CN103487452A - Method for realizing three-dimensional imaging of symmetrical micro/nano sample through single X-ray measurement - Google Patents
Method for realizing three-dimensional imaging of symmetrical micro/nano sample through single X-ray measurement Download PDFInfo
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Abstract
The invention discloses a method for realizing three-dimensional imaging of a symmetrical micro/nano sample through single X-ray measurement. The method comprises the following steps: by taking a coherent X-ray diffraction microscope as imaging equipment and utilizing the characteristics of free electron laser all-coherence, high flux and short pulse of X ray, acquiring two-dimensional diffraction data of the symmetrical micro/nano sample under single pulse irradiation, calculating an equivalent line of the diffraction data of the sample according to crystallographic symmetry characteristics of the symmetrical sample to determine spatial orientation of the sample, performing symmetry operation of a crystallographic point group of the sample on the two-dimensional diffraction data by utilizing the spatial symmetry of the sample to obtain three-dimensional diffraction data of the sample, and performing phase retrieval and image reconstruction on the three-dimensional recombinant diffraction data according to an oversampling and iterative algorithm to finally obtain a three-dimensional structure of the symmetrical sample. According to the method, three-dimensional reconstruction of the sample can be realized through single measurement only, the experiment efficiency of three-dimensional imaging is effectively improved, and a new thought is provided for enriching a three-dimensional imaging method.
Description
Technical field
The invention belongs to x-ray imaging method and technology field, relate in particular to the x-ray imaging method that two-dimentional diffraction data that a kind of single measurement has the micro-nano sample of symmetry is realized the sample structure three-dimensional reconstruction.
Background technology
Along with improving constantly of synchrotron radiation light source brightness, the x-ray imaging method has obtained development fast, and new x-ray imaging method continues to bring out, and imaging resolution is also in further raising.Due to X ray, to have a wavelength short, and the characteristics that penetration capacity is strong, be the perfect light source of carrying out the high-resolution three-dimensional imaging.At present Micro-CT and Nano-CT imaging technique reach its maturity, and can obtain respectively the micron resolution three-dimensional structure of large sample and than the three-dimensional structure of the nanometer resolution of small sample.But, because X ray is difficult to focus on, the highest imaging resolution of current X ray CT is no more than 30nm.Utilize the characteristics of the third generation and the 4th high coherence of generation synchrotron radiation light source and high brightness, relevant X-ray diffraction formation method has obtained paying attention to widely and development fast.Because relevant X-ray diffraction formation method not take lens as image-forming component, broken away from the restriction of lens quality to imaging resolution, can reach atom definition in theory.The X-ray diffraction formation method that will be concerned with combines with the tomoscan formation method, can obtain at present the three-dimensional imaging result of 10nm resolution.
But no matter above which kind of formation method all needs to measure a large amount of projections in the process that realizes the sample three-dimensional imaging, has not only increased data acquisition time, and can produce serious radiation damage to sample due to time exposure.Especially for biological sample, the radiation damage problem is the principal element that restriction biological sample resolution further improves.Current freezing transmission electron microscope individual particle imaging technique has been widely used in the three-dimensional high definition imaging of the full same sex samples such as individual particle virus.It utilizes sample to have the characteristics of the full same sex, by the image of a large amount of measurement samples in the real space, the combining image disposal route is screened and noise reduction process the sample images of transmissive electron microscope, utilize the method for equivalent lines and projection matching determine orientation and the center of sample and realize the normalization between each image, processed electromicroscopic photograph is carried out to two-dimensional Fourier transform, thereby realize the three-dimensional restructuring of a large amount of viral sample diffraction patterns, finally utilize three-dimensional inverse Fourier transform to realize the three-dimensional imaging of viral sample.The method has successfully realized the parsing of multiple viral three-dimensional structure, and is widely used in entirely resolving with the protein body three-dimensional structure.But Cryo electron microscopy needs equally to take a large amount of full real space images with particle in realizing individual particle three-dimensional imaging process, consider that the electron pair biological sample has serious radiation damage simultaneously, need to reduce electron dose, therefore the transmission electron microscope photo contrast obtained is lower, picture quality is poor, need to screen and the noise-reducing processing data, make data process and become loaded down with trivial details.
