阅读说明：本技术 用于x射线散射材料分析的方法及设备 (Method and apparatus for X-ray scattering material analysis ) 是由 P·奥格霍伊 B·兰茨 K·约恩森 S·斯科 于 2019-05-15 设计创作，主要内容包括：本发明涉及一种用于X射线散射材料分析的方法,尤其涉及用于小角度X射线散射材料分析的方法,所述方法包括：-生成入射X射线束并引导所述入射X射线沿传播方向(X)到保持在样本环境中的样本；执行包括以下步骤的样本测量过程：通过布置在所述样本环境下游的区域检测器(10)确定从所述样本散射的X射线的分布；以及通过所述检测器(10)确定透射通过所述样本的X射线束的强度(I_t)；执行包括以下步骤的样本数据处理过程：通过将取决于所述透射强度的校正应用于所述散射X射线分布来确定校正后散射X射线分布；执行包括以下步骤的数据分析过程：基于所述校正后散射X射线分布确定所述样本的至少一个结构特征；以及其特征在于：将所述散射的X射线和所述透射的X射线的采集分为多个采集周期,其中每个采集周期(T_(acq))小于或等于预先确定的最大采集时间(T_(max)),使得所述检测器(10)在线性范围内工作；所述检测器(10)测量包含所述散射的X射线和所述透射的X射线的信号的单个检测器图像帧,其中,在所述多个采集周期中的一个采样周期内内测量每个单个检测器图像帧；在连接到所述检测器(10)的计算机中,将所述单个检测器图像帧叠加成总检测器图像帧；以及根据基于所述总检测器图像帧获得的绝对散射X射线分布来实现确定所述样本的所述至少一个结构特征。本发明还涉及一种适用于执行这种方法的设备。(The invention relates to a method for analysis of X-ray scattering material, in particular for analysis of small angle X-ray scattering material, the method comprising: -generating an incident X-ray beam and directing the incident X-ray beam in a propagation direction (X) to a sample held in a sample environment; performing a sample measurement process comprising the steps of: determining a distribution of X-rays scattered from the sample by a region detector (10) arranged downstream of the sample environment; and determining the intensity (I) of the X-ray beam transmitted through the sample by means of the detector (10) t ) (ii) a Executing a sample data processing procedure comprising the steps of: determining a corrected scattered X-ray distribution by applying a correction dependent on the transmission intensity to the scattered X-ray distribution; performing a data analysis process comprising the following steps: determining at least one structural feature of the sample based on the corrected scattered X-ray distribution; and is characterized in that: dividing the acquisition of the scattered X-rays and the transmitted X-rays into a plurality of acquisition periods, wherein each acquisition period (T;) acq ) Less than or equal to a predetermined maximum acquisition time (T) max ) -operating the detector (10) in a linear range; the detector (10) measures individual detector image frames of signals containing the scattered X-rays and the transmitted X-rays, wherein each individual detector image frame is measured within one sampling period of the plurality of acquisition periods; superimposing, in a computer connected to the detector (10), the single detector image frames into a total detector image frame; and enabling determination of the at least one structural feature of the sample from an absolute scatter X-ray distribution obtained based on the total detector image frame. The invention also relates to a device suitable for carrying out such a method.)

1. A method for X-ray scattering material analysis, in particular small angle X-ray analysis, characterized in that the method comprises:

-generating an incident X-ray beam and directing the incident X-ray beam along a propagation direction (X) to a sample held in a sample environment;

-performing a sample measurement process (400) comprising the steps of:

determining a distribution of X-rays scattered from the sample by a region detector (10) arranged downstream of the sample environment; and

o determining the intensity (I) of the X-ray beam transmitted through the sample by means of the detector (10)_t)；

-performing a sample data processing procedure (500) comprising the steps of:

determining an absolute scatter X-ray distribution by applying an absolute intensity conversion to the scatter X-ray distribution that depends on the transmitted intensity; and

-performing a data analysis process (800) comprising the steps of:

o determining at least one structural feature of the sample from the absolute scatter X-ray distribution;

it is characterized in that the preparation method is characterized in that,

-dividing the acquisition of the scattered X-rays and the transmitted X-rays into a plurality of acquisition periods, wherein each of the acquisition periods (T;)_acq) Less than or equal to a predetermined maximum acquisition time (T)_max) To operate the detector (10) in a linear range;

-the detector (10) measures a single detector image frame containing the scattered X-ray signal and the transmitted X-ray signal, wherein each of the detector image frames is measured in one of the plurality of acquisition periods;

-superimposing, in a computer connected to the detector (10), the single detector image frames into a total detector image frame; and

-enabling a determination of at least one structural feature of the sample from an absolute scatter X-ray distribution obtained based on the total detector image frames.

2. The method of claim 1 wherein the total detector image frames are displayed on a screen connected to the computer and are continuously updated with each additional individual detector image frame.

3. The method according to any of the preceding claims, further comprising a maximum acquisition time determination process (100) performed before the sample measurement process (400), wherein the maximum acquisition time determination process (100) comprises:

o measuring the intensity (I) of the X-ray beam transmitted through the sample_t) (ii) a And

o is based on the intensity (I)_t) Calculating the maximum acquisition time.

