Time-resolved fluorescence is a powerful technique uncovering the temporal evolution and decay of emitting molecular excited states.
Conventional ultrafast time-resolved fluorescence takes advantage of the femtosecond optical gating (FOG) process first demonstrated by Mahr
and Hirsch in 1975.129 In this technique an ultrashort laser pulse excites the chromophore and its emission at 𝜆• is mixed in a nonlinear crystal at a certain time delay after excitation using sum-frequency generation (SFG) process with an ultrashort pulse at known 𝜆– (gate pulse). SFG occurs at a well-defined crystal orientation when phase-matching conditions are met and during the temporal overlap between the gate pulse and fluorescence.
In this case, upconverted light at 𝜆— is generated and detected. By plotting the intensity 𝐼˜™(𝑡) as a function of time delay between pump and gate pulses, a kinetic trace of the fluorescence at the chosen wavelength is obtained.
However, due to the limiting phase matching, upconversion is efficient within a narrow frequency range of the spectrum for a given crystal orientation and alignment of the optics. Therefore, the experiment is conventionally performed in a single-wavelength fashion. If more than dynamics at just a single wavelength are desired, the crystal must be phase-matched for another wavelength, imaging optics realigned, and the measurement repeated. In principle, the full spectrum can be reconstructed from these measurements using the procedures discussed in the literature.108 However, this procedure is prone to errors and artifacts as it relies on both proper fitting the data with multiexponential decays and extraction of the relative intensities at different wavelengths from accurate and reliable steady-state fluorescence spectra. The latter requirement makes reconstruction virtually impossible for fluorophores with such extremely short lifetimes that little to no steady-state fluorescence can be observed. Additionally, the data acquisition procedure is very tedious and realistically only 20-25 spectral points at most can be measured given the high eagerness and motivation of an experimentalist. It is almost impossible to resolve vibrational progressions in spectra reconstructed in such a way and any structure or multiband pattern in the spectra drastically increases the experimental effort to obtain spectral shapes resembling the genuine ones. Additionally, such a lengthy and tedious experimental procedure effectively bans the use of unstable, slowly decomposing or reactive chromophores using this method.
Although the author of this thesis in his early days in the ultrafast science has enjoyed a lot some very long days, evenings and nights performing full spectral reconstruction of the time-resolved fluorescence, no such data are reported in this thesis. Instead, we focus on the much more convenient, facile and, most importantly, accurate and reliable technique, which has become extremely useful and popular in our lab during the last couple of years.
This is a broadband version of the fluorescence upconversion experiment, termed FLuorescence UPconversion Spectroscopy (FLUPS).130–132 This method was developed in the group of Prof. Nikolaus Ernsting (Humboldt University of Berlin). This method allows the limitations of the conventional single-wavelength FOG to be circumvented and the whole fluorescence spectrum to be simultaneously upconverted at once. Instead of recording fluorescence decays point by point in a single-wavelength fashion, simultaneous phase matching over a broad spectral range followed by multiplex detection allows few hundred spectral data points (typically around 250) to be obtained in a single shot.
Figure 2.5 compares time-resolved fluorescence spectra of coumarin 153 in ethanol obtained by the spectral reconstruction method (bottom) and by the FLUPS technique (top). Apart from the superior quality and spectral resolution of the FLUPS spectra, there is an obvious artificial lack of emission on the high-frequency side of the time-resolved area-normalized emission spectra (TRANES) at the earliest time delays. This artifact is a general error obtained with the reconstruction method originating from the improper scaling of the high-frequency side of the spectrum from the steady-state fluorescence spectrum. This leads to a severe underestimation of the total dynamic Stokes shift and might lead to wrong conclusions when exploring short-time dynamics. This is one of the reasons why many groups measuring solvation dynamics via time-dependent fluorescence Stokes shift of the same system under supposedly identical conditions got different absolute values and dynamics of the fluorescence shift using this method (Figure 2.6, left panel).
At the same time, using the broadband upconversion technique recovers not only the same solvation dynamics with a variance of only few
per cent, but also identical absolute values of the fluorescence maxima and spectral shifts are recovered from the samples and setups situated in different parts of the world (Figure 2.6, middle and right panels).
Figure 2.5 | Comparison between normalized FLUPS spectra measured with coumarin 153 dye in ethanol at 6 time delays and Time-Resolved Area-Normalized Emission Spectra (TRANES) obtained by spectral reconstruction from single-wavelength FOG data. Image courtesy of Joseph Beckwith.
The optical scheme of our FLUPS setup is shown in Figure 2.7. The same femtosecond amplified system, as discussed in the previous sub-chapter, is used to pump a collinear OPA (TOPAS-Prime, Light Conversion) producing horizontally polarized gate pulses at 1340 nm with pulse energies of 80 µJ. A Galilean telescope L2/L3 enlarges and recollimates the gate beam. All lenses in the gate pathway have antireflective coating at 1300 nm. A combination of three prisms Pr1-Pr3 is used for compressing the gate pulses. The fourth prism of a normal compressor would be located at point X. The absence of this last prism
results in a pulse front tilt of 3.55° at X due to the action of prism Pr3.131 X is imaged by a thin lens L4 onto the BBO nonlinear crystal, whereby the tilt angle is leveraged to 21° at the crystal (i.e. to the interaction angle a′ of the setup). The pulse front tilt is important to achieve high time resolution in a geometry where the angle between the fluorescence and gate beams is quite large (vide infra).
