Femtosecond infrared absorption difference spectroscopy is an excellent tool to resolve structural dynamics of both cofactors and proteins via changes in the vibrational absorption spectrum. We developed a multi-pulse, full-timescale (100fs – 1 ms) laser instrument that can be operated in both the visible and mid-infrared spectral regions. With this instrument we can re-excite a protein with a time delay of between 50 fs and 1 ms after a first photon absorption event. Through this novel approach we can selectively monitor and compare protein dynamics associated with the first and second catalytic electron transfer events.
This multi-pulse instrument is configured such that the delays between the 1st pump, 1st probe, 2nd pump, 2nd probe pulses are variable between 50 fs and 1 ms. The instrument is based on two synchronized regeneratively-amplified laser systems (Spitfire ACE-35F-1KXP, and a Hurricane), which share a seed oscillator. Pump wavelengths can be varied between 300 nm and 1200 nm with an optical parametric amplifier, and transient absorption signals in the visible range are detected with a CCD line camera. Spectral changes in the mid-infrared spectral region are recorded to resolve light- or reaction-induced changes in vibrational frequencies. As an improvement to a previous instrument, where we were able to resolve absorption changes with sufficient sensitivity to follow reaction-induced structural changes with atomic resolution on a time scale from 100 femtoseconds up to 6 nanoseconds (6.10-9s), we have now implemented a double-64-element MCT array to reduce noise levels even further. The new instrument is unique because, in combination with a home-developed Lissajous sample scanner, we are able to acquire datasets under single pulse conditions, i.e. the sample is moved at every laser shot and a full dataset is acquired before the sample scanner returns to the same position, and we can monitor the absorption changes in the visible part of the spectrum at the same time as in the mid-IR. In combination with global target analysis, it is possible to disentangle dynamics induced by a first or second excitation event. We are therefore uniquely positioned to study protein and chromophore dynamics via the changes in their electronic and vibrational absorption as a function of applied laser pulses.
Ultrafast infrared spectroscopy in photosynthesis. M. Di Donato and M.L. Groot, Biochimica Et Biophysica Acta-Bioenergetics, 2015. 1847(1): p. 2-11.
Femtosecond time-resolved and dispersed infrared spectroscopy on proteins. M.L. Groot, L.J.G.W. van Wilderen, and M. Di Donato, Time-resolved methods in biophysics. 5. Photochemical & Photobiological Sciences, 2007. 6(5): p. 501-507.