Collection of transient absorption spectra A transient absorption experiment proceeds as follows: the time delay between excitation and probe beams is fixed. Before reaching the sample, the excitation beam (that delivers a pulse every 1 ms) passes through a mechanical chopper that is synchronized

to the amplifier Romidepsin ic50 in such a way that every other excitation pulse is blocked. Thus, alternately the sample is being excited and not excited. Consequently, the white-light continuum that is incident on the detector diode array alternately corresponds to a “pumped” and “unpumped” sample, and the detector alternately measures the intensity of the probe beam of a “pumped” and “unpumped” sample, I(λ)pumped and I(λ)unpumped. I(λ)pumped and I(λ)unpumped are stored in separate buffers (while keeping the time delay between pump and probe fixed), and a number of shots that is sufficient for an acceptable signal-to-noise ratio is measured, usually

103–104. With the shot-to-shot detection capability of the multichannel detection system, particular spectra that deviate from the average (“outliers”) can in real time be rejected during data collection, significantly improving signal-to-noise ratio. A second white-light beam (the reference beam) not overlapping with the pump pulse can also be used to further increase the signal-to-noise ratio. From the averaged values of I(λ)pumped and I(λ)unpumped, Selleck GS1101 an absorbance difference spectrum ΔA(λ) is constructed according to $$ \Updelta Tyrosine-protein kinase BLK A(\lambda ) = – \log (I(\lambda )_\textpumped /I(\lambda )_\textunpumped ). $$Then, the delay line is moved to another time delay between pump and probe, and the above procedure is repeated. In total, absorbance difference spectra at approximately 100–200 time points between 0 fs and ~5 ns are collected, along with absorbance difference spectra before time zero to determine the baseline. In addition, many spectra are collected around the

time that pump and probe pulse overlap in time (“zero delay”) to enable accurate recording of the instrument response function. This whole procedure is repeated several times to test reproducibility, sample stability, and long-term fluctuations of the laser system. In this way, an entire dataset ΔA(λ,τ) is collected. Anisotropy experiments in transient absorption spectroscopy In photosynthetic antennae and reaction centers, the pigments are bound in a well-defined way. Energy and electron transfer processes and pathways can be specifically assessed through the use of polarized excitation and probe beams. The time-dependent anisotropy is defined as $$ r(t) = (\Updelta A_\parallel (t)-\Updelta A_ \bot (t))/(\Updelta A_\parallel (t) + 2\Updelta A_ \bot (t)).