Than electronics, X ray is less to the radiation damage of sample, but for biological individual particle samples such as viruses, due to its diffracting power to X ray a little less than, be difficult to obtain high-quality diffracted signal, obtain the reconstructed image of high-resolution.In order further to improve imaging resolution, need to increase the time shutter of relevant X-ray diffraction imaging technique, not only the stability of imaging device had to higher requirement, and easily because long-time x-ray irradiation changes sample structure.At present relevant X ray light-source brightness rises to address this problem the light source basis is provided.The X ray free-electron laser as the 4th generation synchrotron radiation light source have complete relevant, brightness is high, the characteristics of short pulse, its burst length can reach femtosecond, can before destroying, sample record its diffraction data, successful solution the problem of sample radiation damage, can, to the imaging of samples under state of nature, be the perfect light source that realizes original position, dynamic imaging.It is the emphasis of studying at present that the X ray Free Electron Laser Technology combines with relevant X-ray diffraction imaging technique, it realizes that for realizing the micro-nano sample of single measurement symmetry the formation method of three-dimensional reconstruction provides possibility, and resolving symmetry crystal structure and virus structure and noncrystalline complete same grain pattern, as the various kinds of cell membrane complex, molecular chaperones, have important using value in the sample three-dimensional structures such as ribosomes.Yet, take the X ray free-electron laser as light source through retrieval, the micro-nano sample of single fraction irradiation symmetry realizes that the method for sample three-dimensional reconstruction have not been reported.
Summary of the invention
The object of the present invention is to provide a kind of method that realizes the micro-nano sample three-dimensional imaging of symmetry with the X ray single measurement.
The method that realizes the micro-nano sample three-dimensional imaging of symmetry with the X ray single measurement of the present invention, step comprises: sample preparation, obtaining of symmetry sample two dimension diffraction data, individual particle normal incidence two dimension diffraction data is chosen, the processing of individual particle normal incidence diffraction data, the spatial orientation of sample is definite, and the three dimensional diffraction data acquisition of sample, carry out phase bit recovery and three-dimensional reconstruction to the three dimensional diffraction data.Specifically:
1) prepare the monodispersity sample that concentration is moderate: nanocrystal or the viral sample that will have symmetric profile are carried out purification process, obtain the complete same sample that particle diameter is identical with pattern.
Sample after purifying is diluted, utilize ultrasonic or concussion mode dispersion treatment 1～10 minute, fully dispersed sample, drop to pipettor sample thief 1-10 μ L the Si that thickness is 20～200nm
3n
4on film, and make the lip-deep sample rate of film not higher than 0.1/μ m
2, make single dispersed sample.
Preferably tetrahexahedron is nanocrystalline for the above-mentioned nanocrystal with symmetric profile, and trapezohedron is nanocrystalline, and trioctahedron is nanocrystalline or hexoctahedron is nanocrystalline; The above-mentioned preferred icosahedron viruses particle of virus with symmetric profile.
2) utilize relevant X-ray diffraction microscopie unit, use X ray free-electron laser monopulse to irradiate the sample of preparation, directly obtain the relevant X ray diffracting data of the two dimension of sample in Fourier space.
The a set of complete relevant X-ray diffraction imaging device of method needs of realizing the micro-nano sample three-dimensional imaging of symmetry with the X ray single measurement of the present invention, this device is built in high-performance the 4th generation synchrotron radiation light source (X ray free-electron laser), and all, under high vacuum environment, between each device, adopt vacuum pipe to connect.Wherein said microscopie unit internal vacuum is not less than 10
-6torr, be arranged in order and be provided with the X ray free-electron laser light source that pulse width is 6～30fs along optical path direction, response time is not less than the X ray shutter of 0.03s, first group of X ray slit that slit sizes is 0.5～20 μ m, second group of X ray slit that slit sizes is 1～40 μ m, XY direction moving range be 20 ± 1mm and the mobile accuracy sample stage that is 0.3 μ m and be placed in sample stage after 20～8000mm and the single Pixel Dimensions ccd detector that is 50 μ m * 50 μ m.