4. The method according to any one of the preceding claims, further comprising a beam resolution determination process (200) performed prior to the sample measurement process (400), wherein the beam resolution determination process (200) comprises:

measuring at least one detector image frame containing signals of X-ray beams transmitted through the sample environment and signals of X-rays scattered from the sample environment, preferably without retaining a sample and sample solvent in the sample environment;

determining a beam resolution of the transmitted X-ray beam from the at least one detector image frame.

5. The method of claim 4, wherein the step of determining the beam resolution comprises: performing azimuthal averaging on the at least one detector image frame to convert a two-dimensional signal intensity distribution of the single detector image frame or a total detector image frame obtained by superimposing the single detector image frames into a one-dimensional intensity distribution as a function of momentum transfer (Q).

6. The method according to any one of the preceding claims, further comprising a sample environment calibration process (300) performed prior to the sample measurement process (400), wherein the sample environment calibration process (300) comprises:

measuring, by the detector (10), a single detector image calibration frame containing the scattered X-ray signal and the transmitted X-ray signal without preserving a sample in the sample environment, wherein each of the detector image calibration frames is measured within one of a plurality of calibration acquisition periods;

-superimposing, in the computer connected to the detector (10), the single detector image calibration frames into a total detector image calibration frame;

determining the transmitted intensity and the beam center position from the total detector image calibration frame.

7. The method of claim 6, wherein the sample environment calibration process (300) further comprises: performing azimuthal averaging on the total detector image calibration frame to convert a two-dimensional signal intensity distribution of the total detector image calibration frame to a one-dimensional calibration intensity distribution as a function of momentum transfer (Q).

8. Method according to claim 7, characterized in that said conversion is effected continuously between a scattered signal corresponding to a limited momentum transfer Q >0 and a transmitted signal corresponding to a no momentum transfer Q-0 measured along said propagation direction (X).

9. The method according to any one of the preceding claims, wherein the data analysis process (800) comprises:

determining a transmission intensity and a beam center position from the total detector image frame.

10. The method of claim 9, wherein the data analysis process (800) further comprises:

performing azimuthal averaging on the total detector image frames to convert a two-dimensional signal intensity distribution of the total detector image frames to a one-dimensional intensity distribution as a function of momentum transfer (Q).

11. A method according to claim 10, characterized in that said conversion is effected continuously between a scattered signal corresponding to a limited momentum transfer Q >0 and a transmitted signal corresponding to a no momentum transfer Q-0 measured along said propagation direction (X).

12. The method of claim 11 in combination with claim 7 or 8, further comprising correcting the one-dimensional intensity distribution by the one-dimensional calibration intensity distribution.

13. The method according to claim 12, further comprising a data quality control procedure (600) in which a signal-to-noise ratio within a predetermined momentum transfer range in the corrected one-dimensional intensity distribution is compared with a predetermined threshold value and a feedback action is performed depending on the result of the comparison.

14. The method of claim 13, wherein the feedback action comprises at least one of:

o stopping the sample measurement process (400);

o changing the position of the area detector (10);

o changing the opening of one or more collimating holes.

15. Method according to claim 14, characterized in that several additional measurement steps are performed with different measurement parameters, such as detector position or collimation aperture, comprising a final combination step in which several one-dimensional intensity distributions corresponding to different measurement parameters for different momentum transfer ranges are combined together into a combined one-dimensional intensity distribution.

16. The method of claim 15, wherein the combined one-dimensional intensity distribution is associated with a different beam resolution function obtained by a beam resolution determination process performed for each different measurement parameter.

17. The method according to any of the preceding claims, further comprising a data post-processing procedure (700) comprising a background removal step in which background signals are removed from the single detector image frames based on a comparison of the single detector image frames with the total detector image frames, wherein background corrected single detector image frames are superimposed in the computer into a total background corrected detector image frame, and wherein a background correction determination of the at least one structural feature of the sample is achieved based on the background corrected total detector image frame.

18. The method of claim 17 further comprising determining a background corrected transmission intensity and a background corrected beam center position from the total background corrected detector image frame.

19. The method of claim 18 further comprising performing azimuthal averaging on the total background corrected detector image frames to convert a two-dimensional signal intensity distribution of the total background corrected detector image frames to a one-dimensional background corrected intensity distribution as a function of momentum transfer (Q).

20. A method according to claim 19, characterized in that said conversion is effected continuously between a background corrected scattered signal corresponding to a finite momentum transfer Q >0 and a background corrected transmitted signal corresponding to a no momentum transfer Q-0 measured along said propagation direction (X).

21. The method of claim 20, further comprising correcting the one-dimensional background corrected intensity distribution by the one-dimensional calibration intensity distribution.

22. The method according to any of the preceding claims, further comprising performing a UV-Vis analysis of the sample, preferably simultaneously with the sample measurement process.

23. The method of claim 22, wherein the sample environment comprises a capillary tube that houses the sample and a vacuum capillary holder block that holds the capillary tube, wherein a UV-Vis lens is inserted into the capillary holder block, incident UV-Vis light is directed to the sample through the UV-Vis lens and transmitted UV-Vis light is directed to a spectrometer.

24. The method of claim 23, wherein the UV-Vis lens is mounted in a vacuum tight manner into the capillary holder block.

25. The method of any one of the preceding claims, wherein the structural feature is a particle size distribution, a particle surface area distribution, a radius of gyration, and/or a shape of particles within the sample.

26. An apparatus for X-ray scattering material analysis, in particular small angle X-ray scattering material analysis, characterized in that the apparatus is designed to perform the method according to any of the preceding claims.

Technical Field

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 performing such a method.

