Time-gated in vivo autofluorescence imaging of dental caries.

Laser-induced time-resolved autofluorescence from carious lesions of human teeth was studied by means of ultrashort pulsed laser systems, time-correlated single photon counting and time-gated imaging. Carious regions exhibited a slower fluorescence decay with a main 17 ns fluorescence lifetime than healthy hard dental tissue. The long-lived fluorophore present in carious lesions only emits in the red spectral region. Fluorescence decay time and spectral characteristics are typical of fluorescent metal-free porphyrin monomers. The spatial distribution of the long-lived endogenous porphyrin fluorophore within the tooth material was detected by time-gated nanosecond autofluorescence imaging. In particular, high contrast video images were obtained with an appropriate time delay of 15 ns to 25 ns between excitation and detection due to the suppression of short-lived autofluorescence of healthy tissue. First in vivo applications are reported indicating the potential of time-resolved fluorescence diagnostics for early caries- and dental plaque detection.     

Organization and dynamics of tryptophan residues in tetrameric and monomeric soybean agglutinin: Studies by steady-state and time-resolved fluorescence,phosphorescence and chemical modification

We have investigated the organization and dynamics of tryptophan residues in tetrameric, monomeric and unfolded states of soybean agglutinin (SBA) by selective chemical modification, steady-state and time-resolved fluorescence, and phosphorescence. Oxidation with N-bromosuccinimide (NBS) modifies two tryptophans (Trp 60 and Trp 132) in tetramer, four (Trp 8, Trp 203 and previous two) in monomer, and all six (Trp 8, Trp 60, Trp 132, Trp 154, Trp 203 and Trp 226) in unfolded state. Utilizing wavelength-selective fluorescence approach, we have observed a red-edge excitation shift (REES) of 10 and 5 nm for tetramer and monomer, respectively. A more pronounced REES (21 nm) is observed after NBS oxidation. These results are supported by fluorescence anisotropy experiments. Acrylamide quenching shows the Stern–Volmer constant (K_SV) for tetramer, monomer and unfolded SBA being 2.2, 5.0 and 14.6 M⁻¹, respectively. Time-resolved fluorescence studies exhibit biexponential decay with the mean lifetime increasing along tetramer (1.0 ns) to monomer (1.9 ns) to unfolded (3.6 ns). Phosphorescence studies at 77 K give more structured spectra, with two (0,0) bands at 408.6 (weak) and 413.2 nm for tetramer. However, a single (0,0) band appears at 411.8 and 407.2 nm for monomer and unfolded SBA, respectively. The exposure of hydrophobic surface in SBA monomer has been examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, which shows ∼20-fold increase in ANS fluorescence compared to that for tetramer. The mean lifetime of ANS also shows a large increase (12.0 ns) upon binding to monomer. These results may provide important insight into the role of tryptophans in the folding and association of SBA, and oligomeric proteins in general.     

Applying two-photon excitation fluorescence lifetime imaging microscopy to study photosynthesis in plant leaves

This study investigates to which extent two-photon excitation (TPE) fluorescence lifetime imaging microscopy can be applied to study picosecond fluorescence kinetics of individual chloroplasts in leaves. Using femtosecond 860 nm excitation pulses, fluorescence lifetimes can be measured in leaves of Arabidopsis thaliana and Alocasia wentii under excitation-annihilation free conditions, both for the F ₀- and the F _m-state. The corresponding average lifetimes are ~250 ps and ~1.5 ns, respectively, similar to those of isolated chloroplasts. These values appear to be the same for chloroplasts in the top, middle, and bottom layer of the leaves. With the spatial resolution of ~500 nm in the focal (xy) plane and 2 μm in the z direction, it appears to be impossible to fully resolve the grana stacks and stroma lamellae, but variations in the fluorescence lifetimes, and thus of the composition on a pixel-to-pixel base can be observed.     

Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin

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.     

Evidence that the variable chlorophyll fluorescence in Chlamydomonas reinhardtii is not recombination luminescence

Room temperature single photon timing measurements on intact, Chlamydomonas reinhardtii cells at low excitation energies have been analysed using a four exponential kinetic model. Closing the PSII reaction centres produced two major variable lifetime and two minor constant lifetime components. The yield of each component mirrored the changes in lifetime. Such observations indicate the presence of well-connected PSII centres favoring excitation energy transfer. A Chlamydomonas mutant lacking PSII reaction centre proteins exhibited decay components equivalent to those seen at F_M in the wild-type. A titration of in vivo fluorescence, in both the mutant and wild-type algae, using DNB, produced decay components similar to those seen on opening PSII reaction centres. Such observations indicate that the luminescence hypothesis for the origin of the long-lived lifetime component is not the case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DNB m,Dinitrobenzene - PSII photosystem II - RCII PSII recation centre - I^- reduced pheophytin - Q_A primary stable electron ecceptor of PSII - Ch1 chlorophyl1 - LHCII light harvesting Ch1a/b protein complex of PSII - F_O initial fluorescence level - F_M maximum fluorescence level - F_V variable fluorescence (F_M-F_O) - ps picosecond - ns nanosecond     

