In the recent past, our
understanding of elementary events in proteins and genetic materials has
benefited greatly from the application of ultrafast time-resolved spectroscopic
techniques. Elementary reactions in biology generally display a complex
pattern: multiple reaction pathways may occur after initiation, reactions often
exhibit intrinsic multi-exponentiality, or reaction intermediates may remain
hidden as a result of rate-limiting formation times. These properties are
inherent to time-resolved spectroscopy when it is applied in its traditional ‘pump-probe’
way, and preclude a complete understanding of fundamental processes in biology.
With multi-pulse control spectroscopy, the course of events is ‘controlled’ by
applying additional stimuli at well-defined moments during the reaction. The
power of the multi-pulse method lies in the ability to use a second laser pulse
(or third, etc.) to selectively remove or transfer population of a transient
reaction species with carefully timed and color-adjusted laser pulses, thus
disentangling complex elementary events in biology.
Through a recent
investment grant by NWO-ALW of kEur 675 to R. van Grondelle and myself, we are
constructing an all-purpose multi-pulse femtosecond spectrometer. The apparatus
involves a turn-key amplified Ti:sapphire laser system (Coherent broadband
Vitesse-Legend) of 45 fs pulse duration and 2.5 W output power, equipped with
three independently tunable, computer-controlled OPAs (Coherent OperA), of
which two have a visible-near IR output and the 3rd a mid-IR output,
ensuring the wide tunability of its pump and control pulses to address the wide
variety of biological systems. Detection takes place with a white light
continuum, comprising all colors in the visible, near-UV and near-IR.
Importantly, the capability to detect spectral changes in the mid-infrared is
being installed to selectively probe the dynamic structure of the protein
during the imposed sequence of events. This part of the setup is being
completed in collaboration with M.L. Groot.
Additionally, a novel ultrafast vibrational technique called femtosecond stimulated Raman spectroscopy (FSRS) is being developed. In ‘classical’ time-resolved Raman experiments, where two optical pulses are used, the time-resolution is always limited to a few picoseconds since otherwise the spectral resolution would be too low to resolve the individual vibrational frequencies. With FSRS this problem is overcome by using three pulses. A short visible pulse is used to excite the sample, stimulated Raman scattering is induced by a narrow-bandwidth pulse of picosecond duration and probed by a short, broad-bandwidth white-light continuum pulse. The time-resolution in this experiment is determined by the duration of the first pulse and the probe pulse. The spectral resolution is determined by the Raman pump pulse. Temporal and spectral resolutions of 100 fs and <10 cm-1 have recently been demonstrated. Moreover, background-free Raman spectra at high signal to noise are readily obtained (see e.g. Kukura et al., Science 2005). To perform FSRS, the new laser system has been equipped with a narrowband picosecond OPA (TOPAS) to provide tunable Raman excitation.
The
setup, which will be unique in The Netherlands and the EU, will be used to
study a number of important biological processes that are central in our
current research, often in combination with national or international
collaborators. The new spectrophotometer will be accessible to national and
international researchers/collaborators via the VU-Laser center (LCVU).