Background

While wide angle X-ray scattering (WAXS) generally provides information about the crystal structure and phase of the sample to be analyzed, small angle X-ray scattering (SAXS) generally provides information about the nanoscale sample structure (nanostructures). Examples are solid polymer films, alloys or powders that constitute the sample whose nanostructure is analyzed. However, the invention also relates to the case where SAXS is used to obtain information about one of the components of the sample. Such samples analyzed by SAXS are typically analytes diluted in a sample solvent and contained in a sample container (e.g. a capillary). As a typical example, a dispersion of nanoparticle micelles may be included.

Disclosure of Invention

A method for X-ray scattering material analysis, in particular small angle X-ray scattering material analysis, comprising:

-generating an incident X-ray beam and directing the incident X-ray beam in a propagation direction to a sample held in a sample environment;

-performing a sample measurement procedure comprising the steps of:

determining a distribution of X-rays scattered from the sample by a region detector arranged downstream of the sample environment; and

o determining the intensity I of the X-ray beam transmitted through the sample by means of the detector_t；

-performing a sample data processing procedure comprising the steps of:

determining an absolute scattered X-ray distribution by applying to the scattered X-ray distribution a transformation to an absolute intensity dependent on the transmitted intensity; and

-performing a data analysis process comprising the steps of:

o determining at least one structural feature of the sample based on the absolute scatter X-ray distribution;

scattering by X-rays is generally well known to those skilled in the art of material analysis.

Such an X-ray scattering material analysis method requires measurement of the scattering intensity, so-called absolute intensity, in order to determine the desired structural characteristics of the sample from the relationship 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 some correction required to obtain a scatter signal of absolute intensity. However, this presents the problem that the transmission intensity is up to several orders of magnitude stronger than the scatter signal, so that a large transmission intensity easily drives the area detector out of its linear count range, even at risk of damaging the detector. In prior art methods this problem is taken into account, for example by collecting the transmitted signal with an area detector, attenuating the transmitted intensity by an attenuator or a semi-transparent beam blocker, or by using a second detector such as a beam blocker with a pin diode incorporated inside. Alternatively, the absolute intensity is obtained by measuring the scattering intensity of a reference sample.

The area detector may be a one-dimensional or a two-dimensional detector. In the following, the preferred case of a two-dimensional detector will be mainly considered.

Furthermore, SAXS detectors require inherently low noise and, in addition to modern solid state detectors operating in direct detection mode (so-called mixed pixel detectors) with individual pixel counters, these other detectors suffer from low maximum count rates and/or detector damage risks, as found in modern SAXS instruments, with respect to transmitted beam intensity. Thus, in prior art SAXS methods, one or several beam blockers are typically used in front of the two-dimensional detector to assess the intensity of the direct beam (i.e. transmitted beam) passing through the sample, either by a semi-transparent beam blocker or by a completely absorbing beam blocker to completely block the direct X-ray beam from reaching the detector. In the latter case, the determination of the transmission intensity must be carried out before or after the scatterometry with a short counting time and/or with different acquisition devices, for example pin diodes.

The most advanced combination SAXS/WAXS system uses a beam blocker and a hybrid pixel detector, which is preferably placed in a vacuum chamber environment to reduce parasitic scattering of air and vacuum windows.

However, in all these prior art, the translucent beam stop itself causes parasitic scattering and also causes a so-called beam hardening effect, i.e. it affects the spectrum of the incident X-ray beam, so that an accurate determination of the transmitted intensity becomes unreliable. Furthermore, the use of a translucent or fully absorbing beam stop is always difficult, as it requires time consuming positioning of the beam stop accurately in the transmitted beam, thereby minimizing its effect on the scattered signal.

It is therefore an object of the present invention to overcome such problems associated with the use of beam blockers.

According to the invention, this object is achieved by a method for analyzing a small angle X-ray scattering material as described above, wherein:

-the acquisition of the scattered X-rays and the transmitted X-rays is divided into a plurality of acquisition periods, wherein each acquisition time period (T;) is_acq) Less than or equal to a predetermined maximum acquisition time (T)_max) Such that the detector operates in a linear range;

-the detector measures individual detector image frames of signals containing the scattered X-rays and the transmitted X-rays, wherein each individual detector image frame is measured during one of the plurality of acquisition periods;

-superimposing, in a computer connected to the detectors, the single detector image frames into a total detector image frame; and

-enabling a determination of the at least one structural feature of the sample from an absolute scatter X-ray distribution obtained based on the total detector image frame.

According to the invention, each individual detector image frame is measured in a sufficiently short acquisition period to ensure that the detector operates within its linear range. After a certain number of individual detector image frames have been measured, the counting electronics of the detector will be reset so that the detector counts the next individual detector image frame, essentially starting from 0. Depending on the linearity requirements of the detector, its counting electronics may be reset and restarted, for example, after each single detector image frame or after each second single detector image frame, to ensure that the detector is always within its linear operating range. Thus, the total detector image frame obtained by superimposing the individual detector image frames in the computer is also free of any non-linear distortions.

