguish between them. PMID:24738620

Piskorz, Tomasz K; Ochab-Marcinek, Anna

2014-05-01

162

Single-Molecule Fluorescence Spectroscopy using Phospholipid Bilayer Nanodiscs  

PubMed Central

Nanodiscs are a new class of model membranes that are being used to solubilize and study a range of integral membrane proteins and membrane-associated proteins. Unlike other model membranes, the Nanodisc bilayer is bounded by a scaffold protein coat that confers enhanced stability and a narrow particle size distribution. The bilayer diameter can be precisely controlled by changing the diameter of the protein coat. All these properties make Nanodiscs excellent model membranes for single molecule fluorescence applications. In this chapter, we describe our work using Nanodiscs to apply total internal reflection fluorescence microscopy (TIRFM), fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) to study the integral membrane protein cytochrome P450 3A4 and the membrane-binding proteins islet amyloid popypeptide (IAPP) and ?-synuclein, respectively. The monodisperse size distribution of Nanodiscs enhances control over the oligomeric state of the membrane protein of interest, and also facilitates accurate solution-based measurements. Nanodiscs also comprise an excellent system to stably immobilize integral membrane proteins in a bilayer without covalent modification, enabling a range of surface-based experiments where accurate localization of the protein of interest is required.

Nath, Abhinav; Trexler, Adam J.; Koo, Peter; Miranker, Andrew D.; Atkins, William M.; Rhoades, Elizabeth

2012-01-01

163

Size determination of quantum dots with fluorescence correlation spectroscopy  

NASA Astrophysics Data System (ADS)

Semiconductor quantum dots (QDs) are highly interesting fluorophores for a large variety of spectroscopic applications. Although their fluorescence properties are well investigated, accurate size determination of QDs is still a problem. TEM techniques can image the inorganic core/shell system of QDs, but size determination of polymer coated QDs is difficult. SEC (size exclusion chromatography) compares the QD size only with standard polymers and their sizes, and is therefore not easy to use on nanoparticles. As QDs are fluorescent, single molecule spectroscopy methods such as fluorescence correlation spectroscopy (FCS) can be used to determine QDs diffusion coefficients and hence their hydrodynamic radii. Moreover, this method for size determination requires only very low QD concentrations, which is a mayor advantage compared to other techniques such as dynamic light scattering. Within our contribution we present the size determination of commercially available and self-modified QDs with FCS. The commercial QDs (QD525, QD565, QD605, QD655 and QD705 - purchased from Invitrogen Inc.) have a rather thick polymer shell and are functionalized with streptavidin, biotin or carboxylic groups. The self-modified QDs consist of the same commercial core/shell QDs and are modified with a polymer shell and several bio-functionalization groups. For all nanoparticles the diffusion coefficients were measured by FCS and the hydrodynamic radii were calculated according to the Stokes-Einstein equation. The obtained results are in good agreement with the size information provided by Invitrogen Inc., which demonstrates that FCS is an important technique for QD size determination at very low concentrations.

Hill, D.; Ast, C.; Löhmannsröben, H.-G.; Zulqurnain, A.; Parak, W.; Hildebrandt, N.

2011-02-01

164

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

PubMed Central

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

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

2011-01-01

165

Dual-color fluorescence cross correlation spectroscopy on an integrated optofluidic chip  

Microsoft Academic Search

We demonstrate the detection and discrimination of different sized nanobeads on an integrated optofluidic chip by dual-color fluorescence correlation spectroscopy. Singly and doubly labeled nanobeads can be further identified by cross correlation spectroscopy.

A. Chen; M. I. Rudenko; E. J. Lunt; B. S. Phillips; A. R. Hawkins; H. Schmidt

2010-01-01

166

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

PubMed

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

Ishii, Kunihiko; Tahara, Tahei

2013-10-01

167

Fluorescence spectroscopy: considerations for highly absorbing dissolved organic matter samples  

NASA Astrophysics Data System (ADS)

Fluorescence spectroscopy is a robust method for characterizing organic matter (OM). However, proper collection and correction of spectra are necessary to provide useful data. One important correction is the inner-filter correction, which primarily accounts for the inner-filter effect by adjusting for the wavelength dependent attenuation of emitted light by the solution prior to detection by the fluorometer. The most commonly used correction is based on an assumption that light is emitted at the center of the pathlength. Thus, the inner-filter effect is more pronounced in highly absorbing samples, and has the potential to skew the fluorescence spectra. For this study, the terrestrially derived Suwannee River fulvic acid (SRFA) and microbially derived Pony Lake fulvic acid (PLFA), from the International Humic Substances Society (IHSS), were diluted to incremental absorbances at a wavelength of 254 nm from 0.05 to 1.0 at pH 4 and 7. Three dimensional fluorescence spectra were measured and modeled with the Cory and McKnight (2005) parallel factor analysis (PARAFAC) model which resolves the fluorescence spectra into 13 components, including quinone-like and protein-like components. In the absence of inner-filter effects, plots of absorbance vs. loadings should be linear. Using the data from absorbance of 0.05 to 0.3, where the inner-filter affect is least pronounced, a linear regression was created and used as a baseline to predict component loadings at higher absorbance values in the absence of inner-filter effects. Results indicate that at absorbance values greater than 0.3, the commonly-used inner-filter correction is not able to remove the inner-filter effect. Therefore, in order to obtain reliable component loadings and correctly interpret the spectra, samples should be diluted to absorbance values less than 0.3 at 254 nm prior to collection of three dimensional fluorescence scans. The recommendation of a maximum absorbance of 0.3 agrees with the results of a study by Ohno (2002), which investigated several simpler fluorescence metrics. Suwannee River fulvic acid, pH 4, absorbance at 254nm vs. loading of component Q2. At the higher absorbances the component loadings are over-predicted compared to the linear regression, estimated from data with absorbance below 0.3, where the inner-filter effect is minimal.

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

2009-12-01

168

Assessing Raw and Treated Water Quality Using Fluorescence Spectroscopy  

NASA Astrophysics Data System (ADS)

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.

Bridgeman, J.; Baker, A.

2006-12-01

169

The Fluorescent Materials produced in vivo by certain Dermatophytes  

Microsoft Academic Search

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

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

1954-01-01

170

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

PubMed

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. PMID:20459282

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

2010-01-01

171

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

PubMed Central

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

Wachsmann-Hogiu, Sebastian; Weeks, Tyler

2009-01-01

172

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

NASA Astrophysics Data System (ADS)

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

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

1999-04-01

173

Fluorescence intermittency and spectral shifts of single bio-conjugated nanocrystals studied by single molecule confocal fluorescence microscopy and spectroscopy  

Microsoft Academic Search

We have fabricated a combined measurement system capable of confocal microscopy and fluorescence spectroscopy to simultaneously evaluate multiple optical characteristics of single fluorescent nanocrystals. The single particle detection sensitivity is demonstrated by simultaneously measuring the dynamic excitation-time-dependent fluorescence intermittency and the emission spectrum of single cadmium selenide\\/zinc sulfide (CdSe\\/ZnS) nanocrystals (quantum dots, QDs). Using this system, we are currently investigating

HyeongGon Kang; Mathew M. Maye; Dmytro Nykypanchuk; Matthew Clarke; Peter Yim; Jeffrey Krogmeier; Kimberly Briggman; Oleg Gang; Jeeseong Hwang

2007-01-01

174

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

PubMed

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

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

2013-01-01

175

In Vivo Chlorophyll A Fluorescence of Selenastrum capricornutum as a Screening Bioassay in Toxicity Studies  

Microsoft Academic Search

.   A method for the estimation of the effect of specific toxins on phytoplankton photosynthesis (index of toxicity) was investigated\\u000a using in vivo chlorophyll a fluorescence. No meaningful results were obtained with the following substances; copper, cadmium, mercury, and gusathion,\\u000a within a 4-h exposure period. It can, therefore, be concluded that in vivo chlorophyll a fluorescence are not a simple

J. A. van der Heever; J. U. Grobbelaar

1998-01-01

176

Fluorescence spectroscopy, exciton dynamics, and photochemistry of single allophycocyanin trimers  

SciTech Connect

The authors report a study of the allophycocyanin trimer (APC), a light-harvesting protein complex from cyanobacteria, by room-temperature single-molecule measurements of fluorescence spectra, lifetimes, intensity trajectories, and polarization modulation. Emission spectra of individual APC trimers are found to be homogeneous on the time scale of seconds. In contrast, their emission lifetimes are found to be widely distributed because of generation of long-lived exciton traps during the course of measurements. The intensity trajectories and polarization modulation experiments indicate reversible exciton trap formation within the three quasi-independent pairs of strong interacting {alpha}84 and {beta}84 chromophores in APC, as well as photobleaching of individual chromophores. Comparison experiments under continuous-wave and pulsed excitation reveal a two-photon mechanism for generating exciton traps and/or photobleaching, which involves exciton-exciton annihilation. These single-molecule experiments provide new insights into the spectroscopy, exciton dynamics, and photochemistry of light-harvesting complexes.

Ying, L.; Sie, X.S. [Pacific Northwest National Lab., Richland, WA (United States). William R. Wiley Environmental Molecular Sciences Lab.] [Pacific Northwest National Lab., Richland, WA (United States). William R. Wiley Environmental Molecular Sciences Lab.

1998-12-10

177

Aqueous solutions of lower alcohols investigated by pyrene fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

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.

Zhao, Li-Jun; Xiao, Han-Shuang

2012-03-01

178

Investigating lyophilization of lipid nanocapsules with fluorescence correlation spectroscopy.  

PubMed

This paper describes characterization of lyophilized lipid nanocapsules loaded with Alexa 488 by fluorescence correlation spectroscopy (FCS). Fluorimetry analysis of nanocapsules containing self-quenching concentrations of 5- and 6-carboxyfluorescein was performed to establish a point of reference for FCS. FCS results complemented the results obtained by fluorimetry for a bulk nanocapsule solution and provided additional information about the size and dye retention by individual nanocapsules. Using this method, we determined that nanocapsules composed of the thiol-functionalized lipids showed the best dye retention and the most consistent results. Dye retention, size, and photolysis efficiency of these thiol-functionalized nanocapsules doped with a far-red photosensitizer did not change substantially upon lyophilization and storage at -20 degrees C for up to 2 months, making lyophilization a suitable method for the long-term storage of nanocapsules with the appropriate lipid composition. PMID:20302335

Smith, Polina B; Dendramis, Kimberly A; Chiu, Daniel T

2010-06-15

179

Investigating Lyophilization of Lipid Nanocapsules with Fluorescence Correlation Spectroscopy  

PubMed Central

This paper describes characterization of lyophilized lipid nanocapsules loaded with Alexa 488 by fluorescence correlation spectroscopy (FCS). Fluorimetry analysis of nanocapsules containing self-quenching concentrations of 5-(and-6)-carboxyfluorescein was performed to establish a point of reference for FCS. FCS results complemented the results obtained by fluorimetry for a bulk nanocapsule solution and provided additional information about the size and dye retention by individual nanocapsules. Using this method, we determined that nanocapsules composed of the thiol-functionalized lipids showed the best dye retention and the most consistent results. Dye retention, size, and photolysis efficiency of these thiol-functionalized nanocapsules doped with a far-red photosensitizer did not change substantially upon lyophilization and storage at ?20 °C for up to two months, making lyophilization a suitable method for the long-term storage of nanocapsules with the appropriate lipid composition.

Smith, Polina B.; Dendramis, Kimberly A.; Chiu, Daniel T.

2010-01-01

180

Fluorescence correlation spectroscopy of intact nuclear pore complexes.  

PubMed

No methods proposed thus far have the sensitivity to measure the transport of single molecules through single nuclear pore complexes (NPCs) in intact cells. Here we demonstrate that fluorescence correlation spectroscopy (FCS) combined with real-time tracking of the center of mass of single NPCs in live, unperturbed cells allows us to detect the transport of single molecules in a reference system of a pore with high temporal (millisecond) and spatial (limited by diffraction) resolution. We find that the transport of the classical receptor karyopherin-?1 (Kap?1) is regulated so as to produce a peculiar distribution of characteristic times at the NPC. This regulation, which is spatially restricted to the pore, depends on the properties and metabolic energy of Kap?1. As such, this method provides a powerful tool for studying nucleocytoplasmic shuttling at the nanometer scale under physiological conditions. PMID:21843462

Cardarelli, Francesco; Lanzano, Luca; Gratton, Enrico

2011-08-17

181

Single-Molecule Fluorescence Spectroscopy and Microscopy of Biomolecular Motors  

NASA Astrophysics Data System (ADS)

The methods of single-molecule fluorescence spectroscopy and microscopy have been recently utilized to explore the mechanism of action of several members of the kinesin and myosin biomolecular motor protein families. Whereas ensemble averaging is removed in single-molecule studies, heterogeneity in the behavior of individual motors can be directly observed, without synchronization. Observation of translocation by individual copies of motor proteins allows analysis of step size, rate, pausing, and other statistical properties of the process. Polarization microscopy as a function of nucleotide state has been particularly useful in revealing new and highly rotationally mobile forms of particular motors. These experiments complement X-ray and biochemical studies and provide a detailed view into the local dynamical behavior of motor proteins.

Peterman, Erwin J. G.; Sosa, Hernando; Moerner, W. E.

2004-01-01

182

Afterpulsing and its correction in fluorescence correlation spectroscopy experiments  

NASA Astrophysics Data System (ADS)

Afterpulsing arises from feedback in a photon detector. This means that each real signal pulse can be followed by an afterpulse at a later time. This effect is particularly troubling in photon correlation experiments. Few treatments of this effect have appeared in the literature, and few software programs to solve the problem have been written. We demonstrate the afterpulsing effect in fluorescence correlation spectroscopy by using different avalanche photodiodes. We prove theoretically that under simple and reasonable conditions afterpulsing in autocorrelation can be eliminated to the leading order; we have found it easy to program software for the correction. We compare our results with those from cross correlation. We also discuss some experimental parameters that may affect the afterpulsing.

