The present invention relates to a method for X-Ray Scattering material analysis, in particular Small Angle X-ray Scattering material analysis, and an apparatus for carrying out such a method.

While Wide Angle X-ray Scattering (WAXS) typically gives access to information on crystalline structure and phase(s) of a sample to be analyzed, Small Angle X-ray Scattering (SAXS) typically gives access to information about the sample structure on the nano-scale level (nano-structure). Examples are solid polymer films, alloys or powders constituting a sample whose nano-structure is analyzed. However, the invention also relates to cases in which SAXS is used to obtain information about one among a plurality of components of the sample. Often such samples analyzed by SAXS are analytes diluted in a sample solvent and contained in a sample container like e.g. a capillary. As a typical example dispersions of nano-particle micelles can be mentioned.

A method for X-Ray Scattering material analysis, in particular Small Angle X-ray Scattering material analysis, comprising:generating and directing an incident X-ray beam along a propagation direction to a sample held in a sample environment;executing a sample measurement process comprising the following steps:determining a distribution of X rays scattered from said sample by means of an area detector arranged downstream of said sample environment; anddetermining an intensity Itof a beam of X rays transmitted through said sample by means of said detector;executing a sample data treatment process comprising the following step:determining an absolute scattered X-ray distribution by applying to said scattered X-ray distribution a conversion into absolute intensities which depends on said transmitted intensity; andexecuting a data analysis process comprising the following step:determining at least one structural characteristic of said sample on the basis of said absolute scattered X-ray distribution;
is generally known to the skilled person in the field of material analysis by means of X-ray scattering.

Such X-Ray Scattering material analysis methods require measurement of scattered intensity in so-called absolute intensities for determining the desired structural characteristic(s) of the sample on the basis of a relation between the scattered X-ray distribution and the transmitted X-ray intensity. This means that the intensity of the X-ray beam transmitted through the sample needs to be measured to apply certain corrections required to get the scattered signal in absolute intensity. This, however, poses problems in that the transmitted intensity is up to many orders of magnitude stronger than the scattered signal so that the large transmitted intensity easily drives the area detector out of its linear counting range or even risks destroying the detector. In state of the art methods, this problem is taken into account e.g. either by collecting the transmitted signal with the area detector with an attenuation of the transmitted intensity through an attenuator or a semi-transparent beamstop or by using a second detector like a beamstop with a pin diode incorporated inside. Alternatively, the absolute intensity is obtained through the measurement the scattering intensity of a reference sample.

The area detector can be a 1D or a 2D detector. In the following the preferred case of a 2D detector will be predominantly considered.

Furthermore SAXS detectors require a low intrinsic noise and except for modern solid-state detectors working on direct detection mode with individual pixel counters (the so-called hybrid pixel detectors), those detectors suffer from low maximum count rate and/or detector damage risk like for gas detectors with respect to transmitted beam intensities found in modern SAXS instruments. Therefore in prior art SAXS methods typically use is made of one or several beam stops in front of the 2D detector to attenuate the intensity of the direct beam passing through the sample, i.e. the transmitted beam, by a semi-transparent beam stop or to completely prevent the direct X-ray beam from reaching the detector by a fully absorbing beam stop. In the latter case determination of the transmitted intensity then has to be carried out before or after the scattering measurements with short counting times and/or with a different acquisition device, e.g. a pin diode.

State of the art advanced combined SAXS/WAXS systems use a beamstop and a hybrid pixel detector preferably placed inside a vacuum chamber environment in order to reduce air and vacuum-window parasitic scattering.

However, in ail these prior art approaches a semitransparent beam stop itself causes parasitic scattering and furthermore causes so-called beam hardening effects, i.e. it affects the spectrum of the incident X-ray beam, making precise determination of transmitted intensity unreliable. Furthermore use of a semitransparent or completely absorbing beam stop is always laborious in that it requires a time-consuming precise positioning of the beam stop in the transmitted beam minimizing its impact on the scattered signals.

It is therefore an object of the invention to overcome such problems related to the use of a beam stop.