The method according to the invention ensures a linear operating range of the detector while allowing transmission signals, i.e. X-ray beams which pass through the sample and the sample environment and reach the detector without being attenuated and whose spectrum is affected by the beam blockage, to be collected simultaneously during the entire scatterometry. This provides a number of advantages over prior art methods using a semi-transparent beam blocker or a second detector. Since the same detector is used as is used for collecting the scatter signal, the overall measurement accuracy of the scatter intensity in absolute units of the sample is improved, since there is no problem with different detector efficiencies and gains. Furthermore, the simultaneous acquisition method according to the invention is more accurate than the alternative method of acquiring the transmitted intensity using a short and independent acquisition (typically 0.1 seconds) and using different acquisition devices (e.g. pin diodes) before and/or after the scatterometry measurement, as it suppresses the effects of intensity variations of the incident X-ray beam that may occur during the whole sample measurement as well as the effects of different detector efficiencies. Furthermore, by determining the integration time after the experiment, variations in sample properties and scattering can be accounted for.

In a preferred embodiment of the method according to the invention said total detector image frames are displayed on a screen connected to said computer and are continuously updated with each additional single detector image frame. This allows the user to perform SAXS measurements to continuously monitor the experiment.

Preferably, the method according to the present invention further comprises a maximum acquisition time determination procedure performed before the sample measurement procedure, wherein the maximum acquisition time determination procedure comprises:

o measuring the intensity I of the X-ray beam transmitted through the sample_t(ii) a And

o is based on the intensity I_tThe maximum acquisition time is calculated.

The calculation of the maximum acquisition time for the upper time limit for each individual detector image frame typically implies dividing the allowed total count of area detectors, indicated in the detector specifications by the detector manufacturer, by the measured intensity I_t。

Advantageously, the method according to the invention further comprises a beam resolution determination process performed before said sample measurement process, wherein said beam resolution determination process comprises:

measuring individual detector image frames containing at least one of signals of X-rays transmitted through and scattered from the sample environment, preferably in the absence of sample and sample solvent in the sample environment;

determining a beam resolution of the transmitted X-ray beam from the at least one single detector image frame.

This advantageous embodiment allows a very accurate characterization of the X-ray beam profile for the experiment and a quantification of the beam resolution. This is particularly useful for analysis methods such as particle size distribution analysis, where quantification of the beam resolution and its introduction into the modeling process improves the accuracy of the data analysis and thus the determined particle size. Preferably, the beam resolution determination is performed without sample or sample solvent in the beam, but is typically performed with a sample container, such as a capillary, in the beam. However, the beam resolution determination process may also be performed with the sample in place. In this case, the PSF of the direct beam is determined from the central part of the beam, which corresponds to a no-momentum transfer Q of 0, i.e. a scattering vector modulus of zero, and using the first point of the dynamics from the one-dimensional intensity distribution to about 1E 3.

In these advantageous embodiments, it is further preferred that the step of determining the beam resolution comprises performing an azimuthal averaging of the at least one single detector image frame to convert the two-dimensional signal intensity distribution of the single detector image frame or of a total detector image frame obtained by a true superposition of the single detector images into a one-dimensional intensity distribution as a function of the momentum transfer (Q), i.e. the scattering vector modulus.

The Point Spread Function (PSF) of the incident beam can then be obtained as follows. The direct beam image is azimuth averaged over the same cell grid (denoted by Q or 2 θ angles) as used to reduce the sample scatter data and reduced to one-dimensional scatter data (l ═ f (Q)). The convolution kernel is built from the PSF and is further included in the model used to fit the data. The method takes into account the beam smearing effect in any (arbitrary) model, and does not take into account the details and parameters of the model. The data analysis process includes optimizing model parameters, including smearing on experimental data. In other words, the beam convolution effect is taken into account in the forward model, which leads to an improvement in stability compared to an alternative solution of refining the original model on deconvolution experimental data.

Preferably, the method according to the present invention further comprises a sample environment calibration procedure performed before the sample measurement procedure, wherein the sample environment calibration procedure comprises:

measuring, by the detector, a single detector image calibration frame containing the scattered X-ray signal and the transmitted X-ray signal without a sample in the sample environment, wherein each of the detector image calibration frames is measured during one of a plurality of calibration acquisition periods;

o superimposing, in the computer connected to the detector, the single detector image calibration frames into a total detector image calibration frame;

determining the transmitted intensity and the beam center position from the total detector image calibration frame.

Also in this case, the sample environment calibration procedure preferably further comprises performing an azimuthal averaging of the total detector image calibration frame to convert the two-dimensional signal intensity distribution of the total detector image calibration frame into a one-dimensional calibration intensity distribution as a function of momentum transfer (Q).

As a particular advantage of this embodiment, the conversion can be effected continuously between the scattered signal corresponding to a limited momentum transfer Q >0 and the transmitted signal corresponding to a no momentum transfer Q-0 measured along the propagation direction (X).

In all embodiments of the method according to the present invention, said data analysis process preferably comprises:

determining a transmission intensity and a beam center position from the total detector image frame.

Further preferably, the data analysis process further comprises:

performing azimuthal averaging of the total detector image frames to convert a two-dimensional signal intensity distribution of the total detector image frames to a one-dimensional intensity distribution as a function of momentum transfer (Q).

The transition may be effected continuously between the classification signal corresponding to a limited momentum transfer Q >0 and the transmission signal corresponding to a no momentum transfer Q-0 measured along the propagation direction (X).

In all embodiments utilizing the one-dimensional calibration intensity distribution discussed above, it is further preferred that

The one-dimensional calibration intensity profile corrects for the one-dimensional intensity profile. As a simple example, the one-dimensional calibration intensity distribution determined during calibration of the sample environment may be subtracted from the one-dimensional intensity distribution determined during sample data analysis.

