Fluorescence Lifetime Measurement

Introduction

Fluorescence lifetime (FLT) is the time a fluorophore spends in the excited state before emitting a photon and returning to the ground state. FLT can vary from picoseconds to hundreds of nanoseconds depending on the fluorophore.

The lifetime of a population of fluorophores is the time measured for the number of excited molecules to decay exponentially to N/e (36.8%) of the original population via the loss of energy through fluorescence or non-radiative processes.

Fluorescence lifetime is an intrinsic property of a fluorophore. FLT does not depend on fluorophore concentration, absorption by the sample, sample thickness, method of measurement, fluorescence intensity, photo-bleaching, and/or excitation intensity. It is affected by external factors, such as temperature, polarity, and the presence of fluorescence quenchers. Fluorescence lifetime is sensitive to internal factors that are dependent on fluorophore structure.¹

Methods to Determine Fluorescence Lifetime of Fluorophores

Fluorescence lifetime can be measured in either the frequency domain or the time domain.

The time domain method involves the illumination of a sample (a cuvette, cells, or tissue) with a short pulse of light, followed by measuring the emission intensity against time. The FLT is determined from the slope of the decay curve. Several fluorescence detection methods are available for lifetime measurements, of which, time-correlated single photon counting (TCSPC) enables simple data collection and enhanced quantitative photon counting.

The frequency domain method involves the sinusoidal modulation of the incident light at high frequencies. In this method, the emission occurs at the same frequency as the incident light accompanied with a phase delay and change in the amplitude relative to the excitation light (demodulation).

Advantages of Fluorescence Lifetime Measurement Over Intensity-based Measurement²

1. Lifetime measurements do not require wavelength-ratiometric probes to provide quantitative determination of many analytes.
2. The lifetime method expands the sensitivity of the analyte concentration range by the use of probes with spectral shifts.
3. Lifetime measurements may be used for analytes for which there are no direct probes. These analytes include glucose, antigens, or any affinity or immunoassays based on fluorescence energy transfer transduction mechanism.

Applications

Fluorescence Lifetime Assays:

The fluorescence lifetime is a robust parameter for use in several biological assays. It has the potential to replace conventional measurement techniques, such as absorption, luminescence, or fluorescence intensity.³ Any change in the physicochemical environment of the fluorophore leads to changes in the fluorescence lifetime. Lifetime-based assays can be developed by utilizing various mechanisms, such as a simple binding assay that involves the binding of two components (one being fluorescently labeled) to bring about a change in FLT. Another mechanism is a quench-release type assay that involves a quenched species, present in large excess, having low but finite fluorescence. Once the fluorescent compound is released (by an enzymatic reaction or by binding to a complementary DNA), the lifetime of the system changes. FLT can be combined with FRET (Förster resonance energy transfer) assays for energy transfer efficiency measurement.

Fluorescence Lifetime Sensing:

This technique is based on changes in the lifetime or decay time of the probe. Nanosecond (ns) decay times can be measured by phase-modulation. This technique has been extensively used for sensing of pH, Ca²⁺, K⁺, glucose, and other metabolites. There have been recent developments in the application of lifetime-based sensing in tissue and other random media by using optical probes with excitation and emission spectra in the near-infrared region.^4,5

Fluorescence Lifetime Imaging (FLI):

This technique is relatively new and involves the determination of the spatial distribution of fluorescence decay times at every pixel of an image simultaneously. It is based on the fact that the fluorescence lifetime of a fluorophore depends on its molecular environment but not on its concentration. It can be applied in fluorescence microscopy where the local probe concentration cannot be controlled. Fluorescence lifetime imaging microscopy (FLIM) is used in the measurement of molecular environment parameters, protein-interaction by Förster resonance energy transfer (FRET), and the metabolic state of cells and tissue via their autofluorescence. The molecular environment parameters can be measured from lifetime changes induced by fluorescence quenching or conformation changes of the fluorophores. FLIM can be used in biological applications including scanning of tissue surfaces, mapping of tissue type, photodynamic therapy, DNA chip analysis, skin imaging, etc.⁶

Weak emitters have shorter fluorescence lifetimes, while fluorophores with longer lifetimes have low photon turnover rates. They are not very useful for lifetime imaging because of their limited sensitivity and the necessity for long exposition and acquisition time.

Different classes of fluorescent molecules and probes commonly used in lifetime imaging are listed below: