Phase-Modulation Fluorometer Using an Ultraviolet Light-Emitting Diode

We have constructed a phase-modulation fluorometer using a commercially-available ultraviolet light-emitting diode (UV LED) as an excitation light source. The center wavelength of the UV LED is 370 nm and its spectral bandwidth is 10 nm. A 10 mApp modulation-current was superimposed on a bias current of 5 mA with a fixed frequency in the range of 1-20 MHz. The average UV power on the sample was 250 μW. The fluorescence signal was detected by a photomultiplier tube and was fed into a versatile digital oscilloscope. The phase difference between the fluorescence signal and the reference signal obtained from a diffusion plate was directly read out using the operational functions of the digital oscilloscope. To demonstrate the system performance, fluorescence lifetimes of 25 ppm rhodamine 6G in ethanol and of 10 ppm quinine sulfate in 0.1 N H₂SO₄ were measured. The calculated lifetimes of 5.8 ns and 19.1 ns, respectively, agreed with those reported in the literature. The combination of the UV LED and the digital oscilloscope made the fluorometer simple and easy to construct with low cost.     

Fluorescence lifetime characterization of bacteria using total lifetime distribution analysis with the maximum entropy method

Total lifetime distribution analysis was employed to obtain fluorescence lifetime profiles of the intrinsic fluorescence ofPseudomonas fluorescens, Escherichia coli, Bacillus subtilis, andStaphylococcus epidermidis. The lifetimes were measured using a multiharmonic Fourier transform phase-modulation fluorometer which can simultaneously measure the phase shift and demodulation at many modulation frequencies. The 364-nm line from an argon-ion laser and the 325- and 442-nm lines from a helium-cadmium laser were used for sample excitation. Broad emission windows were used to capture as much of the bacterial emission as possible for the lifetime measurements. The maximum entropy method was used to recover lifetime profiles from the multifrequency phasemodulation data. At all three excitation wavelengths, the bacteria exhibited three lifetime components, in the ranges of 0.5-1, 2–3, and 4–8 ns. Using 325-nm excitation, a fourth component, in the range of 9–14 ns, was recovered in all of the bacteria; using 364-nm excitation, the fourth component was resolved only in the two Gram-negative bacteria (P. fluorescens andE. coli). Excitation at 364 nm provided the most reproducible lifetime profiles and showed some differences among the four bacteria.     

High-efficiency photon-counting fluorometer with a channel width of 5.0 ps

We construct a photon-counting, fluorescence lifetime measurement system with a channel width of 5.0 ps. This is a modified version of our previous fluorometer based on a simultaneous detection method of photoelectron pulse trains after pulsed excitation (Iwata in Meas Sci Technol 28:075501, 2017), which can accept multiple photons during a one period of the excitation for building a histogram. Although the resolution time is 2.0 ns as before, the channel width of 5.0 ps is of the same order as that of time-correlated single-photon counting, which is achieved by delaying the trigger timing for the fluorometer by a step to that of the excitation. This results in an increase in the precision for the lifetime estimation. Although the measurement time increases with increasing the number of delay steps, it is still rather shortened owing to the high signal-gathering and histogram-building efficiencies.     

Phase-modulation fluorometer using a phase-modulated excitation light source

We propose a phase-modulation fluorometer (PMF) with a light-emitting diode (LED) or a laser diode (LD) used as an excitation light source (ELS) that is driven in the phase-modulation (PM) mode. The PM-ELS generates many frequency sidebands that spread in the vicinity of carrier frequency f _c with the interval of modulation frequency f _m depending on the maximum phase deviation Δφ. The scheme enables us to derive fluorescence lifetime values of a multicomponent sample at one time. We show a typical numerical simulation result for explaining the principle of operation. To demonstrate the effectiveness of the proposed PMF, we have measured fluorescence lifetimes of three kinds of inorganic fluorescent glasses and that of a mixture solution of 1 × 10^?6M rhodamine 6G and 1 × 10^?6 M coumarin 152 in ethanol with a volume ratio of 1: 1.     