Such a correction of a one-dimensional intensity distribution by means of said one-dimensional calibration intensity distribution can provide further advantages when the method according to the invention further comprises a data quality control procedure in which a signal-to-noise ratio within a predetermined momentum transfer (Q) range in said corrected one-dimensional intensity distribution is compared with a predetermined threshold value and a feedback action is performed depending on the result of said comparison.

Preferably, the feedback action comprises at least one of:

o stopping the sample measurement process;

o changing the position of the area detector;

o changing the opening of one or more collimating holes.

For example, in a first case, the sample measurement process is automatically stopped when the signal-to-noise ratio within a predetermined momentum transfer (Q) range in the corrected one-dimensional intensity distribution exceeds a predetermined threshold. This makes the overall SAXS measurement faster and requires less user interaction. The term "signal-to-noise ratio" is also intended to include similar statistics known to those skilled in the art of data quality assessment.

In all embodiments, the method according to the present invention may further preferably comprise a data post-processing procedure comprising a background removal step in which background signals are removed from the single detector image frames based on a comparison of the single detector image frames with the total detector image frames, wherein the background corrected single detector image frames are combined in a computer into a total background corrected detector image frame, and wherein a background correction determination of the at least one structural feature of the sample is enabled based on the total background corrected detector image frame.

The background signal removed from the single detector image frame is caused inter alia by an unavoidable environmental background, also called a cosmic background. To identify such background signals, the individual detector image frames are preferably compared pixel by pixel with the total detector image frame using a statistical algorithm for comparing their experimental statistics with theoretical statistics expected by the counting process.

The data post-processing may further include determining a background corrected transmission intensity and a background corrected beam center position from the total background corrected detector image frame.

In this case, the data post-processing procedure may preferably further comprise performing an azimuthal averaging on said total background corrected detector image frames to convert a two-dimensional signal intensity distribution of said total background corrected detector image frames into a one-dimensional background corrected intensity distribution as a function of the momentum transfer (Q).

The conversion may be effected continuously between a background corrected scattered signal corresponding to a limited momentum transfer Q >0 and a background corrected transmitted signal corresponding to a no momentum transfer Q-0 measured along the propagation direction (X).

In this embodiment, the method according to the present invention may further comprise correcting said one-dimensional background corrected intensity distribution by said one-dimensional calibration intensity distribution.

In a further preferred embodiment of the method according to the invention, the data post-processing procedure is further applied to a single detector correction image frame from which background signals are removed based on a comparison of the single detector image frame with the total detector image frame, wherein the single background corrected detector correction image frame is superimposed into a total background corrected detector correction image frame.

In said further preferred embodiment of the method according to the invention, the correction of the one-dimensional background-corrected intensity distribution may be performed by the one-dimensional background-corrected calibration intensity distribution.

All embodiments of the method according to the present invention may preferably further comprise performing a UV-Vis analysis on said sample, preferably simultaneously with said sample measurement process.

The UV-Vis analysis used in conjunction with SAXS is useful because it is known that the concentration of biomolecules exposed to SAXS is important for correct molecular weight estimation and SAXS intensity scaling of sample concentrations.

Preferably, the sample environment comprises a capillary containing the sample and a vacuum capillary holder block holding the capillary, wherein a UV-Vis lens through which incident UV-Vis light is directed to the sample and transmitted UV-Vis light is directed to a spectrometer, respectively, is inserted into the capillary holder block.

To obtain a good signal-to-noise ratio on the biological sample, the source X-ray window is evacuated all the way to the detector, and the only X-ray window to the sample should be the capillary itself. The design according to the preferred embodiment achieves this by: the UV-Vis lens is mounted into the capillary holder block, using the UV-visible lens itself as the vacuum window, thereby exposing the lens directly to vacuum, rather than through a separate vacuum window. This also ensures that the UV-Vis intensity is not lost through the vacuum window alone. To vacuum seal the UV-Vis lens, the edge of the lens and the corresponding lens holder of the capillary tube holder block may be trimmed with glue around it. The lens holder itself may be vacuum compatible with a disc O-ring.

Advantageously, the UV-Vis lens is mounted in a vacuum-tight manner in the capillary holder block. As a typical application of the method according to the invention, the structural feature to be determined may be the particle size distribution, the particle surface area distribution, the radius of gyration and/or the shape of the particles within the sample.

In another embodiment, the method according to the invention may be used for studying the evolution of structural features of a sample under changing conditions, such as temperature or humidity changes of the sample, or under continuous flow of the sample in the sample environment, e.g. from an FPLC column (fast protein liquid chromatography), wherein the data analysis process comprises a process for summing individual detector image frames to determine an optimized integration time performed after the sample measurement process, in which the individual detector image frames are iteratively summed and the quality of the obtained one-dimensional intensity distributions of the instantaneous detector image frames are compared to each other to achieve the best possible data quality without affecting the temporal resolution of the experiment. In a preferred embodiment of such a method, the criterion for determining the experimental resolution is based on the analysis of an external parameter, such as a UV-VIS transmission signal from the sample, to determine possible changes in the sample conditions. In a preferred embodiment of the method, the quality of the one-dimensional intensity distribution of the instantaneous detector image frames is evaluated based on an analysis of the signal-to-noise ratio within a predetermined momentum transfer (Q) range or by calculating a main fitting parameter, such as the radius of gyration, from the one-dimensional intensity distribution.