The role of xanthophylls in the supramolecular organization of the photosynthetic complex LHCII in lipid membranes studied by high-resolution imaging and nanospectroscopy

The xanthophyll cycle is a regulatory mechanism operating in the photosynthetic apparatus of plants. It consists of the conversion of the xanthophyll pigment violaxanthin to zeaxanthin, and vice versa, in response to light intensity. According to the current understanding, one of the modes of regulatory activity of the cycle is associated with the influence on a molecular organization of pigment-protein complexes. In the present work, we analyzed the effect of violaxanthin and zeaxanthin on the molecular organization of the LHCII complex, in the environment of membranes formed with chloroplast lipids. Nanoscale imaging based on atomic force microscopy (AFM) showed that the presence of exogenous xanthophylls promotes the formation of the protein supramolecular structures. Nanoscale infrared (IR) absorption analysis based on AFM-IR nanospectroscopy suggests that zeaxanthin promotes the formation of LHCII supramolecular structures by forming inter-molecular β-structures. Meanwhile, the molecules of violaxanthin act as “molecular spacers” preventing self-aggregation of the protein, potentially leading to uncontrolled dissipation of excitation energy in the complex. This latter mechanism was demonstrated with the application of fluorescence lifetime imaging microscopy. The intensity-averaged chlorophyll a fluorescence lifetime determined in the LHCII samples without exogenous xanthophylls at the level of 0.72 ns was longer in the samples containing exogenous violaxanthin (2.14 ns), but shorter under the presence of zeaxanthin (0.49 ns) thus suggesting a role of this xanthophyll in promotion of the formation of structures characterized by effective excitation quenching. This mechanism can be considered as a representation of the overall photoprotective activity of the xanthophyll cycle.     

Excitation transfer and trapping kinetics in plant photosystem I probed by two-dimensional electronic spectroscopy

Photosystem I is a robust and highly efficient biological solar engine. Its capacity to utilize virtually every absorbed photon’s energy in a photochemical reaction generates great interest in the kinetics and mechanisms of excitation energy transfer and charge separation. In this work, we have employed room-temperature coherent two-dimensional electronic spectroscopy and time-resolved fluorescence spectroscopy to follow exciton equilibration and excitation trapping in intact Photosystem I complexes as well as core complexes isolated from Pisum sativum. We performed two-dimensional electronic spectroscopy measurements with low excitation pulse energies to record excited-state kinetics free from singlet–singlet annihilation. Global lifetime analysis resolved energy transfer and trapping lifetimes closely matches the time-correlated single-photon counting data. Exciton energy equilibration in the core antenna occurred on a timescale of 0.5 ps. We further observed spectral equilibration component in the core complex with a 3–4 ps lifetime between the bulk Chl states and a state absorbing at 700 nm. Trapping in the core complex occurred with a 20 ps lifetime, which in the supercomplex split into two lifetimes, 16 ps and 67–75 ps. The experimental data could be modelled with two alternative models resulting in equally good fits—a transfer-to-trap-limited model and a trap-limited model. However, the former model is only possible if the 3–4 ps component is ascribed to equilibration with a “red” core antenna pool absorbing at 700 nm. Conversely, if these low-energy states are identified with the P₇₀₀ reaction centre, the transfer-to-trap-model is ruled out in favour of a trap-limited model.

     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part II. In the isolated light harvesting complex (phycobilisomes)

The transfer of excitation energy between phycobiliproteins in isolated phycobilisomes has been observed on a picosecond time scale. The photon density of the excitation pulse has been carefully varied so as to control the level of exciton interactions induced in the pigment bed. The 530 nm light pulse is absorbed predominantly by B-phycoerythrin, and the fluorescence of this component rises within the pulse duration and shows a mean 1/e decay time of 70 ps. The main emission band, centred at 672 nm, is due to allophycocyanin and is prominent because of the absence of energy transfer to chlorophyll. Energy transfer to this pigment from B-phycoerythrin via R-phycocyanin produces a risetime of 120 ps to the fluorescence maximum. The lifetime of the allophycocyanin fluorescence is found to be about 4 ns using excitation pulses of low photon densities (10(13) photons.cm-2), but decreases to about 2 ns at higher photon densities. The relative quantum yield of the allophycocyanin fluorescence decreases almost 10 fold over the range of laser pulse intensities, 10(13)--10(16) photons-cm-2. Fluorescence quenching by exciton-exciton annihilation is only observed in allophycocyanin and could be a consequence of the long lifetime of the single exciton in this pigment.     

