Nanosecond Fluorescence Lifetime Research Articles

Ultrafast electron dynamics in excited states of conjugated thiophene-fluorene organic polymer (pF8T2) thin films.

The electronic states of poly(9,9-dioctylfluorenyl-alt-bithiophene) pF8T2 on H/Si(100) substrates, prototypical for organic photovoltaics, were investigated by ultrafast photoelectron spectroscopy and by time-resolved fluorescence studies. Occupied and unoccupied electronic states were analysed by ultraviolet photoelectron spectroscopy (UPS), static and dynamic femtosecond two-photon photoemission (2PPE), and time-correlated single photon counting (TCSPC). Time-resolved measurements allow assessment of population lifetimes of intermediate states. The combination of time-resolved photoelectron spectroscopy and fluorescence excitation allows following the electronic dynamics in excited states from the femtosecond to the nanosecond time scale. For this prototypical material the electron kinetic energy resolved lifetimes range from about a few tens of femtoseconds up to hundreds of picoseconds. After annealing these types of organic thin films the efficiency of organic solar cells usually increases. We show that annealing does not influence the initial ultrafast charge generation processes, but the long-lived states. However, the nanosecond scale fluorescence lifetimes measured by TCSPC are prolonged after annealing, which therefore is identified as the cause of a greater exciton diffusion range and thus is beneficial for charge carrier extraction.

Fluorescence lifetime measurements using photon pair correlations generated via spontaneous parametric down conversion (SPDC).

We have used photon pair correlations generated via spontaneous parametric downconversion (SPDC) to measure the fluorescence lifetime of the organic dye rhodamine 6 G, demonstrating that fluorescence lifetime measurements can be achieved using a continuous wave (CW) laser, without pulsed or modulated lasers. Our entangled photon method, quantum fluorescence lifetime (Q-FL) measurements, uses one photon to excite fluorescence and the resulting fluorescence photon is timed and referenced to the arrival time of the other entangled photon. Thus, we can exploit the short timescale of photon pair correlations to conduct experiments that are typically carried out with pulsed lasers and we show that the inherent timing of the photons is fast enough to resolve the nanosecond scale fluorescence lifetime of the sample. This measurement paves the way towards using the time correlations of entangled photons for fluorescence imaging; capitalizing on the presence of fast, sub-100 ps correlations that have not been demonstrated classically.

Ultrafast Intermolecular Energy Transfer in OLED Materials: Excited-State Dynamics of a Blend of Poly(vinylcarbazole) and Oxadiazole Derivative in Solution and Film States

Intermolecular energy transfer dynamics between layers of organic materials controls the efficiency of organic light-emitting diode (OLED) devices. Here, to understand the ultrafast intermolecular energy transfer dynamics occurring in widely used UV-OLED materials of poly(vinylcarbazole) (PVK, donor: hole-transporting material) and synthesized oxadiazole derivative (OXD, acceptor: electron-transporting material), their excited-state relaxation pathways are investigated in tetrahydrofuran (THF) and film. Upon addition of OXD, the nanosecond fluorescence lifetime studies of PVK exhibited efficient quenching of dynamics, supporting the occurrence of Förster resonance energy transfer from PVK to OXD with an efficiency of ∼90%. The femtosecond time-resolved absorption measurements of the mixture of PVK and OXD revealed the intermolecular resonance energy transfer with a time constant of ∼1.32 ps from all of the intermediates in the excited states of PVK to OXD. Interestingly, in the film state, the formation of exciplex beneficial for devices with a lifetime of ∼32.66 ns was observed at ∼516 nm upon excitation of PVK in a blend of OXD and PVK. Nanosecond transient absorption spectra confirmed the occurrence of energy transfer also from the triplet state of PVK. Understanding of these excited-state dynamics occurring in solution and film states is a prerequisite to design and improve the performance of OLED materials.

Direct frequency domain fluorescence lifetime imaging using simultaneous ultraviolet and visible excitation.