The invention also relates to an apparatus for X-ray scattering analysis, in particular small angle X-ray scattering material analysis, designed to perform a method according to any of the preceding claims.

Drawings

Preferred embodiments of the small angle X-ray scattering analysis method and apparatus according to the present invention will be described below with reference to the accompanying drawings, in which:

FIG. 1a shows a schematic side view of an X-ray scattering device according to an embodiment of the present invention;

FIG. 1b shows a schematic perspective view of the X-ray scattering apparatus of FIG. 1a during SAXS measurement;

fig. 2 shows a schematic flow chart of various procedures of a preferred embodiment of the method according to the present invention.

Fig. 3 shows a schematic flow chart of the steps of a maximum acquisition time determination procedure in a preferred embodiment of the method according to the invention.

Fig. 4 shows a schematic flow chart of the steps of a beam resolution determination procedure in a preferred embodiment of the method according to the invention.

Fig. 5 shows a schematic flow chart of the steps of a sample environment calibration procedure in a preferred embodiment of the method according to the invention.

Fig. 6 shows a schematic flow chart of the steps of a sample measurement procedure and a subsequent sample data processing procedure in a preferred embodiment of the method according to the present invention.

FIG. 7 shows a schematic flow chart of the steps of a sample data processing procedure in a preferred embodiment of a method according to the present invention;

fig. 8a shows a total detector image frame displayed on a screen when the SAXS method according to the present invention is performed;

FIG. 8b shows a one-dimensional intensity distribution as a function of momentum transfer Q determined based on the total detector image frame of FIG. 8 a;

FIG. 9a shows a total background corrected detector image frame displayed on a screen after a SAXS method including data post-processing according to the invention has been performed;

fig. 9b shows a one-dimensional background corrected intensity distribution as a function of the momentum transfer Q determined from the total background corrected detector image frame of fig. 9 a.

Fig. 10 shows details of a sample environment of an X-ray scattering device according to the invention, which is suitable for performing UV-Vis analysis.

Detailed Description

Fig. 1a shows a schematic side view of a preferred embodiment of an X-ray scattering device according to the invention. The apparatus comprises a two-dimensional X-ray detector 10 arranged downstream of a sample environment (not shown) for holding a sample 12 to be analyzed by X-ray scattering in a holding position. The terms "upstream" and "downstream" are directed to a propagation direction X of a direct X-ray beam from an X-ray beam transmission system (not shown) arranged upstream of the sample environment for generating and directing the direct X-ray beam along the propagation direction X towards the sample environment. Such X-ray beam delivery systems typically include an X-ray generator, such as a microfocus sealed tube source or a rotating anode tube, a collimating or focusing monochromator (e.g., a multi-layer coated X-ray mirror) for selecting predetermined X-ray wavelengths, and a slit defining a collimating aperture for shaping the profile of the X-ray beam reaching the sample environment and controlling its divergence and shape towards a distal X-ray detector, as well as other X-ray devices known to those skilled in the art. Thus, in FIG. 1a, the upstream to downstream direction is from left to right. The X-ray beam delivery system can generate a one-dimensional conditioned X-ray beam using a line focus source and one-dimensional X-ray beam shaping optics. In a preferred embodiment, the X-ray beam delivery system produces a two-dimensional conditioned X-ray beam using point focusing and two-dimensional X-ray beam shaping optics. In this case, the X-ray detector 10 may have a two-dimensional pixel array suitable for analyzing anisotropic samples. X-ray scatterometry typically requires optical path evacuation under vacuum or helium to reduce the generation of parasitic scattering in air. Thus, the scatter light path generally needs to be under vacuum, and in a preferred embodiment, the detector 10 is located within a vacuum diffraction beam tube that may be connected to a vacuum sample cavity or a portion of a single volume chamber that includes the incident light path and the sample environment.

In contrast to the SAXS arrangement of the prior art, the device according to the present invention shown in fig. 1a does not require a beam blocker.

As indicated by the arrows in fig. 1a, the two-dimensional detector 10 may be mounted on a detector platform 14 so as to be movable in the X-direction and in the Y-direction perpendicular to the X-direction under the control of a central control computer of the SAXS apparatus.

In fig. 1a, an X-ray beam transmitted through the sample and propagating substantially along the original propagation direction X towards the detector 10 is denoted by reference numeral 16.

Furthermore, the X-ray beam scattered by the sample 12 in the upward direction is denoted by reference numeral 18u, while the corresponding X-ray beam scattered in the downward direction is denoted by reference numeral 18 d.

FIG. 1b shows a schematic perspective view of the X-ray scattering device of FIG. 1a during SAXS measurement. The transmitted beam 16 is represented by a dashed line, with wave numbers denoted k₀. The upward scattered light beam 18u and the downward scattered light beam 18d are indicated by solid lines.

As shown in fig. 1b, the two-dimensional detector measures a rotationally symmetric intensity distribution of the scatter signal. All points whose centres are on the circle of the position of the transmitted beam in the respective image frame measured by the detector 10 belong to the scattering process with the same scattering angle 2Q and therefore have the same momentum transfer, as indicated by Q in fig. 1 b.