Redox transients of P680 associated with the incremental chlorophyll‐a fluorescence yield rises elicited by a series of saturating flashes in diuron‐treated photosystem II core complex of Thermosynechococcus vulcanus

Recent chlorophyll‐a fluorescence yield measurements, using single‐turnover saturating flashes (STSFs), have revealed the involvement of a rate‐limiting step in the reactions following the charge separation induced by the first flash. As also shown here, in diuron‐inhibited PSII core complexes isolated from Thermosynechococcus vulcanus the fluorescence maximum could only be reached by a train of STSFs. In order to elucidate the origin of the fluorescence yield increments in STSF series, we performed transient absorption measurements at 819 nm, reflecting the photooxidation and re‐reduction kinetics of the primary electron donor P680. Upon single flash excitation of the dark‐adapted sample, the decay kinetics could be described with lifetimes of 17 ns (～50%) and 167 ns (～30%), and a longer‐lived component (～20%). This kinetics are attributed to re‐reduction of P680^?+ by the donor side of PSII. In contrast, upon second‐flash (with Δt between 5 μs and 100 ms) or repetitive excitation, the 819 nm absorption changes decayed with lifetimes of about 2 ns (～60%) and 10 ns (～30%), attributed to recombination of the primary radical pair P680^?+Pheo^?–, and a small longer‐lived component (～10%). These data confirm that only the first STSF is capable of generating stable charge separation – leading to the reduction of Q_A; and thus, the fluorescence yield increments elicited by the consecutive flashes must have a different physical origin. Our double‐flash experiments indicate that the rate‐limiting steps, detected by chlorophyll‐a fluorescence, are not correlated with the turnover of P680.     

Non-exponential decay of indole fluorescence — the red-edge effect

The fluorescence of indole in glycerol decays exponentially at both 22° and ?80°C for 280 nm excitation. Under 296 nm excitation the 22°C emission decay is exponential, but at ?80°C, a second shorter lived (1.3 ns) component is also detected. The relevance of this result to the determination of protein fluorescence lifetime is described.     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part II. In the isolated light harvesting complex (phycobilisomes)

The transfer of excitation energy between phycobiliproteins in isolated phycobilisomes has been observed on a picosecond time scale. The photon density of the excitation pulse has been carefully varied so as to control the level of exciton interactions induced in the pigment bed. The 530 nm light pulse is absorbed predominantly by B-phycoerythrin, and the fluorescence of this component rises within the pulse duration and shows a mean 1/e decay time of 70 ps. The main emission band, centred at 672 nm, is due to allophycocyanin and is prominent because of the absence of energy transfer to chlorophyll. Energy transfer to this pigment from B-phycoerythrin via R-phycocyanin produces a risetime of 120 ps to the fluorescence maximum. The lifetime of the allophycocyanin fluorescence is found to be about 4 ns using excitation pulses of low photon densities (10¹³ photons · cm^?2), but decreases to about 2 ns at higher photon densities. The relative quantum yield of the allophycocyanin fluorescence decreases almost 10 fold over the range of laser pulse intensities, 10¹³–10¹⁶ photons · cm^?2. Fluorescence quenching by exciton-exciton annihilation is only observed in allophycocyanin and could be a consequence of the long lifetime of the single exciton in this pigment.     

Electron donor properties of the antitumour drug amsacrine as studied by fluorescence quenching of DNA-bound ethidium

The effect of the antitumour acridine derivative amsacrine [4'-(9-acridinylamino)methanesulphon-m-anisidide] on the fluorescence lifetime of DNA-bound ethidium has been investigated using a synchronously pumped cavity dumped dye laser producing picosecond pulses for sample excitation and a time-correlated single photon counting detection system. As the proportion of DNA-bound amsacrine on the synthetic DNA polymer poly[deoxyadenylic-thymidylic acid] is increased, the fluorescence decay curve of ethidium can be accurately resolved into two exponential components. The short lifetime component, whose proportion increases with increasing proportions of DNA-bound amsacrine, has a lifetime of between 3 and 4 ns, significantly longer than that of ethidium in aqueous solution (1.63 ns). The magnitude of the long lifetime component decreases from 25.4 to 14 ns with increasing proportions of bound amsacrine. It is concluded that a new fluorescence state of ethidium (lifetime 3-4 ns) is present, probably resulting from reversible electron transfer between ethidium and amsacrine. The ability of various 9-anilinoacridine derivatives to quench the fluorescence of DNA-bound ethidium appears to be related to the electron donor properties of the substituents on the anilino ring, as well as to experimental antitumour activity. The electron donor properties of DNA-bound amsacrine may therefore be relevant to its antitumour action.     