Due to the complexity, limited practicality, and cost of conventional fluorescence lifetime imaging/microscopy (FLIM) instrumentation, FLIM adoption has been mostly limited to academic settings. We present a novel point scanning frequency-domain (FD) FLIM instrumentation design capable of simultaneous multi-wavelength excitation, simultaneous multispectral detection, and sub-nanosecond to nanosecond fluorescence lifetime estimation. Fluorescence excitation is implemented using intensity-modulated CW diode lasers that are available in a selection of wavelengths spanning the UV-VI-NIR range (375-1064 nm). Digital laser intensity modulation was adopted to enable simultaneous frequency interrogation at the fundamental frequency and corresponding harmonics. Time-resolved fluorescence detection is implemented using low-cost, fixed-gain, narrow bandwidth (100 MHz) avalanche photodiodes, thus, enabling cost-effective fluorescence lifetime measurements at multiple emission spectral bands simultaneously. Synchronized laser modulation and fluorescence signal digitization (250 MHz) is implemented using a common field-programmable gate array (FPGA). This synchronization reduces temporal jitter, which simplifies instrumentation, system calibration, and data processing. The FPGA also allows for the implementation of the real-time processing of the fluorescence emission phase and modulation at up to 13 modulation frequencies (processing rate matching the sampling rate of 250 MHz). Rigorous validation experiments have demonstrated the capabilities of this novel FD-FLIM implementation to accurately measure fluorescence lifetimes in the range of 0.5-12 ns. In vivo endogenous, dual-excitation (375nm/445nm), multispectral (four bands) FD-FLIM imaging of human skin and oral mucosa at 12.5 kHz pixel rate and room-light conditions was also successfully demonstrated. This versatile, simple, compact, and cost-effective FD-FLIM implementation will facilitate the clinical translation of FLIM imaging and microscopy.

Fluorescence and phosphorescence lifetime imaging reveals a significant cell nuclear viscosity and refractive index changes upon DNA damage

Cytoplasmic viscosity is a crucial parameter in determining rates of diffusion-limited reactions. Changes in viscosity are associated with several diseases, whilst nuclear viscosity determines gene integrity, regulation and expression. Yet how drugs including DNA-damaging agents affect viscosity is unknown. We demonstrate the use of a platinum complex, Pt[L]Cl, that localizes efficiently mostly in the nucleus as a probe for nuclear viscosity. The phosphorescence lifetime of Pt[L]Cl is sensitive to viscosity and provides an excellent tool to investigate the impact of DNA damage. We show using Fluorescence Lifetime Imaging (FLIM) that the lifetime of both green and red fluorescent proteins (FP) are also sensitive to changes in cellular viscosity and refractive index. However, Pt[L]Cl proved to be a more sensitive viscosity probe, by virtue of microsecond phosphorescence lifetime versus nanosecond fluorescence lifetime of FP, hence greater sensitivity to bimolecular reactions. DNA damage was inflicted by either a two-photon excitation, one-photon excitation microbeam and X-rays. DNA damage of live cells causes significant increase in the lifetime of either Pt[L]Cl (HeLa cells, 12.5–14.1 µs) or intracellularly expressed mCherry (HEK293 cells, 1.54–1.67 ns), but a decrease in fluorescence lifetime of GFP from 2.65 to 2.29 ns (in V15B cells). These values represent a viscosity change from 8.59 to 20.56 cP as well as significant changes in the refractive index (RI), according to independent calibration. Interestingly DNA damage localized to a submicron region following a laser microbeam induction showed a whole cell viscosity change, with those in the nucleus being greater than the cytoplasm. We also found evidence of a by-stander effect, whereby adjacent un-irradiated cells also showed nuclear viscosity change. Finally, an increase in viscosity following DNA damage was also observed in bacterial cells with an over-expressed mNeonGreen FP, evidenced by the change in its lifetime from 2.8 to 2.4 ns.