Sample is placed on the sample stage of sample chamber, utilizes the built-in optical microscope to regulate sample to X-ray light spot position and (determine Si
3n
4film position), single dispersed sample that on the scanning film, step 1) makes, preferred embodiment: it is 10Hz that X ray free-electron laser pulsed frequency is set, the X ray shutter time shutter is 0.1s, stepping rate mobile example platform according to 5～40 μ m/ steps, and often move the sample stage that moves a step, the fast exposure gate of X ray once, to being prepared in Si
3n
4single dispersed sample on film is carried out the monopulse irradiation and is obtained the relevant X ray diffracting data of the two dimension of sample in Fourier space, and realizes record and the output of ccd detector to diffracted signal; Wherein in relevant X-ray diffraction microscopie unit, the pulse width of described X ray free-electron laser is 6～20fs, first group of X ray slit is of a size of 2～10 μ m, and second group of X ray slit is of a size of 5～20 μ m, and described ccd detector is placed in 500～4000mm place after sample stage.
The part of diffraction data central area 100 pixels of 3) selection step 2) obtaining * 100 pixels～500 pixels * 500 pixels, calculate the average diffracted intensity in this zone, 50%～80% of the maximum intensity of take is intensity threshold, and Van Gogh can be defined as the normal incidence two dimension diffraction data of individual particle sample in the diffraction data of this threshold value.
Because sample is to be randomly distributed in Si
3n
4on film, therefore each exposure x-ray bombardment sample position is random, need to be screened diffraction data, determine the normal incidence two dimension diffraction data of individual particle sample, Van Gogh can be defined as the normal incidence two dimension diffraction data of individual particle sample in the diffraction data of this threshold value, preserves the two-dimentional diffraction data of the individual particle normal incidence filtered out.
4) for the two-dimentional diffraction data of the normal incidence of the individual particle sample of selecting, first deduct background and the CCD dark noise of diffraction data, remove discontinuous line and the discontinuous point of diffraction data, then by diffraction data, adjacent 3 pixels * 3 pixels～25 pixels * 25 pixels are merged into a pixel, and diffraction data is done to the Central Symmetry operation, obtain complete diffractogram.
Because the response of each pixel of multiport ccd detector there are differences, be the dark noise of CCD itself, therefore need to carry out the background correction processing to diffraction data, background is selected and is tested identical CCD dark background of time shutter; Due to what mainly occur in X ray and micro-nano sample interaction process, be elastic scattering, so diffraction pattern is centrosymmetric; Find out its center according to the symmetry of diffraction pattern, and remove discontinuous or asymmetrical point and the line of diffraction pattern according to symmetry, finally obtain correct diffracted signal, then by diffraction data, adjacent 3 pixels * 3 pixels～25 pixels * 25 pixels are merged into a pixel, and diffraction data is done to the Central Symmetry operation, obtain complete diffractogram data.
5) determine the line at a Tiao Guoqi center on the diffractogram obtained in step 4), by at interval of 0.1～2 degree, making the line that the central rotation mode is set another Tiao Guo center, calculate respectively the intensity difference of every two lines, be less than 10% intensity threshold according to intensity difference and determine self equivalent lines, record the corresponding angle occurred of equivalent lines and the quantity of self equivalent lines, for determining the spatial orientation of described sample simultaneously.
For the micro-nano sample of symmetry, its orientation always can be defined in an asymmetric unit, and arbitrary may be orientated of this sample in space, can obtain by it is carried out to symmetry operation.The spatial orientation of symmetry sample can be by Eulerian angle
with centre coordinate (x, y, z) defines, at first determine the coordinate system of symmetrical sample, define respectively x, y, the z axle, the position of any symmetrical sample and orientation can be described by this coordinate system; Then select the line at Yi Tiaoguo center, choose the line that another is crossed the centre line at interval of 0.1～2, two lines are carried out to matching, calculate the intensity difference of two lines, a pair of self equivalent lines that is defined as diffractogram that intensity difference is less than 10%, record the angle that this occurs self equivalent lines; The angle that self equivalent lines occurs is one to one with the spatial orientation of sample, and is to occur in pairs, so can be used for finally determining the spatial orientation of sample according to the angle of equivalent lines.