Proposal for Fourier-Transform Phase-Modulation Fluorometer

I propose a concept of a novel Fourier-transform phase-modulation fluorometer by which a fluorescence decay waveform can be obtained. In the fluorometer, the modulation frequency of the excitation light source is swept continuously from a start frequency f_min to an end frequency f_max with a time duration T. The resultant fluorescence signal waveform is Fourier-transformed to obtain amplitude and phase spectra. The ratio of the amplitude spectrum and the difference of the phase spectrum over those of the reference spectra that are obtained from a non-fluorescent material are calculated, respectively, and the pair of both spectral data is inverse-Fourier-transformed again to obtain the fluorescence decay waveform. To verify and demonstrate the effectiveness of the concept, I carried out (1) numerical simulations, (2) determination of a time constant of a passive resistor-capacitor (RC) differential circuit, and (3) measurement of a fluorescent decay waveform of YAG materials packed in Nichia’s white LED.     

A UV–Visible–NIR fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution

This article describes the design and characterization of a wide-field, time-domain fluorescence lifetime imaging microscopy (FLIM) system developed for picosecond time-resolved biological imaging. The system consists of a nitrogen-pumped dye laser for UV–visible–NIR excitation (337.1–960 nm), an epi-illuminated microscope with UV compatible optics, and a time-gated intensified CCD camera with an adjustable gate width (200 ps-10^-3 s) for temporally resolved, single-photon detection of fluorescence decays with 9.6-bit intensity resolution and 1.4-μm spatial resolution. Intensity measurements used for fluorescence decay calculations are reproducible to within 2%, achieved by synchronizing the ICCD gate delay to the excitation laser pulse via a constant fraction optical discriminator and picosecond delay card. A self-consistent FLIM system response model is presented, allowing for fluorescence lifetimes (0.6 ns) significantly smaller than the FLIM system response (1.14 ns) to be determined to 3% of independently determined values. The FLIM system was able to discriminate fluorescence lifetime differences of at least 50 ps. The spectral tunability and large temporal dynamic range of the system are demonstrated by imaging in living human cells: UV-excited endogenous fluorescence from metabolic cofactors (lifetime ∼1.4 ns); and 460-nm excited fluorescence from an exogenous oxygen-quenched ruthenium dye (lifetime ∼400 ns). Received: 23 February 2003 / Published online: 22 May 2003 RID="*" ID="*"Corresponding author. Fax: +1-734/9361-905, E-mail: mycek@umich.edu     

Detection and characterization of single molecules in aqueous solution

Using a confocal microscope with a single-photon avalanche photodiode as detector, we studied photon bursts of single Rhodamine 6G (R6G) and Rhodamin B-zwitterion (RB) molecules in aqueous solution by excitation of the lowest excited singlet stateS ₁ with a frequency-doubled titanium: sapphire laser. Multichannel scaler traces, the fluorescence autocorrelation function and fluorescence decay times determined by time-correlated single-photon counting have been measured simultaneously. The time-resolved fluorescence signals were analyzed with a maximum likelihood estimator. Fluorescence lifetime patterns in steps of 100 ps were generated by convolution with the excitation pulse. The lifetime of theS ₁ state was derived from the Kullback-Leibler minimum discrimination information. We are able to demonstrate for the first time identification of two different single dye molecules via their characteristic fluorescence lifetimes of 1.79 ± 0.33 ns (RB) and 3.79 ± 0.38 ns (R6G) in aqueous solution.Dedicated to Prof. F. P. Schäfer on the occasion of his 65th birthday.On leave from Department of Physics, Mokwon University, Taejon, 301-729, Korea     

Effective A-state fluorescence lifetime of formaldehyde in atmospheric pressure CH4/air flames

Using excitation pulses of ∼30-ps duration and a fast photomultiplier detector, effective fluorescence lifetimes of the A-stateof formaldehyde after excitation at 355 and 339 nm have been measured in the preheating zone of an atmospheric pressure, premixed methane/air flame. The fluorescence lifetimes were determined as a function of height above the exit of a slot burner and were thus probed in regions of varying gas temperature and composition. The fluorescence lifetimes were independent of the intensity of the excitation pulse and decreased as a function of height in the burner from ∼18±8 ns at 1.2 mm down to 7±1 ns at 3.8 mm. This trend of the effective fluorescence lifetime with composition and temperature in the flame can qualitatively be reproduced using calculated major species mole fractions and species-specific quenching cross sections for CH from the literature. Received: 13 June 2001 / Revised version: 27 September 2001 / Published online: 29 November 2001     

