Single Photon Counting Apparatus Research Articles

Time gated Fourier transform spectroscopy as a technique for disentangling short- and long-lived luminescence

Overlapping short- and long-lived luminescence signals are often encountered and are not trivial to disentangle in a single measurement. Here, we demonstrate for a broad range of emitters that time gated Fourier transform spectroscopy can be used as a technique to simultaneously capture and temporally disentangle overlapping spectral features of luminescence signals. This allows us to concurrently measure the fluorescence and phosphorescence bands of an organic fluorophore, retrieve the emission of a quantum dot that is overshadowed by an autofluorescent serum solution, and for removing residual laser scatter and ambient room light in an optical filter free configuration. Time gated Fourier transform spectroscopy only requires the introduction of a common-path interferometer to the emission path of a standard time-correlated single photon counting setup; it can function as a technique to both discriminate signals as well as characterize properties of new materials.

Precise rise and decay time measurements of inorganic scintillators by means of X-ray and 511 keV excitation

The emergence of new solid-state avalanche photodetectors, e.g. SiPMs, with unprecedented timing capabilities opens new ways to profit from ultrafast and prompt photon emission in scintillators. In time of flight positron emission tomography (TOF-PET) and high energy timing detectors based on scintillators the ultimate coincidence time resolution (CTR) achievable is proportional to the square root of the scintillation rise time, decay time and the reciprocal light yield, CTR∝τrτd∕LY. Hence, the precise study of light emission in the very first tens of picoseconds is indispensable to understand time resolution limitations imposed by the scintillator. We developed a time correlated single photon counting setup having a Gaussian impulse response function (IRF) of 63ps sigma, allowing to precisely measure the scintillation rise time of various materials with 511keV excitation. In L(Y)SO:Ce we found two rise time components, the first below the resolution of our setup <10 ps and a second component being ∼380 ps. Co-doping with Ca2+ completely suppresses the slow rise component leading to a very fast initial scintillation emission with a rise time of <10ps. A very similar behavior is observed in LGSO:Ce crystals. The results are further confirmed by complementary measurements using a streak-camera system with pulsed X-ray excitation and additional 511 keV excited measurements of Mg2+ co-doped LuAG:Ce, YAG:Ce and GAGG:Ce samples.

Solvent Effect, Photochemical and Photophysical Properties of Phthalocyanines with Different Metallic Nuclei

Photophysical and photochemical properties of lithium phthalocyanine ( 1 ), gallium(III) phthalocyanine chloride ( 2 ), titanium(IV) phthalocyanine dichloride ( 3 ) and iron(II) phthalocyanine ( 4 ) were investigated in dimethyl sulfoxide (DMSO), tetrahydrofuran (THF) and DMSO-THF mixtures. The influence of the central metal on these properties was analyzed according to solvent type, axial ligands and their paramagnetic and diamagnetic effect. Fluorescence lifetimes were recorded using a time correlated single photon counting setup (TCSPC) technique. In order to demonstrate the generation of reactive oxygen species under light irradiation, the indirect method (applying 1,3-diphenylisobenzofuran (DPBF) as chemical suppressor) and the direct method (analyzing the phosphorescence decay curves of singlete oxygen at 1270 nm) were employed. Compounds 1 , 2 and 3 showed a monomeric behavior in all media while compound 4 presented low aggregation in DMSO, but a very pronounced aggregation behavior in THF. Steady-state fluorescence anisotropy was compared with emission spectra and complex 4 presented values beyond the expected limits. DOI: http://dx.doi.org/10.17807/orbital.v9i5.1047