Fig. 2 shows a schematic flow diagram of various processes according to a preferred embodiment of the method of the present invention, including a maximum acquisition time determination process 100, a beam resolution determination process 200, a sample environment calibration process 300, a sample measurement process 400, a sample data processing process 500, a data quality control process 600, a data post-processing process 700, and a data analysis process 800. The processes 100 to 600 are performed while the sample is irradiated by the incident X-ray beam. The data post-processing procedure 700 is typically performed after the X-ray sample measurement has terminated. In the embodiment shown in fig. 2, the data analysis process 800 is performed only after the data post-processing process 700. However, in alternative embodiments, the data analysis process 800 may be performed in real-time while the sample measurement is still continuing, without the need for data refinement using any post-processing processes.

These processes will be further explained below in conjunction with fig. 3 to 7.

Fig. 3 shows a schematic flow chart of the steps of a maximum acquisition time determination process 100 in a preferred embodiment of the method according to the invention. In step 101, the light beam transmitted through the sample in the sample environment is measured with a short acquisition time, e.g. 0.1 seconds. The transmitted intensity is then determined by integrating the signal in the detector 10 in the central region of interest, step 102. Finally, in step 103, the maximum allowable total count of detectors 10 provided by the detector manufacturer is divided by the determined value I_tTo calculate the maximum acquisition time T_max. In the subsequent sample measurement process 400, T will be less than or equal to_maxAcquisition period T of_acqDuring which each individual detector is measuredAnd (5) image frames. This ensures that the detector 10 always remains within its linear detection range.

Fig. 4 shows a flow chart of the steps of a beam resolution determination process 200 in a preferred embodiment of the method according to the invention. In step 201, a single detector image frame is measured with a shorter acquisition time, e.g. 0.1s, without a sample 12 in the sample environment. In step 202, azimuthal averaging around the beam center is performed for the single detector image frame to convert the two-dimensional intensity distribution shown on the detector 10 in FIG. 1b to a one-dimensional intensity distribution as a function of the momentum transfer Q. Finally, the beam resolution is determined from the one-dimensional intensity distribution.

Fig. 5 shows a flow chart of the steps of a sample environment calibration procedure 300 in a preferred embodiment of the method according to the invention. In step 301, measurements are made on a plurality of detector image calibration frames. The measurement is made without a sample in the beam. More precisely, step 301 is performed in a substantially empty sample environment without the sample being in the incident beam, without a free standing sample requiring any sample container or solvent.

However, in case of a sample, e.g. a powder sample, requiring a sample container but not a sample solvent, step 301 is performed with an empty sample container in the incident light beam.

Furthermore, in case the sample is an analyte requiring a sample container filled with a sample solvent, step 301 is performed in the incident light beam with a sample container filled with a sample solvent but without an analyte.

In a subsequent step 302, the single detector image calibration frames are superimposed into a total detector image calibration frame.

Then, in step 303, the transmission intensity I is determined from the total detector image calibration frame_tAnd a beam center.

Finally, in step 304, azimuthal averaging around the beam center is performed in a single correction detector image frame to convert the two-dimensional calibration intensity distribution into a one-dimensional calibration intensity distribution as a function of the momentum transfer Q, and based on the value I_tThe one-dimensional intensity distribution is converted into absolute intensity.

Fig. 6 shows a schematic flow chart of the steps of a sample measurement process 400 and a subsequent sample data processing process 500 and a data quality control process 600 in a preferred embodiment of the method according to the present invention. The sample measurement process 400 comprises a step 401 of measuring a new single detector image frame, a step 402 of superimposing said new measured single detector image frame into an instantaneous total detector image frame and calculating an emission intensity I_tAnd a beam center step 403.

The subsequent sample data processing procedure 500 comprises a step 501 in which azimuthal averaging is performed on the total detector image frame obtained in step 402 around the beam center obtained in step 403 to convert the two-dimensional intensity distribution into a one-dimensional intensity distribution as a function of the momentum transfer Q and based on the value I_tThe one-dimensional intensity distribution is converted into absolute intensity.

In a subsequent step 502, this one-dimensional intensity distribution will be corrected by the one-dimensional calibration intensity distribution obtained in step 304.

In the subsequent data quality control process 600, the signal-to-noise ratio in the predetermined momentum transfer Q range in the corrected one-dimensional intensity distribution is compared with a predetermined threshold value, and a feedback action is performed according to the comparison result. In the embodiment shown in fig. 6, if the signal-to-noise ratio is equal to or greater than the threshold, the feedback action is to terminate the measurement process 400 and subsequent sample data processing process 500, otherwise return to step 401.

In another embodiment of the invention, the feedback action may be defining and implementing one or more additional measurement steps to improve the signal-to-noise ratio within a predetermined momentum transfer Q range of said one-dimensional intensity distribution. Such an additional measurement step may be to acquire additional single detector image frames with improved measurement parameters, for example to measure sample scatter with different positions of the area detector 10 or different openings with one or more collimation holes. In this case, the defined additional measurement step may include a process of determining the beam resolution using the improved measurement parameter. In this case, the sample data processing procedure comprises a corrected one-dimensional intensity distribution obtained by azimuthal averaging of total detector image frames corresponding to different sequential acquisitions performed during the complete sample measurement procedure. The merging process to obtain the merged one-dimensional intensity distribution will comprise selecting the point in the corrected one-dimensional intensity distribution with the highest signal-to-noise ratio.

In this case, for each one-dimensional intensity distribution portion constituting such a curve, the resulting combined one-dimensional intensity distribution should be associated with a different beam resolution function, each beam resolution function being obtained by a different beam resolution determination process using each modified measured parameter. Even if dedicated measurement without a sample is preferred as described above in connection with fig. 4, the beam resolution determination process can be applied with the sample in place.

