Peter Kapusta

Fluorescence Lifetime Correlation Spectroscopy

This article explains the basic principles of FLCS, a genuine fusion of Time-Correlated Single Photon Counting (TCSPC) and Fluorescence Correlation Spectroscopy (FCS), using common terms and minimum mathematics. The usefulness of the method is demonstrated on simple FCS experiments. The method makes possible to separate the autocorrelation function of individual components of a mixture of fluorophores, as well as purging the result from parasitic contributions like scattered light or detector afterpulsing.

TCSPC upgrade of a confocal FCS microscope

We extended the measurement capabilities of the Carl Zeiss ConfoCor 1 FCS microscope by (a) using pulsed picosecond diode lasers instead of a continuous wave (CW) laser excitation, (b) introducing a fast single photon avalanche diode detector, and (c) exploiting the capabilities of the PicoQuant TimeHarp 200 board. When the time-tagged time-resolved (TTTR) mode of the TimeHarp is utilized, the complete fluorescence dynamics are recorded. That is, the time-evolution of the fluctuations and the fluorescence decay kinetics are captured simultaneously. Recording individual photon events (without on-the-fly data reduction like in hardware correlators) preserves the full information content of the measurement for virtually unlimited data analysis tasks and provides a much more detailed view of processes happening in the detection volume. For example, autocorrelation functions of dyes in a mixture can be separated and/or their cross-correlation can be investigated. These virtual two-channel measurements are perf...

Fluorescence Lifetime Correlation Spectroscopy (FLCS): Concepts, Applications and Outlook

Fluorescence Lifetime Correlation Spectroscopy (FLCS) is a variant of fluorescence correlation spectroscopy (FCS), which uses differences in fluorescence intensity decays to separate contributions of different fluorophore populations to FCS signal. Besides which, FLCS is a powerful tool to improve quality of FCS data by removing noise and distortion caused by scattered excitation light, detector thermal noise and detector after pulsing. We are providing an overview of, to our knowledge, all published applications of FLCS. Although these are not numerous so far, they illustrate possibilities for the technique and the research topics in which FLCS has the potential to become widespread. Furthermore, we are addressing some questions which may be asked by a beginner user of FLCS. The last part of the text reviews other techniques closely related to FLCS. The generalization of the idea of FLCS paves the way for further promising application of the principle of statistical filtering of signals. Specifically, the idea of fluorescence spectral correlation spectroscopy is here outlined.

Instrument response standard in time-resolved fluorescence

The fluorescence of LDS 798 dye in aqueous solution has a very short lifetime of 24 ps, independent of excitation wavelength. The time response of common photon counting detectors depends on the wavelength of the registered photon. In lifetime measurements, the instrument response function (IRF) is usually approximated by the temporal profile of the scattered excitation light. Because lambda(Exc) is typically much shorter than lambda(Em), a systematic error may be present in these measurements. We demonstrate that the fluorescence decay of LDS 798 is a better approximation of IRF, in particular, for avalanche photodiodes used in the near infrared spectral region.

On the Resolution Capabilities and Limits of Fluorescence Lifetime Correlation Spectroscopy (FLCS) Measurements

Quantitative tests were performed in order to explore the practical limits of FLCS. We demonstrate that: a) FLCS yields precise and correct concentration values from as low as picomolar to micromolar concentrations; b) it is possible to separate four signal components in a single detector setup; c) diffusion times differing only 25% from each other can be resolved by separating a two component mixture based on the different fluorescence lifetimes of both components; d) most of the inherent technical limitations of conventional FCS are easily overcome by FLCS employing a single detector channel confocal detection scheme.

Time-Resolved Fluorescence Anisotropy Measurements Made Simple

Time-resolved anisotropy measurements were performed using simple instrumentation with the aim to demonstrate the speed and ease of the experiment. Subsequent data analysis examples involved common, easily adaptable procedures.

Collisional Quenching of Erythrosine B as a Potential Reference Dye for Impulse Response Function Evaluation

We studied the collisional quenching of the erythrosine B fluorophore by potassium iodide. The quenching follows a Stern–Volmer dependence up to the highest quencher concentration. The lifetime of erythrosine B decreases to 24 ps in 5.02 M of potassium iodide. The quantum yield of erythrosine B in the presence of 5.02 M KI is 0.0035. The relatively high brightness makes this compound attractive as an ultrashort reference in time-resolved measurements. In both frequency- and time-domain fluorescence techniques, there is a need for lifetime standards with extremely short decay times. Mimicking the instantaneous scattering at longer wavelengths allows color-effect-free measurements in the emission region. Another motivation is the problem of obtaining the impulse response function in the case of two-photon excitation. Time-resolved microscopy also benefits from fast-decaying dyes because the impulse response function can be evaluated at the emission wavelength of the investigated specimen without changing filters. We demonstrated that impulse response functions for commonly used detectors are practically the same for scattering as for quenched erythrosine B emission. We also analyzed a complex fluorescence decay using both elastic scattering and quenched erythrosine B emission as a response function.

Dead-time effects in TCSPC data analysis

In Time Correlated Single Photon Counting (TCSPC) the maximum signal throughput is limited by the occurrence of classical pile-up and dead-time effects: At a given photon rate characteristic distortions become visible in the TCSPC histogram. How to describe these distortions in mathematical terms is well known1,2. While the approach of correcting these distortions directly by operations on the raw data has drawbacks, e.g. with respect to calculation effort as well as numerical stability, it is comparably straightforward to include corrections in the models describing the data. We demonstrate the applicability of a model based approach on decay data which are heavily distorted by dead-time and pile-up effects.

Förster resonance energy transfer (FRET)-based picosecond lifetime reference for instrument response evaluation

Forster resonance energy transfer (FRET) can be utilized to achieve ultrashort fluorescence responses in time-domain fluorometry. In a poly(vinyl) alcohol matrix, the presence of 60 mM Rhodamine 800 acceptor shortens the fluorescence lifetime of a pyridine 1 donor to about 20 ps. Such a fast fluorescence response is very similar to the instrument response function (IRF) obtained using scattered excitation light. A solid fluorescent sample (e.g a film) with picosecond lifetime is ideal for IRF measurements and particularly useful for time-resolved microscopy. Avalanche photodiode detectors, commonly used in this field, feature color- dependent-timing responses. We demonstrate that recording the fluorescence decay of the proposed FRET-based reference sample yields a better IRF approximation than the conventional light-scattering method and therefore avoids systematic errors in decay curve analysis.

Fluorescence spectral correlation spectroscopy (FSCS) for probes with highly overlapping emission spectra

We present a fluorescence correlation spectroscopy (FCS) approach to obtain spectral cross-talk free auto- and cross-correlation functions for probes with highly overlapping emission spectra. Confocal microscopes with either a hyperspectral EM-CCD or six-channel PMT array spectral detection were used, followed by a photon filtering correlation approach that results in spectral unmixing. The method is highly sensitive and can distinguish between Atto488 and Oregon Green 488 signals so that auto-correlation curves can be fitted without the need for cross-talk correction. We also applied the approach to the membrane dye Laurdan whose emission is dependent on the lipid order within the bilayer. With fluorescence spectral correlation spectroscopy (FSCS), we could obtain spectral cross-talk free auto- and cross-correlation functions corresponding to Laurdan located in liquid ordered and liquid disordered phases.