Zhao, Ming; Jin, Lei; Chen, Bo; Ding, Yao; Ma, Hui; Chen, Dieyan

2003-07-01

183

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

PubMed Central

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.

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

2013-01-01

184

Preparation and Characterization of Highly Fluorescent, Glutathione-coated Near Infrared Quantum Dots for in Vivo Fluorescence Imaging  

PubMed Central

Fluorescent probes that emit in the near-infrared (NIR, 700–1,300 nm) region are suitable as optical contrast agents for in vivo fluorescence imaging because of low scattering and absorption of the NIR light in tissues. Recently, NIR quantum dots (QDs) have become a new class of fluorescent materials that can be used for in vivo imaging. Compared with traditional organic fluorescent dyes, QDs have several unique advantages such as size- and composition-tunable emission, high brightness, narrow emission bands, large Stokes shifts, and high resistance to photobleaching. In this paper, we report a facile method for the preparation of highly fluorescent, water-soluble glutathione (GSH)-coated NIR QDs for in vivo imaging. GSH-coated NIR QDs (GSH-QDs) were prepared by surface modification of hydrophobic CdSeTe/CdS (core/shell) QDs. The hydrophobic surface of the CdSeTe/CdS QDs was exchanged with GSH in tetrahydrofuran-water. The resulting GSH-QDs were monodisperse particles and stable in PBS (phosphate buffered saline, pH = 7.4). The GSH-QDs (800 nm emission) were highly fluorescent in aqueous solutions (quantum yield = 22% in PBS buffer), and their hydrodynamic diameter was less than 10 nm, which is comparable to the size of proteins. The cellular uptake and viability for the GSH-QDs were examined using HeLa and HEK 293 cells. When the cells were incubated with aqueous solutions of the GSH-QDs (10 nM), the QDs were taken into the cells and distributed in the perinuclear region of both cells. After 12 hrs incubation of 4 nM of GSH-QDs, the viabilities of HeLa and HEK 293 cells were ca. 80 and 50%, respectively. As a biomedical utility of the GSH-QDs, in vivo NIR-fluorescence imaging of a lymph node in a mouse is presented.

Jin, Takashi; Fujii, Fumihiko; Komai, Yutaka; Seki, Junji; Seiyama, Akitoshi; Yoshioka, Yoshichika

2008-01-01

185

Localized in vivo13C NMR spectroscopy of the brain  

PubMed Central

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

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

2006-01-01

186

Advanced detection systems for X-ray fluorescence excitation spectroscopy.  

PubMed

This paper accounts for selected detector developments carried out over the past 15 years within the ESRF X-ray Absorption Spectroscopy group. This includes various types of photodiodes used as integrated current detectors. Special emphasis is put on the long-standing development of a Si drift-diode array suitable for energy-dispersive detection of X-ray fluorescence. This detector, which is now operational, was used to record high-quality XMCD/XAFS spectra on [Fe70Pt30] nanoparticles highly dispersed on a Si wafer. Using numerically deconvoluted spectra, energy resolution was decreased to 82 eV for the Si Kalphabeta line, 126 eV for the Fe Kalpha line and 176 eV for the Pt Lalpha line. A high-vacuum-compatible high-energy-resolution crystal analyzer was also installed on ID12, making it possible to record X-ray fluorescence excitation spectra in the photon-in/photon-out mode over a wide spectral range. Prospects of adapting these methods in order to investigate biological samples are briefly discussed. PMID:15616366

Goulon, J; Rogalev, A; Goujon, G; Gauthier, Ch; Moguiline, E; Solé, A; Feite, S; Wilhelm, F; Jaouen, N; Goulon-Ginet, Ch; Dressler, P; Rohr, P; Lampert, M-O; Henck, R

2005-01-01

187

Fluorescence correlation spectroscopy: Statistical analysis and biological applications  

NASA Astrophysics Data System (ADS)

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

Saffarian, Saveez

2002-01-01

188

Pancreatic tumor detection using hypericin-based fluorescence spectroscopy and cytology  

NASA Astrophysics Data System (ADS)

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

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

2005-04-01

189

Near-infrared in vivo fluorescence sensor with integrated dielectric emission filter  

Microsoft Academic Search

We present a monolithically integrated near-infrared fluorescence sensor incorporating a dielectric emission filter for in vivo applications. We successfully integrated a dielectric emission filter (OD3) onto a low-noise detector and sensed 50 nM fluorescent dye concentration.

Thomas D. O'Sullivan; Elizabeth Munro; Christopher Conca; Natesh Parashurama; Adam de la Zerda; Sanjiv S. Gambhir; James S. Harris; Ofer Levi

2009-01-01

190

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

Microsoft Academic Search

In vivo fluorescence of Chlorophyll-a (Chl-a) is a potentially useful property to study the vertical distribution of phytoplankton biomass. However the technique is presently not fully exploited as it should be, essentially because of the difficulties in converting the fluorescence signal into an accurate Chl-a concentration. These difficulties arise noticeably from natural variations in the Chl-a fluorescence relationship, which is

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

2011-01-01

191

Studies of protein folding and dynamics using single molecule fluorescence spectroscopy.  

PubMed

Single molecule fluorescence spectroscopy is emerging as an extremely powerful and sensitive tool to study complex biological problems. Single molecule fluorescence measurements can extract useful information that is hidden in the ensemble averaged biophysical or biochemical studies by virtue of their wide range of spatial and temporal resolution capabilities. With these advantages, single molecule fluorescence spectroscopy enables us to monitor the conformational states and their dynamics in the form of statistical distribution or time trajectory of physical observables. This review illustrates how the single molecule fluorescence spectroscopy has been used to solve questions on the complexity and heterogeneity of protein folding and dynamics. PMID:24805942

Basak, Sujit; Chattopadhyay, Krishnananda

2014-06-21

192

The effect of variable liposome brightness on quantifying lipid–protein interactions using fluorescence correlation spectroscopy  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) has been increasingly used to study the binding of fluorescently-labeled peptides and proteins to phospholipid vesicles. In this work, we present a new method to analyze partition data obtained by this technique based on the assumption that the number of fluorescently-labeled protein molecules bound per liposome follows a Poisson distribution. To not overestimate the recovered partition

Ana M. Melo; Manuel Prieto; Ana Coutinho

2011-01-01

193

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

PubMed Central

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

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

2013-01-01

194

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

Microsoft Academic Search

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

M. Newville; S. Sutton; M. Rivers

2002-01-01

195

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

Microsoft Academic Search

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

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

2009-01-01

196

Motion of a DNA Sliding Clamp Observed by Single Molecule Fluorescence Spectroscopy*S?  

PubMed Central

DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III ? subunit or ? clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the ? clamp diffusing along DNA is on the order of 10–14 m2/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the ? clamp remains at the 3? end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA.

Laurence, Ted A.; Kwon, Youngeun; Johnson, Aaron; Hollars, Christopher W.; O'Donnell, Mike; Camarero, Julio A.; Barsky, Daniel

2008-01-01

197

Transcutaneous Raman Spectroscopy of Murine Bone In Vivo  

PubMed Central

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.

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

2009-01-01

198

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

PubMed Central

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

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

2013-01-01

199

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

PubMed Central

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

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

2009-01-01

200

Long-term in vivo glucose monitoring using fluorescent hydrogel fibers  

PubMed Central

The use of fluorescence-based sensors holds great promise for continuous glucose monitoring (CGM) in vivo, allowing wireless transdermal transmission and long-lasting functionality in vivo. The ability to monitor glucose concentrations in vivo over the long term enables the sensors to be implanted and replaced less often, thereby bringing CGM closer to practical implementation. However, the full potential of long-term in vivo glucose monitoring has yet to be realized because current fluorescence-based sensors cannot remain at an implantation site and respond to blood glucose concentrations over an extended period. Here, we present a long-term in vivo glucose monitoring method using glucose-responsive fluorescent hydrogel fibers. We fabricated glucose-responsive fluorescent hydrogels in a fibrous structure because this structure enables the sensors to remain at the implantation site for a long period. Moreover, these fibers allow easy control of the amount of fluorescent sensors implanted, simply by cutting the fibers to the desired length, and facilitate sensor removal from the implantation site after use. We found that the polyethylene glycol (PEG)-bonded polyacrylamide (PAM) hydrogel fibers reduced inflammation compared with PAM hydrogel fibers, transdermally glowed, and continuously responded to blood glucose concentration changes for up to 140 days, showing their potential application for long-term in vivo continuous glucose monitoring.

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

2011-01-01

201

Infrared and Raman imaging spectroscopy of ex vivo skin.  

PubMed

This primer describes and illustrates experimental protocols for both Fourier transform infrared (FTIR) spectroscopic imaging and confocal Raman mapping of ex vivo skin and thereby acquaints the reader with these measurement techniques, including the temporal and spatial limitations associated with each technique. The experimental conditions by which the unique 'molecular histology' information obtained from confocal Raman mapping and infrared spectroscopic mapping of ex vivo skin is generated will be described. Raman and FTIR spectra of tissue, when collected in spatially resolved arrays, permit the generation of 'molecular images' of tissue components and tissue organization without the use of fluorescent labels or chemical stains. To illustrate the molecular information from ex vivo skin that can be spectroscopically imaged with confocal Raman and infrared microspectroscopy, we have collected new data using both techniques and generated spectral images which illustrate the capacity of each technique to provide unique insights into skin histology, biochemistry and biophysics. Understanding the measurement possibilities and specific constraints of both approaches is a prerequisite to their meaningful use as powerful research tools in skin research. PMID:23106608

Flach, Carol R; Moore, David J

2013-04-01

202

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

Microsoft Academic Search

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

Guowen Zhang; Qingmin Que; Junhui Pan; Jinbao Guo

2008-01-01

203

Fluorescence spectroscopy of fulvic acids from fen peatlands  

NASA Astrophysics Data System (ADS)

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

Maryganova, Victoria; Wojciech Szajdak, Lech

2010-05-01

204

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

PubMed

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 ?M 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

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

2014-06-30

205

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

PubMed Central

DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal peaked 1 week after transfection and returned to background level within 4 weeks. Katushka expression was not as stable as GFP expression, which was detectable for 8 weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the novel Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability.

2009-01-01

206

Fluorescence Glucose Detection: Advances Toward the Ideal In Vivo Biosensor  

Microsoft Academic Search

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

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

2004-01-01

207

In vivo tomographic imaging of red-shifted fluorescent proteins  

PubMed Central

We have developed a spectral inversion method for three-dimensional tomography of far-red and near-infrared fluorescent proteins in animals. The method was developed in particular to address the steep light absorption transition of hemoglobin from the visible to the far-red occurring around 600 nm. Using an orthotopic mouse model of brain tumors expressing the red-shifted fluorescent protein mCherry, we demonstrate significant improvements in imaging accuracy over single-wavelength whole body reconstructions. Furthermore, we show an improvement in sensitivity of at least an order of magnitude over green fluorescent protein (GFP) for whole body imaging. We discuss how additional sensitivity gains are expected with the use of further red-shifted fluorescent proteins and we explain the differences and potential advantages of this approach over two-dimensional planar imaging methods.

Deliolanis, Nikolaos C.; Wurdinger, Thomas; Pike, Lisa; Tannous, Bakhos A.; Breakefield, Xandra O.; Weissleder, Ralph; Ntziachristos, Vasilis

2011-01-01

208

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

NASA Astrophysics Data System (ADS)

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

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

1997-12-01

209

Longitudinal in vivo two-photon fluorescence imaging.  

PubMed

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

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

2014-06-01

210

In vivo EPR spectroscopy of free radicals in the heart.  

PubMed Central

Electron paramagnetic resonance (EPR) spectroscopy can be applied to directly measure free radicals; however, it has not been possible to measure important biologic radicals in situ because conventional spectrometer designs are not suitable for the performance of measurements on large aqueous structures such as whole organs or tissues. We describe the design, construction, and application of instrumentation developed in an effort to obtain optimum performance in measuring free radicals in intact biologic organs or tissues. This spectrometer consists of a 1- to 2-GHz microwave bridge with the source locked to the resonant frequency of a specially designed recessed gap, loop-gap resonator. The principles of resonator design and construction are analyzed and described. Using this spectrometer radical concentrations as low as 0.4 microM in aqueous solutions could be measured. Studies of isolated beating hearts involving simultaneous real time measurements of free radicals and cardiac contractile function are performed. This in vivo EPR technique is applied to study the kinetics of free radical uptake and metabolism in normally perfused and globally ischemic hearts. In addition, it is demonstrated that this technique can be used to noninvasively measure tissue oxygen consumption. Thus, low frequency EPR spectroscopy offers great promise in the study of in vivo free radical generation and the effects of this radical generation on whole biologic tissues.

Zweier, J L; Kuppusamy, P

1994-01-01

211

In vivo two-dimensional NMR correlation spectroscopy  

NASA Astrophysics Data System (ADS)

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

Kraft, Robert A.

1999-10-01

212

Detection of Protein Expression in Vivo Using Fluorescent Puromycin Conjugates.  

National Technical Information Service (NTIS)

Disclosed is a class of reagents for examining protein expression in vivo that does not require transfection, radiolabeling, or the prior choice of a candidate gene. Further, a series of puromycin conjugates was constructed bearing various labeling moieti...

E. Schuman H. M. Green J. Alberola-ila R. W. Roberts S. R. Starck-Green

2005-01-01

213

Noninvasive determination of cell nucleoplasmic viscosity by fluorescence correlation spectroscopy  

NASA Astrophysics Data System (ADS)

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 (?nu), an important parameter of nucleoplasmic rheology. Our FCS analyses show that ?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 °C. ?nu 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 ?nu of HeLa cells reached the lowest value in the S phase and that there was no significant difference of ?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 ?nu in living cells synchronized in the G1, S, and G2 phases.