Low Temperature Synthesis of Fluorescent Carbon Dots from Pomegranate Peels

In recent times, much attention has been drawn to the synthesis of carbon dots from agro waste since they are a cheap source, readily available, non-toxic and in most instances disposed of as waste. Yet, the synthesis of fluorescent carbon dots at low temperatures from agro waste remain a challenge. Herein, we report for the first time on a sucessful synthesis of flourescent carbon dots, with average size 4.7 nm, at low temperature of only 100 °C using hydrothermal method from pomegranate peels as a sole precursor. The crystal structure and the morphological features of the synthesized dots were characterized by XRD, UV-vis absorption spectroscopy, photoluminescence (PL), nanosecond fluorescence lifetime, FTIR and Zeta potential measurments. The synthesized dots showed bluish emission at 440 nm, when excited at a wavelength of 360 nm, with relatively long decay lifetime of 7.4 ns.

Morphological Evolution of Strongly Fluorescent Water Soluble AIEEgen-Triblock Copolymer Mixed Aggregates with Shape-Dependent Cell Permeability.

Dimethyl-2,5-bis(4-methoxyphenylamino)terephthalate (DBMPT) is a water-insoluble fluorogenic molecule, which has been rendered water-soluble in physiological conditions, by the addition of triblock copolymers (TBPs), P123 (PEO19PPO69PEO19), and F127 (PEO100PPO65PEO100). DBMPT-TBP mixed aggregates, formed in the process, exhibit significant aggregation-induced enhancement of emission, with nanosecond fluorescence lifetimes. Dynamics involved in suppression of nonradiative pathways and consequent enhancement of fluorescence are followed by femtosecond transient absorption and time-resolved fluorescence spectroscopic techniques. Interestingly, shapes of the aggregates formed with the two TBPs are found to be very different, even though they differ only in the length of hydrophilic blocks. DBMPT-P123 aggregates are micrometer-sized and spherical, while DBMPT-F127 aggregates form nanorods. Evolution of their morphologies, as a function of TBP concentration, is monitored using cryo-TEM, FESEM, and fluorescence lifetime imaging microscopy. Fluorescence lifetime distribution provides useful insight into microheterogeneity in these mixed aggregates. Excellent cell permeability is observed for DBMPT-F127 nanorods, in contrast to DBMPT-P123 microspheres. These fluorescent nanorods exhibit the ability to mark lipid droplets within the cell and hence bear the promise for application in intracellular imaging.

Fluorescence lifetime measurements with simple correction for instrument temporal response in the advanced undergraduate laboratory

Observation of time-dependent luminescence from excited states with a wide range of lifetimes allows students to explore the connection between selection rules and transition rates. It is fairly simple to measure microsecond and longer lifetimes with equipment common to undergraduate programs, because the instrument response time of even modest bandwidth systems is insignificant on microsecond and longer time scales. The measurement of nanosecond lifetimes, however, is more challenging, because the instrument response time is comparable to the lifetimes being measured. In this case, the instrument temporal response must be deconvolved from the observed luminescence signals in order to extract the actual excited state lifetime. We describe a method for measuring nanosecond fluorescence lifetimes in the advanced undergraduate laboratory that uses real-time analog luminescence signals instead of traditional photon counting techniques. The detection electronics of this method are fairly simple, consisting of an oscilloscope monitoring the time-dependent output of an inexpensive silicon photomultiplier. We introduce a simple and transparent method for students to characterize the instrument response and deconvolve it from the observed luminescence signals, yielding measured nanosecond fluorescence lifetimes in good agreement with the corresponding literature values obtained by time-correlated single photon counting. The limitations of silicon photomultipliers for this method of measuring nanosecond lifetimes are discussed in detail. Application of this treatment to decay processes that are not single exponential is also discussed.

Dehydrogenase Binding Sites Abolish the "Dark" Fraction of NADH: Implication for Metabolic Sensing via FLIM.