According to the invention this object is achieved in a Small Angle X-ray scattering material analysis method as outlined above in which:acquisition of said scattered and said transmitted X-rays is divided into a plurality of acquisition periods, wherein each acquisition period (Tacq) is shorter than or equal to a previously determined maximum acquisition time (Tmax) in such a way that said detector operates in a linear range;said detector measures individual detector image frames containing signals of said scattered and said transmitted X-rays, wherein each individual detector image frame is measured during one among said plurality of acquisition periods;said individual detector image frames are added up to a total detector image frame in a computer connected to said detector; andsaid determination of said at least one structural characteristic of said sample is realized based on an absolute scattered X-ray distribution obtained on the basis of said total detector image frame.

According to the invention each individual detector image frame is measured during an acquisition period which is short enough to make sure that the detector operates in its linear range. After a certain number of individual detector image frames has been measured the counting electronics of the detector are reset so that the detector starts counting the next individual detector image frame basically from 0. Depending on the linearity requirements of the detector its counting electronics can e.g. be reset and restarted after each individual detector image frame or after every 2ndindividual detector image frame etc. to make sure that the detector always stays in its linear operation range. Thus also the total detector image frame obtained by adding up the individual detector image frames in the computer is free from any nonlinear distortions.

The method according to the invention assures a linear operation range of the detector while allowing to collect simultaneously the transmitted signal, i.e. the X-ray beam passing through the sample and the sample environment and reaching the detector without being attenuated and having its spectrum affected by a beam stop, during the complete scattering measurement. This provides several advantages compared to prior art methods using a semi-transparent beamstop or a second detector. Because one uses the same detector as the one used to collect the scattered signal, accuracy of the overall measurement of the scattering intensity of sample in absolute units is improved as there are no problems with different detector efficiencies and gain. Moreover, compared to alternative methods using a short and separate acquisition (typically 0.1 seconds) and collection of transmitted intensity before and/or after the scattering measurement with a different acquisition device, e.g with a pin diode, the simultaneous collection method according to the invention is more accurate as it suppresses the impact of possible intensity variations of the incoming X-ray beam during the complete sample measurement and the influence of different detector efficiencies. Also, variations in sample characteristics and scattering can be resolved and data used by determining the integration time after the experiment

In a preferred embodiment of the method according to the invention said total detector image frame is displayed and continuously updated with each additional individual detector image frame on a screen connected to said computer. This allows a user carrying out the SAXS measurement to continuously monitor the experiment.

Preferably the method according to the invention furthermore comprises a maximum acquisition time determining process executed before said sample measurement process, wherein said maximum acquisition time determining process comprises:measuring said intensity Itof a beam of X rays transmitted through said sample; andcalculating said maximum acquisition time on the basis of said intensity It.

This calculation of the maximum acquisition time to be used as an upper time limit for each individual detector image frame usually implies dividing an allowable total count number of the area detector as indicated by the manufacturer of the detector in the detector specification by the measured intensity It.

Advantageously the method according to the invention furthermore comprises a beam resolution determining process executed before said sample measurement process, wherein said beam resolution determining process comprises:measuring at least one individual detector image frame containing signals of an X-ray beam transmitted through said sample environment and of X rays scattered from said sample environment preferably in a situation in which no sample and no sample solvent is held in said sample environment; anddetermining a beam resolution of said transmitted X-ray beam from said at least one individual detector image frame.

This advantageous embodiment allows to characterize very accurately the X-ray beam profile used for the experiment and to quantify the beam resolution. This is particularly useful for analysis methods like particle size distribution analysis where the quantification of the beam resolution and its inclusion within the modelling process increases the accuracy of the data analysis and consequently the accuracy of the determined particle size. Preferably this beam resolution determining process is carried out without a sample or a sample solvent in the beam, but usually with a sample container like e.g. a capillary in the beam. However the beam resolution determining process can also be carried out with the sample in place. In this case, the PSF of the direct beam is determined from the central part of the beam corresponding to no momentum transfer Q=0, i.e. a scattering vector modulus of zero, and using the first points from the 1D intensity distribution up to a dynamic of 1E3 approximately.