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

2009-03-01

214

Intraoperative metastases detection by laser-induced fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

1991-06-01

215

Fluorescence Correlation Spectroscopy Evidence for Structural Heterogeneity in Ionic Liquids  

SciTech Connect

Self-aggregation in room temperature ionic liquids (RTILs) has been a subject of intense interest in recent years. In this work, we provide new experimental evidence for chain length-dependent self-aggregation in RTILs using fluorescence correlation spectroscopy (FCS). In studying a homologous series of N-alkyl-N-methylpyrrolidinium bis(trifluoromethylsulfonyl) imide, [CnMPy][Tf2N] RTILs of varying alkyl chain length (n = 3, 4, 6, 8, and 10), biphasic rhodamine 6G solute diffusion dynamics were observed; both the fast and slow diffusion coefficients decrease with increasing alkyl chain length, with the relative contribution from slower diffusion increasing for longer-chained [CnMPy][Tf2N]. We propose that the biphasic diffusion dynamics originate from self-aggregation of the nonpolar alkyl chains in the cationic [CnMPy]+. The presence of this local liquid structuring provides important insight into the behavior of RTILs relevant to their application in photovoltaics, fuel cells, and batteries.

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

2011-01-01

216

DOM transformations in stream biofilms shown by fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

2012-04-01

217

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

PubMed

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

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

2014-04-01

218

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

PubMed

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

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

2013-09-01

219

Fluorescent Pluronic nanodots for in vivo two-photon imaging.  

PubMed

We report the synthesis of new nanosized fluorescent probes based on bio-compatible polyethylene-polypropylene glycol (Pluronic) materials. In aqueous solution, mini-emulsification of Pluronic with a high fluorescent di-stryl benzene-modified derivative, exhibiting a two-photon absorption cross section as high as 2500 Goeppert-Mayer units at 800 nm, leads to nanoparticles exhibiting a hydrodynamic radius below 100 nm. We have demonstrated that these new probes with luminescence located in the spectral region of interest for bio-imaging (the yellow part of the visible spectrum) allow deep (500 microm) bio-imaging of the mice brain vasculature. The dose injected during our experiments is ten times lower when compared to the classical commercial rhodamine-B isothicyanate-Dextran system but gives similar results to homogeneous blood plasma staining. The mean fluorescent signal intensity stayed constant during more than 1 h. PMID:19448291

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

2009-06-10

220

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

PubMed Central

Objectives We used reflectance and fluorescence spectroscopy to noninvasively and quantitatively distinguish benign from dysplastic/malignant oral lesions. We designed diagnostic algorithms to account for differences in the spectral properties among anatomic sites (gingiva, buccal mucosa, etc). Methods In vivo reflectance and fluorescence spectra were collected from 71 patients with oral lesions. The tissue was then biopsied and the specimen evaluated by histopathology. Quantitative parameters related to tissue morphology and biochemistry were extracted from the spectra. Diagnostic algorithms specific for combinations of sites with similar spectral properties were developed. Results Discrimination of benign from dysplastic/malignant lesions was most successful when algorithms were designed for individual sites (area under the receiver operator characteristic curve [ROC-AUC], 0.75 for the lateral surface of the tongue) and was least accurate when all sites were combined (ROC-AUC, 0.60). The combination of sites with similar spectral properties (floor of mouth and lateral surface of the tongue) yielded an ROC-AUC of 0.71. Conclusions Accurate spectroscopic detection of oral disease must account for spectral variations among anatomic sites. Anatomy-based algorithms for single sites or combinations of sites demonstrated good diagnostic performance in distinguishing benign lesions from dysplastic/malignant lesions and consistently performed better than algorithms developed for all sites combined.

McGee, Sasha; Mardirossian, Vartan; Elackattu, Alphi; Mirkovic, Jelena; Pistey, Robert; Gallagher, George; Kabani, Sadru; Yu, Chung-Chieh; Wang, Zimmern; Badizadegan, Kamran; Grillone, Gregory; Feld, Michael S.

2010-01-01

221

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

PubMed

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

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

2013-11-11

222

Spectral features selection and classification for bimodal optical spectroscopy applied to bladder cancer in vivo diagnosis.  

PubMed

This paper describes an experimental study combining spatially resolved autofluorescence (AF) and diffuse reflectance (DR) fibred spectroscopies to discriminate in vivo between healthy and pathological tissues in a preclinical model of bladder cancer. Then, a detailed step-by-step analysis scheme is presented for the extraction and the selection of discriminative spectral features (correlation, linear discriminant, and logistic regression analysis), and for the spectroscopic data final classification algorithms (regularized discriminant analysis and support vector machines). Significant differences between healthy, inflammatory, and tumoral tissues were obtained by selecting a reasonable number of discriminant spectral features from AF, DR, and intrinsic fluorescence spectra, leading to improved sensitivity (87%) and specificity (77%) compared to monomodality (AF or DR alone). PMID:21216703

Péry, Emilie; Blondel, Walter C P M; Tindel, Samy; Ghribi, Maha; Leroux, Agnès; Guillemin, François

2014-01-01

223

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

NASA Astrophysics Data System (ADS)

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

Rujihan, Suparat; Damrongsak, Badin; Kittidachachan, Pattareeya

2013-06-01

224

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

NASA Astrophysics Data System (ADS)

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

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

2014-03-01

225

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

NASA Astrophysics Data System (ADS)

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

Mo, Weirong; Rohrbach, Daniel; Sunar, Ulas

2012-07-01

226

Cutaneous tumors in vivo investigations using fluorescence and diffuse reflectance techniques  

NASA Astrophysics Data System (ADS)

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.

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

2008-06-01

227

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

PubMed

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

Hoffman, Robert M

2014-01-01

228

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

NASA Astrophysics Data System (ADS)

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

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

2011-02-01

229

Inflammation Modulates Murine Venous Thrombosis Resolution In Vivo: Assessment by Multimodal Fluorescence Molecular Imaging  

PubMed Central

Objective Assessment of thrombus inflammation in vivo could provide new insights into deep vein thrombosis (DVT) resolution. Here we develop and evaluate two integrated fluorescence molecular-structural imaging strategies to quantify DVT-related inflammation and architecture, and to assess the effect of thrombus inflammation on subsequent DVT resolution in vivo. Methods and Results Murine DVT were created with topical 5% FeCl3 application to thigh or jugular veins (n=35). On day 3, mice received macrophage and matrix metalloproteinase (MMP) activity fluorescence imaging agents. On day 4, integrated assessment of DVT inflammation and architecture was performed using confocal fluorescence intravital microscopy (IVM). Day 4 analyses showed robust relationships among in vivo thrombus macrophages, MMP activity, and FITC-dextran deposition (r>0.70, p<0.01). In a serial two-timepoint study, mice with DVT underwent IVM at day 4 and at day 6. Analyses revealed that the intensity of thrombus inflammation at day 4 predicted the magnitude of DVT resolution at day 6 (p<0.05). In a second approach, noninvasive fluorescence molecular tomography-computed tomography (FMT-CT) was employed, and detected macrophages within jugular DVT (p<0.05 vs. sham-controls). Conclusions Integrated fluorescence molecular-structural imaging demonstrates that the DVT-induced inflammatory response can be readily assessed in vivo, and can inform the magnitude of thrombus resolution.

Ripplinger, Crystal M.; Kessinger, Chase W.; Li, Chunqiang; Kim, Jin Won; McCarthy, Jason R.; Weissleder, Ralph; Henke, Peter K.; Lin, Charles P.; Jaffer, Farouc A.

2012-01-01

230

The multiple uses of fluorescent proteins to visualize cancer in vivo.  

PubMed

Naturally fluorescent proteins have revolutionized biology by enabling what was formerly invisible to be seen clearly. These proteins have allowed us to visualize, in real time, important aspects of cancer in living animals, including tumour cell mobility, invasion, metastasis and angiogenesis. These multicoloured proteins have allowed the colour-coding of cancer cells growing in vivo and enabled the distinction of host from tumour with single-cell resolution. Visualization of many aspects of cancer initiation and progression in vivo should be possible with fluorescent proteins. PMID:16195751

Hoffman, Robert M

2005-10-01

231

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

PubMed Central

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

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

2013-01-01

232

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

National Technical Information Service (NTIS)

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

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

1991-01-01

233

Sample Preparation of Waste Water to Determine Metallic Contaminants by X-Ray Fluorescence Spectroscopy.  

National Technical Information Service (NTIS)

Trace X-ray fluorescence spectroscopy analysis in liquid samples is preceded by sample preparation, which usually consists in the precipitation of the metallic ions and concentration over a thin cellulose filter. The samples preparation of waste water by ...

J. Gonzalez Olivos

1987-01-01

234

Aldringsstudier av Hydroxylterminerad Polybutadien med Fluorescensspektroskopi (Aging Studies of Hydroxyl-Terminated Polybutadiene by Fluorescence Spectroscopy).  

National Technical Information Service (NTIS)

The aim of the work was to see if fluorescence spectroscopy can be used for determination of the antioxidant protection remaining in a stored sample of hydroxyl terminated polybutadiene (HTPB). The antioxidants Vulcanox BKF and Irganox 565 were studied in...

E. Berggren

1990-01-01

235

Fluorescence and Diffuse Reflectance Spectroscopy for Breast Cancer Diagnosis During Core Needle Biopsy.  

National Technical Information Service (NTIS)

The goal of this project is to explore the potential of using tissue fluorescence and diffuse reflectance spectroscopy for breast cancer detection during a core needle breast biopsy. Both empirically based and model based approaches have been explored for...

C. Zhu

2007-01-01

236

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

PubMed Central

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

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

2009-01-01

237

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

NASA Astrophysics Data System (ADS)

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

Zettergren, Eric William

238

In vivo near-infrared fluorescence imaging of osteoblastic activity  

Microsoft Academic Search

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

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

2001-01-01

239

Biocompatible fluorescent nanoparticles for in vivo stem cell tracking  

NASA Astrophysics Data System (ADS)

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

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

2013-06-01

240

Use of fluorescence spectroscopy for monitoring petroleum hydrocarbon contamination in estuarine and ocean waters  

SciTech Connect

The aromatic hydrocarbon content of extracted water samples were estimated by fluorescence emission spectroscopy. Solutions were excited at 300 nm, the emission scanned from 310-500 nm and the fluorescence emission intensity of the main peaks measured and reported as equivalents of m-terphenyl (lambda/sub em/330 nm) and chrysene (lambda/sub em/ 380 nm). The fluorescence emission of the reagent impurities was measured as the blank. Results show that if fluorescence spectroscopy is to be used to monitor petroleum contamination of estuarine and seawaters, the fluorescence emission of extracts should be measured at least at two wavelenghts (330 nm; 380 nm) to allow the detection of fuel oil, lubricating oil and crude oil inputs. A preliminary clean up of extracts is not usually required, thus the measurement of the fluorescence emission of water extracts provides a rapid means of monitoring petroleum hydrocarbon contamination. (JMT)

Maher, W.A.

1983-04-01

241

In vivo magnetic resonance spectroscopy of liver tumors and metastases  

PubMed Central

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

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

2011-01-01

242

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

PubMed Central

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

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

2011-01-01

243

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

PubMed Central

Purpose: Noninvasive in vivo cell-tracking techniques are necessary to advance the field of cellular-based therapeutics as well as to elucidate mechanisms governing in vivo cell biology. Fluorescence is commonly used for in vitro and postmortem biomedical studies but has been limited by autofluorescence at the whole-animal level. Procedures: In this report, we demonstrate the ability of in vivo fluorescent lifetime imaging to remove autofluorescence and thereby enable in vivo dendritic cell tracking in naïve mice. Specifically, we track mature dendritic cells (DCs) labeled internally with near-infrared-emissive polymersomes (NIR-DCs). Results: We establish the ability to detect labeled cells in vivo and image NIR-DC trafficking after both intravenous and subcutaneous delivery. In addition, we demonstrate the longitudinal capacity of this method by characterizing NIR-DC migration kinetics in the popliteal lymph node. Conclusions: This work provides a tool to evaluate dendritic-cell-based immunotherapy and generates novel opportunities for in vivo fluorescence imaging.

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

2009-01-01

244

Pitfalls and Their Remedies in Time-Resolved Fluorescence Spectroscopy and Microscopy  

Microsoft Academic Search

Time-resolved fluorescence spectroscopy and microscopy in both time and frequency domains provide very useful and accurate information on dynamic processes. Good quality data are essential in obtaining reliable parameter estimates. Distortions of the fluorescence response due to artifacts may have disastrous consequences. We provide here a concise overview of potential difficulties encountered under daily laboratory circumstances in the use of

Martin vandeVen; Marcel Ameloot; Bernard Valeur; Noël Boens

2005-01-01

245

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

NSDL National Science Digital Library

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

PhD Carey K. Johnson (University of Kansas Department of Chemistry); Kenneth D. Osborn (University of Kansas Department of Chemistry); Michael W. Allen (University of Kansas Department of Chemistry); Brian D. Slaughter (University of Kansas Department of Chemistry)

2005-02-01

246

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

NASA Astrophysics Data System (ADS)

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

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

2014-03-01

247

Single molecule fluorescence spectroscopy: approaches toward quantitative investigations of structure and dynamics in living cells  

Microsoft Academic Search

The investigation of the structure and dynamics of biomolecules and biomolecular assemblies in living cells is of current interest in molecular biology. Recent developments in single molecule fluorescence spectroscopy (SMFS) have opened ways for investigating the dynamics and stoichiometry of individual biomolecular complexes e.g., by application of single pair fluorescence resonance energy transfer (spFRET) with alternating laser excitation (ALEX), and

Daniel Siegberg; Christian Michael Roth; Dirk-Peter Herten

2006-01-01

248

In-vivo optical imaging and spectroscopy of cerebral hemodynamics  

NASA Astrophysics Data System (ADS)

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

Zhou, Chao

249

Tunable UV-laser-induced fluorescence spectroscopy of trace plastic compounds dissolved in seawater  

Microsoft Academic Search

Summary form only given. UV-laser-induced fluorescence (LIF) spectroscopy is a well-known technique to detect and measure trace compounds in the air or in liquids. A new fluorescence technique called excitation-emission matrix (EEM) provides enhanced identification of one compound among others by tuning the excitation wavelength and measuring the emitted fluorescence spectrum over a wide spectral range. We show an EEM

C. Luo; V. Nase; D. K. Killinger; J. Boehme

1998-01-01

250

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

PubMed

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

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

2012-11-21

251

MR imaging and in vivo proton spectroscopy of the brain in neonates with hypoxic ischemic encephalopathy  

Microsoft Academic Search

Introduction: A number of studies have suggested the potential utility of in vivo proton MR spectroscopy for the evaluation of brain injury in the asphyxiated neonates. We present our initial experience with in vivo proton MR spectroscopy in neonates who were diagnosed as having hypoxic injury on clinical examination and the severity of the insult was graded using Sarnat staging.