Near-field time-resolved fluorescence studies of poly(phenylmethyl silane) with subwavelength resolution

We present an example of the first time-correlated single-photon counting (TCSPC) near-field optical measurement. The aperture size of our prepared aluminun-coated fiber-optic probe was approximately 50 nm, which represents a spatial resolution of _ex/7 for our UV measurements. Near-field fluorescence decays of poly(phenylmethyl silane) in solid thin films excited in the range 325–360 nm were obtained and the steady-state excitation spectra compared with the excitation spectral information obtained in the far field. Fluorescence decays showed single exponential lifetimes ranging from 45 to 277 ps, which was dependent on the excitation wavelength and the selected near-field tip. The proximity of the metal-coated tip to the sample may be the reason for the modulation in fluorescence lifetime.     

Tyrosyl Fluorescence Decays and Rotational Dynamics in Tyrosine Monomers and in Dipeptides

Picosecond time-correlated single-photon counting was used to measure fluorescence lifetimes and fluorescence anisotropy decays of tyrosine and the tyrosine–alanine and tyrosine–leucine dipeptides. After excitation of tyrosine at 287 nm two emitting species were observed, one at 303 nm with a lifetime of 3.3 ns and another at 340 nm with a lifetime of 360 ps. The rotational correlation time of tyrosine at 303 nm is 38 ps in water at pH 7 and depends linearly on viscosity with a slope of 44 ps/cP, consistent with Stokes–Einstein–Debye theory. We calculated a value of 45 ns for the radiative lifetime of tyrosine, yielding a fluorescence quantum yield of 0.07. The dipeptides Tyr–Ala and Tyr–Leu exhibit two- or three-exponential decays. The amplitudes of the decay components for three-exponential fits correlate closely with the populations of rotamers in these peptides as determined by NMR. The quenching of dipeptide fluorescence is shown to depend on the solvent polarity, strongly supporting the hypothesis that tyrosyl fluorescence in peptides is quenched by charge transfer. The rotational correlation times of tyrosine, Tyr–Ala, and Tyr–Leu increase linearly with the van der Waals volumes. However, rotational relaxation is somewhat faster than expected from Stokes–Einstein–Debye theory with stick boundary conditions.     

Native Fluorescence and Time Resolved Fluorescence Spectroscopic Characterization of Normal and Malignant Oral Tissues Under UV Excitation—an In Vitro Study

The present work aims to investigates the native fluorescence and time resolved fluorescence spectroscopic characterization of oral tissues under UV excitation. The fluorescence emission spectra of oral tissues at 280 nm excitation were obtained. From the spectra, it was observed that the alteration in the biochemical and morphological changes present in tissues. Subsequently, the Full width at Half Maximum (FWHM) of every individual spectra of 20 normal and 40 malignant subjects were calculated. The student’s t-test analysis reveals that the data were statistically significant (p?=?0.001). The fluorescence excitation spectra at 350 nm emission of malignant tissues confirms the alteration in protein fluorescence with respect to normal counterpart. To quantify the observed spectral differences, the two ratio variables R₁?=?I₂₇₅/I₃₁₀ and R₂?=?I₃₁₀/I₃₂₈ were introduced in the excitation spectra. Among them, the Linear Discriminant Analysis (LDA) of R₁ reveals better classification with 86.4 % specificity and 82.5 % sensitivity. The fluorescence decay kinetics of oral tissues was obtained at 350 nm emission and it was found that the decay kinetics was triple exponential. Then the ROC analysis of fractional amplitudes and component lifetime reveals that the average lifetime shows 77 % sensitivity and 70 % specificity with the cut off value 4.85 ns. Briefly, the average lifetime exhibits better statistical significance when compared to fractional amplitudes and component lifetimes.     

