Fluorescence Instrument Response Standards in Two-Photon Time-Resolved Spectroscopy (original) (raw)
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Multi-dimensional fluorescence lifetime measurements
Progress in Biomedical Optics and Imaging, 2007
In this study, we present two different approaches that allow multi-wavelength fluorescence lifetime measurements in the time domain. One technique is based on a streak camera system, the other technique is based on a time-correlated singlephoton- counting (TCSPC) approach. The setup consists of a confocal laser-scanning microscope (LSM 510, Zeiss) and a Titanium:Sapphire-laser (Mira 900D, Coherent) that is used for pulsed one- and two-photon excitation. Fluorescence light emitted by the sample is dispersed by a polychromator (250is, Chromex) and recorded by a streak camera (C5680 with M5677 sweep unit, Hamamatsu Photonics) or a 16 channel TCSPC detector head (PML-16, Becker & Hickl) connected to a TCSPC imaging module (SPC-730/SPC-830, Becker & Hickl). With these techniques it is possible to acquire fluorescence decays in several wavelength regions simultaneously. We applied these methods to Förster resonance energy transfer (FRET) measurements and discuss the advantages over fluorescence techniques that are already well established in the field of confocal microscopy, such as spectrally resolved intensity measurements or single-wavelength fluorescence lifetime measurements.
The measurements of picosecond fluorescence lifetimes with high accuracy and subpicosecond precision
Chemical Physics Letters, 2001
Systematic studies of the¯uorescence picosecond lifetimes determination by laser-excited time-correlated singlephoton-counting (TCSPC) have been undertaken. The results have been used to develop methods for determining lifetimes with much smaller error and much greater reproducibility than any hitherto reported. The error in the determination of the lifetimes (AEthree standard deviations) can be as low as that in simulations and amounts to 0.01 FWHM of the IRF. The lifetimes determined for the second excited singlet state of xanthione in toluene 5:1 AE 0:3 ps and in benzene 8:1 AE 0:3 ps can be treated as reliable standards of picosecond lifetimes. Ó
Fluorescence Lifetime Standards for Time and Frequency Domain Fluorescence Spectroscopy
Analytical Chemistry, 2007
A series of fluorophores with single-exponential fluorescence decays in liquid solution at 20°C were measured independently by nine laboratories using single-photon timing and multifrequency phase and modulation fluorometry instruments with lasers as excitation source. The dyes that can serve as fluorescence lifetime standards for time-domain and frequency-domain measurements are all commercially available, are photostable under the conditions of the measurements, and are soluble in solvents of spectroscopic quality (methanol, cyclohexane, water). These lifetime standards are anthracene, 9-cyanoanthracene, 9,10-diphenylanthracene, N-methylcarbazole, coumarin 153, erythrosin B, N-acetyl-L-tryptophanamide, 1,4-bis(5-phenyloxazol-2-yl)benzene, 2,5-diphenyloxazole, rhodamine B, rubrene, N-(3-sulfopropyl)acridinium, and 1,4-diphenylbenzene. At 20°C, the fluorescence lifetimes vary from 89 ps to 31.2 ns, depending on fluorescent dye and solvent, which is a useful range for modern pico-and nanosecond time-domain or mega-to gigahertz frequencydomain instrumentation. The decay times are independent of the excitation and emission wavelengths. Depend
Double-pulse fluorescence lifetime measurements
Journal of Microscopy-oxford, 1997
It is demonstrated that fluorescence lifetimes in the nanosecond and picosecond time-scale range can be observed with the recently proposed double-pulse fluorescence lifetime imaging technique Double-pulse fluorescence lifetime imaging in confocal microscopy. J. Microsc. 177,[171][172][173][174][175][176][177][178][179].
Multi-dimensional fluorescence lifetime measurements
Multiphoton Microscopy in the Biomedical Sciences VIII, 2008
In this study, we present two different approaches that can be used for multi-wavelength fluorescence lifetime measurements in the time domain. One technique is based on a streak-camera system, the other technique is based on the timecorrelated-single-photon-counting (TCSPC) approach. The setup consists of a confocal laser-scanning microscope and a Titanium:Sapphire-laser that is used for pulsed one-and two-photon excitation. Fluorescence light emitted by the sample is fed back through the scan head and guided to one of the confocal channels, where it is coupled into an optical fiber and directed to a polychromator. The polychromator disperses the emitted light according to its wavelength and focuses the resulting spectrum on the entrance slit of a streak camera or a 16 channel PMT array, which is connected to a TCSPC imaging module. With these techniques it is possible to acquire fluorescence decays in several wavelength regions simultaneously. We applied these methods to Förster resonance energy transfer (FRET) measurements and discuss the advantages and pitfalls of fluorescence lifetime measurements.