G. K Malik; M Pandey; R Kumar; S Chawla; B Rathi; R. K Gupta

2002-01-01

252

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

NASA Astrophysics Data System (ADS)

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

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

2013-03-01

253

Front face fluorescence spectroscopy and visible spectroscopy coupled with chemometrics have the potential to characterise ripening of Cabernet Franc grapes.  

PubMed

The potential of front-face spectroscopy for grape ripening dates discrimination was investigated on Cabernet Franc grapes from three parcels located on the Loire Valley and for six ripening dates. The 18 batches were analysed by front-face fluorescence spectroscopy and visible spectroscopy. The excitation spectra (250-310nm, emission wavelength=350nm) were characterised by a shoulder at 280nm. Grapes spectra were classified by factorial discriminant analysis (FDA). Ripening dates were well predicted by fluorescence spectra: grapes before veraison were separated from grapes after veraison and almost every ripening date was identified. The common spectroscopic space obtained by CCSWA showed that wavelengths corresponding to anthocyanin absorption in the visible were correlated to fluorescence wavelengths around the starting and ending points of the shoulder (263 and at 292nm). Then, regression models were investigated to predict total soluble solids (TSS), total acidity, malvidin-3G, total anthocyanins and total phenolics content from visible and fluorescence spectra. To predict technological indicators (TSS and total acidity), the PLS model with visible spectra (RMSECV=0.82 degrees Brix or 0.96gL(-1) H(2)SO(4)) was better than those with fluorescence one (RMSECV=1.39 degrees Brix or 2.06gL(-1) H(2)SO(4)). For malvidin-3G and total anthocyanins, all R(c)(2) and R(cv)(2) were superior to 0.90 and RMSECV were low. Visible and fluorescence spectroscopies succeeded in predicting anthocyanin content. Concerning total phenolic, the best prediction was provided by fluorescence spectroscopy. PMID:18573364

Le Moigne, Marine; Dufour, Eric; Bertrand, Dominique; Maury, Chantal; Seraphin, Denis; Jourjon, Frédérique

2008-07-21

254

Fast and Reversible Photoswitching of the Fluorescent Protein Dronpa as Evidenced by Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

Controlling molecular properties through photoirradiation holds great promise for its potential for noninvasive and selective manipulation of matter. Photochromism has been observed for several different molecules, including green fluorescent proteins, and recently the discovery of a novel photoswitchable green fluorescent protein called Dronpa was reported. Dronpa displays reversible and highly efficient on\\/off photoswitching of its fluorescence emission, and reversible switching

Peter Dedecker; Jun-ichi Hotta; Ryoko Ando; Atsushi Miyawaki; Yves Engelborghs; Johan Hofkens

2006-01-01

255

Bent fluorescent solar concentrators (BFSCs): Spectroscopy, stability and outdoor performance  

Microsoft Academic Search

The spectroscopic properties of a coumarin derivative dye (macrolex fluorescent red G) impregnated in a poly(methylmethacrylate)\\/silica hybrid matrix were studied. The stability of the hybrid against both light irradiation and raised temperature showed excellent weathering durability for use in fluorescent solar concentrator (FSC) applications. A new geometry for FSCs was introduced, which we call a “bent plate configuration”. Each BFSC

M. G. El-Shaarawy; S. M. El-Bashir; M. Hammam; M. K. El-Mansy

2007-01-01

256

Fluorescence spectroscopy for estimation of anticancer drug sonodestruction in vitro  

NASA Astrophysics Data System (ADS)

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

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

2013-11-01

257

Conventional and Confocal Epi-Reflection and Fluorescence Microscopy of the Rat Kidney in vivo  

Microsoft Academic Search

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

Alan Boyde; Giovambattista Capasso; Robert J. Unwin

1998-01-01

258

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

PubMed Central

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

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

259

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

Microsoft Academic Search

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

ZHANG Yi; HAN Yu; ZHAO Chun-Lin

260

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

PubMed Central

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

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

2008-01-01

261

A fluorescence spectroscopy study of traditional Chinese medicine Angelica  

NASA Astrophysics Data System (ADS)

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

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

2013-10-01

262

In Vivo Fluorescent Labeling of Tumor Cells with the HaloTag® Technology.  

PubMed

Many fluorescent sensors are currently available for in vitro bio-physiological microscopic imaging. The ability to label cells in living animals with these fluorescent sensors would help translate some of these assays into in vivo applications. To achieve this goal, the first step is to establish a method for selectively labeling target cells with exogenous fluorophores. Here we tested whether the HaloTag® protein tagging system provides specific labeling of xenograft tumors in living animals. After systemic delivery of fluorophore-conjugated ligands, we performed whole animal planar fluorescent imaging to determine uptake in tag-expressing HCT116 xenografts. Our results demonstrate that HaloTag ligands containing red or near-infrared fluorophores have enhanced tumor uptake and are suitable for non-invasive in vivo imaging. Our proof-of-concept results establish feasibility for using HaloTag technology for bio-physiological imaging in living animals. PMID:23115613

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

2012-01-01

263

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

PubMed Central

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.

Lu, Yujie; 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

264

Fluorescent viral nanoparticles with stable in vitro and in vivo activity.  

PubMed

We synthesized fluorescent capsid nanoparticles (FCNPs) by genetically inserting fluorescent protein (FP) (DsRed or eGFP) into each of 240 surface spike tips of hepatitis B virus (HBV) capsid particles. That is, when expressed in E. coli, FCNPs formed spherical nanoparticles with uniform diameter of about 40 nm owing to the self-assembly function of HBV core protein (i.e. basic assembly unit of capsid) and were successfully purified through Ni(+2) affinity- and sucrose gradient based purification. We also added the glycine-rich fexible linker peptides in between DsRed (or eGFP) and capsid to reduce fluorescence quenching among the densely displayed DsReds (or eGFPs) on the capsid surface. As compared to cognate fluorescent monomer proteins, it is notable that FCNPs showed a significantly amplified (160-170-fold) fluorescence intensity and enhanced conformational stability even in 50% serum solutin at 37 °C. The high conformational stability of FCNPs seems to result both from the highly stable structure of HBV capsid particles and from the well oriented insertion of fluorescent protein into capsid spike tip to keep native conformation of DsRed or eGFP. When estimated with continuous exposure to strong excitation light, FCNPs also showed much higher photostability than DsRed, eGFP, and a commonly used organic fluorescent dye, which happened presumably because the enhanced conformational stability of FCNPs significantly reduced photobleaching of fluorophores. Especially, it is notable that rFCNPs stably emitted high-level fluorescence inside mouse for a prolonged period, thereby showing high in vivo stability. The developed FCNPs are likely to have a great potential to be used as an effective and non-cytotoxic tool for in vivo optical imaging as well as in vitro fluorescent reporter in various biomolecular detection assays. PMID:22677189

Yoo, Lina; Park, Jin-Seung; Kwon, Koo Chul; Kim, Seong-Eun; Jin, Xiong; Kim, Hyunggee; Lee, Jeewon

2012-09-01

265

Evaluation of lipopeptide (daptomycin) aggregation using fluorescence, light scattering, and nuclear magnetic resonance spectroscopy.  

PubMed

The aggregation behavior and critical aggregation concentration (CAC) values of daptomycin in aqueous solutions were evaluated under the external factors of pH, temperature, daptomycin concentration, and calcium ions concentration by using the complementary characterization techniques, fluorescence, dynamic and static light scattering, and nuclear magnetic resonance (NMR) spectroscopy. On the basis of the intrinsic fluorescence resonance energy transfer of daptomycin, the CAC values were identified by an upward inflection of the fluorescence emission from Kyn-13 at 460 nm. The pH-dependent CAC values were determined to be 0.14 mM at pH 3.0, 0.12 mM at pH 4.0, and 0.20 mM at pH 2.5 and 5.0. The CAC values obtained by fluorescence spectroscopy were confirmed by dynamic light scattering and NMR spectroscopy. PMID:24464772

Qiu, Jiang; Kirsch, Lee E

2014-03-01

266

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

PubMed

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 nm 10 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. PMID:18404192

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

2008-04-10

267

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

NASA Astrophysics Data System (ADS)

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.

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

1998-04-01

268

Fluorescence spectroscopy of individual semiconductor nanoparticles in different ethylene glycols.  

PubMed

The optical properties of single colloidal semiconductor nanoparticles (NPs) are considerably influenced by the direct environment of the NPs. Here, the influence of different liquid and solid glycol matrices on CdSe-based NPs is investigated. Since the fluorescence of individual NPs varies from one NP to another, it is highly desirable to study the very same individual NPs in different matrices. This was accomplished by immobilizing NPs in a liquid cell sample holder or in microfluidic devices. The samples have been investigated by space-resolved wide-field fluorescence microscopy and energy- and time-resolved confocal scanning fluorescence microscopy with respect to fluorescence intensities, emission energies, blinking behavior, and fluorescence decay dynamics of individual NPs. During the measurements the NPs were exposed to air, to liquid ethylene glycols H(OCH2CH2)nOH (also called EGn) with different chain lengths (1 ? n ? 7), to liquid 2-methylpentane-2,3-diol, or to solid polyethylene oxide. It was found that EG6-7 (also known as PEG 300) is very well suited as a liquid matrix or solvent for experiments that correlate chemical and physical modifications of the surface and of the immediate environment of individual NPs to their fluorescence properties since it leads to intense and stable fluorescence emission of the NPs. PMID:24788878

Flessau, Sandra; Wolter, Christopher; Pöselt, Elmar; Kröger, Elvira; Mews, Alf; Kipp, Tobias

2014-06-14

269

In vivo time-gated fluorescence imaging with biodegradable luminescent porous silicon nanoparticles.  

PubMed

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

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

2013-01-01

270

Study of photosensitizers pharmacokinetics in mouse tumor model by transillumination fluorescence imaging in vivo  

NASA Astrophysics Data System (ADS)

In this work we demonstrate an efficiency of transillumination fluorescence imaging for intravital study of photosensitizers pharmacokinetics in tumor-bearing mice. Experiments were performed on CBA mice with subcutaneously transplanted cervical carcinoma. Photosensitizer fotoditazin was used in the therapeutic dose of 10 mg/kg, i.v. Measuring the fluorescence in the whole tumors at different times after injection of photosensitizer, we obtained in vivo data about the drug accumulation and elimination. It was found in vivo that the period of maximum uptake of the photosensitizer in the mouse tumor is from 1 to 8 h post-injection, and its concentration in the tumor tissue at this time is about 2 ?g/g. An accumulation of the photosensitizer in the tumor was confirmed by standard methods ex vivo. Noninvasive assessment of pharmacokinetics by transillumination imaging setup provides an opportunity for rapid and cost-effective studies of newly developed photosensitizers.

Shirmanova, Marina V.; Balalaeva, Irina V.; Sirotkina, Marina A.; Lekanova, Natalya Yu.; Turchin, Ilya V.; Zagainova, Elena V.

2011-02-01

271

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

NASA Astrophysics Data System (ADS)

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

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

2013-04-01

272

Fluorescence spectroscopy: A promising tool for carbonate petrology  

SciTech Connect

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

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

1992-01-01

273

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

PubMed Central

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

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

2010-01-01

274

Femtosecond broadband fluorescence spectroscopy by down- and up-conversion in ?-barium borate crystals  

NASA Astrophysics Data System (ADS)

Broadband transient fluorescence spectroscopy is performed by difference- and sum-frequency generation in ?-barium borate crystals at a fixed geometry. Phase matching is spectrally broadened by using (i) 1340 nm gate pulses, (ii) a fluorescence angle of ~4° extended at the crystal, and (iii) angular dispersion of the fluorescence in a calcite prism. The latter provides 1° angular separation between the two spectral wings of the fluorescence. By combining down- and up-conversion processes, a 26 000 cm-1 wide multiplex window is realized with ~100 fs time resolution.

Sajadi, M.; Quick, M.; Ernsting, N. P.

2013-10-01

275

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

PubMed Central

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

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

2009-01-01

276

Time-Resolved Single Vibronic Level Fluorescence Spectroscopy: Glyoxal.  

National Technical Information Service (NTIS)

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

E. Photos G. H. Atkinson

1975-01-01

277

Simple model for plasmon enhanced fluorescence correlation spectroscopy.  

PubMed

Metallic nano-antennas provide strong field confinement and intensity enhancement in hotspots and thus can ultimately enhance fluorescence detection and provide ultra small detection volumes. In solution-based fluorescence measurements, the diffraction limited focus driving the nano-antenna can outshine the fluorescence originating from the hotspot and thus render the benefits of the hotspot negligible. We introduce a model to calculate the effect of a nano-antenna, or any other object creating a nontrivial intensity distribution, for fluorescence fluctuation measurements. Approximating the local field enhancement of the nano-antenna by a 3D Gaussian profile, we show which hotspot sizes and intensities are the most beneficial for an FCS measurement and compare it to realistic antenna parameters from literature. PMID:24977800

Langguth, Lutz; Femius Koenderink, A

2014-06-30

278

Laser-induced fluorescence spectroscopy for combustion diagnostics  

NASA Technical Reports Server (NTRS)

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.

