Using an OptistatDry Cryostat for Measuring Ultrafast Transient Absorption Spectra at Cryogenic Temperatures

Cryostat in transient absorption set-up.

Figure 1. Cryostat in transient absorption set-up.

This article outlines a new experimental approach to determine ultrafast transient absorption spectra of a biologically related chromophore at cryogenic temperatures. This experiment shows the viability of such measurements, which were performed using Oxford Instruments’ OptistatDry cryogenic system in a broadband ultrafast pump-probe transient absorption spectrometer (Figure 1).

Background

Flavoproteins generally occur in nature. The proteins’ key chromophore is either flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). Proteins have various functions, including acting as photoreceptors, and more commonly as enzymes. The FMN and FAD cofactors are essential for protein to function.

As protein movements freeze at cryogenic temperatures, only the photochemistry continues, which lacks the movements of atoms, enabling a better understanding of protein mechanism to be obtained.

These experiments can be carried out by defining the dynamics of the chomophore at low temperatures through cryogenic transient absorption methods.

Experimental Set-Up

In this analysis, the broadband ultrafast pump-probe transient absorbance spectrometer ‘Helios’ (Ultrafast systems LLC) was powered with a Ti:sapphire amplifier system (Spectra Physics Solstice Ace) that creates 100 fs, 800 nm pulses at 1 kHz. This spectrometer was used to obtain data (at arbitrary time points) from approximately 0.2 ps to 2.9 ns.

A white light continuum created in a sapphire crystal forms the probe beam, and the change in absorbance was monitored between 460 to 640 nm. A part of the amplifier output, pumping a Topas Prime OPA with related Nir UVis unit, generates the pump beam, 0.2 nJ at 375 nm.

After setting up the OptistatDry cryogenic system in the transient absorption enclosure (Figure 1), the position and height were modified, making sure that the position of the sample remains at the focus of the probe and pump beams where they superimposed. At the sample, the beam diameters were on the order of 300 µm.

A cuvette of solvent was scanned using the same conditions to those in the cryostat, and the coherent artefacts were noted (Figure 2). This gave an estimate of the time resolution of ~ 470 fs (typically ~ 250 fs without the cryostat).

A mixture of 50% pH7 phosphate buffer and 50% glycerol was used to dissolve FMN. A 2 mm pathlength cell with quartz windows was filled with ~ 800 ul of this mixture.

Coherent artefacts in buffer solution in cryostat.

Figure 2. Coherent artefacts in buffer solution in cryostat.

Experimental Results

Figures 3 and 4 display the data obtained for a stirred FAD solution at 10 K (in the cryostat but without the external front window) and at room temperature (RT). The spectra display the well-defined transient spectral features of FAD at RT and which are a negative feature centered at ~ 450 nm.

This is due to the loss of population of the ground state, a negative feature at ~ 570 nm as a result of stimulated emission and a positive feature at ~ 500 nm due to excited state absorption. Although the intensity is reduced and there is a considerable reduction in the stimulated emission feature, the same features were observed at 10 K.

It was also observed that the decay kinetics are similar on longer, ns, timescales yet the relaxation takes place more rapidly at 10 K and at short timescales.

Transient absorption data of FAD after excitation at 375 nm at room temperature and at 10K.

Figure 3. Transient absorption data of FAD after excitation at 375 nm at room temperature and at 10K.

Transient absorption data of FAD after excitation at 375 nm.

Figure 4. Transient absorption data of FAD after excitation at 375 nm.

Conclusion

Transient absorption spectra were effectively recorded at 10 K for FAD, which displayed distinct kinetic behavior and spectral features from those quantified at room temperature. As these measurements were recorded on a solid, frozen sample, the sample was not damaged by the laser pulse that constantly irradiating the same spot. This provides a new method to measure flavoproteins at cryogenic temperatures.

This information has been sourced, reviewed and adapted from materials provided by Oxford Instruments Nanoscience.

For more information on this source, please visit Oxford Instruments Nanoscience.

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