This technique of time-resolved fluorescence spectroscopy is based on nonlinear optical tricks rather than cutting-edge electronics. The experimental layout of upconversion experiment, shown in fig. 15, resembles the autocorrleator (fig. 11B). To obtain fluorescence signal from the sample, the sample is placed into one of the branches of the autocorrelator and cross-correlation between the excitation laser pulse and sample fluorescence is recorded, by adding the frequencies of both light pulses in the nonlinear crystal. More specifically, the pulse from the laser is split into two, one of which is delayed in the optical delay line (it is called gating, or gate pulse). The rest of the light is used (usually after additional devices to produce the correct excitation wavelength) to excite the sample. The emitted fluorescence is collected using off-axis parabolic (or elliptical) reflectors or lenses and is focused into nonlinear crystal . Mirrors instead of lenses are used to get the best time resolution, because they do not introduce additional dispersion in the signal. Gate pulse is overlapped in the crystal with the fluorescence pulse. Crystal orientation is chosen to satisfy the phase matching condition for sum frequency generation between the gate and the emission wavelengths.
Sum frequency generation is a quadratic nonlinear optical phenomenon (see above), and the intensity of resulting sum frequency is proportional to the product of intensities of the two added fields:
Therefore, if the gate pulse intensity is kept constant, the measured signal is proportional to the fluorescence intensity of the sample at the time instance when sample fluorescence is overlapped with the gate pulse (fig. 16). In other words, the short gating pulse cuts time slices out of long fluorescence pulse. These slices are registered by the detector as time-frozen fluorescence intensities. The instance, at which they are frozen, depends on the delay of the gating pulse. This implies that varying this delay allows recording fluorescence intensity as a function of time. The frequencies of fluorescence and gate pulses are added in the nonlinear crystal; therefore, with the wavelength of gate pulse known, one can recover the fluorescence wavelengths using equation
Fig. 1) Fluorescence up-conversion experiment: the pulse is split into two, one of which (gate) is delayed by the optical delay line, whereas the other is used (after second harmonic generation in BBO1) to excite the sample. The fluorescence emitted by the sample is collected using off-axis parabolic reflectors and focused into a nonlinear crystal BBO2 together with the delayed gate pulse. BBO2 generates sum frequency between the gate and the fluorescence, the spectrum of which is spread by the monochromator and recorded by the CCD detector.
because the wavelength of the recorded signal is measured experimentally.
Fluorescence upconversion is a purely optical method, and its time resolution is essentially limited only by the pulse duration of the employed laser pulses. This resolution can easily be 150 fs and less, when Ti:Sapphire lasers are used as excitation and gate light sources. Therefore, fluorescence upconversion is the method of choice, when the investigated processes are extremely fast (faster than 1 ps). Other methods cannot compete for time resolution. However, there are also disadvantages. Frequency upconversion (sum frequency generation) is a nonlinear process, necessitating the use of intense excitation pulses, often damaging to biological samples. Due to the uncertainties of phasematching condition, different upconversion efficiencies at different wavelengths and other subtle nuances that are hard to control during the experiment, it is tricky to measure undistorted fluorescence spectra when using upconversion technique. Finally, this technique only records fluorescence at a single time point and a single spectral point. To get spectro-temporal data, one has to scan both wavelength and delay, which takes time.