full “white” beam. The pink-Laue method eliminates the need for the thousands of
pump-probe cycles required in the stroboscopic method used for monochromatic
data sets [33]. Because of the reduced time for the experiment, the crystal deterioration caused by laser and X-ray exposure may be reduced and the heating caused by
the repeated laser pulses may also be reduced. In the best possible case, a single
synchrotron pulse is sufficient to record a good enough diffraction pattern to solve
the crystal structure [34]. In other examples several pump-probe cycles are required
before a sufficiently intense diffraction pattern is obtained.
For pink-Laue data, a wavelength dependence correction has to be applied to the
intensity data, because diffraction spots are being obtained from many wavelengths,
and there also has to be a fitting of intensities of equivalent reflections that appear.
With molecular crystals having relatively small unit cells, this scaling of intensities is
challenging because there are relatively few of them. To avoid this difficulty, the
RATIO method can be used [35], a method that will be explained in further detail
below. A second complication with pink-Laue data is that there is a steep slope of
wavelength distribution on the high-energy side of the pink-Laue spectrum. A small
change in the unit cell dimensions on exposure, as would be expected with excitation, might lead to anomalous values of the on/off ratios of reflections scattered by
wavelengths in this narrow region. The effect would be small if the conversion
percentage from the ground to the excited state is small and also if the temperature
increase is small. The affected reflections can be identified in the analysis of
equivalent reflections and confirmed by checking the calculated wavelength from
the Bragg angle and the hkl index after indexing. These can be removed from the
averaging procedures and used to establish the structural changes that have taken
place in the excited state.
As indicated above, the most sensitive measure of the structural change in a
crystal when it is photoactivated into an excited state is the observed change in
intensity of each of the reflections. In the RATIO method [35], this intensity
difference is identified by using the on/off ratios as the observables in the activated
structure refinement program LASER [36]. As explained in the previous paragraph,
the advantage of the RATIO method when used in a Laue data collection is the
elimination of the need to have a spectral curve to determine the wavelength at
which each reflection is observed. To exploit the method, the laser-on and laser-off
intensities for each reflection need to be collected immediately after one another.
This eliminates variations in the intensity of the X-ray beam over time. This, in turn,
adds an error to the intensity of the individual reflections and also eliminates the
effect of any slow deterioration of the crystalline sample. In addition, slight differences in the absorption correction may occur if the laser-on and laser-off reflections
were collected at different times in different settings. Finally, scaling is not required
as the paired frames are collected at the same temperature and under the same
conditions.
Even with these advances, it is often necessary to use multiple crystals of a
crystalline sample, because of crystal deterioration, in order to obtain a complete
data set. Scaling of the data sets is then required before electron density maps can be
calculated and the structure solved and refined. Various scaling methods have been
248
P. R. Raithby
pump-probe cycles required in the stroboscopic method used for monochromatic
data sets [33]. Because of the reduced time for the experiment, the crystal deterioration caused by laser and X-ray exposure may be reduced and the heating caused by
the repeated laser pulses may also be reduced. In the best possible case, a single
synchrotron pulse is sufficient to record a good enough diffraction pattern to solve
the crystal structure [34]. In other examples several pump-probe cycles are required
before a sufficiently intense diffraction pattern is obtained.
For pink-Laue data, a wavelength dependence correction has to be applied to the
intensity data, because diffraction spots are being obtained from many wavelengths,
and there also has to be a fitting of intensities of equivalent reflections that appear.
With molecular crystals having relatively small unit cells, this scaling of intensities is
challenging because there are relatively few of them. To avoid this difficulty, the
RATIO method can be used [35], a method that will be explained in further detail
below. A second complication with pink-Laue data is that there is a steep slope of
wavelength distribution on the high-energy side of the pink-Laue spectrum. A small
change in the unit cell dimensions on exposure, as would be expected with excitation, might lead to anomalous values of the on/off ratios of reflections scattered by
wavelengths in this narrow region. The effect would be small if the conversion
percentage from the ground to the excited state is small and also if the temperature
increase is small. The affected reflections can be identified in the analysis of
equivalent reflections and confirmed by checking the calculated wavelength from
the Bragg angle and the hkl index after indexing. These can be removed from the
averaging procedures and used to establish the structural changes that have taken
place in the excited state.
As indicated above, the most sensitive measure of the structural change in a
crystal when it is photoactivated into an excited state is the observed change in
intensity of each of the reflections. In the RATIO method [35], this intensity
difference is identified by using the on/off ratios as the observables in the activated
structure refinement program LASER [36]. As explained in the previous paragraph,
the advantage of the RATIO method when used in a Laue data collection is the
elimination of the need to have a spectral curve to determine the wavelength at
which each reflection is observed. To exploit the method, the laser-on and laser-off
intensities for each reflection need to be collected immediately after one another.
This eliminates variations in the intensity of the X-ray beam over time. This, in turn,
adds an error to the intensity of the individual reflections and also eliminates the
effect of any slow deterioration of the crystalline sample. In addition, slight differences in the absorption correction may occur if the laser-on and laser-off reflections
were collected at different times in different settings. Finally, scaling is not required
as the paired frames are collected at the same temperature and under the same
conditions.
Even with these advances, it is often necessary to use multiple crystals of a
crystalline sample, because of crystal deterioration, in order to obtain a complete
data set. Scaling of the data sets is then required before electron density maps can be
calculated and the structure solved and refined. Various scaling methods have been
248
P. R. Raithby