Testing Fluorescence Lifetime Standards using Two-Photon Excitation and Time-Domain Instrumentation: Rhodamine B,Coumarin 6 and Lucifer Yellow

Having good information about fluorescence lifetime standards is essential for anyone performing lifetime experiments. Using lifetime standards in fluorescence spectroscopy is often regarded as a straightforward process, however, many earlier reports are limited in terms of lifetime concentration dependency, solvents and other technical aspects. We have investigated the suitability of the fluorescent dyes rhodamine B, coumarin 6, and lucifer yellow 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 which wavelengths we should use for the excitation and an appropriate detector range. We also measured lifetimes for different concentrations, ranging from 10^?2– 10^?6 M, in both water, ethanol and methanol solutions. We observed that rhodamine B lifetimes depend strongly on concentration. Coumarin 6 provided the most stable lifetimes, with a negligible dependency on concentration and solvent. Lucifer yellow lifetimes were also found to depend little with concentration. Finally, we found that a mix of two fluorophores (rhodamine B/coumarin 6, rhodamine B/lucifer yellow, and coumarin 6/lucifer yellow) all yielded very similar lifetimes from a double-exponential decay as the separate lifetimes measured from a single-exponential decay. All lifetime measurements were made using two-photon excitation and obtaining lifetime data in the time-domain using time-correlated single-photon counting.     

Picosescond fluorescence lifetime standards for frequency- and time-domain fluorescence

We characterized a series of dimethylamino-stilbene derivatives as standards for time-domain and frequency-domain lifetime measurements. The substances have reasonable quantum yields, are soluble in solvents available with a high purity, and do not show significant sensitivity to oxygen quenching. All the fluorophores displayed single exponential intensity decays, as characterized by frequency-domain measurements to 10 GHz. The decay times vary from 880 to 57 ps, depending on structure, solvent, and temperature, which is a useful range for modern picosecond time-domain or gigahertz frequency-domain instruments. These fluorophores may be used either to test an instrument or as reference compounds to eliminate color effects. We also characterized two-fluorophore mixtures, with the decay times spaced twofold (150 and 300 ps), with varying proportions. These mixtures are useful for testing the resolution of other time- and frequency-domain instrumentation. The excitation wavelength ranges from 260 to 430 nm, and the emission from 350 to 550 nm. The decay times are independent of the excitation and emission wavelengths.     

Long Wavelength Fluorescence Lifetime Standards for Front-Face Fluorometry

With the increased development and use of fluorescence lifetime-based sensors, fiber optic sensors, fluorescence lifetime imaging microscopy (FLIM), and plate and array readers, , calibration standards are essential to ensure the proper function of these devices and accurate results. For many devices that utilize a “front face excitation” geometry where the excitation is nearly coaxial with the direction of emission, scattering-based lifetime standards are problematic and fluorescent lifetime standards are necessary. As more long wavelength (red and near-infrared) fluorophores are used to avoid background autofluorescence, the lack of lifetime standards in this wavelength range has only become more apparent . We describe an approach to developing lifetime standards in any wavelength range, based on Förster resonance energy transfer (FRET). These standards are bright, highly reproducible, have a broad decrease in observed lifetime, and an emission wavelength in the red to near infrared making them well suited for the laboratory and field applications as well. This basic approach can be extended to produce lifetime standards for other wavelength regimes.     

Fluorescence-lifetime-based tomography for turbid media

We derive a novel algorithm to recover the in vivo distributions of fluorophores based on an asymptotic life-time analysis of time-domain fluorescence measurements with turbid tissue. We experimentally demonstrate the advantage offered by this method in localizing fluorophores with distinct lifetimes. This algorithm has wide applicability for diagnostic fluorescence imaging in the presence of several-centimeter-thick biological tissue, since fluorescence lifetime is a sensitive indicator of local tissue environment and interactions at the molecular level.     

Different preparation methods and characterization of magnetic maghemite coated with chitosan

In this report, we investigated the so-called plasmonic platforms prepared to target ultra-short fluorescence and accurate instrumental response function in a time-domain spectroscopy and microscopy. The interaction of metallic nanoparticles with nearby fluorophores results in the increase of the dye fluorescence quantum yield, photostability and decrease of the lifetime parameter. The mentioned properties of platforms were applied to achieve a picosecond fluorescence lifetime (21 ps) of erythrosin B, used later as a better choice for deconvolution of fluorescence decays measured with “color” sensitive photo-detectors. The ultra-short fluorescence standard based on combination of thin layers of silver film, silver colloidal nanoparticles (about 60 nm in diameter), and top layer of erythrosin B embedded in 0.2 % poly(vinyl) alcohol. The response functions were monitored on two photo-detectors; microchannel plate photomultiplier and single photon avalanche photodiode as a Rayleigh scattering and ultra-short fluorescence. We demonstrated that use of the plasmonic base fluorescence standard as an instrumental response function results in the absence of systematic error in lifetime measurements and analysis.     