Structural Dynamics of Proteins using Novel Visible Fluorescence Probes

Fluorescence spectroscopy has been widely used in life science. In this paper, some fluorescent molecules have been designed and synthesized to interact with biological thiol groups, in order to further study protein structural changes at different environment. 4-(2-(1,10c-dihydropyren-1-yl)vinyl)-1-methylpyridinium iodide (4-PBPMI) and 2-(2-(1,10c-dihydropyren-1-yl)vinyl)-1-methylpyridinium iodide (2-PBPMI), whose absorption and fluorescence spectra are in ultraviolet and visible region respectively, have been shown to very sensitively probe L-Cysteine (L-Cys) due to the strong interaction between thiols and vinyl. We reported here the systematic measurements of steady state fluorescence of PBPMIs in the mixtures with various proportions of glycerol and water, and time resolved fluorescence decay using a time correlated single photon counting apparatus on the 100ps to 30ns time scale. The probes are excellent to monitor the L-Cys concentrations and environment simultaneously. For example, with the increasement of the concentration of L-Cys, the emission intensities of PBPMIs in ultraviolet got a great enhancement as well as absorptions. Meanwhile, in visible region, the intensities were decreased sharply. Further experiments and discussion will be made to study structural dynamics of proteins with disulfide bondsand free thiols. The results may motivate additional experimental and theoretical research on the molecular-level design of luminescent probes in biophysics.

Time-Resolved Fluorescence Studies of Fullerene Derivatives

Fullerene (nano-C60) and its water-soluble derivatives have several clinical applications including use as a drug carrier to bypass the blood-ocular and blood-brain barriers. However, in vitro and in vivo detection of these nanomaterials is limited by their very low fluorescence quantum yield. The accumulation of fullerene and its derivatives in cells is particularly difficult to measure using standard fluorescence microscopy because their fluorescence is barely detectable in aqueous media. We have developed a time-correlated single-photon counting apparatus with which we were not only able to detect the fluorescence of fullerene and its derivatives in water but could also measure fluorescence temporal decays and determine lifetimes in the range of tens of picoseconds. The compounds studied in this report are C60 (fullerene), the partially hydrogenated hydride C60H36, a monomeric cyclodextrin complexed fullerene [(γ-CyD)2/C60], and C60(OH)24 (fullerol). In addition, we examined the effect of aggregation on photophysical properties and identified a very short lifetime component belonging to the fluorescence decay of monomeric fullerene, which is lost with increasing aggregation. These data will help to design nanoparticles that have the appropriate structural and photophysical properties to ultimately be of use in a clinical setting.

Femtosecond Fluorescence Dynamics of Trp-Trp Dipeptide in Water and its Interaction with DNA

We reported here the systematic measurements of fluorescence decay and quenching of tryptophan dipeptide Trp-Trp in water, and its interaction with DNA by using an ultraviolet upconversion fluorescence measurement system with time resolution better than 100 fs together with a time correlated single photon counting apparatus on the 100 ps to 30 ns time scale. We analyzed the effect of acidity and phosphate ion etc upon Trp-Trp fluorescence decay profiles. Fluorescence data reveal multiexponential decay of Trp-Trp dipeptide in water, and Trp-Trp can interact with DNA. It is shown that Trp-Trp is subject to ultrafast quenching with DNA and also senses nearby water dynamics. The time constant for the bulk water “solvent relaxation” remains near 1 ∼ 2 ps, but a new ultrafast decay, fit to an exponential time constant of ∼ 10 ps with associated amplitude positive or negative (>380 nm) depending on the emission wavelength, has been found. Candidate mechanisms (including ET to nearby acceptors and/or collisional quenching etc.) and the analysis of time resolved emission spectra will be discussed.

Structural Basis for the 14-3-3 Protein-dependent Inhibition of the Regulator of G Protein Signaling 3 (RGS3) Function

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction. We have used small angle x-ray scattering, hydrogen/deuterium exchange kinetics, and Förster resonance energy transfer measurements to determine the low-resolution solution structure of the 14-3-3ζ·RGS3 complex. The structure shows the RGS domain of RGS3 bound to the 14-3-3ζ dimer in an as-yet-unrecognized manner interacting with less conserved regions on the outer surface of the 14-3-3 dimer outside its central channel. Our results suggest that the 14-3-3 protein binding affects the structure of the Gα interaction portion of RGS3 as well as sterically blocks the interaction between the RGS domain and the Gα subunit of heterotrimeric G proteins.

