Fluorescence Lifetime

An autoclavable sensing film was developed for monitoring dissolved CO 2. The sensing film, based on fluorescence resonance energy transfer (FRET), consisted of a fluorescent donor, an acceptor, and a quaternary ammonium hydroxide, which were doped in a two-component silicone film. As no aqueous solution was used in the sensing film matrix, the sensing film was unaffected by osmotic pressure. Fluorescence lifetime was selected as the sensing parameter, and measured in frequency domain using phase fluorometry. Upon exposure to 20% CO 2-saturated water, a 43°increase in phase angle was observed at 100 MHz. The process was fully reversible when the sensing film was exposed to nitrogen-saturated water. The estimated response and recovery times for 90% signal change were 1 min (for a step change from 0 to 6.7% CO 2-saturated water) and 1.5 min (for a step change from 6.7 to 3.3% CO 2-saturated water). When used for on-line monitoring of dissolved CO 2 produced by a culture of Escherichia coli, the sensing film showed a similar trend to that obtained from off-line measurements using a wet chemistry analyzer.

Ultrasensitive, near-infrared (NIR), time-resolved fluorescence is evaluated as a detection method for reading DNA hybridization events on solid surfaces for microarray applications. In addition, the potential of mulitiplexed analyses using time-resolved identification protocols is described. To carry out this work, a NIR time-resolved confocal imager was constructed to read fluorescence signatures from the arrays. The device utilized a 780-nm pulsed diode laser, a single-photon avalanche diode (SPAD), and a high-numerical-aperture microscope objective mounted in an epi-illumination format. Due to the small size of the components that are required to construct this imager, the entire detector could easily be mounted on high-resolution translational stages and scanned over the stationary arrays. The instrument response function of the device was determined to be 275 ps (fwhm), which is adequate for measuring fluorophores with subnanosecond lifetimes. To characterize the system, NIR dyes were deposited directly on different substrate materials typically used for DNA microarrays, and the fluorescence lifetimes of two representative dyes were measured. The fluorescence lifetime for aluminum tetrasulfonated naphthalocyanine was found to be 1.92 ns, and a value of 1.21 ns was determined for the tricarbocyanine dye, IRD800, when it was deposited onto poly(methyl methacrylate) (PMMA) and measured in the dry state. Finally, the imager was used to monitor hybridization events using probe oligonucleotides chemically tethered to a PMMA substrate via a glutardialdehyde linkage to an aminated-PMMA surface. The limit of detection for oligonucleotides containing a NIR fluorescent reporter was determined to be 0.38 molecules/µm 2 , with this detection limit improving by a factor of 10 when a time-gate was implemented. Fluorescence lifetime analysis of the hybridization events on PMMA indicated a lifetime value of 1.23 ns for the NIR-labeled oligonucleotides when using maximum-likelihood estimators.

The lifetimes and relative quantum yields of the three fluorescence components of the PI (red-absorbing) form of 124-kDa oat phytochrome in H 2 0 and D 2 0 solutions have been determined by single-photon-timing techniques. Lifetime and quantum yield of the main component, which is the shortest lived (about 40 ps a t low concentration) and reflects the photochromic properties of P,, are not affected by D20. The medium-lifetime component (about 180 ps at low concentration), which is attributable to a photochromic source behaving comparable to that responsible for the main component, is not affected

We report the demonstration of time-correlated single-photon counting (TCSPC) fluorescence lifetime imaging (FLIM) to ex vivo decayed and healthy dentinal tooth structures, using a white-light supercontinuum excitation source. By using a 100 fs-pulsed Ti:Sapphire laser with a low-frequency chirp to pump a 30-cm long section of photonic crystal fibre, a ps-pulsed white-light supercontinuum was created. Optical bandpass interference filters were then applied to this broad-bandwidth source to select the 488-nm excitation wavelength required to perform TCSPC FLIM of dental structures. Decayed dentine showed significantly shorter lifetimes, discriminating it from healthy tissue and hard, stained and thus affected but noninfected material. The white-light generation source provides a flexible method of producing variable-bandwidth visible and ps-pulsed light for TCSPC FLIM. The results from the dental tissue indicate a potential method of discriminating diseased tissue from sound, but stained tissue, which could be of crucial importance in limiting tissue resection during preparation for clinical restorations.

