Fluorescence Lifetime Imaging
6.3.1. Intensified CCD Camera
The structure of an intensified CCD (ICCD) system is illustrated in Fig. 6.10. After being impacted by a photon, the photocathode creates photoelectrons that are amplified by the micro channel plate (MCP); the amplified electrons are converted back into photons by a phosphor screen. These photons are relayed to a CCD by either a fiber-optic bundle or a relay lens; the CCD creates the photoelectrons that are measured. The biggest advantage of ICCD is its ability of gating that allows the luminescent lifetime imaging over a painted area. Electronic shutter action can be produced by pulsing the MCP voltage and the gain can be modulated by simply changing the voltage on the intensifier. Figure 6.11 illustrates the luminescent lifetime imaging method with an ICCD.
For the pulse excitation light, the gain function is typically a top-hat function or a square function. The luminescent signal is gated in two different intervals during an exponential decay of luminescence and the gated intensity ratio is related to the luminescent lifetime by Eq. (6.29). This approach was employed for PSP measurements by Goss et al. (2000), Bencic (2001), Bell (2001), Baker (2001), and Mitsuo et al. (2002). Another approach uses the sinusoidal excitation light combined with either the square gain function (Holmes 1998) or sinusoidal gain function (Lakowicz and Berndt 1991). Consider the sinusoidally modulated excitation light E(t) = Am[1 + H sin( m t)] and the corresponding luminescent signal from PSP is I(t) = Am z [1 + HM ef sin(mt – f)] , where the effective amplitude modulation index is M eff = (1 + z2 m2) ~1/2 = cos( у). When the gain
function has a square-waveform, the gated intensity ratio is given by Eq. (6.25).
Instead of using the square function, Lakowicz and Berndt (1991) adopted the sinusoidal gain function for modulating the intensifier. When the MCP is sinusoidally modulated, the gain function of the detector is
G(t) = G0 [ 1 + mDsin(mt – вD)] , where G0 is the intensifier gain without applying a modulating signal, mD is the gain modulation depth, and QD is the detector phase angle relative to the modulated illumination light. The CCD collecting photons over an integration time actually serves as an integrator; thus, the signal output from the CCD is represented by a time-averaged intensity over an integration time TINT
1 Г tint
< I > =——– I I( t, r)G( t)dt = Am z G0[ 1 + 0.5Mef mD cos( (p – 6D)] .
To extract the phase angle or lifetime from the CCD output < I > , several values of < I > are obtained by changing the detector phase angle вD. Therefore, a system of equations is given for eliminating Am and G0 . These equations can be solved using least-squares method to determine the phase angle ^ that is related to the luminescent lifetime T. In the simplest case where only two different detector phase angles dD1 and вD2 are chosen, a ratio between the two time – averaged intensities at dD1 and вD2 is
Once the parameters mD, 0D1, and вD2 are given, the ratio in Eq. (6.32) is only related to the phase angle ^. Lakowicz and Berndt (1991) used three different detector phase angles to recover the luminescent lifetime. One shortcoming of the intensifier CCD camera is that the SNR may be reduced due to quantum losses and additive noise in the multiple-step photon-electron transfer processes.