Limits...
Single-molecule imaging with longer X-ray laser pulses.

Martin AV, Corso JK, Caleman C, Timneanu N, Quiney HM - IUCrJ (2015)

Bottom Line: One of the key reasons for this success is the 'self-gating' pulse effect, whereby the X-ray laser pulses do not need to outrun all radiation damage processes.As a result, serial femtosecond crystallography does not need to be performed with pulses as short as 5-10 fs, but can succeed for pulses 50-100 fs in duration.The results suggest that sub-nanometre single-molecule imaging with 30-50 fs pulses, like those produced at currently operating facilities, should not yet be ruled out.

View Article: PubMed Central - HTML - PubMed

Affiliation: ARC Centre of Excellence for Advanced Molecular Imaging, School of Physics, University of Melbourne, Parkville, Victoria 3010, Australia.

ABSTRACT
During the last five years, serial femtosecond crystallography using X-ray laser pulses has been developed into a powerful technique for determining the atomic structures of protein molecules from micrometre- and sub-micrometre-sized crystals. One of the key reasons for this success is the 'self-gating' pulse effect, whereby the X-ray laser pulses do not need to outrun all radiation damage processes. Instead, X-ray-induced damage terminates the Bragg diffraction prior to the pulse completing its passage through the sample, as if the Bragg diffraction were generated by a shorter pulse of equal intensity. As a result, serial femtosecond crystallography does not need to be performed with pulses as short as 5-10 fs, but can succeed for pulses 50-100 fs in duration. It is shown here that a similar gating effect applies to single-molecule diffraction with respect to spatially uncorrelated damage processes like ionization and ion diffusion. The effect is clearly seen in calculations of the diffraction contrast, by calculating the diffraction of the average structure separately to the diffraction from statistical fluctuations of the structure due to damage ('damage noise'). The results suggest that sub-nanometre single-molecule imaging with 30-50 fs pulses, like those produced at currently operating facilities, should not yet be ruled out. The theory presented opens up new experimental avenues to measure the impact of damage on single-particle diffraction, which is needed to test damage models and to identify optimal imaging conditions.

No MeSH data available.


Related in: MedlinePlus

The function D(q) for different pulse durations for 8 keV photon energy, 100 × 100 nm spot size and constant pulse energy of 2 mJ.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4645111&req=5

fig5: The function D(q) for different pulse durations for 8 keV photon energy, 100 × 100 nm spot size and constant pulse energy of 2 mJ.

Mentions: The statistical analysis of diffraction contrast can be used to measure the amount of damage in single-molecule experiments. The average change to the atomic structure factors, characterized by A(q), can readily be measured by summing diffraction patterns. This provides some information about ionization levels but not ion motion. There is more information to be gained by analysing the fluctuations of the diffraction signal. It is not convenient to measure SNRND(q), because B(q) cannot be measured directly without resolving the issue of unknown orientations and assembling a three-dimensional data set, effectively accomplishing a full imaging experiment. An experimentally simpler proposition, which is independent of the imaging experiment, is to measure the standard deviation of the signal within each resolution ring, averaged over all of the measured diffraction patterns. The standard deviation is proportional to and is a measure of the speckle contrast. It will contain contributions from both the average structure of the sample and the damage noise. Unfortunately it is not clear how to separate those two contributions experimentally. Nevertheless, the standard deviation is a sensitive measure of any dynamic change in the sample structure because it will drop relative to the mean scattering signal, as has been shown for averages of molecular conformation (Maia et al., 2009 ▸). To isolate the effect of damage-induced structural change, we create a measure that first subtracts the expected contribution of shot noise, which is equal to μpix(q), and then normalizes by the mean intensity as followswhere μpix(q) is the average intensity at a pixel in resolution ring q averaged over the whole data set and σpix(q) is the corresponding standard deviation. The mean and standard deviation are calculated from the ensemble of experimental data of molecules measured individually in random orientations. It possible to show thatwhere is given in Appendix D. It is possible to show that 0 < D(q) < 1, because 〈f(q, t)f(q, t′)〉2 < 〈f2(q, t)〉 〈f2(q, t′)〉. Fig. 5 ▸ shows D(q) for variations in pulse duration at constant pulse energy (2 mJ). The large variations at high scattering angle indicate the sensitivity of D(q) to ion motion and inner shell ionization, thereby providing complementary information to a measurement of A(q). The term D(q) provides a new means of comparing damage simulations with experiment, and testing the assumptions that underpin damage models for the single-molecule case.