Results of absorption, emission and fluorescence lifetime (for the 4 F 3/2 -4 I 11/2 transition) are presented for a new lead fluoroborate glass doped with different concentrations of Nd 2 O 3 varying from 0.04 up to 3.49 wt%. The calculated Judd-Ofelt parameters are: (1.770.1) Â 10 À20 cm 2 , (3.470.3) Â 10 À20 cm 2 , (3.670.3) Â 10 À20 cm 2 . Branching ratios calculations show that the 4 F 3/2 -4 I 11/2 transition has the most potential for laser applications. The best spectroscopic performance was obtained for the sample doped with 1.75 wt% of Nd 2 O 3 : emission cross-section of 3.6 Â 10 À20 cm 2 at 1060 nm, fluorescence lifetime of 0.06 ms and effective fluorescence bandwidth of 30.43 nm. The samples have high refractive index of 2.2 and a density of about 6.9 g/cm 3 . Concentration quenching was observed at 3.49 wt%. The results obtained suggest that this new material can be considered as an interesting candidate for laser applications with similar properties when compared to other known glasses used as laser media. r

We have investigated the absorption, the fluorescence and phosphorescence emission and the fluorescence lifetimes of adenine in low-temperature argon and nitrogen matrices at 15 K. Compared to other environments the absorption spectrum shows higher intensity at the shortest wavelengths, and a weak apparent absorption peak is observed at 280 nm. The resolved fluorescence excitation spectrum has five peaks at positions corresponding to those observed in the absorption spectrum. The position of the fluorescence maximum depends on the excitation wavelength. Excitation below 220 nm displays a fluorescence maximum at 305 nm, while for excitations at higher wavelengths the maximum occurs at 335 nm. The results suggest that multiple-emission excited electronic states are populated in low-temperature gas matrices. Excitation at 265 nm produces a phosphorescence spectrum with a well-resolved vibrational structure and a maximum at 415 nm. The fluorescence decays corresponding to excitation at increasing energy of each resolved band could be fit with a double exponential, with the shorter and longer lifetimes ranging from 1.7 to 3.3 ns and from 12 to 23 ns, respectively. Only for the excitation at 180 nm one exponential is required, with the calculated lifetimes of 3.3 ns. The presented results provide an experimental evidence of the existence of multiple site-selected excited electronic states, and may help elucidate the possible deexcitation pathways of adenine. The additional application of synchrotron radiation proved to result in a significant enhancement of the resolution and spectral range of the phenomena under investigation.

The 4T~ + 'A emission band of Mn " in Zn2Si04:Mn shows a red shift and an increase of the oscillator strength as a function of the Mn2+ concentration. In this paper it is shown that these phenomena are due to exchange interactions between Mn2+ ions.

Objective: To investigate the potential of endogenous multispectral fluorescence lifetime imaging microscopy (FLIM) for biochemical characterization of human coronary atherosclerotic plaques. Methods: Endogenous multispectral FLIM imaging was performed on the lumen of 58 segments of postmortem human coronary artery. The fluorescence was separated into three emission bands targeting the three main arterial endogenous fluorophores (390 ± 20 nm for collagen, 452 ± 22.5 nm for elastin, and 550 ± 20 for lipids). The fluorescence normalized intensity and average lifetime from each emission band was used to classify each pixel of an image as either "High-Collagen", "High-Lipids" or "Low-Collagen/Lipids" via multiclass Fisher's linear discriminant analysis. Results: Classification of plaques as either "High-Collagen", "High-Lipids" or "Low-Collagen/Lipids" based on the endogenous multispectral FLIM was achieved with a sensitivity/specificity of 96/98%, 89/99%, and 99/99%, respectively, where histopathology served as the gold standard. Conclusion: The endogenous multispectral FLIM approach we have taken, which can readily be adapted for in vivo intravascular catheter based imaging, is capable of reliably identifying plaques with high content of either collagen or lipids.

The fluorescence properties of Alexa 488, Oregon Green 488, and Oregon Green 514 (Molecular Probes (Eugene, OR)) are compared when conjugated to biomolecules and as model compounds free in solution. We show that these relatively new, green fluorescence probes are excellent probes for investigation of the thermodynamics of protein-protein and protein-nucleic acid interactions by fluorescence anisotropy. Unlike fluorescein, the emission of these dyes has minimal pH dependence near neutrality and is significantly less susceptible to photobleaching. Steady-state and time-resolved fluorescence anisotropy data are compared for two interacting proteins of different size and for the association of a transcription factor with a DNA oligonucleotide containing a specific binding site. The temperature dependence of the fluorescence lifetimes of the probes is reported, and the effects of molecular size and probe motion on steady-state anisotropy data are discussed. The critical interplay among correlation time, fluorescence lifetime, and the observed steady-state anisotropy is evaluated. Ó

In live tissue, alterations in metabolism induce changes in the fluorescence decay of the biological coenzyme NAD(P)H, the mechanism of which is not well understood. In this work, the fluorescence and anisotropy decay dynamics of NADH and NADPH were investigated as a function of viscosity in a range of water-glycerol solutions. The viscosity dependence of the non-radiative decay is well described by Kramers and Kramers-Hubbard models of activated barrier crossing over a wide viscosity range. Our combined lifetime and anisotropy analysis indicates common mechanisms of non-radiative relaxation in the two emitting states (conformations) of both molecules. The low frequencies associated with barrier crossing suggest that non-radiative decay is mediated by small scale motion (e.g. puckering) of the nicotinamide ring. Variations in the fluorescence lifetimes of NADH and NADPH when bound to different enzymes may therefore be attributed to differing levels of conformational restriction upon binding.

