Time-resolved Fluorescence Imaging Research Articles

Using Phasors in Interpreting One- and Two-Photon Fluorescence Lifetime Images of Fruit and Polymer Interfaces

Phasors prove to be an elegant way of characterizing time-resolved fluorescence images, (Digman et al., Biophys. J., 94, 1483-96, 2008). Fast Flim micro- and macro imaging (Biophys. J., 82, 502a) was applied to: 1. the pre-symptom and early detection of biotic and abiotic stress as well as surface defects and physiological disorders in fruit tissue using photosystem II Chlorophyll a fluorescence and 2. the characterization of conjugated polymer film produced under various conditions for biosensor development. Both Olympus and Zeiss imaging systems were used in conjunction with one photon 488 nm and 80 MHz, typically 15 mW two-photon illumination. For comparison overview color or transmission images were also collected. Several spots spread over the surfaces were used. Images have been analysed using phasors with Globals for Images, aka. SimFCS (LFD, UCI, CA, USA). The potential of the phasor approach as analysis tool for detection of both ageing and physiological stress progression (biological surfaces) and the influence of bleaching and preparation methods (polymer interfaces) is discussed.

3P327 タンパク質の時間分解蛍光イメージング法の開発(バイオイメージング,第48回日本生物物理学会年会)

3P327 タンパク質の時間分解蛍光イメージング法の開発(バイオイメージング,第48回日本生物物理学会年会)

Excitation emission and time-resolved fluorescence spectroscopy of selected varnishes used in historical musical instruments

The analysis of various varnishes from different origins, which are commonly found on historical musical instruments was carried out for the first time with both fluorescence excitation emission spectroscopy and laser-induced time-resolved fluorescence spectroscopy. Samples studied include varnishes prepared using shellac, and selected diterpenoid and triterpenoid resins from plants, and mixtures of these materials. Fluorescence excitation emission spectra have been collected from films of naturally aged varnishes. In parallel, time-resolved fluorescence spectroscopy of varnishes provides means for discriminating between short- (less than 2.0 ns) and long-lived (greater than 7.5 ns) fluorescence emissions in each of these complex materials. Results suggest that complementary use of the two non destructive techniques allows a better understanding of the main fluorophores responsible for the emission in shellac, and further provides means for distinguishing the main classes of other varnishes based on differences in fluorescence lifetime behaviour. Spectrofluorimetric data and time resolved spectra presented here may form the basis for the interpretation of results from future in situ fluorescence examination and time resolved fluorescence imaging of varnished musical instruments.

Lateral Distribution of the Transmembrane Domain of Influenza Virus Hemagglutinin Revealed by Time-resolved Fluorescence Imaging

Influenza virus hemagglutinin (HA) has been suggested to be enriched in liquid-ordered lipid domains named rafts, which represent an important step in virus assembly. We employed Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy to study the interaction of the cytoplasmic and transmembrane domain (TMD) of HA with agly co sylphos pha tidyl ino si tol (GPI)-anchored peptide, an established marker for rafts in the exoplasmic leaflet of living mammalian plasma membranes. Cyan fluorescent protein (CFP) was fused to GPI, whereas the HA sequence was tagged with yellow fluorescent protein (YFP) on its exoplasmic site (TMD-HA-YFP), avoiding any interference of fluorescent proteins with the proposed role of the cytoplasmic domain in lateral organization of HA. Constructs were expressed in Chinese hamster ovary cells (CHO-K1) for which cholesterol-sensitive lipid nanodomains and their dimension in the plasma membrane have been described (Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., Rao, M., and Mayor, S. (2004) Cell 116, 577-589). Upon transfection in CHO-K1 cells, TMD-HA-YFP is partially expressed as a dimer. Only dimers are targeted to the plasma membrane. Clustering of TMD-HA-YFP with GPI-CFP was observed and shown to be reduced upon cholesterol depletion, a treatment known to disrupt rafts. No indication for association of TMD-HA-YFP with GPI-CFP was found when palmitoylation, an important determinant of raft targeting, was suppressed. Clustering of TMD-HA-YFP and GPI-CFP was also observed in purified plasma membrane suspensions by homoFRET. We concluded that the pal mit oy lated TMD-HA alone is sufficient to recruit HA to cholesterol-sensitive nanodomains. The corresponding construct of the spike protein E2 of Semliki Forest virus did not partition preferentially in such domains.