Single-molecule imaging with longer X-ray laser pulses.

Martin AV, Corso JK, Caleman C, Timneanu N, Quiney HM - IUCrJ (2015)

The function D(q) for different pulse durations for 8 keV photon energy, 100 × 100 nm spot size and constant pulse energy of 2 mJ.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4645111&req=5

fig5: The function D(q) for different pulse durations for 8 keV photon energy, 100 × 100 nm spot size and constant pulse energy of 2 mJ.
Mentions: The statistical analysis of diffraction contrast can be used to measure the amount of damage in single-molecule experiments. The average change to the atomic structure factors, characterized by A(q), can readily be measured by summing diffraction patterns. This provides some information about ionization levels but not ion motion. There is more information to be gained by analysing the fluctuations of the diffraction signal. It is not convenient to measure SNRND(q), because B(q) cannot be measured directly without resolving the issue of unknown orientations and assembling a three-dimensional data set, effectively accomplishing a full imaging experiment. An experimentally simpler proposition, which is independent of the imaging experiment, is to measure the standard deviation of the signal within each resolution ring, averaged over all of the measured diffraction patterns. The standard deviation is proportional to and is a measure of the speckle contrast. It will contain contributions from both the average structure of the sample and the damage noise. Unfortunately it is not clear how to separate those two contributions experimentally. Nevertheless, the standard deviation is a sensitive measure of any dynamic change in the sample structure because it will drop relative to the mean scattering signal, as has been shown for averages of molecular conformation (Maia et al., 2009 ▸). To isolate the effect of damage-induced structural change, we create a measure that first subtracts the expected contribution of shot noise, which is equal to μpix(q), and then normalizes by the mean intensity as followswhere μpix(q) is the average intensity at a pixel in resolution ring q averaged over the whole data set and σpix(q) is the corresponding standard deviation. The mean and standard deviation are calculated from the ensemble of experimental data of molecules measured individually in random orientations. It possible to show thatwhere is given in Appendix D. It is possible to show that 0 < D(q) < 1, because 〈f(q, t)f(q, t′)〉2 < 〈f2(q, t)〉 〈f2(q, t′)〉. Fig. 5 ▸ shows D(q) for variations in pulse duration at constant pulse energy (2 mJ). The large variations at high scattering angle indicate the sensitivity of D(q) to ion motion and inner shell ionization, thereby providing complementary information to a measurement of A(q). The term D(q) provides a new means of comparing damage simulations with experiment, and testing the assumptions that underpin damage models for the single-molecule case.

Bottom Line: One of the key reasons for this success is the 'self-gating' pulse effect, whereby the X-ray laser pulses do not need to outrun all radiation damage processes.As a result, serial femtosecond crystallography does not need to be performed with pulses as short as 5-10 fs, but can succeed for pulses 50-100 fs in duration.The results suggest that sub-nanometre single-molecule imaging with 30-50 fs pulses, like those produced at currently operating facilities, should not yet be ruled out.

View Article: PubMed Central - HTML - PubMed

Affiliation: ARC Centre of Excellence for Advanced Molecular Imaging, School of Physics, University of Melbourne, Parkville, Victoria 3010, Australia.

ABSTRACT
During the last five years, serial femtosecond crystallography using X-ray laser pulses has been developed into a powerful technique for determining the atomic structures of protein molecules from micrometre- and sub-micrometre-sized crystals. One of the key reasons for this success is the 'self-gating' pulse effect, whereby the X-ray laser pulses do not need to outrun all radiation damage processes. Instead, X-ray-induced damage terminates the Bragg diffraction prior to the pulse completing its passage through the sample, as if the Bragg diffraction were generated by a shorter pulse of equal intensity. As a result, serial femtosecond crystallography does not need to be performed with pulses as short as 5-10 fs, but can succeed for pulses 50-100 fs in duration. It is shown here that a similar gating effect applies to single-molecule diffraction with respect to spatially uncorrelated damage processes like ionization and ion diffusion. The effect is clearly seen in calculations of the diffraction contrast, by calculating the diffraction of the average structure separately to the diffraction from statistical fluctuations of the structure due to damage ('damage noise'). The results suggest that sub-nanometre single-molecule imaging with 30-50 fs pulses, like those produced at currently operating facilities, should not yet be ruled out. The theory presented opens up new experimental avenues to measure the impact of damage on single-particle diffraction, which is needed to test damage models and to identify optimal imaging conditions.

No MeSH data available.


Related in: MedlinePlus