Lifetime measurement of the 9s level of atomic francium

We use two-photon resonant excitation and time-correlated single-photon counting techniques on a sample of 210Fr atoms confined and cooled in a magneto-optical trap to measure the lifetime of the 9s excited level. Direct measurement of the decay through the 7P(3/2) level at 851 nm yields a lifetime of 107.53 +/- 0.80 ns.     

某些稠环芳香碳氢化合物的荧光寿命测量

应用时间相关单光子计算仪器,测量了八个对荧光光谱学和生物化学重要的稠环芳烃.所有溶液在测量前均通高纯氮除氧,对所有化合物都使用337nm激发波长,对荧蒽还在351nm激发,以验证上述两组作者所给数值之间的分歧.我们的实验对于荧蒽在337nm激发给出荧光寿命39ns,在351nm激发给出36ns,表明两个激发波长不应导致寿命数值的实质性差异.我们对其余稠环芳烃所测得的寿命值或者与文献相符,或者看起来是合理的.     

Reverse-saturable absorption in aluminophthalocyanine-doped xerogels

Aluminophthalocyanines were encaged in different silica xerogel matrices. Nonlinear reverse-saturable absorption behavior was observed for both nanosecond and picosecond pulse excitation at 532 nm. In order to determine the relaxation paths of the molecules after optical excitation, we measured the evolution of the transmission of a continuous probe beam through the samples following absorption induced by a pump pulse. The transmission recovery was a few tens of microseconds. The fluorescence lifetime was about 10 ns.     

Kinetics of Er3+-doped fluorozirconate optical fiber upconversion fluorescence and laser emission under 800 nm excitation

Applying single- and double-pulse excitation at 800 nm, the kinetics of the upconversion fluorescence in the green, as well as the upconversion laser at 543 nm was studied. No significant delay between the pumping pulse and the laser emission was found. In the erbium doped (1000 ppm) optical fiber, the mechanism responsible for the upconversion is purely of the excited state absorption (ESA) type. The double-pulse technique enables also a determination of the lifetime of the intermediate metastable state ⁴ I _11/2 (7±0.3 ms). Some other basic properties of the upconverted fluorescence and of the laser itself (fluorescence spectrum, optical gain, laser threshold) are also described. Received: 11 December 2000 / Revised version: 18 February 2001 / Published online: 18 July 2001     

Two-photon-excited fluorescence in KTiOPO4 polycrystals

The spectra of two-photon-excited fluorescence in KTiOPO₄ were obtained at room temperature. A coppervapor laser was used as a source of excitation light and provided two emission lines (λ=510.6 and 578.2 nm). The laser operated at a high pulse-repetition rate (～ 10⁴ Hz) and featured a peak power of about 10⁴ W, average power of 1 W, and pulse duration of 20 ns. The fluorescence spectra of crystalline KTiOPO₄ are compared with the resonance fluorescence spectra of KTiOPO₄ at 4.2 K. The measured decay time of fluorescence was found to be less than 16 ns. The efficiency of conversion of the laser radiation to fluorescence was about 10^?10 under saturation conditions.     

Photo-physical properties of anisole: temperature, pressure, and bath gas composition dependence of fluorescence spectra and lifetimes

Anisole is a promising candidate for use as fluorescent tracer for gas-phase imaging diagnostics. Its high-fluorescence quantum yield (FQY) and its large Stokes shift lead to improved signal intensity (up to 100 times stronger) compared with the often used toluene. Fluorescence spectra and effective fluorescence lifetimes of gaseous anisole were investigated after picosecond laser excitation at 266 nm as a function of temperature (296–977 K) and bath gas composition (varying amounts of N₂ and O₂) at total pressures in the range of 1–10 bar to provide spectroscopic data and FQY for applications, e.g., in in-cylinder measurements in internal combustion engines. Fluorescence spectra of anisole extend from roughly 270–360 nm with a peak close to 290 nm at 296 K. The spectra show a red-shift with increasing temperature (0.03 nm/K) and O₂ partial pressure (5 nm from N₂ to air). In the investigated temperature range and in pure N₂ at 1 bar total pressure the effective fluorescence lifetime drops with increasing temperature from 13.3 ± 0.5 to 0.05 ± 0.01 ns. Increasing the total pressure of N₂ leads to a small decrease of the lifetime at temperatures above 400 K (e.g., at 525 K from 4.2 ± 0.2 ns at 1 bar to 2.7 ± 0.2 ns at 10 bar). At constant temperature and in the presence of O₂ the lifetimes decrease significantly (e.g., at 296 K from 13.3 ± 0.5 ns in N₂ to 0.40 ± 0.02 ns in air), with this trend diminishing with increasing temperature (e.g., at 675 K from 1.02 ± 0.08 ns in N₂ to 0.25 ± 0.05 ns in air). A phenomenological model that predicts fluorescence lifetimes, i.e., relative quantum yields as a function of temperature, pressure, and O₂ concentration is presented. The photophysics of anisole is discussed in comparison with other aromatics.     