Absorption and fluorescence spectra of alizarin (1,2-dihydroxyanthraquinone) and purpurin (1,2,4trihydroxyanthraquinone) were investigated as a function of pH in water-dioxane (2:1, v/v) in the pH range 2-14. Absorbance changes with pH are accompanied by color changes; by increasing the pH, the spectra shift to the red. These molecules exhibit fluorescence emissions over the whole pH range explored; fluorescence quantum yields increase with increasing pH and are greater for purpurin (Φ F = 10 À3 -10 À2 ) than for alizarin (Φ F = 10 À4 -10 À3 ). The fluorescence lifetimes were measured by the phase-shift technique for the three species derived from purpurin (neutral, t = 150 ps; mono-anion, t = 2600 ps and di-anion, t = 380 ps), and only for the di-anion of alizarin (t = 80 ps). Both spectrophotometric and fluorimetric titrations gave pKs of the first and second dissociation steps in the ground state. Excited-state pK*s were calculated by the Förster cycle. In the ground state, purpurin (pK 1 = 4.7; pK 2 = 9.5) is a stronger acid than alizarin (pK 1 = 6.6; pK 2 = 12.4) in both the first and second dissociation steps; in the excited state, purpurin is a weaker acid in both dissociation steps.

It is demonstrated that fluorescence lifetimes in the nanosecond and picosecond time-scale range can be observed with the recently proposed double-pulse fluorescence lifetime imaging technique Double-pulse fluorescence lifetime imaging in confocal microscopy. J. Microsc. 177,[171][172][173][174][175][176][177][178][179].

The polarity of biological mediums controls a host of physiological processes such as digestion, signaling, transportation, metabolism, and excretion. With the recent widespread use of near-infrared (NIR) fluorescent dyes for biological imaging of cells and living organisms, reporting medium polarity with these dyes would provide invaluable functional information in addition to conventional optical imaging parameters. Here, we report a new approach to determine polarities of macro-and microsystems for in vitro and potential in vivo applications using NIR polymethine molecular probes. Unlike the poor solvatochromic response of NIR dyes in solvents with diverse polarity, their fluorescence lifetimes are highly sensitive, increasing by a factor of up to 8 on moving from polar to nonpolar mediums. We also established a correlation between fluorescence lifetime and solvent orientation polarizability and developed a lifetime polarity index for determining the polarity of complex systems, including micelles and albumin binding sites. Because of the importance of medium polarity in molecular, cellular, and biochemical processes and the significance of reduced autofluorescence and deep tissue penetration of light in the NIR region, the findings reported herein represent an important advance toward using NIR molecular probes to measure the polarity of complex biological systems in vitro and in vivo.

The reaction products of 1,2-indanedione (a new fluorescent fingerprint reagent) with glycine in methanol, at room temperature have been studied using excitation and emission and time-resolved fluorescence spectroscopy. Gas chromatography-mass spectroscopy (GC/MS) has also been used to determine which compounds are formed. Reaction products were identified using GC/MS as 2-carboxymethyliminoindanone (MW = 203 g) and 1,2-di(carboxymethylimino)indane (MW = 260 g). Identified compounds show room temperature fluorescence lifetimes of t 1 = 7.69 ns and t 2 = 1.27 ns. It is not clear yet which compound is having fluorescence lifetime of 7.69 ns and which one is showing 1.27 ns.

A synthesis of a novel zinc phthalocyanine with four 3-[(2-diethylamino) ethyl]-7-oxo-4-methylcoumarin dye groups at the non-peripheral positions was performed and characterized. The photophysical and photochemical properties of the peripherally (p) and ...