Statistical analysis of time resolved single molecule fluorescence data without time binning

We depict two algorithms to calculate correlation functions from two different time resolved single molecule fluorescence experiments without the need of time binning. Our first procedure allows to calculate the reduced linear dichroism from polarization resolved fluorescence data. Since we process single photon counts instead of time binned data, considerably faster fluctuations of the dichroism can be analyzed than with conventional methods. With our second procedure time resolved fluorescence obtained with a time correlated single photon counting setup can be analyzed with respect to fluorescence lifetime fluctuations. Again this new algorithm processes single photon events making time binning of photon counts obsolete. Both methods presented are characterized by enhanced time resolution thus allowing to study fast fluctuations of either single molecular orientation or fluorescence life times, respectively.

Femtosecond Fluorescence Spectra of Tryptophan in Human γ-Crystallin Mutants: Site-Dependent Ultrafast Quenching

The eye lens Crystallin proteins are subject to UV irradiation throughout life, and the photochemistry of damage proceeds through the excited state; thus, their tryptophan (Trp) fluorescence lifetimes are physiologically important properties. The time-resolved fluorescence spectra of single Trps in human gammaD- and gammaS-Crystallins have been measured with both an upconversion spectrophotofluorometer on the 300 fs to 100 ps time scale, and a time correlated single photon counting apparatus on the 100 ps to 10 ns time scale, respectively. Three Trps in each wild type protein were replaced by phenylalanine, leading to single-Trp mutants: W68-only and W156-only of HgammaD- and W72-only and W162-only of HgammaS-Crystallin. These proteins exhibit similar ultrafast signatures: positive definite decay associated spectra (DAS) for 50-65 ps decay constants that indicate dominance of fast, heterogeneous quenching. The quenched population (judged by amplitude) of this DAS differs among mutants. Trps 68, 156 in human gammaD- and Trp72 in human gammaS-Crystallin are buried, but water can reach amide oxygen and ring HE1 atoms through narrow channels. QM-MM simulations of quenching by electron transfer predict heterogeneous decay times from 50-500 ps that agree with our experimental results. Further analysis of apparent radiative lifetimes allow us to deduce that substantial subpopulations of Trp are fully quenched in even faster (sub-300 fs) processes for several of the mutants. The quenching of Trp fluorescence of human gammaD- and gammaS-Crystallin may protect them from ambient light induced photo damage.

Quasi-Static Self-Quenching of Trp-X and X-Trp Dipeptides in Water: Ultrafast Fluorescence Decay

Time-resolved fluorescence decay profiles of N-acetyl-l-tryptophanamide (NATA) and tryptophan (Trp) dipeptides of the form Trp-X and X-Trp, where X is another aminoacyl residue, have been investigated using an ultraviolet upconversion spectrophoto fluorometer with time resolution better than 350 fs, together with a time-correlated single photon counting apparatus on the 100 ps to 20 ns time scale. We analyzed the set of fluorescence decay profiles at multiple wavelengths using the global analysis technique. Nanosecond fluorescence transients for Trp dipeptides all show multiexponential decay, while NATA exhibits a monoexponential decay near 3 ns independent of pH. In the first 100 ps, a time constant for the water "bulk relaxation" around Trp, NATA and Trp dipeptides are seen near 1-2 ps, with an associated preexponential amplitude that is positive or negative, depending on emission wavelength, as expected for a population conserving spectral shift. The initial brightness (sub-picosecond) we measure for all these dipeptides is less than that of NATA, implying even faster (<200 fs) intramolecular (quasi-) static quenching occurs within them. A new, third, ultrafast decay, bearing an exponential time constant of 20-30 ps with positive amplitude, has been found in many of these dipeptides. We believe it verifies our previous predictions of dipeptide QSSQ ("quasi-static self-quenching")-the loss of quantum yield to sub-100-ps decay process (Chen, R. F.; et al. Biochemistry 1991, 30, 5184). Most important, this term is found in proteins as well (Xu, J.; et al. J. Am. Chem. Soc. 2006, 128, 1214; Biophys. J. 2008, 94, 546; 2009, 96, 46a), suggesting an ultrafast quenching mechanism must be common to both.