A Single-Photon Avalanche Diode Array for Fluorescence Lifetime Imaging Microscopy.

We describe the design, characterization, and demonstration of a fully integrated single-photon avalanche diode (SPAD) imager for use in time-resolved fluorescence imaging. The imager consists of a 64-by-64 array of active SPAD pixels and an on-chip time-to-digital converter (TDC) based on a delay-locked loop (DLL) and calibrated interpolators. The imager can perform both standard time-correlated single-photon counting (TCSPC) and an alternative gated-window detection useful for avoiding pulse pile-up when measuring bright signal levels. To illustrate the use of the imager, we present measurements of the decay lifetimes of fluorescent dyes of several types with a timing resolution of 350 ps.

Multispectral fluorescence lifetime imaging of feces-contaminated apples by time-resolved laser-induced fluorescence imaging system with tunable excitation wavelengths

We recently developed a time-resolved multispectral laser-induced fluorescence (LIF) imaging system capable of tunable wavelengths in the visible region for sample excitation and nanosecond-scale characterizations of fluorescence responses (lifetime imaging). Time-dependent fluorescence decay characteristics and fluorescence lifetime imaging of apples artificially contaminated with a range of diluted cow feces were investigated at 670 and 685 nm emission bands obtained by 418, 530, and 630 nm excitations. The results demonstrated that a 670 nm emission with a 418 nm excitation provided the greatest difference in time-dependent fluorescence responses between the apples and feces-treated spots. The versatilities of the time-resolved LIF imaging system, including fluorescence lifetime imaging of a relatively large biological object in a multispectral excitation-emission wavelength domain, were demonstrated.

Examination of laser microbeam cell lysis in a PDMS microfluidic channel using time-resolved imaging.

We use time-resolved imaging to examine the lysis dynamics of non-adherent BAF-3 cells within a microfluidic channel produced by the delivery of single highly-focused 540 ps duration laser pulses at lambda = 532 nm. Time-resolved bright-field images reveal that the delivery of the pulsed laser microbeam results in the formation of a laser-induced plasma followed by shock wave emission and cavitation bubble formation. The confinement offered by the microfluidic channel constrains substantially the cavitation bubble expansion and results in significant deformation of the PDMS channel walls. To examine the cell lysis and dispersal of the cellular contents, we acquire time-resolved fluorescence images of the process in which the cells were loaded with a fluorescent dye. These fluorescence images reveal cell lysis to occur on the nanosecond to microsecond time scale by the plasma formation and cavitation bubble dynamics. Moreover, the time-resolved fluorescence images show that while the cellular contents are dispersed by the expansion of the laser-induced cavitation bubble, the flow associated with the bubble collapse subsequently re-localizes the cellular contents to a small region. This capacity of pulsed laser microbeam irradiation to achieve rapid cell lysis in microfluidic channels with minimal dilution of the cellular contents has important implications for their use in lab-on-a-chip applications.

The effect of annealing/quenching on the performance of polymer light-emitting electrochemical cells

We report the effect of thermal annealing and quenching on the film morphology and device performance of polymer light-emitting electrochemical cells (LECs). The polymer films of LECs consist of a luminescent polymer, an ion-conducting polymer, and a lithium salt. The LECs studied have an extremely large planar configuration, which enables time-resolved fluorescence imaging of both doping and emission profiles of the devices. Annealing at temperatures above 350K leads to the disappearance of many visible “white dots” initially present in the LEC film, and a much smoother surface. Annealed and quenched devices exhibit dramatically improved initial and peak current, peak electroluminescence (EL) intensity, doping propagation speed and response time. In addition, the emission zone of annealed devices is more centered than un-annealed devices. These improvements are attributed to the melting of electrolyte domains in the LEC film, which leads to better film quality and enhanced ion conductivity. Our results demonstrate that the simple annealing/quenching technique can be used to achieve the desired phase morphology in LEC films, which are often severely phase-separated due to incompatibility between the luminescent polymer and the electrolyte polymer.