The fluorescence of dinucleotide NADH has been exploited for decades to determine the redox state of cells and tissues in vivo and in vitro. Particularly, nanosecond (ns) fluorescence lifetime imaging microscopy (FLIM) of NADH (in free vs bound forms) has recently offered a label-free readout of mitochondrial function and allowed the different "pools" of NADH to be distinguished in living cells. In this study, the ultrafast fluorescence dynamics of NADH-dehydrogenase (MDH/LDH) complexes have been investigated by using both a femtosecond (fs) upconversion spectrophotofluorometer and a picosecond (ps) time-correlated single photon counting (TCSPC) apparatus. With these enhanced time-resolved tools, a few-picosecond decay process with a signatory spectrum was indeed found for bound NADH, and it can best be ascribed to the solvent relaxation originating in "bulk water". However, it is quite unlike our previously discovered ultrafast "dark" component (∼26 ps) that is prominent in free NADH (Chemical Physics Letters 2019, 726, 18-21). For these two critical protein-bound NADH exemplars, the decay transients lack the ultrafast quenching that creates the "dark" subpopulation of free NADH. Therefore, we infer that the apparent ratio of free to bound NADH recovered by ordinary (>50 ps) FLIM methods may be low, since the "dark" molecule subpopulation (lifetime too short for conventional FLIM), which effectively hides about a quarter of free molecules, is not present in the dehydrogenase-bound state.

Fluorescence lifetime estimation by 1-bit photon autocorrelation procedure from time-series data recorded using a high-speed digitizer

We propose a photon-counting fluorescence lifetime measurement scheme that uses a 1-bit autocorrelation procedure. The proposed scheme is an extension of the previously reported lifetime measurements using the autocorrelation analysis (Nishimura et al. in: Phys Med Biol 48:N283–N290, 2003; Nishimura et al. in: J Biomed Opt 12:020503, 2007). The fluorescence photons obtained from a high-repetition-rate, pulsed excitation light source are fed into a photodetector, and its output photoelectron pulse train is stored in a transient memory composed of a high-speed digitizer that retains time information over a large number of excitation cycles. After the stored data have been binarized, a 1-bit autocorrelation procedure is performed to estimate the fluorescence lifetimes through a repetitive curve-fitting process. We could successfully demonstrate this scheme in nanosecond fluorescence lifetime measurements with a progressed high-speed digitizer, which requires no synchronization with the excitation timing. To date, the minimum sampling interval and the maximum number of sample points of the digitizer are 400 ps and ~ 1 × 109, respectively. We present the results of some fluorescence lifetime measurements of standard samples that were performed as a proof of principle experiment.

A Novel Viewpoint to Analyze Structured Illumination Microscopy (Sim) Data

Structured Illumination Microscopy (SIM) is an important tool among the Super Resolution Microscopy techniques [1]. SIM stands out for its capability to perform optical sectioning, multicolor channel acquisition and live cell imaging, thanks to its low phototoxicity. In SIM, the specimen is illuminated with a high-frequency grating pattern, instead of a uniform field. The multiplication of sample features and this pattern gives rise to Moiré fringes.The obtained interference pattern allows encoding of sub-diffraction sample's features into lower frequencies collected by the objective lens. The reconstruction process then decodes otherwise unresolvable high-frequency information creating the super-resolved image.Here we aim to analyze SIM data from a rather different standpoint, based on the idea of Separation of Photons by LIfetime Tuning (SPLIT)[2]. In SPLIT the sub-diffraction spatial information is encoded into an additional channel of the microscope. The information encoded within this channel is visualized and analyzed in the phasor plot and allows separating a fractional image component of higher spatial resolution corresponding to the center of the PSF.In the context of Stimulated Emission Depletion (STED) microscopy, we recently demonstrated that SPLIT can be used to decode spatial information encoded in the nanosecond fluorescence lifetime [2] or variations of the depletion power [3]. We show here that in the case of SIM it is possible to use SPLIT to analyze the information encoded into the images acquired at different illumination patterns. In particular an important aspect of SPLIT is that the Phasor Plot provides a visual, intuitive and direct evaluation of the acquired data [3].We demonstrate that knowledge of the illumination pattern can be used to perform SPLIT analysis and to reconstruct a super-resolved image bypassing the traditional Fourier reconstruction.As an application, we use the SPLIT-SIM approach to perform super-resolution imaging of chromatin-related structures.