The fluorescence decay kinetics of in vivo chlorophyll measured using low intensity excitation

We report fluorescence lifetimes for in vivo chlorophyll a using a time-correlated single-photon counting technique with tunable dye laser excitation. The fluorescence decay of dark-adapted chlorella is almost exponential with a lifetime of 490 ps, which is independent of excitation from 570 nm to 640 nm.Chloroplasts show a two-component decay of 410 ps and approximately 1.4 ns, the proportion of long component depending upon the fluorescence state of the chloroplasts. The fluorescence lifetime of Photosystem I was determined to be 110 ps from measurements on fragments enriched in Photosystem I prepared from chloroplasts with digitonin.     

Energy dissipation efficiency in the CP43 assembly intermediate complex of photosystem II

Photosystem II in oxygenic organisms is a large membrane bound rapidly turning over pigment protein complex. During its biogenesis, multiple assembly intermediates are formed, including the CP43-preassembly complex (pCP43). To understand the energy transfer dynamics in pCP43, we first engineered a His-tagged version of the CP43 in a CP47-less strain of the cyanobacterium Synechocystis 6803. Isolated pCP43 from this engineered strain was subjected to advanced spectroscopic analysis to evaluate its excitation energy dissipation characteristics. These included measurements of steady-state absorption and fluorescence emission spectra for which correlation was tested with Stepanov relation. Comparison of fluorescence excitation and absorptance spectra determined that efficiency of energy transfer from β-carotene to chlorophyll a is 39 %. Time-resolved fluorescence images of pCP43-bound Chl a were recorded on streak camera, and fluorescence decay dynamics were evaluated with global fitting. These demonstrated that the decay kinetics strongly depends on temperature and buffer used to disperse the protein sample and fluorescence decay lifetime was estimated in 3.2–5.7 ns time range, depending on conditions. The pCP43 complex was also investigated with femtosecond and nanosecond time-resolved absorption spectroscopy upon excitation of Chl a and β-carotene to reveal pathways of singlet excitation relaxation/decay, Chl a triplet dynamics and Chl a → β-carotene triplet state sensitization process. The latter demonstrated that Chl a triplet in the pCP43 complex is not efficiently quenched by carotenoids. Finally, detailed kinetic analysis of the rise of the population of β-carotene triplets determined that the time constant of the carotenoid triplet sensitization is 40 ns.     

Effect of photosystem II reaction center closure on nanosecond fluorescence relaxation kinetics

The fluorescence decay of chlorophyll in spinach thylakoids was measured as a function of the degree of closure of Photosystem II reaction centers, which was set for the flowed sample by varying either the preillumination by actinic light or the exposure of the sample to the exciting pulsed laser light. Three exponential kinetic components originating in Photosystem II were fitted to the decays; a fourth component arising from Photosystem I was determined to be negligible at the emission wavelength of 685 nm at which the fluorescence decays were measured. Both the lifetimes and the amplitudes of the components vary with reaction center closure. A fast (170–330 ps) component reflects the trapping kinetics of open Photosystem II reaction centers capable of reducing the plastoquinone pool; its amplitude decreases gradually with trap closure, which is incompatible with the concept of photosynthetic unit connectivity where excitation energy which encounters a closed trap can find a different, possibly open one. For a connected system, the amplitude of the fast fluorescence component is expected to remain constant. The slow component (1.7–3.0 ns) is virtually absent when the reaction centers are open, and its growth is attributable to the appearance of closed centers. The middle component (0.4–1.7 ns) with approximately constant amplitude may originate from centers that are not functionally linked to the plastoquinone pool. To explain the continuous increase in the lifetimes of all three components upon reaction center closure, we propose that the transmembrane electric field generated by photosynthetic turnover modulates the trapping kinetics in Photosystem II and thereby affects the excited state lifetime in the antenna in the trap-limited case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PQ plastoquinone - PSI and PSII Photosystem I and II - Q_A and Q_B primary and secondary quinone acceptor of PSII     

Determination of the metabolic index using the fluorescence lifetime of free and bound nicotinamide adenine dinucleotide using the phasor approach