6) spatial symmetry of definite sample space orientation and this sample according to step 5), in rectangular coordinate system, described sample two dimension diffraction data being carried out to the symmetric rotation of crystallographic point groups and Central Symmetry under the symmetry sample processes, obtain the three dimensional diffraction data of described sample, wherein in rectangular coordinate system, without the interpolation of numerical value lattice point position, select the method for linear interpolation.
Three dimensions rectangular coordinate system of model, the two-dimentional diffractogram data that will obtain by step 4) are placed in three-dimensional coordinate system according to the determined spatial orientation of step 5), then determine according to the crystallographic point groups under the symmetry sample all space symmetry operations that it has, two-dimentional diffraction data is rotated according to its space symmetry operation, wherein in rectangular coordinate system, without the interpolation of numerical value lattice point position, select the method for linear interpolation, finally obtain the three dimensional diffraction data of described sample.
7) the three dimensional diffraction data of utilizing oversampling smoothness reconstruction algorithm to obtain step 6) are carried out phase bit recovery and three-dimensional reconstruction, the three dimensional diffraction data are carried out to iterative computation; First in iterative process, the initial phase information of three dimensional diffraction data is selected random phase information, in each iterative process, the phase information of three dimensional diffraction data is selected a front resulting phase place of iterative computation afterwards, and its strength information is selected the three dimensional diffraction data of obtaining by step 6); Set the square constraint condition identical with sample size in the real space in iterative process, the negatron density in constraint condition and the electron density outside constraint condition are by being multiplied by coefficient 0.05～0.3 cancellation gradually; Select the three-dimensional gauss low frequency filter of gradual change to carry out the gradient noise reduction process to the three dimensional diffraction data, its high frequency percent of pass threshold interval is 0.1～0.8; Through 1000-10000 iteration, every through an iteration, phase place is all upgraded, and finally obtains the three-dimensional structure image of the micro-nano sample of symmetry.
The present invention be take the X ray free-electron laser and has been set up as light source the method that the micro-nano sample of single fraction irradiation symmetry realizes the sample three-dimensional reconstruction, has effectively improved the three-dimensional imaging conventional efficient, for enriching three-D imaging method, provides new approaches.
The X ray free-electron laser advantages coherent diffraction imaging method of relevant, ultrashort pulse and superpower intensity has entirely overcome diffraction limit restriction in imaging process, and the method that realizes the micro-nano sample three-dimensional imaging of symmetry with the X ray single measurement is had to important using value; For the micro-nano sample of symmetry, utilize the method only to need its two-dimentional diffraction data in Fourier space of single-pulse measurement by the X ray free-electron laser, the symmetry of itself, can reconstruct its three-dimensional structure per sample.The advantage of utilizing the extremely short pulse of X ray free-electron laser of success of the present invention, can before changing, sample structure record its diffraction data, realized the home position observation of sample structure, especially the research for biological samples such as symmetric viruses has important application prospect.
Formation method of the present invention only needs the two-dimentional diffraction data of one-shot measurement sample to realize the sample three-dimensional reconstruction, can effectively improve the sample utilization factor, improve data acquisition efficiency, aspect the inorganic micro-nano crystal structure of research symmetry, considerable using value is being arranged.
The accompanying drawing explanation
Fig. 1 is for the device schematic diagram of the relevant X-ray diffraction imaging microscope of imaging experiment in the present invention, be aligned in sequence with 1-X ray light source along the light path working direction, 2-X ray shutter, the 3-the first group X ray slit, the 4-the second group X ray slit, 5-sample stage, 6-vacuum pipe, 7-multiport ccd detector.
The symmetric points system that Fig. 2 is trioctahedron gold-monocrystal nano particle and the polar stereographic projection figure of axis of symmetry system, crystallographic point groups is
, three 4 solid axles are arranged, four 3 solid axles, six 2 solid axles.
Fig. 3 is that obtain in the present invention representational appears face for the { two dimension of the trioctahedron gold-monocrystal nano particle of the 552} diffraction pattern that is concerned with.