Influence of Nonenzymatic Glycation in Dentinal Collagen on Dental Caries

Advanced glycation end-products (AGEs) are generated via nonenzymatic glycation of dentinal collagen, resulting in accumulation of AGEs in dentin tissue. Since accumulated AGEs cause crosslinking between amino acid polypeptides in the collagen molecule and modify mechanical properties of dentinal collagen, the authors assumed that there would be a significant interaction between the generation of AGEs and progression of caries in dentin. To confirm such an interaction, spectroscopic imaging analyses (i.e., nanosecond fluorescence lifetime imaging and second harmonic generation light imaging) were performed in addition to biochemical and electron microscopic analyses in the present study. Seven carious human teeth were fixed in paraformaldehyde and cut longitudinally into 1-mm sections using a low-speed diamond saw for the following analyses. In transmission electron microscopy (TEM) analysis, nondecalcified specimens were embedded in epoxy resin and sliced into thin sections for observation. For the immunohistochemical analysis, the specimens were paraffin embedded after decalcification for 2 wk and sectioned with a microtome. Resultant sections were stained with anti-AGE and anticollagen antibodies. The demineralized specimens were used for spectroscopic analyses without additional treatment. For Western blotting analysis, specimens were separated into carious and sound dentin. Each specimen was homogenized with a bead crusher and an ultrasonic homogenizer and then treated with hydrochloric acid. In carious dentin, the collagen fibers showed an amorphous structure in the TEM image, and the AGEs were localized in the areas of bacterial invasion in the immunostaining image. The total amount of AGEs in carious dentin was higher than in sound dentin in Western blotting. The ultrastructure of type I collagen and total amount of AGEs varied markedly in the dentinal caries region. The fluorescence lifetime was shorter in the carious area than that in the sound areas, indicating an increase of AGEs in the carious area. The increase of AGEs could influence the progression of dentinal caries.

Outright Green Synthesis of Fluorescent Carbon Dots from Eutrophic Algal Blooms for In Vitro Imaging

Carbon dots (CDs) synthesized from biological sources have attracted much interest in bioimaging and biomedical applications due to their excellent biocompatibility, and thus, a facile synthesis of CDs with high fluorescence quantum yield (QY) is requisite for practical applications. In this work, we report a simple, rapid, and green approach to synthesize photoluminescent CDs using eutrophic algal blooms as the carbon source. This method offers a possibility for large scale production of highly luminescent CDs (QY = 13%) with the average particle size ∼8 nm. These CDs are highly water-soluble and exhibit nanosecond fluorescence lifetime with high photostability, luminescence stability in different environments, low cytotoxicity, and excellent cell permeability. Laser scanning confocal microscopy shows the uptake of CDs by MCF-7 cells, and the destined application of these CDs as a potential biomarker is demonstrated.

Time-Resolved Fluorescence of Water-Soluble Pyridinium Salt: Sensitive Detection of the Conformational Changes of Bovine Serum Albumin.