Figure 2.6 | The left panel (adapted from130) shows the solvent correlation function measured with coumarin 153 in methanol measured by six different groups over two decades by means of spectral reconstruction (first five entries) and by broadband fluorescence (Ernsting). The functions are set to a common value at 200 fs when the IRF was over. A large variance and even different shapes of the kinetic curves are evident. The middle panel shows the position of the fluorescence spectral maximum and the right panel depicts the peak shift of the fluorescence of coumarin 153 in DMSO measured with broadband fluorescence upconversion setups by the research groups in three different laboratories and as compared to the classical work of Horng et al.108 where the spectral reconstruction was used with great care to avoid all the possible artifacts. The middle and right panels are adapted from Ref. 133.
For optical pumping, part of the amplifier output is frequency-doubled to produce 400 nm pulses, which are sent through the optical delay line to a zero-order half-wave plate to control their polarization. After passing a thin lens L1, the laser pulses (pulse energies at this stage in the range of 50-500 nJ) are focused onto the sample cell to a spot diameter of <100 µm.
The same quartz cuvettes with 1.0 mm optical pathlength as used for TA experiments (see previous sub-chapter) are utilized in FLUPS. The sample solution is bubbled with a steam of nitrogen gas just above the laser spot to ensure that the sample volume is refreshed between laser shots but not to interfere with the measurement. An off-axis Schwarzschild objective M1/M2 refocuses the generated fluorescence with sevenfold magnification onto pinhole P. The transmitted pump light is blocked by a small mask
before M1. Lens L5 and a CMOS camera are used for monitoring and alignment of the fluorescence beam (after removing the prism C). The calcite prism C is cut with the calcite optical axis orthogonal to the refracting edge and in the input plane. It separates the vertically polarized fluorescence from the horizontally polarized one that is blocked afterward.
In addition, the prism causes angular dispersion between different colors of the emitted light, which helps for phase-matching. The prism is placed in the beam in such a way that the amount of the dispersive material traversed is minimal. Both pump and low-frequency Raman scattering are reduced with a dielectric longpass filter (cut-off at 420 nm).
Figure 2.7 | Optical scheme of our FLUPS setup.132
The fluorescence emerging from P is imaged onto the BBO SFG crystal by concave mirror M3. The crystal is cut at Q = 40° and has an antireflection coating for 532 nm and 1300 nm on the input face and at 360 nm on the output face. The central rays of the fluorescence and the gate beam form an external gating angle a′ = 21° at the crystal. Type II phase matching is chosen because it provides the broadest spectral window.
Vertically polarized fluorescence at 𝜆• interacts with horizontally polarized gate pulses at 𝜆– = 1340 nm to generate horizontally polarized upconverted light at 𝜆—. Phase matching requires minimizing the wavevector mismatch over a wide range of 𝜆• in the whole visible range of frequencies. The
efficiency of the upconversion depends on the thickness of the crystal and the external angle a′.
The upconverted signal passes through a polarizer G which is used to suppress direct fluorescence, pump scatter and gate harmonics. The concave mirror M4 collects and focuses the signal light on the entrance of a fiber bundle coupling it into the spectrograph. The spectrograph-camera detection system is mounted above the optical table and has ‘no footprint’.
The spectrograph employs a ruled plane grating (Newport) in a Czerny-Turner mount. Photon loss to unused orders is reduced and the UV efficiency is maximized by the choice of blaze angle. The linear wavelength calibration 𝜆— 𝑝 as function of pixel number 𝑝 was obtained by coupling the spectrograph through a fiber to a calibrated steady-state spectrofluorimeter (Cary Eclipse) and selecting consecutively many different wavelengths by tuning the monochromator of the latter. This provides many more points than a mercury lamp. The spectral range 𝜆— = 307-520 nm (corresponding to 𝜆• = 400-850 nm) is mapped onto a 21 mm wide spectrum. It is recorded with a CCD camera (Andor). The resolution of the spectrograph is 50 cm-1 (FWHM of spectral instrument response). We use a full-vertical and 3-horizontal pixel binning and the integration time is set to 1-2 seconds. For a scan in the temporal dimension, the background is first recorded four times at negative delay (-3 ps), averaged and subsequently subtracted from each recorded spectrum while the delay is scanned. Results from 15 time scans are individually corrected for cosmic spikes and then averaged. Measurements in time are done in two different modes. The first range goes up to 2 ps and contains time points evenly spread on a linear grid, while the second range goes from 2 ps up to 1500 ps (maximum available delay) with time points equally spaced on a logarithmic grid. In general, we performed all the FLUPS experiments reported in this thesis under magic angle conditions.
FLUPS data also require some additional preprocessing prior to inspection and analysis. Photometric correction is performed with standard commercial dyes130,132 (BBOT (2,5-bis[5-tert-butylbenzoxazol-2-yl]thiophene), coumarin 6H, coumarin 153 and DCM) to compensate for the different efficiency of the upconversion process for different
wavelengths as well as for the inherently different detection efficiency due to the fiber, spectrograph and CCD. The references are freshly prepared, sealed in 1.0 mm quartz cuvettes and kept in the dark. This procedure ensures that they can be reused during a timespan of approximately 2 weeks. Steady-state absorption and fluorescence spectra of the references are checked before use.
Before the grating, fluorescence passes some dispersive material in the exit window of the cell, polarizer and filter causing group velocity dispersion. Therefore, like for transient absorption, this chirp has to be corrected. This is done by performing a FLUPS measurement with BBOT in the solvent of interest. It is crucial that the dye used as a reference for this purpose does not undergo any solvation dynamics and therefore no spectral shifts should be observed.
As a corollary, this setup could be considered as a photometrically correct spectrofluorimeter with ~170 fs time resolution (which could be improved if the pump laser pulse duration is shortened) allowing to watch fluorescence evolving up to 1.5 ns.