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

1983-01-01

279

Multiphoton fluorescence spectroscopy of flourescent bioprobes and biomolecules  

Microsoft Academic Search

Multi-photon fluorescence spectra of a number of commonly used biological probes were measured in this study. Significant spectral variation has been detected between single and multi- photon excitation. The result is important for the proper selection of spectral setting\\/dichroic beam splitter in the set- up of a multi-photon fluorescence microscope. The information can also be useful in the detection of

Ping-chin Cheng; Bai-Ling Lin; Fu-Jen Kao; Chi-Kuang Sun; Yung-Shun Wang; TzuMing Liu; YiMin Wang; JianCheng Chen; Mao-Kuo Huang; Iain Johnson

2000-01-01

280

Real-time background-free selective imaging of fluorescent nanodiamonds in vivo.  

PubMed

Recent developments of imaging techniques have enabled fluorescence microscopy to investigate the localization and dynamics of intracellular substances of interest even at the single-molecule level. However, such sensitive detection is often hampered by autofluorescence arising from endogenous molecules. Those unwanted signals are generally reduced by utilizing differences in either wavelength or fluorescence lifetime; nevertheless, extraction of the signal of interest is often insufficient, particularly for in vivo imaging. Here, we describe a potential method for the selective imaging of nitrogen-vacancy centers (NVCs) in nanodiamonds. This method is based on the property of NVCs that the fluorescence intensity sensitively depends on the ground state spin configuration which can be regulated by electron spin magnetic resonance. Because the NVC fluorescence exhibits neither photobleaching nor photoblinking, this protocol allowed us to conduct long-term tracking of a single nanodiamond in both Caenorhabditis elegans and mice, with excellent imaging contrast even in the presence of strong background autofluorescence. PMID:23066639

Igarashi, Ryuji; Yoshinari, Yohsuke; Yokota, Hiroaki; Sugi, Takuma; Sugihara, Fuminori; Ikeda, Kazuhiro; Sumiya, Hitoshi; Tsuji, Shigenori; Mori, Ikue; Tochio, Hidehito; Harada, Yoshie; Shirakawa, Masahiro

2012-11-14

281

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

NASA Astrophysics Data System (ADS)

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

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

2011-12-01

282

Fluorescence spectroscopy of anisole at elevated temperatures and pressures  

NASA Astrophysics Data System (ADS)

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

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

2014-06-01

283

Diffuse reflectance spectroscopy for in vivo pediatric brain tumor detection  

NASA Astrophysics Data System (ADS)

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

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

2010-11-01

284

In Vivo Blood Glucose Quantification Using Raman Spectroscopy  

PubMed Central

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

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

2012-01-01

285

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

PubMed Central

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

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

2012-01-01

286

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

NASA Astrophysics Data System (ADS)

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

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

2012-08-01

287

In vivo method to monitor changes in HER2 expression using near-infrared fluorescence imaging.  

PubMed

Human epidermal growth factor receptor type 2 (HER2) is a well-known biomarker that is overexpressed in many breast carcinomas. HER2 expression level is an important factor to optimize the therapeutic strategy and monitor the treatment. We used albumin binding domain-fused HER2-specific Affibody molecules, labeled with Alexa Fluor750 dye, to characterize HER2 expression in vivo. Near-infrared optical imaging studies were carried out using mice with subcutaneous HER2-positive tumors. Animals were divided into groups of five: no treatment and 12 hours and 1 week after treatment of the tumors with the Hsp90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). The compartmental ligands-receptor model, describing binding kinetics, was used to evaluate HER2 expression from the time sequence of the fluorescence images after the intravenous probe injection. The normalized rate of accumulation of the specific fluorescent biomarkers, estimated from this time sequence, linearly correlates with the conventional ex vivo enzyme-linked immunosorbent assay (ELISA) readings for the same tumor. Such correspondence makes properly arranged fluorescence imaging an excellent candidate for estimating HER2 overexpression in tumors, complementing ELISA and other ex vivo assays. Application of this method to the fluorescence data from HER2-positive xenografts reveals that the 17-DMAG treatment results in downregulation of HER2. Application of the AngioSense 750 probe confirmed the antiangiogenic effect of 17-DMAG found with Affibody-Alexa Fluor 750 conjugate. PMID:22554482

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

2012-06-01

288

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

NASA Astrophysics Data System (ADS)

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

Bartlett, Matthew Allen

289

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

NASA Astrophysics Data System (ADS)

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

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

1999-02-01

290

In vivo imaging of zebrafish retinal cells using fluorescent coumarin derivatives  

PubMed Central

Background The zebrafish visual system is a good research model because the zebrafish retina is very similar to that of humans in terms of the morphologies and functions. Studies of the retina have been facilitated by improvements in imaging techniques. In vitro techniques such as immunohistochemistry and in vivo imaging using transgenic zebrafish have been proven useful for visualizing specific subtypes of retinal cells. In contrast, in vivo imaging using organic fluorescent molecules such as fluorescent sphingolipids allows non-invasive staining and visualization of retinal cells en masse. However, these fluorescent molecules also localize to the interstitial fluid and stain whole larvae. Results We screened fluorescent coumarin derivatives that might preferentially stain neuronal cells including retinal cells. We identified four coumarin derivatives that could be used for in vivo imaging of zebrafish retinal cells. The retinas of living zebrafish could be stained by simply immersing larvae in water containing 1 ?g/ml of a coumarin derivative for 30 min. By using confocal laser scanning microscopy, the lamination of the zebrafish retina was clearly visualized. Using these coumarin derivatives, we were able to assess the development of the zebrafish retina and the morphological abnormalities induced by genetic or chemical interventions. The coumarin derivatives were also suitable for counter-staining of transgenic zebrafish expressing fluorescent proteins in specific subtypes of retinal cells. Conclusions The coumarin derivatives identified in this study can stain zebrafish retinal cells in a relatively short time and at low concentrations, making them suitable for in vivo imaging of the zebrafish retina. Therefore, they will be useful tools in genetic and chemical screenings using zebrafish to identify genes and chemicals that may have crucial functions in the retina.

2010-01-01

291

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

PubMed Central

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

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

2011-01-01

292

In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers  

PubMed Central

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

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

2012-01-01

293

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

PubMed

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

Brand, Christian; Abdel-Atti, Dalya; Zhang, Yachao; Carlin, Sean; Clardy, Susan M; Keliher, Edmund J; Weber, Wolfgang A; Lewis, Jason S; Reiner, Thomas

2014-07-16

294

Uptake of Gold Nanoparticles in Several Rat Organs after Intraperitoneal Administration In Vivo: A Fluorescence Study  

PubMed Central

Background. The gold nanoparticles (GNPs) have potential applications in cancer diagnosis and therapy. In an attempt to characterise the potential toxicity or hazards of GNPs as a therapeutic or diagnostic tool, the fluorescence spectra in several rat organs in vivo were measured after intraperitoneal administration of GNPs. Methods. The experimental rats were divided into control and six groups (G1A, G1B, G2A, G2B, G3A, and G3B; G1: 20?nm; G2: 10?nm; G3: 50?nm; A: infusion of GNPs for 3 days; B: infusion of GNPs for 7 days). The fluorescence measurements were investigated in the liver, kidney, heart, and lung organs of rats after intraperitoneal administration of GNPs for periods of 3 and 7 days in vivo. Results. The 10 and 20?nm GNPs exhibited spherical morphology shape, while the 50?nm GNPs exhibited hexagonal shape. A sharp decrease in the fluorescence intensity induced with the larger 50?nm GNPs in the liver, kidney, heart, and lung organs of rats at the exposure duration of 3 and 7 days in vivo compared with the smaller 10 and 20?nm GNPs was observed. Conclusions. The decrease in fluorescence intensity may be attributed to occurrence of strong quenching, decrease in number and surface area of GNPs, and high clearance of GNPs via urine and bile. Moreover, decreasing size may lead to an exponential increase in surface area relative to volume, thus making GNPs surface more reactive on aggregation and to its surrounding biological components. The size, shape, surface area, number, and clearance of GNPs play a key role in toxicity and accumulation in the different rat organs. This study demonstrates that fluorescence peak intensity is particle size and exposure duration dependent. This study suggests that fluorescence intensity can be used as a useful tool for pointing to bioaccumulation and toxicity induced by GNPs in the different rat organs.

Abdelhalim, Mohamed Anwar K.

2013-01-01

295

Correlation analysis of fluorescence intensity and fluorescence anisotropy fluctuations in single-molecule spectroscopy of conjugated polymers.  

PubMed

Single-molecule spectroscopy techniques are used to investigate time fluctuations of the fluorescence properties of two different types of conjugated polymer, a polythiophene derivative (PDOPT) and a phenylene vinylene derivative (MEH-PPV), at 100 and 293 K. Linear correlation coefficients between fluorescence intensity and polarization are used to characterize fluctuations. We are able to distinguish between different blinking and bleaching effects on the polarization. Furthermore, the polarization data reveal clear differences in the topology of these two polymers, which is related to the ordered conformation of the MEH-PPV. Plots of correlation coefficients appear to be very different for the two polymers and are also very sensitive to temperature. These observations prove that correlation analysis is a useful tool to understand fluorescence fluctuations in multi-chromophoric systems. PMID:20087921

Thomsson, Daniel; Lin, Hongzhen; Scheblykin, Ivan G

2010-03-15

296

Time-resolved Hyperspectral Fluorescence Spectroscopy using Frequency Modulated Excitation  

SciTech Connect

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

,; Neill, M

2012-07-01

297

Activatable near-infrared fluorescent probe for in vivo imaging of fibroblast activation protein-alpha.  

PubMed

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

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

2012-08-15

298

Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy  

Microsoft Academic Search

Dynamic structural changes of macromolecules undergoing biochemical reactions can be studied using novel single molecule spectroscopy tools. Recent advances in applying such distance and orientation molecular rulers to biological systems are reviewed, and future prospects and challenges are discussed.

Shimon Weiss

2000-01-01

299

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

NASA Astrophysics Data System (ADS)

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.

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

2014-03-01

300

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

SciTech Connect

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

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

1986-01-01

301

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

National Technical Information Service (NTIS)

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

J. W. Daily

1981-01-01

302

Integrated optical measurement system for fluorescence spectroscopy in microfluidic channels  

Microsoft Academic Search

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

Jorg Hubnera; Klaus B. Mogensen; Anders M. Jorgensen; Peter Friis; Pieter Telleman; Jorg P. Kutter

2001-01-01

303

Tailor-Made Dyes for Fluorescence Correlation Spectroscopy (FCS)  

Microsoft Academic Search

Two new fluorescent labels are presented that are op- timized for excitation with He\\/Ne laser and red diode lasers. Application in FCS and labeling of proteins and oligomers are demonstrated. A strong rise of quantum yield and emission life time upon binding to biomolecules are characteristic features of the dyes.

Peter Czerney; Frank Lehmann; Matthias Wenzel; Volker Buschmann; Anja Dietrich; Gerhard J. Mohr

2001-01-01

304

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

NASA Astrophysics Data System (ADS)

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

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

2000-04-01

305

Visible-super-resolution infrared microscopy using saturated transient fluorescence detected infrared spectroscopy  

Microsoft Academic Search

A scanning visible-super-resolution microscope based on the saturation behaviour of transient fluorescence detected infrared (TFD-IR) spectroscopy is proposed. A Gaussian IR beam, a Gaussian visible beam and a Laguerre–Gaussian (LG) visible beam are used to obtain two separate two-color excitation fluorescence (2CF) images of the sample. The final image is obtained as the difference between the two recorded images. If

Nándor Bokor; Keiichi Inoue; Satoshi Kogure; Masaaki Fujii; Makoto Sakai

2010-01-01

306

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

SciTech Connect

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

Rutan, S.C.

1989-01-01

307

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

PubMed Central

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

Scherrer, Gregory; Tryoen-Toth, Petra; Filliol, Dominique; Matifas, Audrey; Laustriat, Delphine; Cao, Yu Q.; Basbaum, Allan I.; Dierich, Andree; Vonesh, Jean-Luc; Gaveriaux-Ruff, Claire; Kieffer, Brigitte L.

2006-01-01

308

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

NASA Astrophysics Data System (ADS)

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

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

2006-03-01

309

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

PubMed Central

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

Chiara Munisso, Maria; Yamaoka, Tetsuji

2014-01-01

310

A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy.  