The fluorescence lifetime of nicotinamide adenine dinucleotide (NADH) is commonly used in conjunction with the phasor approach as a molecular biomarker to provide information on cellular metabolism of autofluorescence imaging of cells and tissue. However, in the phasor approach, the bound and free lifetime defining the phasor metabolic trajectory is a subject of debate. The fluorescence lifetime of NADH increases when bound to an enzyme, in contrast to the short multiexponential lifetime displayed by NADH in solution. The extent of fluorescence lifetime increase depends on the enzyme to which NADH is bound. With proper preparation of lactate dehydrogenase (LDH) using oxalic acid (OA) as an allosteric factor, bound NADH to LDH has a lifetime of 3.4 ns and is positioned on the universal semicircle of the phasor plot, inferring a monoexponential lifetime for this species. Surprisingly, measurements in the cellular environments with different metabolic states show a linear trajectory between free NADH at about 0.37 ns and bound NADH at 3.4 ns. These observations support that in a cellular environment, a 3.4 ns value could be used for bound NADH lifetime. The phasor analysis of many cell types shows a linear combination of fractional contributions of free and bound species NADH.     

FLIM and emission spectral analysis of caspase-3 activation inside single living cell during anticancer drug-induced cell death

Two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM) and emission spectral imaging (ESI) are powerful tools for fluorescence resonance energy transfer (FRET) measurement. In this study, we use these two techniques to analyze caspase-3 activation inside single living cells during anticancer drug-induced human lung adenocarcinoma (ASTC-a-1) cell death. TPE-ESI of SCAT3, a caspase-3 indicator based on FRET, was performed inside single living cell stably expressing SCAT3. The TPE-ESI measurement was performed using 780 nm excitation which was considered to selectively excite the donor ECFP of SCAT3 by measuring the emission ratio of 526 to 476 nm. The emission peak at 526 nm disappeared and that of 476 nm increased after STS or bufalin treatment, but taxol treatment did not induce a significant change for the SCAT3 emission spectra, indicating that caspase-3 was activated during STS- or bufalin-induced cell apoptosis, but was not involved in taxol-induced PCD. Fluorescence lifetime of ECFP inside living cells was acquired using FLIM. The lifetime of ECFP was the same as that of the control group after taxol treatment, but increased from 1.83 ± 0.02 to 2.05 ± 0.03 and 1.90 ± 0.03 ns, respectively after STS and bufalin treatment, which agree with the results obtained using TPE-ESI. Taken together, TPE-FLIM and ESI analysis were proved to be valuable approaches for monitoring caspase-3 activation inside single living cells. W. Pan and J. Qu contributed equally to this study.     

Time-resolved fluorescence spectroscopy of spinach chloroplast

Picosecond fluorescent kinetics and time-resolved spectra of spinach chloroplast were measured at room temperature and low temperatures. The measurement is conducted with 530 nm excitation at an average intensity of 2 · 10¹⁴ photons/cm², pulse and at a pulse separation of 6 ns for the 100 pulses used. The 685 nm fluorescent kinetics was found to decay with two components, a fast component with a 56 ps lifetime, and a slow component with a 220 ps lifetime. The 730 nm fluorescent kinetics at room temperature is a single exponential decay with a 100 ps lifetime. The 730 nm fluorescence lifetime was found to increase by a factor of 6 when the temperature was lowered from room temperature to 90 K, while the 685 and 695 nm fluorescent kinetics were unchanged. The time-resolved spectra data obtained within 10 ps after excitation is consistent with the kinetic data reported here. A two-level fluorescence scheme is proposed to explain the kinetics. The effect of excitation with high light intensity and multiple pulses is discussed.     

Multispectral fluorescence lifetime imaging system for intravascular diagnostics with ultrasound guidance: in vivo validation in swine arteries

Fluorescence lifetime technique has demonstrated potential for analysis of atherosclerotic lesions and for complementing existing intravascular imaging modalities such as intravascular ultrasound (IVUS) in identifying lesions at high risk of rupture. This study presents a multimodal catheter system integrating a 40 MHz commercial IVUS and fluorescence lifetime imaging (FLIm) using fast helical motion scanning (400 rpm, 0.75 mm/s), able to acquire in vivo in pulsatile blood flow the autofluorescence emission of arterial vessels with high precision (5.08 ± 0.26 ns mean average lifetime over 13 scans). Co‐registered FLIm and IVUS data allowed 3D visualization of both biochemical and morphological vessel properties. Current study supports the development of clinically compatible intravascular diagnostic system integrating FLIm and demonstrates, to our knowledge, the first in vivo intravascular application of a fluorescence lifetime imaging technique. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)