Self-quenching of uranin: Instrument response function for color sensitive photo-detectors

Concentration is a key determining factor in the fluorescence properties of organic fluorophores. We studied self-quenching of disodium fluorescein (uranin) fluorescence in polyvinyl alcohol (PVA) thin films. The concentration dependent changes in brightness and anisotropy were followed by a lifetime decrease. We found that at a concentration of 0.54 M, the lifetime decreases to 7 ps. At a concentration of 0.18 M the lifetime was 10 ps with the relatively high quantum yield of 0.002. In these conditions the fluorescence intensity decay was homogeneous (well approximated by a single lifetime). We realized that such a sample was an ideal fluorescence lifetime standard for spectroscopy and microscopy, and therefore characterized instrument response functions for a time-domain technique. We show that self-quenched uranin enables measurements free of the color effect, making it a superior choice for a lifetime reference over scattered light.     

Light propagation for time-domain fluorescence diffuse optical tomography by convolution using lifetime function

Time-domain light propagation in biological tissue is studied by solving the forward problem for fluorescence diffuse optical tomography using a convolution of the zero-lifetime emission light and the exponential function for a finite lifetime. We firstly formulate the fundamental equations in a time-domain assuming that the fluorescence lifetime is equal to zero, and then the solution including the lifetime is obtained by convolving the emission light and the lifetime function. The model is a two-dimensional (2-D) 10 mm-radius circle with the optical properties simulating biological tissue for the near infrared light, and contains some inclusions with fluorophores. Temporal and spatial profiles of excitation and emission light are calculated and discussed for several models with different inclusions. The results are physically reasonable and will be used for the inverse problem of fluorescence diffuse optical tomography.     

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     

Uranyl Soil Extraction and Fluorescence Enhancement by Nanoporous Silica Gel: pH effects

Nanoporous silica gel was employed to extract uranyl from contaminated soil and to enhance the fluorescence intensity and lifetime. The fluorescence lifetime and intensity of uranyl ions absorbed within nanoporous silica gel was measured from pH 1?C13. The results show that the uranyl fluorescence intensity can be enhanced by approximately two orders of magnitude by the silica nanoporous matrix from pH 4?C12 with the greatest enhancement occurring from pH 4?C7. The enhanced fluorescence lifetime can be used in time-gated measurements to help minimize the influence of background environmental fluorophores.     

Fluorescence lifetime system for microscopy and multiwell plate imaging with a blue picosecond diode laser

We report a wide-field fluorescence lifetime imaging (FLIM) system that uses a blue picosecond pulsed diode laser as the excitation source. This represents a significant miniaturization and simplification compared with other time-domain FLIM instruments that should accelerate the development of clinical and real-world applications of FLIM. We have demonstrated this instrument in two configurations: a macroimaging setup applied to multiwell plate assays of chemically and biologically interesting fluorophores and a microscope system that has been applied to imaging of tissue sections. The importance of the adjustable repetition rate of this laser source is discussed with respect to noise reduction and precision in the lifetime determination, illustrating a further significant advantage over conventional mode-locked solid-state lasers.     

A FRET Fluorescent Sensor for Ratiometric and Visual Detection of Sulfide Based on Carbon Dots and Silver Nanoclusters

In this work, the fluorescent sensor based on fluorescence resonance energy transfer (FRET) and electrostatic interaction (EI) was prepared for the ratiometric and visual detecting S^2–. The FRET fluorescent sensor consists of two fluorophores, with carbon dots (CDs) as energy donors and silver nanoclusters (Ag NCs) as acceptors. At 390 nm excitation, CDs and Ag NCs showed two well-separated peaks at 445 nm and 660 nm, separately. The existence of S^2– caused the red fluorescence at 660 nm to be quenched, whereas the blue fluorescence at 445 nm was restored, and the fluorescence color of the ratiometric sensor changed from pink to blue. It could be employed in ratiometric and visual detecting S^2–. The linear range of quantitative detection S^2– was 0.5–100 μM, and its detection limit was 0.35 μM. CDs-Ag NCs could be used for detecting S^2– in mineral water and tap water. The results showed that the FRET ratiometric fluorescent sensor exhibits good anti-interference and high selectivity for detecting S^2– in environmental water samples.