The spectrophotometric study of luminol (LH 2 ) in dimethyl sulfoxide (DMSO), DMSO-water solutions, and alkaline DMSO and DMSO-water solutions has been done, focusing on the effect of the KOH additon on LH 2 absorption and fluorescence properties. The absorption spectra indicate an acid-base equilibrium, and the luminol dianion (L 2Ϫ ) formation at 3 ϫ 10 Ϫ4 Ϫ 2.4 ϫ 10 Ϫ3 M KOH. The decrease of the fluorescence intensity and the variation of the excitation spectra of LH 2 -DMSO-KOH solutions with KOH concentration have been similarly explained. The acid-base process is reversible. The addition of HCl to the solution with 3.0 ϫ 10 Ϫ3 M KOH leads to an increase of the fluorescence intensity to its highest value, observed in pure DMSO. The addition of HCl to the LH 2 -DMSO solution leads to the decrease of the fluorescence intensity as a result of the LH 3 ϩ cation formation. In LH 2 -DMSO-water, the fluorescence band is shifted from 405 nm to 424 nm and increased in the intensity. In the presence of KOH (in LH 2 -DMSO-water-KOH solution) a new band appears, with the maximum at 485 nm and the band at 405 nm decreased. The changes in fluorescence lifetimes also evidence the different chemical species formed.

Advancements in single molecule detection (SMD) continue to unfold powerful ways to study the behavior of individual and complex molecular systems in real time. SMD enables the characterization of complex molecular interactions and reveals basic physical phenomena underlying chemical and biological processes. We present here a systematic study of the quenching efficiency of Förster-type energy-transfer (FRET) for multiple fluorophores immobilized on a single antibody. We simultaneously monitor the fluorescence intensity, fluorescence lifetime, and the number of available photons before photobleaching as a function of the number of identical emitters bound to a single IgG antibody. The detailed studies of FRET between individual fluorophores reveal complex through-space interactions. In general, even for two or three fluorophores immobilized on a single protein, homo-FRET interactions lead to an overall non-linear intensity increase and shortening of fluorescence lifetime. Over-labeling of protein in solution (ensemble) results in the loss of fluorescence signal due to the self-quenching of fluorophores making it useless for assays applications. However, in the single molecule regime, over-labeling may bring significant benefits in regards to the number of available photons and the overall survival time. Our investigation reveals possibilities to significantly increase the observation time for a single macromolecule allowing studies of macromolecular interactions that are not obscured by ensemble averaging. Extending the observation time will be crucial for developing immunoassays based on single-antibody.

Spectral characteristics and prototropic reactions of 2-(2-hydroxyphenyl)benzimidazole (HPBI) in Tween-20, Tween-40, Tween-60, Tween-80, and dioxane-water mixtures of different compositions have been studied. Comparison of the fluorescence band maxima of the tautomer band in nonionic micelles with the correlation diagram, drawn between the fluorescence band maxima and the dielectric constants of the dioxane-water mixtures, have shown that the effective dielectric constant (⑀ eff) at the binding site of HPBI is 18 ؎ 2 for all the Tweens. Fluorescence lifetimes (f) and fluorescence quantum yields (fl) have shown that the hydrophobicity of these micelles is maximum for Tween-80 and at a minimum for Tween-20. Similar results have also been observed from the pK a values for the deprotonation of HPBI, whereas the protonation reaction of HPBI occurs at the site in nonionic micelles which is more hydrophilic than that where the deprotonation reaction takes place. The protonation reaction in 2-(2-methoxyphenyl)benzimidazole (MPBI) has shown that the value of ⑀ eff in Tweens where this reaction occurs is less hydrophobic than the site where the same reaction occurs in HPBI.

Temperature (T) is the most fundamental parameter in science. Respective sensors also are widely used in daily life. Besides conventional thermometers, optical sensors are considered to be attractive alternatives in sensing and on-line monitoring of T. This review focuses on all kinds of lumines-cent probes and sensors for measurement of T and summarizes the recent progress in their design and application formats. In the introduction, we cover the significance of optical probes for T, the origin of their T-dependent spectra, and the various detection modes.This is followed by a suryey on (a) molecular probes, (b) nanomaterials, and (c) bulk materials for sensing T, a discussion of polymeric matrices for immobilizing probes and an overview on the various application formats of T-sensors. The review closes with a discussion on the prospects, challenges, and new directions in the design of new probes and sensors. 248 references.
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Membrane microdomains and their involvement in cellular processes are part of the current paradigm of biomembranes. However, a better characterization of domains, namely lipid rafts, is needed. In this review, it is shown how the use of time-resolved fluorescence, with the adequate parameters and probes, helps elucidating the type, number, fraction, composition and size of lipid phases and domains in multicomponent model systems. The determination of phase diagrams for lipid mixtures containing sphingolipids and/or cholesterol is exemplified. The use of fluorescence quenching and Förster resonance energy transfer (FRET) are also illustrated. Strategies for studying protein-induced domains are presented. The advantages of using single point microscopic decays and fluorescence lifetime imaging microscopy (FLIM) in systems with three-phase coexistence are explained. Finally, the introduction of FLIM allows studies in live cell membranes, and the nature of the microdomains observed is readily elucidated due to the information retrieved from fluorescence lifetimes.