Ultrafast Decay of Trp in Biological Macromolecules

Femtosecond (<300fs fwhm) measurements of fluorescence decay and quenching of Tryptophan (Trp) were performed in a variety of proteins, including GB1, Thioredoxin (both wild type from two species and a human Trx mutant with a single Trp), Cyanovirin and Interleukin-1beta using an upconversion spectrophotofluorometer combined with a time correlated single photon counting apparatus to span the ∼200fs to 20ns time scale. Trp is subject both to ultrafast quenching in proteins and spectral energy loss coupled to nearby water dynamics. All fluorescence transients of tryptophan in proteins reveal complex, i.e. multiexponential behavior. In addition to a “bulk water” relaxation (∼2 ps), a 50 ps fluorescence decay was found in single-Trp thioredoxin which matched the component we had found previously in two-Trp Anabaena and E. coli thioredoxins . In fact, a sub-100ps component is consistently found in all but one of these proteins with positive amplitudes even at longer wavelengths (e.g., 390nm). The exception is GB1, a protein which Toptygin and Brand previously found carried a negative preexponential term near 390nm. Since the lifetime associated with that negative was 65ps, it was just within the edge of TCSPC detection. The more prevalent positive amplitude DAS (decay-associated spectra) we see in the other proteins on these timescales are indicative of ultrafast quenching processes depleting the partly relaxed singlet state. Candidate mechanisms include ET to nearby acceptors and/or collisional quenching. This is similar to our prior observation (J. Am. Chem. Soc., 2006, 128, 1214) that ultrafast quenching, not desorbing water, dominates the time-resolved emission spectra (TRES) of monellin. We will discuss the −/+ data signatures for both water relaxation and fast quenching, including simulations to lay out the circumstances where a fast relaxation accompanied by a direct radiative rate reduction might mask the negative term.

Time-correlated single-photon counting based method for submillimeter transillumination imaging of objects embedded in tissue-phantoms

We test the performances of an imaging system in revealing either opaque, light diffusing, or transparent objects embedded in tissue phantoms. The method relies on measuring the time-of-flight distributions of near-infrared photons emerging from the phantom within a collection angle of 0.6 mrad about the direction of the incident light by means of a time-correlated single-photon counting apparatus. The apparatus is based on new generation single photon avalanche photodiodes, which are endowed with ≤35 ps resolution, low dark-count rate, short dead time and relatively high quantum efficiency in the near-infrared. The weakly-scattered (ballistic/snake) photons, bringing the relevant image information, are efficiently discriminated from the multiply scattered ones. Thus, the profiles of the embedded objects can be reconstructed and their nature can be assessed. Opaque objects are localized with ≤220 µm spatial resolution. The potential of the new detectors in medical imaging applications is discussed.

Optical profiles with 180μm resolution of objects hidden in scattering media

We measure the time-of-flight distributions of near-infrared photons emerging from thick scattering media within a collection angle of 0.6mrad about the incident light direction by means of a time-correlated single-photon counting apparatus endowed with &amp;lt;35ps resolution. These measurements, which are performed with a picosecond laser beam, allow us to isolate the weakly scattered (ballistic∕snake) photons from the multiply scattered ones. By scanning the incidence position across a target we find variations in the fraction of detected unscattered photons that are significative of local changes in the optical parameters of the target. In particular, if either opaque, light diffusing, or transparent objects are embedded in the scattering medium, their profiles can be reconstructed and their nature can be assessed. Opaque objects embedded in realistic tissue phantoms are detected with &amp;lt;180μm spatial resolution.

