X-ray detectors must be able to capture faint scattering signals. Scientists currently use the following technologies (5):
- Imaging-plate detectors
- Charged-coupled device detectors
- Wire detectors
- Diode-array detectors.
The pixel resolution determines the required distance between the detector and the sample. High-resolution detectors can be
brought closer to the sample, but low-resolution detectors should be positioned further away from the sample.
The intensity of the scattered X-rays decreases with as distance increases, according to the following quadratic law:
A detector with a distance 2d to the sample receives only 25% of the intensity of a detector with a distance d to the sample at the same photon flux.
Typically, a beamstop protects the detector from exposure to the direct beam, which is several magnitudes more intense than
the scattered X-rays.
Dilute systems allow the analysis of particlate and intraparticlate structure. Interaction between particles can be observed
with highly concentrated samples.
Scientists place the prepared measuring sample into a quartz capillary or a cell with Kapton (DuPont, Wilmington, DE) or polycarbonate
All electrons in the path of the beam interact with the X-ray photons. Application of a vacuum prevents the unwanted scattering
by gas molecules. The contributions of capillary material, windows, and the solvent are significant. These factors need to
be considered separately.
The majority of the scattered intensity comes from electrons that are not part of the molecule or particle of interest. To
remove this unwanted scattering, scientists conduct a second measurement with identical setup, but without the protein or
particle in solution. The difference between the two measurements is the contribution of the molecule or sample under investigation.
This process is called background subtraction.
For the SAXS experiment, the sample holder, containing the sample to be measured is inserted into the camera system, between
the collimation system and detector. The shutter of the X-ray source is then opened for a certain amount of time, thus exposing
the sample to X-rays.
The exposure time depends on the X-ray photon flux through the sample, the sample-detector distance, and the efficiency of
the detector at converting the scattered X-ray photons into measurable electrical signals. The exposure time typically ranges
from fractions of a second to seconds on a synchrotron beam line and from minutes to hours on laboratory-based SAXS systems.
Primary data handling.
Upon completion of the experiment, the resulting intensity map is analyzed. In the first step, the scattering image is reduced
to a scattering curve as a function of the scattering vector q by integrating the image data over a pie slice area (i.e., in point collimation) or box area (i.e., in line collimation).
The same is done for the background experiment.
Next, scientists subtract the background curve from the sample measurement. The remaining curve is then ready for further
evaluation (see Figure 4).
Figure 4: Scattering image of a protein solution (red) and buffer (green). Subtracting the buffer from the solution yields
the information of the actual protein molecule (blue).
Analysis and evaluation
After background subtraction and desmearing (if applicable) the data are ready for the following analytical steps:
- Determining the radius of dyration
- Calculating the surface–volume ratio
- Finding the peak spacing for determining liquid crystalline structure
- Indirect Fourier transform
- Fitting to theoretical models
- Deconvolution of the radial electron-density profile
Ab initio 3D shape and domain modeling.