In this paper, we report a pyridinium salt "turn-on" fluorescent probe, 4-[2-(4-Dimethylamino-phenyl)-vinyl]-1-methylpyridinium iodide (p-DASPMI), and applied its time-resolved fluorescence (TRF) to monitor the protein conformational changes. Both the fluorescence lifetime and quantum yield (QY) of p-DASPMI were increased about two orders of magnitude after binding to the protein bovine serum albumin (BSA). The free p-DASPMI in solution presents an ultrashort fluorescence lifetime (12.4 ps), thus it does not interfere the detection of bound p-DASPMI which has nanosecond fluorescence lifetime. Decay-associated spectra (DAS) show that p-DASPMI molecules bind to subdomains IIA and IIIA of BSA. The TRF decay profiles of p-DASPMI can be described by multi-exponential decay function ([Formula: see text]), and the obtained parameters, such as lifetimes ([Formula: see text]), fractional amplitudes ([Formula: see text]), and fractional intensities ([Formula: see text]), may be used to deduce the conformational changes of BSA. The pH and Cu2+ induced conformational changes of BSA were investigated through the TRF of p-DASPMI. The results show that the p-DASPMI is a candidate fluorescent probe in studying the conformational changes of proteins through TRF spectroscopy and microscopy in the visible range.

(Invited) Multiple Photosynthetic Reaction Centers of Porphyrinic Polypeptide-Li+@C60 Supramolecular Complexes

Extensive efforts have so far been devoted to mimic light harvesting and charge separation processes in natural photosynthesis.1 Multiporphyrin arrays have been employed as light-harvesting units. Light-harvesting and charge-separation units have been combined by coordination bonds between metalloporphyrins and electron acceptor moieties bearing Lewis base ligands. Thus, metal centres have been required for construction of supramolecular complexes between porphyrins acting as light harvesting units and electron acceptors containing Lewis base ligands for coordination to the metal centers. As such there has been only few report on supramolecular complexes of free base porphyrin arrays and electron acceptors without Lewis base ligands, in which the binding is rather weak. We report herein construction of supramolecular complexes of free base porphyrin polypeptides (P(H2P) n : n = 4, 8) with lithium ion-encapsulated C60 (Li+@C60)2,3 (Figure 1), in which binding is much stronger than the caes of C60 in benzonitirle (PhCN). The photodynaimcs were studied by femtosecond and nanosecond laser-induced transient absorption and fluorescence lifetime measurements.4 Upon mixing a PhCN solution of Li+@C60 with that of P(H2P)8, the intensity of the Soret band decreased with increasing concentration of Li+@C60. A Soret band was slightly red-shifted from 425 to 427 nm by the addition of Li+@C60 to the solution. From the net absorption change at 425 nm in which the absorption due to Li+@C60 was subtracted, a linear Benesi-Hildebrand plot was obtained, indiating that each porphyrin unit of P(H2P)8 forms a 1:1 supramolecular charge-transfer complex with Li+@C60 independently with approximately the same binding constant of the unit of M–1. The binding constant at 298 K was determined to be 2.1 ´ 104 M–1, which is significantly larger than that of C60 (5.3 ´ 103 M–1). The stronger binding of Li+@C60 as compared with C60 may result from the stronger elecron acceptor ability of Li+@C60, which facilitates the charge-transfer interaction as reported for the stronger charge-transfer binding of Li+@C60 with corranulerene. Similary the binding constant of the supramolecular complex of P(H2P)4 was determined from the spectral titration to be 6.2 × 103 M–1. In the case of P(H2P)2 and P(H2P)1, howerver, the spectral change was too small to be able to determine the binding constants accurately. Thus, multiporphyris may facilitate charge-transfer interactions through encapsulation of Li+@C60 by multiple porphyrins.4 Nanosecond laser-induced transient absorption spectra of P(H2P)8 with Li+@C60 (40 mM) at the excitation wavelength of 532 nm showed the transient absorption band at 730 nm due to the triplet excited state of Li+@C60 (3Li+@C60 *) observed together with the absorption band at 1035 nm due to Li+@C60 •–. The decay of the absorption at 730 nm coincides with the appearance of Li+@C60 •–. Thus, electron transfer from P(H2P)8 to 3Li+@C60 * occurs to produce the triplet charge-separate (CS) state of P(H2P)8 •+ and Li+@C60 •–. The transient absorption due to P(H2P)8 •+ is overlapped with that of 3Li+@C60 * in the 600-700nm region. The photoexcitation of 532 nm resulted in the excitation of Li+@C60 leading to the formation of 3Li+@C60 * via intersystem crossing. The decay of the absorbance at 1035 nm due to Li+@C60 •– obeyed first-order kinetics with the lifetime of 210 ms. Thus, back electron transfer from Li+@C60 •– to P(H2P)8 •+ occurs in the supramolecular complex. The lifetime is long because of the spin-forbidden back electron transfer in the triplet CS state.4 References [1] Fukuzumi, S.; Ohkubo, K.; Suenobu, T. Acc. Chem. Res. 2014, 47, 1455. [2] Kawashima, Y.; Ohkubo, K.; Fukuzumi, S. Chem. Asian J. 2015, 10, 44. (Review) [3] (a) N. Solladié , A. Hamel and M. Gross, Tetrahedron Lett., 2000, 41, 6075; (b) F. Aziat, R. Rein, J. Peon, E. Rivera and N. Solladié, J. Porphyrins Phthalocyanines, 2008, 12, 1232 [4] Ohkubo, K.; Hasegawa, T.; Rein, R.; Solladié, N.; Fukuzumi, S. Chem. Commun. 2015, 51. 17517. Figure 1