Laser-induced fluorescence detection of acetylene in low-pressure propane and methane flames

Laser-induced fluorescence is used to detect and record profiles of acetylene formed as an intermediate species in 10-Torr premixed propane and methane flames. In low-temperature regions of the flames, excitation spectra confirm acetylene as the spectral carrier. The spectra of acetylene overlap those of O₂ and NO in terms of both excitation and detection wavelengths, however, acetylene can be detected with relatively little interference in the vicinity of 228 nm, using a detection wavelength of 260 nm. The fluorescence lifetime of acetylene in the flame conditions studied is approximately 20 ns, much shorter than the radiative lifetime, due to a high quenching rate for all the colliders investigated. This can be exploited in low-pressure flames to avoid interference from acetylene in monitoring nitric oxide. The acetylene mole fraction in propane flames reaches its peak value at nearly the same location as that of HCO, slightly closer to the burner than the peak CH mole fraction. The acetylene fluorescence signal is easily detected in propane flames over equivalence ratios from 0.6 to 1.2, although it increases under fuel-rich conditions. In methane flames, the acetylene signal is much weaker and is undetectable for fuel-lean conditions. Received: 5 August 2002 / Revised version: 30 September 2002 / Published online: 20 December 2002 RID="*" ID="*"Corresponding author. Fax: +1-202/767-1716, E-mail: brad@code6185.nrl.navy.mil     

Room temperature stable single-photon source

We report on the realization of a stable solid state room temperature source for single photons. It is based on the fluorescence of a single nitrogen-vacancy (NV) color center in a diamond nanocrystal. Antibunching has been observed in the fluorescence light under both continuous and pulsed excitation. Our source delivers 2×10⁴ s^-1 single-photon pulses at an excitation repetition rate of 10 MHz. The number of two-photon pulses is reduced by a factor of five compared to strongly attenuated coherent sources. Received 1st August 2001 and Received in final form 2 October 2001     

Testing Fluorescence Lifetime Standards using Two-Photon Excitation and Time-Domain Instrumentation: Fluorescein,Quinine Sulfate and Green Fluorescent Protein

It is essential for everyone working with experimental science to be certain that their instruments produce reliable results, and for fluorescence lifetime experiments, information about fluorescence lifetime standards is crucial. A large part of the literature on lifetime standards dates back to the 1970s and 1980s, and the use of newer and faster measuring devices may deem these results unreliable. We have tested the three commonly used fluorophores fluorescein, quinine sulfate and green fluorescent protein for their suitability to serve as lifetime standards, especially to be used with two-photon excitation measurements in the time-domain. We measured absorption and emission spectra for the fluorophores to determine optimal wavelengths to use for excitation and detector settings. Fluorescence lifetimes were measured for different concentrations, ranging from 10^??3 ??10^??5 M, as well as for various solvents. Fluorescein was soluble in both ethanol, methanol and sulfuric acid, while quinine sulfate was only soluble in sulfuric acid. Green fluorescent protein was prepared in a commercial Tris-HCl, EDTA solution, and all three fluorophores produced stable lifetime results with low uncertainties. No siginificant variation with concentration was measured for any of the fluorophores, and all showed single-exponential decays. All lifetime measurements were carried out using two-photon excitation and lifetime data was obtained in the time-domain using time-correlated single-photon counting.