In these advantageous embodiments it is further preferred that said step of determining a beam resolution comprises executing an azimuthal averaging of said at least one individual detector image frame such as to convert a 2D signal intensity distribution in said individual detector image frame or in a total detector image frame obtained by adding up said individual detector image frames into a 1D intensity distribution as a function of momentum transfer (Q), i.e. scattering vector modulus.

The point spread function (PSF) of the incoming beam can then be obtained as follows. A direct beam image is azimuthally averaged and reduced to 1D scattering data (I=f(Q)) on the same units grid (in Q or 2θ angle) as the one used for reducing the sample scattering data. A convolution kernel is built from the PSF and further on included in the models used to fit the data. This method accounts for the beam smearing effects in any (arbitrary) model, irrespective of the model's details and parameters. The data analysis process comprises optimizing the model parameters including smearing effects with respect to the experimental data. In other words, the beam convolution effects are considered in the forward model, which yields an increase in the stability of the results as compared to the alternative of refining the raw model on the deconvoluted experimental data.

Preferably the method according to the invention furthermore comprises a sample environment calibration process executed before said sample measurement process, wherein said sample environment calibration process comprises:measuring individual detector image calibration frames containing signals of said scattered and said transmitted X-rays by means of said detector in a situation in which no sample is held in said sample environment, wherein each individual detector image calibration frame is measured during one among a plurality of calibration acquisition periods;adding up said individual detector image calibration frames to a total detector image calibration frame in said computer connected to said detector;determining a transmitted intensity and a beam center position from said total detector image calibration frame.

Also in this case said sample environment calibration process preferably furthermore comprises executing an azimuthal averaging of said total detector image calibration frame such as to convert a 2D signal intensity distribution in said total detector image calibration frame into a 1D calibration intensity distribution as a function of momentum transfer (Q).

As a particular advantage of this embodiment, said conversion may be realized continuously between scattered signals corresponding to a finite momentum transfer Q>0 and transmitted signals measured along said propagation direction (X) corresponding to no momentum transfer Q=0.

In all embodiments of the method according to the invention, said data analysis process preferably comprises:determining a transmitted intensity and a beam center position from said total detector image frame.

It is further preferred that said data analysis process furthermore comprises:executing an azimuthal averaging of said total detector image frame such as to convert a 2D signal intensity distribution in said total detector image frame into a 1D intensity distribution as a function of momentum transfer (Q).

Said conversion may be realized continuously between scattered signals corresponding to a finite momentum transfer Q>0 and transmitted signals measured along said propagation direction (X) corresponding to no momentum transfer Q=0.

In all embodiments that make use of the above-discussed 1D calibration intensity distribution it is furthermore preferred to correct said 1D intensity distribution by means of said 1D calibration intensity distribution. As a simple example, the 1D calibration intensity distribution determined in the sample environment calibration process can be subtracted from the 1D intensity distribution determined during the sample data analysis process.

This correction of the 1D intensity distribution by means of said 1D calibration intensity distribution allows to provide a further advantage when the method according to the invention furthermore comprises a data quality control process in which a signal to noise ratio in a predetermined range of momentum transfer (Q) in said corrected 1D intensity distribution is compared to a predetermined threshold and a feedback action is executed depending on the result of said comparison.

Preferably said feedback action comprises at least one of the following:stopping said sample measurement process;changing a position of said area detector;changing an opening of one or more collimating apertures.

In the 1stcase, for example, said sample measurement process is automatically stopped when a signal to noise ratio in a predetermined range of momentum transfer (Q) in said corrected 1D intensity distribution exceeds a predetermined threshold. This renders the entire SAXS measurement faster and requires less user interaction. The term “signal to noise ratio” is intended to also comprise similar statistics known to the skilled person in the field of data quality evaluation.

In all embodiments the method according to the invention can preferably furthermore comprise a data posttreatment process, comprising a background removal step in which background signals are removed from said individual detector image frames based on a comparison with said total detector image frame, wherein said background corrected individual detector image frames are added up to a total background corrected detector image frame in said computer, and wherein a background corrected determination of said at least one structural characteristic of said sample is realized based on said total background corrected detector image frame.