Three-dimensional time-resolved fluorescence imaging by multifocal multiphoton microscopy for a photosensitizer study in living cells

Two-photon fluorescence microscopy is widely applied to biology and medicine to study both the structure and dynamic processes in living cells. The main issue is the slow acquisition rate due to the point scanning approach limiting the multimodal detection (x, y, z, t). To extend the performances of this powerful technique, we present a time-resolved multifocal multiphoton microscope (MMM) based on laser amplitude splitting. An array of 8 x 8 foci is created on the sample that gives a direct insight of the fluorescence localization. Four-dimensional (4D) imaging is obtained by combining simultaneous foci scanning, time-gated detection, and z displacement. We illustrate time-resolved MMM capabilities for 4D imaging of a photosensitizer inside living colon cancer cells. The aim of this study is to have a better understanding of the photophysical processes implied in the photosensitizer reactivity.

Blinking suppression and intensity recurrences in single CdSe-oligo(phenylene vinylene)nanostructures: experiment and kinetic model

We report time-resolved single molecule fluorescence imaging of individual CdSe quantumdots that are functionalized with oligomeric conjugated organic ligands. The fluorescenceintensity trajectories from these composite nanostructures display both a strong degree ofblinking suppression and intensity fluctuations with characteristic recurrence times on theorder of 10–60 s. In addition, fluorescence decay rate measurements of individual hybridnanostructures indicate significantly modified non-radiative quantum dot decay ratesrelative to conventional ZnS-capped CdSe quantum dots. We show that a modified diffusivereaction coordinate model with slow fluctuations in quantum dot electron energies(1Se,1Pe) can reproduce the experimentally observed behaviour.

Two-photon absorption cross-sections and time-resolved fluorescence imaging using porphyrin photosensitisers

Three porphyrin systems have been characterised for use in two-photon fluorescence imaging of biological samples. We have determined the two-photon absorption cross sections (sigma(2)) of the di-cation, free-base and metallated forms of hematoporphyrin derivative (HpD), hematoporphyrin IX (Hp9) and a boronated protoporphyrin (BOPP) using the open-aperture Z-scan and the two-photon induced fluorescence (TPIF) techniques at an excitation wavelength of 800 nm. The insertion of either protons or a metal ion into the macrocycle is shown not to significantly influence the sigma(2) of the porphyrins. Two-photon time-resolved fluorescence images of C6 glioma cells transfected with a free-base form of the BOPP have been obtained as a function of the porphyrin concentration. These studies reveal a maximum useful porphyrin concentration for fluorescence imaging purposes of approximately 30 microg mL(-1).

Time-resolved fluorescence spectroscopy and imaging of proteinaceous binders used in paintings

The differentiation of proteins commonly found as binding media in paintings is presented based on spectrally resolved and time-resolved laser-induced fluorescence (LIF) and total emission spectroscopy. Proteins from eggs and animal glue were analysed with pulsed laser excitation at 248 nm (KrF excimer) and 355 nm (third harmonic of Nd:YAG) for spectrally resolved measurements, and at 337 nm (N2) and 405 nm (N2 pumped dye laser) for spectrally resolved lifetime measurements and fluorescence lifetime imaging (FLIM). Total emission spectra of binding media are used for the interpretation of LIF spectra. Time-resolved techniques become decisive with excitation at longer wavelengths as fluorescence lifetime permits the discrimination amongst binding media, despite minimal spectral differences; spectrally resolved measurements of fluorescence lifetime have maximum differences between the binding media examined using excitation at 337 nm, with maximum observed fluorescence at 410 nm. FLIM, which measures the average lifetime of the emissions detected, can also differentiate between media, is non-invasive and is potentially advantageous for the analysis of paintings.

Phase-locked 10MHz reference signal for frequency domain time-resolved fluorescence measurements

A complete electronic system that is suitable for use in megahertz frequency domain time-resolved fluorescence instruments based on mode-locked lasers is described. The circuit produces a 10 MHz signal, phase locked to the mode-locked laser pulse frequency, which is required by many commercial frequency synthesizers as the external reference signal. This device is particularly useful in conjunction with ultrafast gated intensified charge coupled device cameras capable of being frequency modulated for time-resolved fluorescence imaging.