PubMed

Steady-state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e. g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well-defined protocol is presented for the investigation of the thermal conformational stability of proteins. This allows the determination of the denaturation temperature as a function of solution conditions. Using intrinsic tryptophan fluorescence spectroscopy for monitoring the denaturation and folding of proteins, it is crucial to understand the influence of different formulation parameters on the intrinsic fluorescence probes of proteins. Therefore, we have re-evaluated and re-assessed the influence of temperature, pH, ionic strength, buffer composition on the emission spectra of tryptophan, phenylalanine and tyrosine to correctly analyse and evaluate the data obtained from thermal-induced protein denaturation as a function of the solution parameters mentioned above. The results of this study are a prerequisite for using this method as a screening assay for analysing the conformational stability of proteins in solution. The data obtained from intrinsic protein fluorescence spectroscopy are compared to data derived from calorimetry. The advantage, challenges and applicability using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology are discussed. PMID:18702089

Garidel, Patrick; Hegyi, Matthias; Bassarab, Stefan; Weichel, Michael

2008-10-01

311

Implantation of human colorectal carcinoma cells in the liver studied by in vivo fluorescence videomicroscopy  

Microsoft Academic Search

In vivo fluorescence videomicroscopy (IVFM) was used to analyse the behavior of weakly and highly metastatic human colorectal carcinoma (CRC) cells during implantation in the liver. A highly metastatic human CRC cell line, CX-1, and a weakly metastatic line, Clone A, were double-labeled with rhodamine B isothiocyanate-dextran (Rd-Dx) to locate cells and with calcein AM to assess cell metabolic activity

Seiichi Ishii; Takayuki Mizoi; Katsunori Kawano; Osman Cay; Peter Thomas; Alexander Nachman; Rosilyn Ford; Yutaka Shoji; John B. Kruskal; Glenn Steele Jr; J. Milburn Jessup

1996-01-01

312

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

Microsoft Academic Search

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

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

1999-01-01

313

Selective Fluorescent Imaging of Superoxide in vivo Using Ethidium-Based Probes  

Microsoft Academic Search

The putative oxidation of hydroethidine (HE) has become a widely used fluorescent assay for the detection of superoxide in cultured cells. By covalently joining HE to a hexyl triphenylphosphonium cation (Mito-HE), the HE moiety can be targeted to mitochondria. However, the specificity of HE and Mito-HE for superoxide in vivo is limited by autooxidation as well as by nonsuperoxide-dependent cellular

Kristine M. Robinson; Michael S. Janes; Mariana Pehar; Jeffrey S. Monette; Meredith F. Ross; Tory M. Hagen; Michael P. Murphy; Joseph S. Beckman

2006-01-01

314

A fluorescence correlation spectroscopy study of macromolecular tracer diffusion in polymer solutions.  

PubMed

We discuss the manner in which the dynamics of tracer polystyrene chains varies with the concentration of matrix polystyrene chains dissolved in toluene. Using fluorescence correlation spectroscopy and theory, it is shown that the cooperative diffusion coefficient of the matrix polystyrene chains can be measured by fluorescence correlation spectroscopy in the semidilute entangled concentration regime. In addition the self-diffusion coefficient of the tracer polystyrene chains can be detected for arbitrary concentrations. The measured cooperative diffusion coefficient is independent of the molecular weight of the tracer polystyrene chains because it is a characteristic feature of the transient entanglement network. PMID:21406777

Zettl, Ute; Ballauff, Matthias; Harnau, Ludger

2010-11-23

315

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

NASA Astrophysics Data System (ADS)

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

Cerussi, Albert Edward

1999-09-01

316

Salt Tolerance in Crop Plants Monitored by Chlorophyll Fluorescence In Vivo  

PubMed Central

The potential of measurements of chlorophyll fluorescence in vivo to detect cellular responses to salinity and degrees of salt stress in leaves was investigated for three crop plants. Sugar beet (Beta vulgaris L.) (salt tolerant), sunflower (Helianthus annuus L.) (moderately salt tolerant), and bean (Phaseolus Vulgaris L. cv Canadian Wonder) (salt intolerant) were grown in pots and watered with mineral nutrient solution containing 100 millimolar NaCl. The fast rise in variable chlorophyll fluorescence yield that is correlated with photoreduction of photosystem II acceptors increased in leaves of sugar beet plants treated with salt suggesting stimulation of photosystem II activity relative to photosystem I. In sunflower, this fast rise was depressed by approximately 25% and the subsequent slow rate of quenching of the chlorophyll fluorescence was stimulated. These differences were more marked in the older mature leaves indicating an increasing gradient of salt response down the plant. The salt effect in vivo was reversible since chloroplasts isolated from mature leaves of salt-treated and control sunflower plants gave similar photosystem II activities. Unlike in sugar beet and sunflower, leaves of salt-treated bean progressively lost chlorophyll. The rate of slow quenching of chlorophyll fluorescence decreased indicating development of a partial block after photosystem II and possible initial stimulation of photosystem II activity. With further loss of chlorophyll photosystem II activity declined. It was concluded that measurements of chlorophyll fluorescence in vivo can provide a rapid means of detecting salt stress in leaves, including instances where photosynthesis is reduced in the absence of visible symptoms. The possible application to screening for salt tolerance is discussed.

Smillie, Robert M.; Nott, Robyn

1982-01-01

317

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

NASA Astrophysics Data System (ADS)

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

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

2007-03-01

318

Dendrimer-Based Fluorescent Indicators: In Vitro and In Vivo Applications  

PubMed Central

Background The development of fluorescent proteins and synthetic molecules whose fluorescence properties are controlled by the environment makes it possible to monitor physiological and pathological events in living systems with minimal perturbation. A large number of small organic dyes are available and routinely used to measure biologically relevant parameters. Unfortunately their application is hindered by a number of limitations stemming from the use of these small molecules in the biological environment. Principal Findings We present a novel dendrimer-based architecture leading to multifunctional sensing elements that can overcome many of these problems. Applications in vitro, in living cells and in vivo are reported. In particular, we image for the first time extracellular pH in the brain in a mouse epilepsy model. Conclusion We believe that the proposed architecture can represent a useful and novel tool in fluorescence imaging that can be widely applied in conjunction with a broad range of sensing dyes and experimental setups.

Albertazzi, Lorenzo; Brondi, Marco; Pavan, Giovanni M.; Sato, Sebastian Sulis; Signore, Giovanni; Storti, Barbara; Ratto, Gian Michele; Beltram, Fabio

2011-01-01

319

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

PubMed

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

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

2014-01-01

320

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

Microsoft Academic Search

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

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

1999-01-01

321

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

NASA Astrophysics Data System (ADS)

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

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

2011-08-01

322

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

NASA Astrophysics Data System (ADS)

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

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

2011-04-01

323

MoI density measurements by laser induced fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

1999-01-01

324

Time-resolved fluorescence spectroscopy for chemical sensors  

NASA Astrophysics Data System (ADS)

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

Draxler, Sonja; Lippitsch, Max E.

1996-07-01

325

In vivo biosensing via tissue-localizable near-infrared-fluorescent single-walled carbon nanotubes.  

PubMed

Single-walled carbon nanotubes 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 single-walled carbon nanotubes have been used as highly sensitive detectors for various compounds, their use as in vivo biomarkers requires the simultaneous optimization of various parameters, including biocompatibility, molecular recognition, high fluorescence quantum efficiency and signal transduction. Here we show that a polyethylene glycol ligated copolymer stabilizes near-infrared-fluorescent single-walled carbon nanotubes sensors in solution, enabling intravenous injection into mice and the selective detection of local nitric oxide concentration with a detection limit of 1 µM. The half-life for liver retention is 4 h, with sensors clearing the lungs within 2 h 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 nitric oxide 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 demonstrate that alginate-encapsulated single-walled carbon nanotubes can function as implantable inflammation sensors for nitric oxide detection, with no intrinsic immune reactivity or other adverse response for more than 400 days. PMID:24185942

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

2013-11-01

326

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

PubMed Central

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

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

2010-01-01

327

Fluorescence-based transient state monitoring for biomolecular spectroscopy and imaging  

PubMed Central

To increase read-out speed, sensitivity or specificity, an often applied strategy in fluorescence-based biomolecular spectroscopy and imaging is to simultaneously record two or more of the fluorescence parameters: intensity, lifetime, polarization or wavelength. This review highlights how additional, to-date largely unexploited, information can be extracted by monitoring long-lived, photo-induced transient states of organic dyes and their dynamics. Two major approaches are presented, where the transient state information is obtained either from fluorescence fluctuation analysis or by recording the time-averaged fluorescence response to a time-modulated excitation. The two approaches combine the detection sensitivity of the fluorescence signal with the environmental sensitivity of the long-lived transient states. For both techniques, proof-of-principle experiments are reviewed, and advantages, limitations and possible applications for biomolecular cellular biology studies are discussed.

Widengren, Jerker

2010-01-01

328

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

PubMed Central

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

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

2013-01-01

329

Naphthenic acids quantification in organic solvents using fluorescence spectroscopy.  

PubMed

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

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

2014-01-01

330

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

Microsoft Academic Search

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

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

2003-01-01

331

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

NASA Astrophysics Data System (ADS)

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

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

2011-02-01

332

Classification evaluation of tobaccos using LED-induced fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

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

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

2014-02-01

333

Vibrationally resolved photoabsorption spectroscopy of red fluorescent protein chromophore anions.  

PubMed

Photoabsorption studies of red fluorescent protein chromophore anions have been performed at the ELISA electrostatic heavy-ion storage ring. The broad absorption band due to electronic excitation of the chromophores is tuned to a longer wavelength (redshifted) by extending the electronic conjugation of the molecule. A clear vibrational progression is resolved with E(vib) approximately 380 and 520 cm(-1) for two different forms of the chromophore. The vibrational modes correspond to collective motions of the entire molecular structure. It is argued that the excited electronic state has an equilibrium configuration far from that of the electronic ground state, i.e., poor Franck Condon overlap. PMID:12688970

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

2003-03-21

334

2-Dimensional Fluorescence Spectroscopy: Determining the Temperature-Dependent Conformations of Porphyrin Dimers and Nucleic Acids  

NASA Astrophysics Data System (ADS)

I will describe spectroscopic studies on a covalently-linked zinc tetraphenylporphyrin dimer embedded in a phospholipid bilayer membrane. Using phase-modulation 2-Dimensional Fluorescence Spectroscopy (2D FS, a fluorescence-detected version of 2D electronic spectroscopy) along with linear absorption and fluorescence spectroscopy, it was found that the dimer adopts two predominant conformations in the membrane, and that the relative populations of these two states change as a function of temperature. Simultaneously fitting the linear absorption spectrum and the 2D FS spectra at four different excitation wavelengths revealed a wealth of information about these two states, including their relative populations, relative fluorescence quantum yields, the strength of the exciton coupling present in each state, and the approximate angles between the electronic transition dipole moments of the two porphyrins. Ongoing analysis focuses on elucidating the relaxation and energy transfer dynamics of this system through the population time dependence of the 2D spectra. Finally, I will present preliminary results from experiments in which 2D FS was performed with ultraviolet excitation to study the conformations of DNA constructs labeled with a fluorescent analogue of guanine.

Widom, Julia; Perdomo-Ortiz, Alejandro; Aspuru-Guzik, Alan; Marcus, Andrew

2012-02-01

335

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

PubMed

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

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

2014-07-22

336

Focal Volume Optics and Experimental Artifacts in Confocal Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

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

Samuel T. Hess; Watt W. Webb

2002-01-01

337

Fluorescence and Diffuse Reflectance Spectroscopy for Breast Cancer Diagnosis During Core Needle Biopsy.  

National Technical Information Service (NTIS)

The goal of this project is to exploit the potential of using fluorescence and diffuse reflectance spectroscopy for breast cancer detection during a core needle breast biopsy. A novel side-firing fiber optic probe has been developed for use in a vacuum-as...

C. Zhu

2006-01-01

338

Using Fluorescence Correlation Spectroscopy to Study Conformational Changes in Denatured Proteins  

Microsoft Academic Search

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement

Eilon Sherman; Anna Itkin; Yosef Yehuda Kuttner; Elizabeth Rhoades; Dan Amir; Elisha Haas; Gilad Haran

2008-01-01

339

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

Microsoft Academic Search

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

Petra Schwille; Jan Bieschke; Frank Oehlenschläger

1997-01-01

340

Picosecond time-resolved fluorescence spectroscopy of phytochrome and stentorin  

NASA Astrophysics Data System (ADS)

Phytochrome is a tetrapyrrole chromoprotein. It serves as a sensitive photosensor for red lightmediated gene expression and other developmental/morphological responses in plants. In this paper photochemical dynamics of the phytochrome molecule have been described in terms of photoisomerization of the tetrapyrrole chromophore in its singlet excited state and subsequent thermal processes in the Pr Pfr phototransformation of phytochrome. Stentorin acts as the photosensor molecule in the ciliate Stentor coeruleus. This unicellular protozoan is most sensitive to red light (610-620 urn). Stentor also senses the direction of light propagation as evidenced by their light-avoiding and negative phototactic swimming behaviors. This aneural photosensory phenomenon is triggered by the photoreceptor stentorin. The possible involvement of a light-induced transient proton release from the photoreceptor as the primary mechanism of light-signal processing has been discussed on the basis of picosecond fluorescence decays and time-resolved fluorescence spectra of stentorin in solution. An initial sensory signal generated by the primary photoprocess of stentorin then triggers subsequent transduction steps that include calcium ion influx from the extracellular medium. Calcium ion influx from the extracellular medium to the cytosol causes the Stentor cell to reverse its ciliary beating and subsequently steer away from the light trap. II.

Song, Pill-Soon

1991-05-01

341

In-vivo diffusing-wave-spectroscopy measurements of the ocular fundus  

NASA Astrophysics Data System (ADS)

We present what is to our knowledge the first observation of a diffusing-wave-spectroscopy signal recorded in-vivo on the ocular fundus. A modified ophthalmic microscope was developed which can acquire diffusing-wave-spectroscopy signal from the eye fundus. The diffusing-wave-spectroscopy signal was recorded in-vivo on a rabbit eye during transpupillary thermotherapy. Experimental results show the ability of the system to detect motion of the scattering sites in the ocular fundus layers during laser thermal heating.

Rovati, L.; Cattini, S.; Zambelli, N.; Viola, F.; Staurenghi, G.

2007-04-01

342

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

NASA Astrophysics Data System (ADS)

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.

Pestana, Noah Benjamin

343

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

NASA Astrophysics Data System (ADS)

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

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

2013-06-01

344

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

PubMed

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

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

2013-01-01

345

Dynamics of Nanoconfined Fluids measured by combined Force Microscopy and Fluorescence Correlation Spectroscopy  

NASA Astrophysics Data System (ADS)

We present work performed on a model liquid, tetrakis(2-ethylhexoxy)silane (TEHOS), using Atomic Force microscopy (AFM) and Fluorescence Correlation Spectroscopy (FCS) to study its dynamical structure at the nanoscale. A novel homebuilt interferometer-based small amplitude AFM was used to measure directly the stiffness and damping coefficient of TEHOS film. Oscillations in stiffness and damping coefficient with period ˜1 nm (TEHOS molecular size) were observed. Translational diffusion in spin-coated TEHOS films was measured using Fluorescence Correlation Spectroscopy (FCS). Diffusion was found to be heterogeneous. Finally we present the ongoing work on an integrated platform of AFM and FCS to perform simultaneous measurements of nanoconfined fluids. Recent results using this new setup on a fluorescently labelled nanoparticle solution in confinement will be discussed.

