Materials, Methods and Instrumentations
2.0. Introduction
2.4.4. Time Resolved Spectrofluorimeter
Fluorescence lifetime is the time required by a population of N electronically excited fluorophores to decrease exponentially by a factor of e to N/e via the loss of energy through fluorescence and other nonradiative processes. The fluorescence lifetime may vary from tens of femtoseconds to nanoseconds. It is an intrinsic property of a fluorophore and is independent the way it is measured. It is considered as a state function, as it is independent of the initial perturbation conditions like excitation wavelength, one or multiphoton excitation, duration of light exposure, fluorescence intensity and fluorophore concentration. Moreover, it TH-1151_07612201
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is not affected by photobleaching. Since fluorescence occurs from an energetically unstable state, fluorescence lifetime is sensitive to internal and external factors. The internal factor comprises of the fluorophore structure and the external factors include temperature, polarity, viscosity and the presence of fluorescence quenchers.
Figure 2.5. Block diagram of a TCSPC instrument.
There are two ways to determine the fluorescence lifetime of the fluorophores, frequency-domain and time-domain.242-248 They have different instrumentation setups and follow different data acquisition methods, however, both approaches are mathematically equivalent and their data can be interconverted by Fourier transform. In frequency domain method, the incident light is sinusoidally modulated at high frequencies such that the emission occurs at the same frequency as the incident light. The difference between the incident and emitted light is that the emitted light experiences a phase delay and change in the amplitude relative to the excitation light (demodulation). Data are acquired with photomultipliers or charge-coupled devices (CCD) equipped with a gain modulator.
In time-domain, the sample to be analyzed is excited with a short light pulse from a light source with sufficient delay between pulses. The light source can be flash lamp, pulsed laser, laser diode, or LED. Various methods are available for data collection. The most common technique is the time correlated single photon counting (TCSPC) which is applied in TH-1151_07612201
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this work.247,248 Figure 2.5 explains schematically the working principle of a TCSPC instrument. In TCSPC method, the light source beam is split into start and stop signal pulses.
The start signal pulse travels to a PMT or micro-channel plate (MCP) photomultiplier which activates the time-to-amplitude converter (TAC). The stop signal pulse travels through the sample. The growth of ramp signal in TAC is stopped by this pulse. The TAC output can be amplified by an amplifier, and this analogue pulse of height corresponding to a measured time of the signal goes through further processing to convert to digital pulse through the analogue to digital converter (ADC).
Figure 2.6. Exponential decay model for three components.
Since the fluorophores emit photons at different relaxation times following their excitation by radiation, the decay time of single molecules must have a certain rate rather than occurring at a specific time with excitation. The principle of TCSPC is the detection of single photons and the measurement of their arrival times in respect to a reference signal from the light source. The TCSPC method needs a high repetitive light source to accumulate a sufficient number of photons since this is a statistical method and requires many numbers of statistical data precision. The time measurement of the start and stop sequence is represented by an increase of a memory value in a histogram. Thus, this experiment must be repeated many times to gather sufficient photons in the full range of delays between excitation and emission. The resulting histogram counts versus the time channels on the x-axis represents
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FTIR, FT NMR, Single Crystal XRD 48
the curve of fluorescence decay profiles. A typical emission decay of a fluorophore consisting of three components obtained from TCSPC method is shown in Figure 2.6.
In most cases, the TCSPC technique has limits for the temporal resolution and lifetime range measurable for the fluorescence lifetimes. Therefore, for the curve fittings the method involving linearization of the fitting function and least-squares fitting is the most widely used deconvolution technique. The fluorescence temporal profiles were derived by deconvolution procedures with the instrument response using nonlinear least-squares fittings.
In the entire work, fluorescence lifetimes were measured with the use of LifeSpec II from Edinburgh Instruments. LifeSpec II employs Hamamatsu MCP detector that has response time of 50 ps. The light sources used to excite the sample were 308 nm LED from PicoQuant and 375 nm laser diode from Edinburgh with full pulse widths of 635 ps and 2 ps, respectively at half-maximum. Time-resolved data were analyzed with reconvolution method based on discrete components analysis model using the FAST software249 developed by the Edinburgh Instruments Ltd. The goodness of fit was determined by the reduced χ2 values and weighted residuals (X) which were between the range of ± 6. The reduced χ2 and weighted residuals (X) are given by the following equations.
2
2 2[ i i]
i i
S F
w n
χ =
∑
− (2.10)( )
i i i i
X =w S −F (2.11)
where wi, Fi, and Si are the weighting factor, measured value, and fitted value, respectively and n is the number of fitted data points subtracted by the number of lifetime parameters used in the fit. The error limits in the lifetime values were ± 0.05 ns.