The synthesis, photophysical and photochemical properties of soluble 3,4-(methylendioxy)-phenoxy substituted oxo-titanium(IV) phthalocyanines (4–6) are reported for the first time. The new compounds have been characterized by elemental analysis, FT-IR, 1H NMR spectroscopy, electronic spectroscopy and mass spectra. General trends are described for fluorescence lifetimes and fluorescence, photodegradation and singlet oxygen quantum yields, of these compounds in dimethylsulfoxide (DMSO) and toluene.

In this work we report on the fluorescence lifetime characterization of the experimentally prepared Tm-doped silica optical fibers with increased quantum conversion efficiency (QE). Optical fibers were drawn from preforms prepared by conventional solution-doping of thulium and aluminium chlorides and by deposition of dispersed alumina nanoparticles with thulium chloride. Prepared preforms and optical fibers were characterized by means of thulium and aluminium concentrations, refractive index profiles, optical spectral attenuations (absorptions) and fluorescence lifetimes. Highly aluminium-codoped optical fiber prepared from alumina nanoparticles exhibited fluorescence lifetime of about 690 μs, which is about 40% higher compared to the conventionally prepared Tm-doped silica fiber. Downloaded From: http://proceedings.spiedigitallibrary.org/ on 01/16/2015 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 9450 945017-2 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 01/16/2015 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 9450 945017-6 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 01/16/2015 Terms of Use: http://spiedl.org/terms

The quenching of the excited singlet and triplet states of resazurin and resorufin by p-benzoquinones was investigated in methanol and in aqueous solution at pH 10. Fluorescence lifetimes were determined for the dyes and from the fluorescence quenching experiments rate constants were obtained for the singlet state quenching in MeOH. The rate constants were nearly diffusion controlled. Triplet state processes and transient absorption spectra were investigated by laser flash photolysis. Triplet quenching rate constants were obtained from the decay of the triplet at 825 nm for resazurin and at 700 nm for resorufin as a function of the quinone concentration. In the case of resazurin, the triplet quenching rate constants were lower than those of the singlet state. For resorufin, the values of the singlet and triplet were in the same order. From the transient absorption spectrum determinations it was concluded that the quenching proceeded by an electron-transfer reaction from the dye excited-states to the quinones. The results suggest that these dyes may function as very efficient photochemical-reductants.

We report spectra of various benzene isotopomers and their dimers in helium nanodroplets in the region of the first Herzberg-Teller allowed vibronic transition 6 1 0 1 B2u← 1 A1g (the A 0 0 transition) at ∼260 nm. Excitation spectra have been recorded using both beam depletion detection and laserinduced fluorescence. Unlike for many larger aromatic molecules, the monomer spectra consist of a single "zero-phonon" line, blueshifted by ∼30 cm −1 from the gas phase position. Rotational band simulations show that the moments of inertia of C6H6 in the nanodroplets are at least 6 times larger than in the gas phase. The dimer spectra present the same vibronic fine structure (though modestly compressed) as previously observed in the gas phase. The fluorescence lifetime and quantum yield of the dimer are found to be equal to those of the monomer, implying substantial inhibition of excimer formation in the dimer in helium.

Phosphates Rare-earths in glasses Phosphors Structure Medium-range order Short-range order a b s t r a c t Various fluoride, phosphate and borosilicate glasses with known properties and global structure have been doped with Eu 3+ (4f 6 ) at doping concentrations between 10 18 and 10 21 cm À3 . By applying reductive melting conditions Eu 3+ could partially be transformed to Eu 2+ (4f 7 ). Four fluoroaluminate glasses with varying phosphate content between 0 and 20 mol% (FPx), a pure phosphate glass (P100) and two borosilicate glasses with low (DURAN Ò -like) and high optical basicity (NBS1) have been used for these investigations. The time-resolved fluorescence in the visible range has been studied for both ions. Although static and dynamic fluorescence of Eu 3+ and Eu 2+ are dramatically different (f-f-transitions for Eu 3+ ; df-transitions for Eu 2+ ), glass structure changes have a similar influence on the dynamic fluorescence behavior of both ions. A strongly ionic surrounding due to fluorine ligands as in fluoroaluminate glass samples provides the longest fluorescence lifetime (about 7 ms for Eu 3+ ; about 1.3 ls for Eu 2+ ). Increasing phosphate content decreases the fluorescence lifetime due to more oxygen ligands. Interesting differences have been found for the two borosilicate glasses due to the difference in their optical basicity (Na 2 O/B 2 O 3 ratio). Measurements indicate a homogeneous distribution of europium ions in most FP samples. NBS1 measurements suggest that two different local europium sites are formed. For Duran-like samples only one specific europium site was found, although these samples show phase separation at high doping concentrations into a SiO 2 -rich phase and borate-and europium-rich droplets. Fluorescence quenching due to energy transfer from Eu 2+ to Eu 3+ could be found for co-doped samples; Eu 3+ -doped samples show no fluorescence quenching.

It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages, for example high sensitivity and multiple transduction approaches (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimermonomer light switching). These multiple signaling options combined with the design flexibility of the recognition element (DNA, RNA, PNA, LNA) and various labeling strategies contribute to development of numerous selective and sensitive bioassays. This review covers fundamentals of the design and engineering of oligonucleotide probes, describes typical construction approaches, and discusses examples of probes used both in hybridization studies and in aptamer-based assays.

The PS II-LHC II supercomplex is a novel type of oxygen evolving Photosystem II (PS II) core particle that contains the light harvesting complex proteins Lhcb1/2/4/5 in addition to the PS II reaction centre, oxygen evolving complex (OEC) and inner antennae [Hankamer et al. (1997) Eur J Biochem 243: 422-429]. The 33 and 23 kDa extrinsic proteins in these particles have been localised by image analysis of electron micrographs and averaging techniques [Boekema et al. (1998) Eur J Biochem 252: 268-276].

A novel Al 2 O 3 fluorescent nuclear track detector (FNTD), recently developed by Landauer, Inc., has demonstrated sensitivity and functionality superior to that of existing nuclear track detectors. The FNTD is based on single crystals of aluminum oxide doped with carbon and magnesium, and having aggregate oxygen vacancy defects (Al 2 O 3 :C,Mg). Radiation-induced color centers in the new material have an absorption band at 620 nm and produce fluorescence at 750 nm with a high quantum yield and a short, 75 ± 5 ns, fluorescence lifetime. Non-destructive readout of the detector is performed using a confocal fluorescence microscope. Scanning of the three-dimensional spatial distribution of fluorescence intensity along the track of a heavy charged particle (HCP) permits reconstruction of particle trajectories through the crystal and the LET can be determined as a function of distance along the trajectory based on the fluorescence intensity. Major advantages of Al 2 O 3 :C,Mg FNTD over conventionally processed CR-39 plastic nuclear track detector include superior spatial resolution, a wider range of LET sensitivity, no need for post-irradiation chemical processing of the detector and the capability to anneal and reuse the detector. Preliminary experiments have demonstrated that the material possesses a low-LET threshold of <1 keV/lm, does not saturate at LET in water as high as 1800 keV/lm, and is capable of irradiation to fluences in excess of 10 6 cm À2 without saturation (track overlap).

A system for advanced fluorescence investigation of works of art has been assembled and integrated in a characterization procedure that allows one to localize and identify organic compounds that are present in artworks. At the beginning of the investigation, fluorescence lifetime imaging and spectroscopy address a selective microsampling of the artwork. Then analytical measurements of microsamples identify the chemical composition of the materials under investigation. Finally, on the basis of fluorescence lifetime and amplitude maps, analytical data are extended to the whole artwork. In such a way, information on the spatial distribution of organic materials can be inferred. These concepts have been successfully applied in an extensive campaign for analysis of Renaissance fresco paintings in Castiglione Olona, Italy. Residue of various types of glue and stucco left from a restoration carried out in the early 1970s was localized and classified. Insight into the technique used by the painter to make gilded reliefs was also obtained.

An imager for time-resolved optical sensing was fabricated in CMOS technology. The sensor comprises an array of 128 2 128 single-photon pixels, a bank of 32 time-to-digital-converters, and a 7.68 Gbps readout system. Thanks to the outstanding timing precision of single-photon avalanche diodes and the optimized measurement circuitry, a typical resolution of 97 ps was achieved within a range of 100 ns. To the best of our knowledge, this imager is the first fully integrated system for photon time-of-arrival evaluation. Applications include 3-D imaging, optical rangefinding, fast fluorescence lifetime imaging, imaging of extremely fast phenomena, and, more generally, imaging based on time-correlated single photon counting. When operated as an optical rangefinder, this design has enabled us to reconstruct 3-D scenes with milimetric precisions in extremely low signal exposure. A laser source was used to illuminate the scene up to 3.75 m with an average power of 1 mW, a field-of-view of 5 and under 150 lux of constant background light. Accurate distance measurements were repeatedly achieved based on a short integration time of 50 ms even when signal photon count rates as low as a few hundred photons per second were available. Index Terms-Depth sensor, FCS, flash laser camera, fluorescence lifetime imaging microscopy (FLIM), Förster Resonance Energy Transfer (FRET), Geiger-mode avalanche photodiode, rangefinder, range imaging, single-photon avalanche diode, single-photon detector, solid-state 3-D imaging, SPAD, TCSPC, time-correlated single-photon counting, time-of-flight, 3-D image sensor.