Subba Rao, Venkatesh; Pantea, Mircea; Grabowski, Christopher; Mukhopadhyay, Ashis; Hoffmann, Peter

2009-03-01

346

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

PubMed

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

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

2010-01-24

347

High-throughput single-molecule fluorescence spectroscopy using parallel detection  

PubMed Central

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

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

2011-01-01

348

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

PubMed

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

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

2012-01-01

349

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

PubMed Central

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

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

2012-01-01

350

Assessment of the unidentified organic matter fraction in fogwater using fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

Dissolved organic matter (DOM) in fogwaters from southeastern Louisiana and central-eastern China has been characterized using excitation-emission matrix (EEM) fluorescence spectroscopy. The results demonstrate that fluorescence spectroscopy can be used to obtain a qualitative assessment of the large fraction of fogwater organic carbon (~40 - 80% by weight) that cannot be identified in terms of specific chemical compounds. The method has the principle advantage that it can be applied at natural abundance concentrations, thus eliminating the need for large sample volumes required to isolate DOM for characterization by other spectroscopic (NMR, FTIR) and chemical (elemental) analyses. It was anticipated that the fogwater organic matter fluorescence spectra would resemble those of surface and rain waters, containing peaks indicative of both humic substances and fluorescent amino acids. Humic- and protein-like fluorophores were observed in the fogwater spectra and fluorescence-derived indices had values comparable to other natural waters. Biological character (intensity of tyrosine and tryptophan peaks) was found to increase with organic carbon concentration. Fogwater organic matter appears to contain a mixture of terrestrially- and microbially-derived material. The fluorescence results show that most of the unidentified fogwater organic carbon can be represented by humic-like and biologically-derived substances similar to those present in other aquatic systems.

Valsaraj, K.; Birdwell, J.

2010-07-01

351

[Monitoring of photodynamic therapy of port wine stain by fluorescence spectroscopy].  

PubMed

The blood drug level and the formation of photoproduct were monitored during photodynamic therapy (PDT) of port wine stain (PWS) by fluorescence spectroscopy. The irradiation was implemented by a 532 nm double-frequency Nd:YAG laser, and the collection of fluorescence spectra was completed with the use of spectrograph and ICCD. In the experiment for validation of the system, the fluorescence basis spectra of hematoporphyrin monomethyl ether (HMME)-sensitized mouse normal skin were constructed, and, by least-square fitting, HMME fluorescence (624 nm) could be discriminated from that of photoproduct (652 nm). The fitting of fluorescence spectra measured from PWS patients skin containing PSD-007 was performed with the same basis spectra as those from the mouse skin. The curves of blood drug level of different patients with significant variance, as well as those of formation and bleaching of photoproduct were obtained. Fluorescence spectroscopy monitoring system and fitting method presented here can provide technical means for rigorous quantitative PDT dosimetry method, and the results obtained here will make for the individual scheme of PDT. PMID:18051530

Wang, Lei; Gu, Ying; Li, Xiao-Song; Liu, Fan-Guang; Yu, Chang-Qing

2007-09-01

352

Challenges of Using MR Spectroscopy to Detect Neural Progenitor Cells In Vivo  

PubMed Central

SUMMARY A recent report of detection of neural progenitor cells (NPCs) in living human brain by using in vivo proton MR spectroscopy (1H-MR spectroscopy) has sparked great excitement in the field of biomedicine because of its potential influence and utility in clinical neuroscience research. On the other hand, the method used and the findings described in the report also caused heated debate and controversy. In this article, we will briefly detail the reasons for the debate and controversy from the point of view of the in vivo 1H-MR spectroscopy methodology and will propose some technical strategies in both data acquisition and data processing to improve the feasibility of detecting NPCs in future studies by using in vivo 1H-MR spectroscopy.

Dong, Z.; Dreher, W.; Leibfritz, D.; Peterson, B.S.

2010-01-01

353

Organ transplant tissue rejection: detection and staging by fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

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.

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

354

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

NASA Astrophysics Data System (ADS)

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

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

2011-02-01

355

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

PubMed Central

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

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

2013-01-01

356

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

Microsoft Academic Search

Reactive oxygen species (ROS) are believed to be involved in many diseases and injuries to the brain, but the molecular processes are not well understood due to a lack of in vivo imaging techniques to evaluate ROS. The fluorescent oxidation products of dihydroethidium (DHE) can monitor ROS production in vivo. Here we demonstrate the novel optical imaging of brain in

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

2009-01-01

357

The long-term stability and biocompatibility of fluorescent nanodiamond as an in vivo contrast agent.  

PubMed

Nanocarbon is a promising type of biomaterial for diagnostic and therapeutic applications. Fluorescent nanodiamond (FND) containing nitrogen-vacancy centers as built-in fluorophores is a new addition to the nanocarbon family. Here, we study the long-term stability and biocompatibility of 100-nm FNDs in rats through intraperitoneal injection over 5 months and develop the potential application of this biomaterial for sentinel lymph node mapping in a mouse model. From both in vivo and ex vivo fluorescence imaging as well as transmission electron microscopy, we found that the intradermally administered FND particles can be drained from the injection sites by macrophages and selectively accumulated in the axillary lymph nodes of the treated mice. Our measurements of water consumption, fodder consumption, body weight, and organ index showed no significant difference between control and FND-treated groups of the rats. Histopathological analysis of various tissues and organs indicated that FNDs are non-toxic even when a large quantity, up to 75 mg/kg body weight, of the particles was administered intraperitoneally to the living animals. With the properties of wide-ranging biocompatibility and perfect chemical and photophysical stability, FND is well suited for use as a contrast agent for long-term in vivo imaging. PMID:22863379

Vaijayanthimala, V; Cheng, Po-Yun; Yeh, Shih-Hua; Liu, Kuang-Kai; Hsiao, Cheng-Hsiang; Chao, Jui-I; Chang, Huan-Cheng

2012-11-01

358

A novel method for monitoring tumor proliferation in vivo using fluorescent dye DiD.  

PubMed

Monitoring single cell proliferation in vivo is difficult, but optimizing this technique is essential in order to expand our knowledge of the regulation of tumor proliferation. In this study, we used a lipophilic fluorescent dye, DiD, that rapidly and stably integrates into the phospholipid cell membrane. We cultured DiD-stained prostate cancer cell lines for 10 days and isolated cells by flow cytometry based on expression levels of DiD. We found that a decrease in DiD intensity was correlated to the reduction of EdU, where the DiD-high population proliferated more slowly than the DiD-low population and the DiD-low population exhibited a higher mitotic index. We also found that DiD was detected after 3 weeks of implantation in an in vivo setting. Importantly, DiD dye did not have any effect on normal cell growth, whereas a gold standard fluorescent dye for measuring cell proliferation, CFSE, slowed cell proliferation. Although further study is indicated, DiD can be useful for identifying the molecular mechanisms underlying tumor proliferation in vivo. © 2014 International Society for Advancement of Cytometry. PMID:24700602

Yumoto, Kenji; Berry, Janice E; Taichman, Russell S; Shiozawa, Yusuke

2014-06-01

359

Fluorescence and absorption spectroscopy of the weakly fluorescent chlorophyll a in cytochrome b6f of Synechocystis PCC6803.  

PubMed Central

A spectroscopic characterization of the chlorophyll a (Chl) molecule in the monomeric cytochrome b6f complex (Cytb6f) isolated from the cyanobacterium Synechocystis PCC6803 is presented. The fluorescence lifetime and quantum yield have been determined, and it is shown that Chl in Cytb6f has an excited-state lifetime that is 20 times smaller than that of Chl in methanol. This shortening of the Chl excited state lifetime is not caused by an increased rate of intersystem crossing. Most probably it is due to quenching by a nearby amino acid. It is suggested that this quenching is a mechanism for preventing the formation of Chl triplets, which can lead to the formation of harmful singlet oxygen. Using site-selected fluorescence spectroscopy, detailed information on vibrational frequencies in both the ground and Qy excited states has been obtained. The vibrational frequencies indicate that the Chl molecule has one axial ligand bound to its central magnesium and accepts a hydrogen bond to its 13(1)-keto carbonyl. The results show that the Chl binds to a well-defined pocket of the protein and experiences several close contacts with nearby amino acids. From the site-selected fluorescence spectra, it is further concluded that the electron-phonon coupling is moderately strong. Simulations of both the site-selected fluorescence spectra and the temperature dependence of absorption and fluorescence spectra are presented. These simulations indicate that the Huang-Rhys factor characterizing the electron-phonon coupling strength is between 0.6 and 0.9. The width of the Gaussian inhomogeneous distribution function is 210 +/- 10 cm-1.

Peterman, E J; Wenk, S O; Pullerits, T; Palsson, L O; van Grondelle, R; Dekker, J P; Rogner, M; van Amerongen, H

1998-01-01

360

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

NASA Astrophysics Data System (ADS)

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

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

2008-06-01

361

Optimization of holographic optical tweezers for multiplexed fluorescence spectroscopy  

NASA Astrophysics Data System (ADS)

We are developing a multiplexed spectroscopy technique that employs holographic optical tweezers to trap and excite multiple sensor particles. Our goal is to develop a lab-on-a-chip measurement platform for monitoring pH and other ion concentrations with high spatial resolution in a microfluidic device or within biological cells. To ensure efficient use of the available laser power required to trap multiple particles, we address three aspects of the spatial light modulator (SLM) used in the holographic technique. We measure and optimize the input and output polarizations used before and after the birefringent SLM. We reduce optical aberrations by adding appropriate Zernike polynomials to the computed hologram. We optimize the diffraction efficiency of the SLM by adjusting the gray scale input-to-output table to account for the nonlinear phase response of the SLM.

Cibula, Matthew; McIntyre, David

2012-10-01

362

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

NASA Astrophysics Data System (ADS)

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.

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

2009-07-01

363

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

PubMed Central

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

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

2013-01-01

364

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

PubMed Central

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.

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

2011-01-01

365

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

NASA Astrophysics Data System (ADS)

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

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

2013-04-01

366

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

PubMed Central

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

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

2014-01-01

367

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

NASA Astrophysics Data System (ADS)

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

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

2013-03-01

368

Fluorescence lifetime imaging microscopy: in vivo application to diagnosis of oral carcinoma.  

PubMed

A compact clinically compatible fluorescence lifetime imaging microscopy (FLIM) system was designed and built for intraoperative disease diagnosis and validated in vivo in a hamster oral carcinogenesis model. This apparatus allows for the remote image collection via a flexible imaging probe consisting of a gradient index objective lens and a fiber bundle. Tissue autofluorescence (337 nm excitation) was imaged using an intensified CCD with a gate width down to 0.2 ns. We demonstrate a significant contrast in fluorescence lifetime between tumor (1.77+/-0.26 ns) and normal (2.50+/-0.36 ns) tissues at 450 nm and an over 80% intensity decrease at 390 nm emission in tumor versus normal areas. The time-resolved images were minimally affected by tissue morphology, endogenous absorbers, and illumination. These results demonstrate the potential of FLIM as an intraoperative diagnostic technique. PMID:19572006

Sun, Yinghua; Phipps, Jennifer; Elson, Daniel S; Stoy, Heather; Tinling, Steven; Meier, Jeremy; Poirier, Brian; Chuang, Frank S; Farwell, D Gregory; Marcu, Laura

2009-07-01

369

Fluorescence lifetime imaging microscopy: In vivo application to diagnosis of oral carcinoma  

PubMed Central

A compact clinically compatible fluorescence lifetime imaging microscopy (FLIM) system was designed and built for intraoperative disease diagnosis and validated in vivo in a hamster oral carcinogenesis model. This apparatus allows for the remote image collection via a flexible imaging probe consisting of a gradient index objective lens and a fiber bundle. Tissue autofluorescence (337 nm excitation) was imaged using an intensified CCD with a gate width down to 0.2 ns. We demonstrate a significant contrast in fluorescence lifetime between tumor (1.77±0.26 ns) and normal (2.50±0.36 ns) tissues at 450 nm and an over 80% intensity decrease at 390 nm emission in tumor versus normal areas. The time-resolved images were minimally affected by tissue morphology, endogenous absorbers, and illumination. These results demonstrate the potential of FLIM as an intraoperative diagnostic technique.

Sun, Yinghua; Phipps, Jennifer; Elson, Daniel S.; Stoy, Heather; Tinling, Steven; Meier, Jeremy; Poirier, Brian; Chuang, Frank S.; Farwell, D. Gregory; Marcu, Laura

2014-01-01

370

Prediction of cell culture media performance using fluorescence spectroscopy.  

PubMed

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

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

2010-02-15

371

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

PubMed Central

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

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

2013-01-01

372

Hematoporphyrin-mediated fluorescence reflectance imaging: application to early tumor detection in vivo in small animals.  

PubMed

The in vivo early detection of subcutaneous human tumors implanted in small animals was studied by laser-induced fluorescence reflectance imaging (FRI), with a hematoporphyrin (HP) compound as an exogenous optical contrast agent. Tumor detection was shown to be possible just 3 days after the inoculation of tumor cells, when tumors were neither visible nor palpable. However, this detection capability is limited to a temporal window of approximately 100 h from HP administration and to a low optical contrast of the tumor (<2). PMID:18324434

Autiero, Maddalena; Cozzolino, Rosanna; Laccetti, Paolo; Marotta, Marcello; Quarto, Maria; Riccio, Patrizia; Roberti, Giuseppe

2009-03-01

373

[Study on determination of carbaryl content in duck meat based on synchronous fluorescence spectroscopy].  