Multifunctional nanoparticles possessing magnetic, long-lived fluorescence and bio-affinity properties for time-resolved fluorescence cell imaging

Multifunctional nanoparticles possessing magnetic, long-lived fluorescence and bio-affinity properties have been prepared by copolymerization of a conjugate of (3-aminopropyl)triethoxysilane bound to a fluorescent Eu 3+ complex, 4,4′-bis(1″,1″,1″-trifluoro- 2″,4″-butanedion-4″-yl)chlorosulfo- o-terphenyl-Eu 3+ (APS-BTBCT-Eu 3+), free (3-aminopropyl)triethoxysilane (APS) and tetraethyl orthosilicate (TEOS) in the presence of poly(vinylpyrrolidone) (PVP) stabilized magnetic Fe 3O 4 nanoparticles (∼10 nm) with aqueous ammonia in ethanol. The nanoparticles were characterized by transmission electron microscopy (TEM), spectrofluorometry and vibrating sample magnetometry methods. The direct-introduced amino groups on the nanoparticle's surface by using free APS in nanoparticle preparation facilitated the surface modification and bioconjugation of the nanoparticles. The nanoparticle-labeled transferrin was prepared and used for staining the cultured Hela cells. A time-resolved fluorescence imaging technique that can fully eliminate the fast-decaying background noises was developed and used for the fluorescence imaging detection of the cells. A distinct image with the high ratio of signal to noise (S/N) was obtained.

Time‐Resolved Fluorescence Imaging of Islet Cell Autoantibodies

Time-resolved fluorescence of lanthanide chelates has been widely used in bioanalytical assays. Long fluorescence time, large Stokes shift, and minute fading out of the fluorescence over years are major advantages of the lanthanides over the conventional fluorescent dyes. We have now applied time-resolved fluorescence imaging (TRFI) also for measurement of type 1 diabetes mellitus (T1DM)-related islet cell autoantibodies (ICA). Retaining the accuracy of conventional ICA, TRFI has over 10 times better signal-to-noise ratio than the conventional fluorochromes. The technology allows objective determination of fluorescence intensity with the camera and computer software, and serial dilutions for obtaining the antibody titer in autoantibody-positive samples are unnecessary. We now describe the TRFI as a method and its application for measurement of ICA.

A Novel Method for Time‐Resolved Fluorimetric Determination and Imaging of the Activity of Peroxidase, and Its Application to an Enzyme‐Linked Immunosorbent Assay

A new type of fluorescence assay for the determination of peroxidase (POx) activity is presented. The assay is based on the indication of the enzymatic consumption of H(2)O(2) (HP), using a fluorescent europium-tetracycline (Eu(3)TC) complex as indicator. On addition of HP, this complex forms a highly fluorescent adduct (Eu(3)TC-HP), which is decomposed in the presence of POx to form the weakly fluorescent europium-tetracycline (Eu(3)TC). Hence, the activity of the enzyme can be directly determined by means of the luminescent Eu(3)TC complex as indicator. The POx assay demonstrated herein was elaborated starting from a spectral characterization of the complex systems involved. Due to the long lifetime of lanthanide luminescence, both steady-state and time-resolved luminescence assays can easily be performed. The time-resolved assay can quantify POx in the range from 4.0 x 10(-5) to 5.9 x 10(-3) U mL(-1), with a limit of detection of 1.0 x 10(-5) U mL(-1). The effects of POx inhibitors such as cyanide, hydroxylamine, and azide have also been studied. In addition, a time-resolved fluorescent detection method for a POx-based enzyme-linked immunosorbent assay (ELISA) has been developed, which is demonstrated in a sandwich model assay with bovine IgG serving as analyte. Furthermore, a time-resolved fluorescent imaging method is demonstrated that makes use of a straightforward imaging set-up adjusted to the optical properties of the europium reagent.

Development of an ultrafast single photon counting imager for single molecule imaging.