a b s t r a c t La 1.90 Eu 0.10 TeO 6 :RE 3+ (RE = Gd, Sm) and Y 2 TeO 6 :Eu 3+ nanophosphors were prepared by the Pechini sol-gel process, using lanthanide sesquioxides and telluric acid as precursors. X-ray diffraction (XRD), scanning electron microscopy (SEM), photoluminescence spectra (PL) and fluorescence lifetime were used to characterize the resulting phosphors. The results of XRD indicate that all samples crystallized completely at 1073 K and are isostructural with orthorhombic Ln 2 TeO 6 . The SEM study reveals that the samples have a strong tendency to form agglomerates with an average size ranging from 40 to 65 nm. The luminescence decay curves suggest for all samples a monoexponential behavior. The photoluminescence intensity and chromaticity were improved for excitation at 395 nm when the co-doping concentration reaches the 1% mol. The optimized phosphorsLa 1.88 Eu 0.10 Gd 0.02 TeO 6 and La 1.88 Eu 0.10 Sm 0.02 TeO 6 , could be considered an efficient red-emitting phosphor for solid-state lighting devices based on InGaN LEDs.

Er 3+ / Yb 3+ codoped transparent phosphate precursor glasses and glass ceramics have been fabricated and characterized. The formation of ErPO 4 and YbPO 4 nanocrystals was studied using x-ray diffraction. The intensity parameters, the spontaneous radiative transition probabilities, the branching ratios, and the radiative lifetimes of Er 3+ in both glass ceramics and precursor glasses were calculated based on the Judd-Ofelt theory, and the crystallization process in the glass ceramics is confirmed by the optical parameters obtained. Under 975 nm wavelength excitation intense 1.54 m fluorescence and upconversion luminescence were observed in the glass ceramics, the quantum efficiency of the Er 3+ 4 I 13/2 → 4 I 15/2 transition is 95%, and the transition mechanisms of the upconversion luminescence are due to a two-photon process.

We review fluorescence lifetime techniques including timeresolved laser-induced fluorescence spectroscopy ͑TR-LIFS͒ and fluorescence lifetime imaging microscopy ͑FLIM͒ instrumentation and associated methodologies that allow for characterization and diagnosis of atherosclerotic plaques. Emphasis is placed on the translational research potential of TR-LIFS and FLIM and on determining whether intrinsic fluorescence signals can be used to provide useful contrast for the diagnosis of high-risk atherosclerotic plaque. Our results demonstrate that these techniques allow for the discrimination of important biochemical features involved in atherosclerotic plaque instability and rupture and show their potential for future intravascular applications.

Following the considerable impact of the application of convenient ultrafast lasers to multiphoton microscopy on biomedical imaging, it seems to us that FLIM and MDFI continue the trend in which advances in instrumentation will facilitate new discoveries — and modes of discovery — in biology and medicine. We hope we have shown the reader that fluorescence lifetime can provide intrinsic molecular contrast in unstained tissue and that the prospects for in vivo application are exciting. We believe that the capability to excite fluorophores at almost any excitation wavelength and the opportunities to extract more information from fluorescence signals by resolving with respect to lifetime, excitation and emission spectrum and also polarisation, will have a major impact on the ability to identify and exploit intrinsic contrast and on investigations of molecular biology. There the combination of new fluorescence probe technology, including genetically-expressed labels and nano-engineered devices, with new modes of interrogation and analysis, will continue to fuel the astounding advances in this field. There is a real prospect that our ability to ask and test biological questions will cease to be limited by the availability of suitable instrumentation. Rather it is likely to be limited by our ability to analyse and comprehend the (rapidly increasing volume of) data that we collect.

This work reports on the synthesis and fluorescence behaviour of novel unsymmetrically substituted monocarboxy germanium ((OH) 2 GeMCPc, 3), titanium (OTiMCPc 4) and tin ((ac) 2 SnMCPc, 5) phthalocyanines. The fluorescence quantum yields ranged from 0.09 to 0.14. The fluorescence lifetimes were found to be higher for the complex with higher fluorescence quantum yield value. Higher fluorescence quantum yields and lifetimes were obtained for the (ac) 2 SnMCPc complex (5), followed by OTiMCPc complex (4), and the lowest fluorescence quantum yield and lifetime were observed for (OH) 2 GeMCPc (3).