PubMed

For the rapid detection of carbaryl residue in duck meat, synchronous fluorescence spectroscopy was used, and GA combined with SVR was used to establish regression forecasting mode for the application of forecasting carbaryl residue in duck meat. Firstly, fluorescence spectrophotometer was used to get the 3D synchronous fluorescence spectra of carbaryl hydrolysate and duck solution containing carbaryl, and 140 nm was selected as the optimum wavelength difference delta lambda; Secondly, some concentration quenching was analysed. Finally, GA was used to optimize and choose the 3D synchronous fluorescence spectra. According to the root mean square error of cross-validation (RMSECV) 21 characteristic wavelengths were chosen, then the full wavelength and 21 characteristic wavelengths were used as input characteristic variables of SVR regression forecasting model respectively. At last the results showed that characteristic wavelengths chosen by GA can get better forecasting results, and the correlation coefficient of the prediction samples set and the root mean squared error (RMSEP) were 0.976 4 and 12.232 2, respectively. The results of experiments showed that the synchronous fluorescence spectroscopy could be used to detect carbaryl residue in duck meat efficiently and rapidly when combined with GA-SVR. PMID:23387179

Xiao, Hai-Bin; Liu, Mu-Hua; Yuan, Hai-Chao; Xu, Jiang; Zhao, Jin-Hui

2012-11-01

374

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

PubMed Central

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

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

2013-01-01

375

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

PubMed Central

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

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

2010-01-01

376

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

NASA Astrophysics Data System (ADS)

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

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

2013-10-01

377

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

NASA Astrophysics Data System (ADS)

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

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

2014-02-01

378

Picosecond fluorescence spectroscopy of purple membrane in Halobacterium halobium with a photon-counting streak camera  

NASA Astrophysics Data System (ADS)

Fluorescence lifetimes and spectra of native and deionized purple membranes of Halobacterium halobium at 22°C were measured to be <3 and 12±4 ps, respectively, with a photon-counting streak camera system. The results confirmed that the blue-shifted transient previously found by absorption spectroscopy is attributed to bacteriorhodopsin in the lowest excited-singlet state. Ultraweak fluorescence of the light-adapted purple membrane with 2.5 × 10 -4 quantum yield could be detected even though the excitation pulse energy at 570 nm was reduced to 0.88 pJ (72 ?W average power).

Ohtani, Hiroyuki; Ishikawa, Mitsuru; Itoh, Hiroyasu; Takiguchi, Yoshihiro; Urakami, Tsuneyuki; Tsuchiya, Yutaka

1990-05-01

379

Profile of a focussed collimated laser beam near the focal minimum characterized by fluorescence correlation spectroscopy  

SciTech Connect

Central to the application of fluorescence correlation spectroscopy, to measure the self-diffusion coefficients and average concentration of fluorescent molecules in a volume determined by a focussed laser beam, is the determination of the focal spot size. As the focal spot size in the sample plane is varied by displacing either the focusing lens or sample position along the beam axis, the diffusion time and average number of molecules vary in a parabolic manner. Analysis of the parameters of the parabola leads to estimates of the beam radius at the waist. The results agree with theoretical predictions and provide an independent measurement of the beam profile.

Sorscher, S.M.; Klein, M.P.

1980-01-01

380

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

PubMed

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

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

2013-11-27

381

Fluorescence resonance energy transfer mediated large Stokes shifting near-infrared fluorescent silica nanoparticles for in vivo small-animal imaging.  

PubMed

Fluorescent dye-doped silica nanoparticles are increasingly used for in vivo imaging due to their unique biocompatibility and easy surface modification. However, the utility of existing fluorescent dye-doped silica nanoparticles for in vivo imaging is still limited because most studies are focused on doping single near-infrared (NIR) dyes in the silica matrix, which would cause background and crosstalk between the excitation light and the emitting signals due to the small Stokes shift of the traditional NIR dyes. To address this issue, we present a novel large Stokes shifting NIR fluorescent silica nanoparticles (LSS-NFSiNPs) based on the principle of fluorescence resonance energy transfer. Two highly water-soluble dyes, tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate (RuBpy) and methylene blue (MB), were chose as the model donor-acceptor pair. The LSS-NFSiNPs were prepared by synchronously doping RuBpy and MB in the silica nanoparticles. By optimizing the molar ratio of RuBpy and MB for doping in the silica nanoparticles, the energy transfer from RuBpy to MB occurred in the silica matrix, resulting in a near-infrared fluorescent silica nanoparticles with strong fluorescence and large Stokes shift (>200 nm). As a result, it can effectively help to increase the discrimination of fluorescence signal of interest over other background signals. With a combination of excellent stability, large Stokes shift, and near-infrared spectral properties, this novel LSS-NFSiNPs provides real-time, deep-tissue fluorescent imaging of live animals. More importantly, the LSS-NFSiNPs can also be gradually cleared from the body through the urinary clearance system. We anticipate this design concept can lay a foundation for further development of in vivo optical nanoparticulate contrast toward clinical applications. PMID:23017033

He, Xiaoxiao; Wang, Yushuang; Wang, Kemin; Chen, Mian; Chen, Suye

2012-11-01

382

Fluorescent properties and spontaneous Raman spectroscopy of new ketocyanine probes in organic solvents  

Microsoft Academic Search

We have used fluorescence spectroscopy and spontaneous Raman spectroscopy to study the characteristics of two ketocyanine\\u000a dyes: 2,5-di[(E)-1-(4-diethylaminophenyl)methylidene]-1-cyclopentanone (CPET) and 2-[(E)-1-(4-diethylaminophenyl)methylidene]-5-{(E)-1-[4-(4,7,10,13-tetraoxa-1-azacyclopentadecalin)\\u000a phenyl]methylidene}-1-cyclopentanone (CPMR) in organic solvents. The position of their electronic spectra depends strongly\\u000a on the polarity of the solvent. We measured the dipole moments of the dyes in the equilibrium ground state and the Franck-Condon\\u000a excited state. In mixtures

N. A. Nemkovich; A. N. Sobchuk; I. A. Khodasevich

2006-01-01

383

Fluorescent properties and spontaneous Raman spectroscopy of new ketocyanine probes in organic solvents  

Microsoft Academic Search

We have used fluorescence spectroscopy and spontaneous Raman spectroscopy to study the characteristics of two ketocyanine dyes: 2,5-di[(E)-1-(4-diethylaminophenyl)methylidene]-1-cyclopentanone (CPET) and 2-[(E)-1-(4-diethylaminophenyl)methylidene]-5-{(E)-1-[4-(4,7,10,13-tetraoxa-1-azacyclopentadecalin) phenyl]methylidene}-1-cyclopentanone (CPMR) in organic solvents. The position of their electronic spectra depends strongly on the polarity of the solvent. We measured the dipole moments of the dyes in the equilibrium ground state and the Franck-Condon excited state. In mixtures

N. A. Nemkovich; A. N. Sobchuk; I. A. Khodasevich

2006-01-01

384

In-Vivo NMR Spectroscopy of the Brain at High Fields  

Microsoft Academic Search

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

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

385

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

PubMed

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

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

1997-02-01

386

In vivo validation of high frequency ultrasound-guided fluorescence tomography system to improve delivery of photodynamic therapy  

NASA Astrophysics Data System (ADS)

Photodynamic therapy (PDT) for skin cancer is sometimes only partially effective, due to inadequate levels of the fluorescent drug (photosensitizer, PS) and due to heterogeneous distribution of PS within the tissue. To image the PS distribution within skin tumors, we have developed a fluorescence tomography system (FTS) that combines a fluorescence detection array with a high frequency ultrasound (HFUS) transducer. In this paper we describe in vitro and in vivo validation of this new system. The target fluorophore for detection was Protoporphyrin IX (PPIX). Validation experiments were performed in vivo using a subcutaneous tumor model in which A431 tumor-bearing mice were treated with 5-aminolevulinic acid to induce production of PPIX. FTS reconstructions were compared with standard histology and with data from bulk tumor slices imaged ex vivo on a fluorescence scanner. Reconstructed images obtained from the FTS were correlated with the histology and the ex vivo scans, confirming several-fold increases in PPIX fluorescence in the skin and in the tumor relative to the surrounding tissues. Our data demonstrate the feasibility of using the FTS for subsurface imaging of PPIX in skin carcinoma in vivo. Future aims are to use this device for individualized treatment planning, in order to improve overall patient responses to PDT.

Paliwal, Akshat; Torosean, Sason; Gruber, Josiah; O'Hara, Julia; Hasan, Tayyaba; Pogue, Brian; Maytin, Edward V.

2011-02-01

387

Dynamic dual-tracer MRI-guided fluorescence tomography to quantify receptor density in vivo  

PubMed Central

The up-regulation of cell surface receptors has become a central focus in personalized cancer treatment; however, because of the complex nature of contrast agent pharmacokinetics in tumor tissue, methods to quantify receptor binding in vivo remain elusive. Here, we present a dual-tracer optical technique for noninvasive estimation of specific receptor binding in cancer. A multispectral MRI-coupled fluorescence molecular tomography system was used to image the uptake kinetics of two fluorescent tracers injected simultaneously, one tracer targeted to the receptor of interest and the other tracer a nontargeted reference. These dynamic tracer data were then fit to a dual-tracer compartmental model to estimate the density of receptors available for binding in the tissue. Applying this approach to mice with deep-seated gliomas that overexpress the EGF receptor produced an estimate of available receptor density of 2.3 ± 0.5 nM (n = 5), consistent with values estimated in comparative invasive imaging and ex vivo studies.

Davis, Scott C.; Samkoe, Kimberley S.; Tichauer, Kenneth M.; Sexton, Kristian J.; Gunn, Jason R.; Deharvengt, Sophie J.; Hasan, Tayyaba; Pogue, Brian W.

2013-01-01

388

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

PubMed

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

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

2014-01-01

389

Fluorescence-Based Monitoring of In Vivo Neural Activity Using a Circuit-Tracing Pseudorabies Virus  

PubMed Central

The study of coordinated activity in neuronal circuits has been challenging without a method to simultaneously report activity and connectivity. Here we present the first use of pseudorabies virus (PRV), which spreads through synaptically connected neurons, to express a fluorescent calcium indicator protein and monitor neuronal activity in a living animal. Fluorescence signals were proportional to action potential number and could reliably detect single action potentials in vitro. With two-photon imaging in vivo, we observed both spontaneous and stimulated activity in neurons of infected murine peripheral autonomic submandibular ganglia (SMG). We optically recorded the SMG response in the salivary circuit to direct electrical stimulation of the presynaptic axons and to physiologically relevant sensory stimulation of the oral cavity. During a time window of 48 hours after inoculation, few spontaneous transients occurred. By 72 hours, we identified more frequent and prolonged spontaneous calcium transients, suggestive of neuronal or tissue responses to infection that influence calcium signaling. Our work establishes in vivo investigation of physiological neuronal circuit activity and subsequent effects of infection with single cell resolution.

Granstedt, Andrea E.; Szpara, Moriah L.; Kuhn, Bernd; Wang, Samuel S. -H.; Enquist, Lynn W.

2009-01-01

390

Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins  

PubMed Central

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

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

2014-01-01

391

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

NASA Astrophysics Data System (ADS)

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

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

2011-09-01

392

An Array of Planar Apertures for Near-Field Fluorescence Correlation Spectroscopy  

PubMed Central

We have developed a method of performing near-field fluorescence correlation spectroscopy via an array of planarized circular apertures of 50 nm diameter. This technique provides 1 ?s and 60 nm resolution on proximal samples, including live cells, without incorporating a scanning probe or pulsed lasers or requiring penetration of the sample into the aperture. Millions of apertures are created in an array within a thin film of aluminum on a coverslip and planarized to achieve no height distinction between the apertures and the surrounding metal. Supported lipid bilayers and plasma membranes from live cells adhere to the top of this substrate. We performed fluorescence correlation spectroscopy to demonstrate the sub-diffraction-limited illumination with these devices.

Kelly, Christopher V.; Baird, Barbara A.; Craighead, Harold G.

2011-01-01

393

Liver uptake of gold nanoparticles after intraperitoneal administration in vivo: A fluorescence study  

PubMed Central

Background One particularly exciting field of research involves the use of gold nanoparticles (GNPs) in the detection and treatment of cancer cells in the liver. The detection and treatment of cancer is an area in which the light absorption and emission characteristics of GNPs have become useful. Currently, there are no data available regarding the fluorescence spectra or in vivo accumulation of nanoparticles (NPs) in rat liver after repeated administration. In an attempt to characterise the potential toxicity or hazards of GNPs in therapeutic or diagnostic use, the present study measured fluorescence spectra, bioaccumulation and toxic effects of GNPs at 3 and 7 days following intraperitoneal administration of a 50 ?l/day dose of 10, 20 or 50 nm GNPs in rats. Methods The experimental rats were divided into one normal group (Ng) and six experimental groups (G1A, G1B, G2A, G2B, G3A and G3B; G1: 20 nm; G2: 10 nm; G3: 50 nm; A: infusion of GNPs for 3 days; B: infusion of GNPs for 7 days). A 50 ?l dose of GNPs (0.1% Au by volume) was administered to the animals via intraperitoneal injection, and fluorescence measurements were used to identify the toxicity and tissue distribution of GNPs in vivo. Seventy healthy male Wistar-Kyoto rats were exposed to GNPs, and tissue distribution and toxicity were evaluated after 3 or 7 days of repeated exposure. Results After administration of 10 and 20 nm GNPs into the experimental rats, two fluorescence peaks were observed at 438 nm and 487 nm in the digested liver tissue. The fluorescence intensity for 10 and 20 nm GNPs (both first and second peaks) increased with the infusion time of GNPs in test rats compared to normal rats. The position of the first peak was similar for G1A, G2A, G1B, G2B, G3B and the normal (438 nm); that