Synthesis of Blue-, Green-, Yellow-, and Red-Emitting Graphene-Quantum-Dot-Based Nanomaterials with Excitation-Independent Emission

A one-pot method is described for the preparation of graphene quantum dots/graphene oxide (GQDs/GO) hybrid composites with emission in the visible region, through heteroatom doping and hydroxyl-radical-induced decomposition of GO. The NH4OH- and thiourea-mediated dissociation of H2O2 produces hydroxyl radicals. Treatment of GO with hydroxyl radicals results in the production of small-sized GO sheets and GQDs, which self-assemble to form GQDs/GO through strong π–π interactions. For example, the reaction of GO with a mixture of NH4OH and H2O2 for 40, 120, and 270 min generates yellow-emitting GQDs/GO (Y-GQDs/GO), green-emitting GQDs/GO, and blue-emitting GQDs, while red-emitting GQDs/GO (R-GQDs/GO) are prepared by incubating GO with a mixture of thiourea and H2O2. From the analysis of these four GQD-based nanomaterials by transmission electron microscopy, atomic force microscopy, and fluorescence lifetime spectroscopy, it is found that this tunable fluorescence wavelength results from the differences in particle size. All four GQD-based nanomaterials exhibit moderate quantum yields (1–10%), nanosecond fluorescence lifetimes, and excitation-independent emissions. Except for R-GQDs/GO, the other three GQD-based nanomaterials are stable in a high-concentration salt solution (e.g., 1.6 m NaCl) and under high-power irradiation, enabling the sensitive (high-temperature resolution and large activation energy) and reversible detection of temperature change. It is further demonstrated that Y-GQD/GO can be used to image HeLa cells.

A portable time-domain LED fluorimeter for nanosecond fluorescence lifetime measurements.

Fluorescence lifetime measurements are becoming increasingly important in chemical and biological research. Time-domain lifetime measurements offer fluorescence multiplexing and improved handling of interferers compared with the frequency-domain technique. In this paper, an all solid-state, filterless, and highly portable light-emitting-diode based time-domain fluorimeter (LED TDF) is reported for the measurement of nanosecond fluorescence lifetimes. LED based excitation provides more wavelengths options compared to laser diode based excitation, but the excitation is less effective due to the uncollimated beam, less optical power, and longer latency in state transition. Pulse triggering and pre-bias techniques were implemented in our LED TDF to improve the peak optical power to over 100 mW. The proposed pulsing circuit achieved an excitation light fall time of less than 2 ns. Electrical resetting technique realized a time-gated photo-detector to remove the interference of the excitation light with fluorescence. These techniques allow the LED fluorimeter to accurately measure the fluorescence lifetime of fluorescein down to concentration of 0.5 μM. In addition, all filters required in traditional instruments are eliminated for the non-attenuated excitation/emission light power. These achievements make the reported device attractive to biochemical laboratories seeking for highly portable lifetime detection devices for developing sensors based on fluorescence lifetime changes. The device was initially validated by measuring the lifetimes of three commercial fluorophores and comparing them with reported lifetime data. It was subsequently used to characterize a ZnSe quantum dot based DNA sensor.