Ultrafast Fluorescence Dynamics of Tryptophan in the Proteins Monellin and IIAGlc

The complete time-resolved fluorescence of tryptophan in the proteins monellin and IIA(Glc) has been investigated, using both an upconversion spectrophotofluorometer with 150 fs time resolution and a time-correlated single photon counting apparatus on the 100 ps to 20 ns time scale. In monellin, the fluorescence decay displays multiexponential character with decay times of 1.2 and 16 ps, and 0.6, 2.2, and 4.2 ns. In contrast, IIA(Glc) exhibited no component between 1.2 ps and 0.1 ns. For monellin, surprisingly, the 16 ps fluorescence component was found to have positive amplitude even at longer wavelengths (e.g., 400 nm). In conjunction with quantum mechanical simulation of tryptophan in monellin, the experimental decay associated spectra (DAS) and time-resolved emission spectra (TRES) indicate that this fluorescence decay time should be ascribed to a highly quenched conformer. Recent models (Peon, J.; et al. Proc. Natl.Acad. Sci. U.S.A. 2002, 99, 10964) invoked exchange-coupled relaxation of protein water to explain the fluorescence decay of monellin.

Application of time- and space-resolved fluorescence spectroscopy to the distribution of guest species into micrometer-sized zeolite crystals

We measured the fluorescence decays and spectra of perylene adsorbed from solution into zeolite X crystals of 2-3 microm in diameter at the level of individual crystals by the application of a microscopy method coupled with a single photon counting apparatus and a multichannel spectrophotometer. We found that both decays and spectra are particle-dependent, i.e. a particle-to-particle difference was observed for the fluorescence decay curves at a fixed loading level along with a particle-dependent spectral change due to the various contribution of excimer emission band relative to those of three monomers. These findings are due to a non-homogeneous distribution which is confirmed by the various emission intensities of perylene-loaded zeolite crystals observed by fluorescence microscopy. Previously, a homogeneous distribution of the guest between zeolite crystals has been just taken for granted and not justified by experiment. The present result suggests that commonly employed collective measurements such as UV-VIS absorption and emission spectroscopies, IR and Raman spectroscopies, and NMR of bulk zeolite powders provide only averaged results and may sometimes suffer from acquiring precise molecular level pictures.

Elucidation of the Nature of the Conformational Changes of the EF-interhelical Loop in Bacteriorhodopsin and of the Helix VIII on the Cytoplasmic Surface of Bovine Rhodopsin: A Time-resolved Fluorescence Depolarization Study

The conformation of the AB-loop and EF-loop of bacteriorhodopsin and of the fourth cytoplasmic loop (helix VIII) of bovine rhodopsin were assessed by a combination of time-resolved fluorescence depolarization and site-directed fluorescence labeling. The fluorescence anisotropy decays were measured employing a tunable Ti:sapphire laser/microchannel plate based single-photon counting apparatus with picosecond time resolution. This method allows measurement of the diffusional dynamics of the loops directly on a nanosecond time-scale. We implemented the method to study model peptides and two-helix systems representing sequences of bacteriorhodopsin. Thus, we systematically analyzed the anisotropic behavior of four different fluorescent dyes covalently bound to a single cysteine residue on the protein surface and assigned the anisotropy decay components to the modes of motion of the protein and its segments. We have identified two mechanisms of loop conformational changes in the functionally intact proteins bacteriorhodopsin and bovine rhodopsin. First, we found a surface potential-dependent transition between two conformational states of the EF-loop of bacteriorhodopsin, detected with the fluorescent dye bound to position 160. A transition between the two conformational states at 150 mM KCl and 20 °C requires a surface potential change that corresponds to Δσ≈−1.0 e −/bacteriorhodopsin molecule. We suggest, that the surface potential-based switch of the EF-loop is the missing link between the movement of helix F and the transient surface potential change detected during the photocycle of bacteriorhodopsin. Second, in the visual pigment rhodopsin, with the fluorescent dye bound to position 316, a particularly striking pH-dependent conformational change of the fourth loop on the cytoplasmic surface was analyzed. The loop mobility increased from pH 5 to 8. The midpoint of this transition is at pH 6.2 and correlates with the midpoint of the pH-dependent equilibrium between the active metarhodopsin II and the inactive metarhodopsin I state.