Fig. 7 shows a schematic flow chart of the steps of a data post-processing procedure in a preferred embodiment of the method according to the invention.

In step 701, a single detector image frame of all measurements under test is compared to a total detector image frame. In a subsequent step 702, a background signal (sometimes referred to as a cosmic background) is removed from the individual detector image frames according to the comparison performed in step 701. In a subsequent step 703, the background corrected individual detector image frames are superimposed to obtain a total background corrected detector image frame. The background corrected transmission intensity I is then calculated in a subsequent step 704_tAnd a background corrected beam center.

According to the acquisition period T_acqAnd in view of limiting the computation time, the comparison 701 and the further correction by removal 702 may be applied to each single detector image frame, or they may be applied to intermediate detector image frames obtained by superimposing a plurality of single detector image frames. Typically, the comparison 701 and further correction 702 are applied to a single detector image frame for several seconds (e.g., from 1 second to 10 seconds) to properly detect and remove the cosmic background. In a subsequent step 705, the total background corrected detector image frames obtained in step 703 are paired around the background corrected beam center obtained in step 704To convert the two-dimensional intensity distribution into a one-dimensional background calibration intensity distribution as a function of the momentum transfer Q, and to correct the transmitted intensity I based on the background_tThe value of (d) converts the one-dimensional background calibration intensity distribution to absolute intensity.

Finally, in a further step 706, such a one-dimensional background-corrected intensity distribution with absolute intensity is corrected by a one-dimensional background-corrected calibration intensity distribution with absolute intensity, which is similar to the one-dimensional calibration intensity distribution with absolute intensity obtained in step 304 and which may be obtained by applying the background removal of the data post-processing procedure 700 not only to a single detector image frame, but also to a single detector image calibration frame obtained in step 301.

Fig. 8a shows a total detector image frame displayed on a screen when the SAXS method according to the present invention is performed on a sample. The screen is connected to a computer, which is in turn connected to the detector. Which may or may not be the central control computer of the device according to the invention. The transmitted beam and scattered X-rays can be seen in the upper left corner of the screen. The white vertical stripe in the middle of the screen is an artifact caused by invalid detector pixels.

Fig. 8b shows a one-dimensional intensity distribution as a function of the momentum transfer Q determined by performing azimuthal averaging from the total detector image frame of fig. 8 a. In this particular case, the sample contains SiO₂And (3) nanoparticles.

Fig. 9a shows a total background corrected detector image frame displayed on a screen after the SAXS method including data post-processing procedure according to the present invention has been performed. Many of the streaks caused by the cosmic background, visible in fig. 8a, for example near positions X-80, Z-390, have been successfully eliminated, thus showing the data quality improvement achieved by background correction. Fig. 9b shows a one-dimensional background corrected intensity distribution as a function of the momentum transfer Q determined on the basis of the total background corrected detector image frame of fig. 9 a. The upper solid grey curve shows the one-dimensional background corrected intensity distribution as a function of the momentum transfer Q determined by performing the azimuthal averaging from the total background corrected detector image frame of fig. 9 a.

The lower, grey dashed curve in fig. 9b shows the corresponding one-dimensional calibration intensity distribution determined in step 304. The resulting solid black difference curve corresponding to the difference between the upper and lower curves is also shown in fig. 9 b.

Fig. 10 shows details of an X-ray scattering device according to the invention suitable for performing UV-Vis analysis.

When biomolecules are separated by molecular mass using FPLC (fast protein liquid chromatography) or other techniques, the sample is pushed through a size exclusion column filled with packed beads, such that low molecular weight molecules bounce back in the column for longer than high molecular weight molecules, resulting in size separation when the sample is pushed through the column with sample buffer. The individual separated species in the constant stream were identified by an increase in uv absorption as they passed through a uv exposure cell at the exit of the size exclusion column on the FPLC instrument. As the individual species flow through the FPLC uv cell over time, the large species pass first, followed by the smaller species, they generate an absorption peak. A constant flow from the FPLC may be connected to the SAXS flow cell for SAXS exposure. Due to the taylor dispersion effect (caused by the parabolic flow profile in the conduit), the separated molecules flowing in the conduit will start to mix again, which can be seen as a broadening of the absorption peak compared to the absorption peak obtained at the outlet of the size exclusion column.

UV-Vis at exactly the same position as SAXS measurement ensures correct concentration of the sample exposed to X-rays. It is important to choose the correct X-ray acquisition time. It should be long enough to obtain enough signal for SAXS data analysis, but short enough to obtain data for completely separated molecules (top of absorption peak), rather than data for multiple peaks or average of entire peaks.

The time to obtain the correct acquisition depends on the concentration and molecular weight of the substance, as this is proportional to the scattering intensity. In practice, the acquisition time is set at some fixed time, which is considered to be the optimal time for a particular sample.

2a) By choosing a smaller acquisition time, a number of frames can be acquired and then superimposed or integrated together, and the best balance between separation resolution and scatter data quality is obtained by adjusting the sum of the data detector images around a single UV-VIS absorption peak.

2b) To improve SAXS signal-to-noise ratio while still maintaining the desired separation resolution, the same sample can be acquired in a SEC-SAXS run, and SAXS frames corresponding to exactly the same concentration on a single absorption peak can be merged together.