The background signals removed from said individual detector image frames result in particular from inevitable ambient background, also called cosmic background. The comparison of individual detector image frames with the total detector image frame in order to identify such background signals is preferably carried out on a pixel-by-pixel basis using statistical algorithms confronting its experimental statistics to the theoretical statistics expected for the counting process.

The data posttreatment process can furthermore comprise determining a background corrected transmitted intensity and a background corrected beam center position from said total background corrected detector image frame.

In this case the data posttreatment process can preferably furthermore comprise executing an azimuthal averaging of said total background corrected detector image frame such as to convert a 2D signal intensity distribution in said total background corrected detector image frame into a 1D background corrected intensity distribution as a function of momentum transfer (Q).

This conversion may be realized continuously between background corrected scattered signals corresponding to a finite momentum transfer Q>0 and background corrected transmitted signals measured along said propagation direction (X) corresponding to no momentum transfer Q=0.

In this embodiment the method according to the invention can furthermore comprise correcting said 1D background corrected intensity distribution by means of said 1D calibration intensity distribution.

In a further preferred embodiment of the method according to the invention, the data posttreatment process is also applied to the individual detector calibration image frames in which background signals are removed from said individual detector image frames based on a comparison with said total detector image frame, wherein said background corrected individual detector calibration image frames are added up to a total background corrected detector calibration image frame.

In this further preferred embodiment of the method according to the invention correcting said 1D background corrected intensity distribution can be executed by means of said 1D background corrected calibration intensity distribution

All embodiments of the method according to the invention can preferably furthermore comprise executing UV-Vis analysis on said sample, preferably simultaneously with said sample measurement process.

UV-Vis analysis in combination with SAXS is useful since knowing the concentration of the biomolecules exposed to SAXS is important for correct molecular weight estimation and SAXS intensity scales with the sample concentration.

Preferably said sample environment comprises a capillary containing said sample and an evacuated capillary holder block holding said capillary, wherein UV-Vis lenses through which incoming UV-Vis light is directed to said sample and transmitted UV-Vis light is directed to a spectrometer, respectively, are inserted into said capillary holder block.

In order to get a good signal-to-noise ratio on biological samples, there should be vacuum all the way from the source X-ray window to the detector, and the only X-ray window to the sample should be the capillary itself. The design according to this preferred embodiment achieves this by mounting the UV-Vis lenses into the capillary holder block, using the UV-Vis lenses themselves as vacuum windows, thus exposing the lenses directly to vacuum and not through a separate vacuum window. This also ensures no UV-Vis intensity is lost through a separate vacuum window. To make the UV-Vis lenses vacuum tight they can be modified with glue around the edges of the lenses and a respective lens holder of the capillary holder block. The lens holder itself can be made vacuum compatible using a disc O-ring.

Advantageously said UV-Vis lenses are mounted into said capillary holder block in a vacuum tight manner.

As a typical application of the method according to the invention, said structural characteristic to be determined can be a particle size distribution, a particle surface area distribution, a radius of gyration and/or a shape of particles within said sample.

In a further embodiment the method according to the invention can be applied to study the evolution of a sample structural characteristic during changing conditions such as a temperature or humidity change of the sample, or a continuous flow of the sample within the sample environment for example coming from a FPLC column (Fast Protein Liquid Chromatography) wherein the data analysis process includes a process to determine an optimized integration time for summing the individual detector image frames performed after the sample measurement process in which individual detector image frames are summed iteratively and quality of the 1D intensity distribution of the momentary detector image frames obtained are compared to each other for the purpose of achieving the best possible data quality without affecting the time resolution of the experiment. In a preferred embodiment of such method, the criteria determining the resolution of the experiment are based on an analysis of an external parameter such as the UV-VIS transmission signal from the sample to determine a possible change of sample conditions. In a preferred embodiment of such method the quality of the 1D intensity distribution of the momentary detector image frames are evaluated based on the analysis of the signal to noise ratio in a predetermined range of momentum transfer (Q) or by calculating primary fit parameters from 1D intensity distribution such as Radius of Gyration.

The invention furthermore relates to an apparatus for X-Ray Scattering material analysis, in particular Small Angle X-ray Scattering material analysis, designed to execute the method according to any of the preceding claims.