in vivo fluorescence lifetime imaging; autofluorescence; eye; excitation and emission spectra of endogenous fluorophores ABSTRACT Functional alterations are first signs of a starting pathological process. A device that measures parameter for the characterization of the metabolism at the human eye-ground would be a helpful tool for early diagnostics in stages when alterations are yet reversible. Measurements of blood flow and of oxygen saturation are necessary but not sufficient. The new technique of auto-fluorescence lifetime measurement (FLIM) opens in combination with selected excitation and emission ranges the possibility for metabolic mapping. FLIM not only adds an additional discrimination parameter to distinguish different fluorophores but also resolves different quenching states of the same fluorophore. Because of its high sensitivity and high temporal resolution, its capability to resolve multi-exponential decay functions, and its easy combination with laser scanner ophthalmoscopy, multi-dimensional time-correlated single photon counting was used for fundus imaging. An optimized set up for in vivo lifetime measurements at the human eye-ground will be explained. In this, the fundus fluorescence is excited at 446 or 468 nm and the time-resolved autofluorescence is detected in two spectral ranges between 510 and 560 nm as well as between 560 and 700 nm simultaneously. Exciting the fundus at 446 nm, several fluorescence maxima of lifetime t 1 were detected between 100 and 220 ps in lifetime histograms of 408 fundus images. In contrast, excitation at 468 nm results in a single maximum of lifetime t 1 ¼ 190 6 16 ps. Several fundus layers contribute to the fluorescence intensity in the short-wave emission range 510-560 nm. In contrast, the fluorescence intensity in the long-wave emission range between 560 and 700 nm is dominated by the fluorescence of lipofuscin in the retinal pigment epithelium. Comparing the lateral distribution of parameters of a tri-exponential model function in lifetime images of the fundus with the layered anatomical fundus structure, the shortest component (t 1 ¼ 190 ps) originates from the retinal pigment epithelium and the second lifetime (t 2 ¼ 1,000 ps) from the neural retina. The lifetime t 3 & 5.5 ns might be influenced by the long decay of the fluorescence in the crystalline lens. In vitro analysis of the spectral properties of expected fluorophores under the condition of the living eye lightens the interpretation of in vivo measurements. Taking into account the transmission of the ocular media, the excitation of NADH is unlikely at the fundus. Microsc. Res. Tech. 70:410-419, 2007.

Nicotinamide adenine dinucleotide is an important coenzyme involved in the production of ATP, the fuel of energy, in every cell. It alternates between the oxidized form NAD + and the reduced form dihydronicotinamide adenine dinucleotide (NADH) and serves as a hydrogen and electron carrier in the cellular respiratory processes. In the present work, the catalytic effect of gold nanoparticles on the oxidization of NADH to NAD + was investigated. The addition of gold nanoparticles was found to quench the NADH fluorescence intensities but had no effect on the fluorescence lifetime. This suggested that the fluorescence quenching was not due to coupling with the excited state, but due to changing the ground state of NADH. The intensity of the 340 nm absorption band of NADH was found to decrease while that of the 260 nm band of NAD + was found to increase as the concentration of gold nanoparticles increased. This conversion reaction was further supported by nuclear magnetic resonance and mass spectroscopy. The effect of the addition of NADH was found to slightly red shift and increase the intensity of the surface plasmon absorption band of gold nanoparticles at 520 nm. This gives a strong support that the conversion of NADH to NAD + is occurring on the surface of the gold nanoparticles, i.e. NADH is surface catalyzed by the gold nanoparticles. The catalytic property of this important reaction might have important future applications in biological and medical fields.

Thin films of Er 3+ -doped gallium lanthanum sulfide (GLS) glass were prepared by RF magnetron sputtering onto fused silica and BK7 glass substrates. The films were characterized by use of SEM imaging, X-ray diffractometry, and energy dispersive spectroscopy. They are shown to be glassy, uniform in composition, and to exhibit an RMS surface roughness of b 8 Å. The fluorescence lifetimes of the 4 I 11 / 2 → 4 I 13 / 2 and 4 I 13 / 2 → 4 I 15 / 2 transitions of Er 3+ were measured at each processing stage.

Cholesterol with BODIPY at carbon-24 of the side chain (BCh2) has recently been introduced as new cholesterol probe with superior fluorescence properties. We compare BCh2 with the intrinsically fluorescent dehydroergosterol (DHE), a well-established marker for cholesterol, by introducing simultaneous imaging of both sterols in model membranes and living cells. BCh2 had a lower affinity than DHE for the biologically relevant liquid-ordered phase in model membranes. Still, DHE and BCh2 trafficked from the plasma membrane to the endocytic recycling compartment (ERC) of BHK cells with identical kinetics. This transport pathway was strongly reduced after energy depletion of cells or expression of the dominantnegative clathrin heavy chain. The partitioning into lipid droplets of BHK and HeLa cells was higher for BCh2 than for DHE. Within droplets, the photodegradation of BCh2 was enhanced and followed a stretched exponential decay, while the fluorescence lifetime of BCh2 was comparable in various cellular regions.