Fig. 4 utilizes single measurement to realize that it is { the three-dimensional reconstruction result of the trioctahedron gold-monocrystal nano particle of 552} that the x-ray imaging method of the micro-nano sample three-dimensional imaging of symmetry is rebuild the face that appears obtained, be respectively along 2 secondary axes, 3 secondary axes, the reconstructed results image of 4 minor axis directions.
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment, only in order to explain the present invention, is not intended to limit the present invention as described below.
Embodiment 1:
In the described x-ray imaging method that realizes symmetrical sample three-dimensional imaging with single measurement, the present embodiment selects the high symmetry trioctahedron gold-monocrystal nano particle to having 48 symmetry operations to implement three-dimensional imaging, and step is as follows:
1), sample preparation:
In this embodiment, the symmetry sample selects to have the symmetric trioctahedron gold-monocrystal nano of height space particle; This sample belongs to
crystallographic point groups, comprise 34 solid axles, 3 planes of symmetry, 43 heavy rotation-inversion axis, 62 solid axles, 6 planes of symmetry; The sample average size is 150nm; The face that appears of sample is { 552};
To synthesize uniform trioctahedron gold-monocrystal nano particle and dilute with deionized water, and utilize supercentrifuge to select 10000 rev/mins to carry out centrifugally, wash the surfactant of gold-monocrystal nano particle, filter out the uniform monocrystalline gold grain of particle diameter simultaneously; Nanogold particle is diluted to the concentration of setting, utilizes Ultrasound Instrument that dilute sample is carried out to ultrasonic processing 5 minutes, the gold-monocrystal nano particle is disperseed fully; Drawing 3 μ l with pipettor, to drop to thickness be 100nm, is of a size of the Si of 2mm * 2mm
3n
4on film, the gold-monocrystal nano uniform particles is distributed on film, concentration is about 0.01/μ m
2;
2), utilize relevant X-ray diffraction microscopie unit, use X ray free-electron laser monopulse to irradiate the sample of preparation, directly obtain the relevant X ray diffracting data of the two dimension of sample in Fourier space:
Described relevant X-ray diffraction imaging device, as shown in Figure 1, show along light beam working direction coaxial row successively: X ray light source 1,3, the second groups of X ray slits 4 of 2, the first groups of X ray slits of X ray shutter, sample stage 5, vacuum pipe 6, multiport ccd detector 7; In above-mentioned relevant X-ray diffraction imaging device, it is 10fs that the X ray light source is selected its one pulse time, and the monopulse flux can reach 10
11the SACLA X ray free-electron laser light source of photons, the X ray energy is chosen as 7KeV; The response time of X ray shutter is 0.1s; Second group of slit location of first group of X ray slot distances is 300mm, and the slit opening size is 5 μ m; Second group of slit location is 150mm apart from the sample stage position, and the slit opening size is 7 μ m; Sample stage distance C CD is 2700mm; The equipment set internal vacuum is 10
-6torr; The mono-pixel size of multiport CCD is 50 μ m * 50 μ m;
By the Si that is loaded with the gold-monocrystal nano particulate samples prepared
3n
4film is fixed on the sample stage in sample chamber, utilizes the built-in optical microscope to regulate sample to the X-ray light spot position, and whole equipment is vacuumized to processing, treats that vacuum tightness reaches 10
-6during torr, tested; Experimental data adopts the pattern of scanning samples platform to obtain the diffraction data of individual particle sample, and it is 10Hz that X ray free-electron laser pulsed frequency is set, and the X ray shutter time shutter is 0.1s, and the sample stage moving step length is 10 μ m; The X ray shutter is opened, and single X ray pulse, by shutter, is irradiated Si
3n
4sample on film, ccd detector exposes simultaneously, records the diffraction data of sample, closes the X ray shutter, and the mobile example platform, to next position, repeats said process, obtains the diffraction pattern of sample by the pattern of scanning samples platform; The dark noise of a ccd detector of a data recording of every survey, noise as a setting;
3), data screening, determine the normal incidence two dimension diffraction data of individual particle sample:
Because sample is to be randomly distributed in Si
3n
4on film, therefore the position of each exposure x-ray bombardment sample is random, need to be screened diffraction data, and determine the normal incidence two dimension diffraction data of individual particle sample: in the present embodiment, average diffracted intensity method is used to carry out data screening; Choose the part of diffraction data central area 300 pixels * 300 pixels, calculate the average diffracted intensity in this zone, 70% of the maximum intensity of take is intensity threshold, Van Gogh can be defined as the normal incidence two dimension diffraction data of individual particle sample in the diffraction data of this threshold value, preserves the two-dimentional diffraction data of the individual particle normal incidence filtered out;