We have begun developing an innovative ultra-fast single-photon counting imager which comprises a mega-pixel CMOS array and a newly-designed Image Intensifier. It is expected to have single photon sensitivity with 100 psec time resolution, operational at a total counting rate exceeding 1MHz. The readout is based on dead-time-free flash ADC, running at 1-2GS/s, followed by a FPGA for real-time parallel data processing. Such a device has not been realized before and is expected to revolutionize time-resolved fluorescence imaging and spectroscopy from a single-molecule to whole animal level. To evaluate the design principle, an Image Intensifier with a GaAsP photocathode (>40% quantum efficiency at 400-600 nm) followed by double MCP was evaluated together with an existing CMOS camera. In our future design, the image from CMOS Camera will be combined with the MCP output, followed by a set of FPGA and CPU for real time data processing. This stream line method will allow ultra fast single-photon counting with 100 psec time resolution and 20 µm position resolution (1M pixel imaging). In this paper, we present the design principle and preliminary results on its performance. Our future plan and the design goals are also described.

A201 紫外パルスレーザーによる紙上潜在指紋の可視化

We have studied inherent fluorescence spectra of fingerprints and various papers in the deep UV region with a nanosecond pulsed laser system and have visualized latent fingerprints on papers with the time-resolved fluorescence imaging method. The excitation wavelength is 230 nm and 280 nm. Fingerprints emit fluorescence from 300 nm to 500 nm. Since papers also emit fluorescence above 400 nm and it is quite strong, it is necessary to suppress paper fluorescence using filters for imaging latent fingerprints. For printed papers, inks or toners often emit fluorescence. By using appropriate filters, latent fingerprints on papers were visualized for all white and printed papers examined. We have also studied absorption images of fingerprints and obtained clearest images with 215-nm illumination for latent fingerprints on white papers.

Optical characterization of Pseudomonas fluorescens on meat surfaces using time-resolved fluorescence

A scanning optical system for the detection of bacteria on meat surfaces based on fluorescence lifetime and intensity measurements is described. The system detects autofluorescent light emitted by naturally occurring fluorophores in bacteria. The technique only requires minimal sample preparation and handling, thus the chemical properties of the specimen are preserved. This work presents the preliminary results obtained from a time-resolved fluorescence imaging system for the characterization of a nonpathogenic gram-negative bacteria, Pseudomonas fluorescens. Initial results indicate that the combination of fluorescence lifetime and intensity measurements provides a means for characterizing biological media and for detecting microorganisms on surfaces.

Fluorescence imaging of petroleum accelerants by time-resolved spectroscopy with a pulsed Nd-YAG laser

In this paper, fluorescence of petroleum accelerants such as kerosene, motor gasoline and diesel fuel were measured by the nanosecond time-resolved spectroscopy with a pulsed Nd-YAG laser. The excitation wavelengths are 266 nm (fourth harmonic generation) and 355 nm (third harmonic generation). Fluorescence was detected by a cooled CCD camera with an image intensifier. From these measurements, fluorescence lifetime was determined. We have also shown that the principal component of fluorescence spectra of kerosene is dimethylnaphthalene when excited with 266 nm laser light. For gasoline and diesel fuel, there exist other hydrocarbons which emit fluorescence. There is a significant difference among fluorescence spectra of kerosene, gasoline and diesel fuel. These results can be used for the identification of petroleum accelerants. We have also measured fluorescence spectra of various background materials such as newspaper, cardboard, carpet, cotton glove, wood board and various kinds of paper. We have shown that there exists a significant difference between petroleum accelerants and these background materials in fluorescence spectra and their lifetimes. By using such a difference, petroleum accelerants can be detected effectively even when they are on the fluorescent background materials which sometimes disturb the detection by emitting strong fluorescence. We have shown that petroleum accelerants on various background materials can be visualized by the time-resolved fluorescence imaging. Taking actual fire cases into account, the influence of heat to kerosene was studied. By heating, the fluorescence intensity of kerosene reduced for 266-nm excitation, but it increased for 355-nm excitation. For spectral profile, no change was observed for 266-nm excitation but there is a difference between non-heated kerosene and heated kerosene for 355-nm excitation. The lifetime seems to increase slightly by heating for 266-nm excitation but it is almost constant for 355-nm excitation. Imaging heated kerosene on burned background materials was examined and it is confirmed that heated kerosene can be detected on burned materials by the time-resolved method with an appropriate band-pass filter.