     

Multiphoton Ligand-Enhanced Excitation of Lanthanides

We describe multiphoton excitation of the lanthanides europium (Eu³⁺) and terbium (Tb³⁺) when these ions are complexed with nucleic acids, proteins, and fluorescent chelators. In all cases excitation occurs by multiphoton absorption of the sensitizers. For the nucleotide GDP and an oligonucleotide with several guanines, the sensitized emission of Tb³⁺ excited at 776 nm indicated a three-photon process. For Tb³⁺ bound to the wild-type troponin C and a single tryptophan mutant (26W), excitation at 794 nm was also close to a three-photon process. For lanthanide chelators containing various sensitizers, we observed three-photon excitation in the case of methyl anthranilate, a mixuture of two- and three-photon excitation for carbostyril 124, and a two-photon process with a coumarin derivative. In the case of coumarin-sensitized emission of Eu³⁺ varied from a two- to a three-photon process at wavelengths ranging from 780 to 880 nm. The sensitized luminescence also shows significantly higher photostability compared to the fluorescence from the organic fluorophores alone. These results suggest the use of multiphoton-induced sensitized lanthanide fluorescence in biochemistry and cellular imaging.On leave from the Institute of Experimental Physics     

Development of Low-Cost Instrumentation for Single Point Autofluorescence Lifetime Measurements

Autofluorescence lifetime measurements, which can provide label-free readouts in biological tissues, contrasting e.g. different types and states of tissue matrix components and different cellular metabolites, may have significant clinical potential for diagnosis and to provide surgical guidance. However, the cost of the instrumentation typically used currently presents a barrier to wider implementation. We describe a low-cost single point time-resolved autofluorescence instrument, exploiting modulated laser diodes for excitation and FPGA-based circuitry for detection, together with a custom constant fraction discriminator. Its temporal accuracy is compared against a “gold-standard” instrument incorporating commercial TCSPC circuitry by resolving the fluorescence decays of reference fluorophores presenting single and double exponential decay profiles. To illustrate the potential to read out intrinsic contrast in tissue, we present preliminary measurements of autofluorescence lifetime measurements of biological tissues ex vivo. We believe that the lower cost of this instrument could enhance the potential of autofluorescence lifetime metrology for clinical deployment and commercial development.     

Photon-counting technique for rapid fluorescence-decay measurement

We report on a novel laser-induced fluorescence triple-integration method (LIFTIME) that is capable of making rapid, continuous fluorescence lifetime measurements by a unique photon-counting technique. The LIFTIME has been convolved with picosecond time-resolved laser-induced fluorescence, which employs a high-repetition-rate mode-locked laser, permitting the eventual monitoring of instantaneous species concentrations in turbulent flames. We verify the technique by application of the LIFTIME to two known fluorescence media, diphenyloxazole (PPO) and quinine sulfate monohydrate (QSM). PPO has a fluorescence lifetime of 1.28 ns, whereas QSM has a fluorescence lifetime that can be varied from 1.0 to 3.0 ns. From these liquid samples we demonstrate that fluorescence lifetime can currently be monitored at a sampling rate of up to 500 Hz with less than 10% uncertainty (1sigma) .     

Two-Photon Excited Fluorescence Energy Transfer: A Study Based on Oligonucleotide Rulers

The use of two-photon excitation of fluorescence for detection of fluorescence resonance energy transfer (FRET) was studied for a selected fluorescent donor–acceptor pair. A method based on labeled DNA was developed for controlling the distance between the donor and the acceptor molecules. The method consists of hybridization of fluorescent oligonucleotides to a complementary single-stranded target DNA. As the efficiency of FRET is strongly distance dependent, energy transfer does not occur unless the fluorescent oligonucleotides and the target DNA are hybridized. A high degree of DNA hybridization and an excellent FRET efficiency were verified with one-photon excited fluorescence studies. Excitation spectra of fluorophores are usually wider in case of two-photon excitation than in the case of one-photon excitation [1]. This makes the selective excitation of donor difficult and might cause errors in detection of FRET with two-photon excited fluorescence. Different techniques to analyze the FRET efficiency from two-photon excited fluorescence data are discussed. The quenching of the donor fluorescence intensity turned to be the most consistent way to detect the FRET efficiency. The two-photon excited FRET is shown to give a good response to the distance between the donor and the acceptor molecules.     