Journal of Biomedical Optics, 2003
Fluorescence lifetime images are obtained with the laser scanning microscope using two methods: the time-correlated singlephoton counting method and the frequency-domain method. In the same microscope system, we implement both methods. We perform a comparison of the performance of the two approaches in terms of signal-to-noise ratio (SNR) and the speed of data acquisition. While in our practical implementation the time-correlated single-photon counting technique provides a better SNR for low-intensity images, the frequency-domain method is faster and provides less distortion for bright samples.
Analytical Chemistry, 2008
Time-gated techniques are useful for the rapid sampling of excited-state (fluorescence) emission decays in the time domain. Gated detectors coupled with bright, economical, nanosecond-pulsed light sources like flashlamps and nitrogen lasers are an attractive combination for bioanalytical and biomedical applications. Here we present a calibration approach for lifetime determination that is noniterative and that does not assume a negligible instrument response function (i.e., a negligible excitation pulse width) as does most current rapid lifetime determination approaches. Analogous to a transducer-based sensor, signals from fluorophores of known lifetime (0.5-12 ns) serve as calibration references. A fast avalanche photodiode and a GHz-bandwidth digital oscilloscope is used to detect transient emission from reference samples excited using a nitrogen laser. We find that the normalized time-integrated emission signal is proportional to the lifetime, which can be determined with good reproducibility (typically <100 ps) even for data with poor signalto-noise ratios (∼20). Results are in good agreement with simulations. Additionally, a new time-gating scheme for fluorescence lifetime imaging applications is proposed. In conclusion, a calibration-based approach is a valuable analysis tool for the rapid determination of lifetime in applications using time-gated detection and finite pulse width excitation.
Single photon timing system for picosecond fluorescence lifetime measurementsa)
Review of Scientific Instruments, 1983
A single-photon timing system is described which is capable of extracting fluorescence lifetimes as short as 25 ps. The system is an improved version of an earlier apparatus. The new system uses a synchronously pumped, mode-locked dye laser with 10-ps pulses operating at 82-MHz repetition rate. A fast photodetector and a leading-edge discriminator were developed to use with this light source. Also, a special rate reduction circuit was built to eliminate large oscillations in fluorescence decay spectra due to the excessive stop rates that overload commercial time-to-amplitude converters.
Proceedings of SPIE, 1994
The Center for Fluorescence Spectroscopy (CFS) is a multi-user facility providing state-of-the-art time-resolved fluorescence instrumentation and software for scientists, whose research can be enhanced by such experimental data. The CFS is a national center, supported by the National Center for Research Resources Division of the National Institutes of Health, and in part by the National Science Foundation. Both time-domain (TD) and frequency-domain (FD) measurements (10 MHz to 10 GHz) are available, with a wide range of excitation and emission wavelengths (UV to NIR). The excitation sources for both domains are cavity dumped and frequency-doubled ps dye lasers. Time-correlated single photon counting is accomplished with a 6J1 red-sensitive microchannel plate (MCP) -PMT, to provide an instrument response near 60 ps. Frequency-domain measurements are possible up to 10 GHz using the Center's frequency-domain instrument, which has 6 micrometer UV and red-sensitive MCP-PMT. Available excitation wavelengths range from 280 to 850 nm. High resolution fluorescence intensity or anisotropy decays can be obtained with the assistance of CFS personnel. The data can be used to recover distances and site-to-site diffusion in protein, interactions between macromolecules, accessibility of fluorophores to quenchers, and the dynamic properties of proteins, membranes and nucleic acids. Current software provides for analysis of multi-exponential intensity and anisotropy decays, lifetime distribution, distance distributions for independent observation of fluorescence donors and acceptors, transient effects in collisional quenching, phase-modulation spectra and time-resolved emission spectra. Most programs provide for global analysis of multiple data sets obtained under similar experimental conditions. Data can be analyzed on-site by connection with the CFS computers through the internet. During six years of operation we have established scientific collaborations with over 30 academic and industrial groups in the United States. These collaborations have resulted in 63 scientific papers.