Advanced non-invasive fluorescence spectroscopy and imaging for mapping photo-oxidative degradation in acrylonitrile–butadiene–styrene: A study of model samples and of an object from the 1960s

Complementary use of fluorescence multispectral imaging and fluorescence lifetime imaging (FLIM) for the assessment of the degradation of a historical design object made in acrylonitrile–butadiene–styrene (ABS) is demonstrated. Model samples of ABS, exposed to UV irradiation to simulate photodegradation, have been analysed with fluorescence spectroscopy and fluorescence lifetime spectroscopy. Results demonstrate rapid modifications of the picosecond and nanosecond fluorescence lifetimes following short irradiation times, which are accompanied with significant modifications in fluorescence excitation-emission spectra. Changes in spectra and lifetimes in samples are ascribed to the accumulation of α,β unsaturated aldehydes and α,β diketones and the loss of volatile organic components (including acetophenone). Complementary analyses of samples with Fourier transform infrared spectroscopy suggest that modifications in fluorescence are correlated with photo-oxidative degradation of ABS. Fluorescence lifetime and fluorescence multispectral images of the Grillo telephone (1965) made in ABS were acquired. Analysis of images allowed the rapid mapping of differences in fluorescence which are attributed to heterogeneous degradation on the object surface. Implications for the assessment, monitoring and conservation of objects in ABS and other polymers are suggested.

Overview of Live‐Cell Imaging: Requirements and Methods Used

Overview of Live‐Cell Imaging: Requirements and Methods Used

Time-resolved nanosecond fluorescence lifetime imaging and picosecond infrared spectroscopy of combretastatin A-4 in solution and in cellular systems**DedicationThis paper is dedicated to the memory of Professor Christopher G Morgan (1949–2011). He was a valued colleague and friend at the University of Salford and made significant contributions to the development and applications of fluorescence lifetime imaging.

Fluorescence lifetime images of intrinsic fluorescence obtained with two-photon excitation at 630 nm are shown following uptake of a series of E-combretastatins into live cells, including human umbilical vein endothelial cells (HUVECs) that are the target for the anticancer activity of combretastatins. Images show distribution of the compounds within the cell cytoplasm and in structures identified as lipid droplets by comparison with images obtained following Nile red staining of the same cells. The intracellular fluorescent lifetimes are generally longer than in fluid solution as a consequence of the high viscosity of the cellular environment. Following incubation, the intracellular concentrations of a fluorinated derivative of E-combretastatin A-4 in HUVECs are between two and three orders of magnitude higher than the concentration in the surrounding medium. Evidence is presented to indicate that at moderate laser powers (up to 6 mW), it is possible to isomerize up to 25% of the combretastatin within the femtolitre focal volume of the femtosecond laser beam. This suggests that it may be possible to activate the E-combretastatin (with low cellular toxicity) to the Z-isomer with high anticancer drug activity using two-photon irradiation. The isomerization of Z- and E-combretastatins by 266 nm irradiation has been probed by ultrafast time-resolved infrared spectroscopy. Results for the E-isomer show a rapid loss of excess vibrational energy in the excited state with a lifetime of 7 ps, followed by a slower process with a lifetime of 500 ps corresponding to the return to the ground state as also determined from the fluorescence lifetime. In contrast, the Z-isomer, whilst also appearing to undergo a rapid cooling of the initial excited state, has a much shorter overall excited state lifetime of 14 ps.