Photon-Counting Phase-Modulation Fluorometer

We propose a phase-modulation fluorometer that is applicable to a very weak fluorescence intensity level. In order to counter the single-photon event situation, we have introduced a combination of a time-to-amplitude converter and a pulse height analyzer (PHA) to the fluorometer, the combination of which is usually used in the single-photon correlation method to measure fluorescence decay waveforms with pulsed excitation. In the proposed fluorometer, a sinusoidal response waveform that is shifted in phase over the reference one is obtained statistically as a histogram in the PHA memory, and then the fluorescence lifetime can be calculated by the same procedure as the conventional analog phase-modulation method. The excitation light source used was a current-modulated ultraviolet light-emitting diode, whose center wavelength was 370 nm and whose spectral bandwidth was 10 nm. Fluorescence lifetimes of 17.5 ns and 5.7 ns obtained for 10 ppb quinine sulfate in 0.1 N H₂SO₄ and for 10 ppb rhodamine 6G in ethanol, respectively, agreed well with those reported in the literature. In passing, we report that the fluorescence lifetime of benzo[a]pyrene in dichloromethane is 14.6 ns, which is known as one of the carcinogenic, environmental-pollution materials.This paper was originally presented at the 2001 International Conference (2nd Joint OSJ-SPIE Conference) on Optical Engineering for Sensing and Nanotechnology, ICOSN 2001 which was held June 6#x2013;8, 2001 at the Pacifico-Yokohama Conference Center, Yokohama, Japan.     

Quick Microwave Assisted Synthesis and <Emphasis Type="Italic">In Vitro</Emphasis> Imaging Application of Oxygen Doped Fluorescent Carbon Dots

In this paper, a fast and simplest one-pot tactic was used to synthesis fluorescent oxygen doped carbon dots from Tween-20 (TTO-CDs) is reported. The TTO-CDs were microwavically synthesized by using Tween-20 as both the carbon precursor and the oxygen dopant as well. The surface morphology, crystalline and/or amorphous nature, composition and optical assets of synthesized TTO-CDs were studied by means of existing techniques. From the results, it was confirmed that the as-synthesized TTO-CDs are amorphous in nature, monodispersed, sphere-shaped and the typical particle size range is 5?±?1.5 nm. The synthesized TTO-CDs emits strong blue fluorescence at 390 nm under excitation of 335 nm. Most interestingly, the excitation dependent emission property of synthesized TTO-CDs was exposed from fluorescence results. The synthesized TTO-CDs have quantum yield of about 14% against quinine sulfate as reference standard. The biotoxicity of synthesized TTO-CDs on HeLa cells was assessed through cytotoxicity assay. These results implied that the fluorescent TTO-CDs showed less biotoxicity, and further which was efficaciously applied as a multicolor staining and bioimaging probe for the confocal imaging of HeLa cells.     

Spectroscopic and nonlinear characteristics of anthracene-containing silicon-organic polymers in solution

Spectroscopic characteristics, i.e. absorption, fluorescence, fluorescence excitation spectra, fluorescence decay time, fluorescence polarization degree of novel silicon-containing organic polymers including main chain anthracene groups were investigated. Three kinds of emission spectra were revealed and assigned to polaron–exciton, anthracene and anthracene dimer. The measured fluorescence polarization spectra gave evidence of directed excitation energy migration along the disordered polymeric chain. Strong quenching of anthracene fluorescence during the polaron–exciton lifetime was interpreted as a result of the interaction between two excitations that causes anthracene anion-radical formation. The third-order nonlinear susceptibility of the polymers in solution measured by the Z-scan technique at 1054 nm is 190×10⁻¹⁴ cm² W⁻¹.     

The Fluorescence Bioassay Platforms on Quantum Dots Nanoparticles

In this paper, we present the optical properties and the platforms on fluorescent quantum dots for biological labeling, biomedical engineering and biosensor in molecular imaging. Quantum dots possess several properties that make them very attractive for fluorescent tagging: broad excitation spectrum, narrow emission spectrum, precise tunability of their emission peak, longer fluorescence lifetime than organic fluorophores and negligible photobleaching. We describe how to take such advantages of quantum dots to develop the technology and employ it to build assay platforms. Finally, ultrasensitivity, multicolor, and multiplexing of the technology of semiconductor quantum dots open up promising and interesting possibilities for bioassay platform.     

Molecular Dynamics of Biological Probes by Fluorescence Correlation Microscopy with Two-Photon Excitation

We report on the application of fluorescence correlation microscopy under two-photon excitation of fluorophores of biological interest: FITC–dextran (MW, from 20 to 150 kDa), green fluorescent protein (MW, 27 kDa), and fluorescein (MW, 330 Da). Under these experimental conditions, the translational diffusion coefficients of these molecules in aqueous solutions derived from the fluorescence intensity autocorrelation function were determined for the first time and were found to be 24 × 10^–7, 8.2 × 10^–7, and 3 × 10^–7 cm² s^–1 for 150-kDa FITC–dextran, green fluorescent protein, and fluorescein, respectively. These results are discussed in connection with previously reported results obtained by different methods. The great sensibility of the system has been applied to single-molecule detection of the smaller fluorophore, fluorescein.