Delayed Fluorescence and Phosphorescence from Polyphenylquinoxalines

We report on phosphorescence and delayed fluorescence from polyphenylquinoxalines in frozen solution and in the film at 77 K. Upon pulsed optical excitation, the following types of experiments have been carried out: (a) optical spectra detected by means of a gated optical multichannel analyzer, (b)-transient measurements employing a single photon counting setup, and (c) photoluminescence excitation spectra. We will argue that in a polymer in which the quinoxaline units are directly linked together via C−C-bonds, the delayed fluorescence is due to the recombination of Coulombically bound electron−hole pairs, which on their part are generated by direct optical excitation. This is deduced from the facts that (a) the intensities of delayed fluorescence, phosphorescence and prompt fluorescence all vary linearly with the pump intensity; that (b) both delayed fluorescence and phosphorescence decay according to a power law up to 1 s, i.e., at times exceeding the triplet lifetime; and that (c) the photoluminescen...

Hypercholesterolemia and chronic anxiety disorder

This chapter discusses the oxystat technique in study of reactive oxygen species. The oxystat system allows samples to be collected from the filtrate as well as directly from the incubation chamber. Thus, the formation of products of oxidative reactions in biological material, such as malon- dialdehyde, can easily be determined during the course of incubation. Furthermore, by putting the incubation chamber directly in front of the photomultiplier of a single-photon counting apparatus, low-level chemiluminescence emitted by electronically excited species such as singlet molecular oxygen (1O2) and triplet carbonyls (3RO) can be detected. Finally, the stimulation of O2 uptake may serve as an additional indicator for the formation of reactive oxygen species. The oxystat system consists of an incubation chamber, an O2 sensor, a pump, and a computer. The water-jacketed incubation chamber is made of plexiglass. It is equipped with a magnetic stirrer and a port for the O2 sensor. At the top two stainless steel needles serve as entrances. The inlet for the O2-saturated infusion medium ends directly above the magnetic stirring bar. The second needle, which does not project into the chamber, is used as an injection port for the addition of reagents and for taking samples of the incubation mixture. The bottom of the incubation chamber is equipped with a membrane holder covered by a filter membrane. The actual PO2 in the incubation chamber is determined by a polarographic O2 sensor.

Self quenching of 1-naphthol. Connection between time-resolved and steady-state measurements

The photochemistry of 1-naphthol following its electronic excitation in aqueous solution was investigated by a single-photon counting apparatus. It was found that following the proton dissociation the excited naphtholate anion decays non-exponentially with a t − sol1 2 dependence over time. This we attribute to a self-quenching reaction of the naphtholate anion by its geminate proton. The quenching rate on contact is 3.5 × 10 10 s −1. We also found that some of the protons interact adiabatically with a geminate recombination rate in D 2O fo 8 × 10 9 s −1. The time-dependent escape probability of the naphtholate iopn-par was sam;ed by carrying out the experiment in presence of a proton scavenger. We show that the leading term in the ultimate scavenging probability is given by the time integral of the homogeneous scavenging rate of the isolated pair at relative long times following its creation.

Rotational diffusion of fluorene in hydroxylic solvents

Rotational diffusion of fluorene was studied by means of fluorescence anisotropy measurements using a time-correlated single photon counting apparatus. The measured rotational relaxation times in various alcohol solvents were compared with hydrodynamic predictions for prolate spheroidal and asymmetric ellipsoidal shapes in the «stick» and «slip» limits. The rotational dynamics of this molecular system was not predicted by slip or stick hydrodynamics assuming the friction which is proportionalto the solvents' viscosities. Some factors that might be responsible for this phenomenon are discussed