4), two-dimentional diffraction data is processed:
For the normal incidence two dimension diffraction data of the individual particle sample of selecting, these data are processed, comprise the background of deducting diffraction data, remove discontinuous line and the discontinuous some point of diffraction data; Wherein to select the dark noise of CCD be back end to background, because the time shutter is consistent, therefore can directly deduct back end, obtains the uniform diffracted signal of background; Under this X ray energy, the individual particle diffraction data meets the Central Symmetry condition, utilizes the symmetry of diffraction data to calculate the center of diffraction data, and diffraction pattern is carried out to the Central Symmetry processing; In addition, because the excessive sampling rate of diffraction pattern is higher, can carry out the pixel overlap-add procedure to diffraction pattern, in the present embodiment, we are superposed to a pixel by 9 pixels of diffraction data * 9 pixels, further strengthen the signal to noise ratio (S/N ratio) of diffracted signal, reduce matrix, increase arithmetic speed;
5), the dimensional orientation of crystal prototype:
Process step 4) process is processed, and has obtained the high s/n ratio two dimension diffraction data of individual particle nm of gold monocrystalline, as shown in Figure 3, in order to realize three-dimensional reconstruction, two-dimentional diffraction data need to be integrated into to the three dimensional diffraction data; The trioctahedron gold-monocrystal nano particle of selecting in the present embodiment belongs to
crystallographic point groups, its polar stereographic projection figure is as shown in Figure 2; Wherein shadow region is its asymmetric unit, and arbitrary may the orientation in space can obtain by the symmetry operation of its axis of symmetry; At first need to determine the spatial orientation of crystal in order to obtain the three dimensional diffraction pattern, the XYZ axle of setting space rectangular coordinate system is respectively three 4 turning axles of trioctahedron, and centre coordinate is trioctahedral center; Arbitrary spatial orientation of this crystal all can pass through attitude
mean; Therefore need to determine the spatial orientation that attitude is determined crystal;
The spatial orientation of crystal is to utilize the method for self equivalent lines to determine; The line at first selected Yi Tiaoguo center, choose the line that another is crossed the centre line at interval of 1 °, two lines are carried out to matching, calculate the intensity difference of two lines, a pair of self equivalent lines that is defined as diffractogram that intensity difference is less than 9%, record the angle that this occurs self equivalent lines; Final definite this crystal one co-exists in 14 to self equivalent lines; The angle that self equivalent lines occurs is one to one with the spatial orientation of crystal, so has finally determined the spatial orientation of crystal according to the angle of equivalent lines;
6) obtaining of three dimensional diffraction data:
Three dimensions rectangular coordinate system of model, obtained two-dimentional diffraction data is placed in described three-dimensional coordinate system according to determined spatial orientation, then determine according to the crystallographic point groups under the symmetry sample 48 space symmetry operations that it has, two-dimentional diffraction data is rotated according to its space symmetry operation, wherein in rectangular coordinate system, without the interpolation of numerical value lattice point position, select the method for linear interpolation, finally obtained this complete trioctahedral three dimensional diffraction pattern;
7), three-dimensional reconstruction:
The three-dimensional reconstruction process of gold-monocrystal nano particle comprises the phase bit recovery of three dimensional diffraction data and the three-dimensional imaging of sample structure, the oversampling smoothness(OSS that in the present embodiment, we adopt excessive sampling to combine with iteration) algorithm carry out three-dimensional reconstruction:
Step 1, first in iterative process, the initial phase information of three dimensional diffraction data is selected random phase information, at first gives restructuring three dimensional diffraction data one random initial phase, obtains complete three dimensional diffraction pattern; In each iterative process, the phase information of three dimensional diffraction data is selected a front resulting phase place of iterative computation afterwards, and its strength information is selected the three dimensional diffraction data of obtaining by step 6);
Repeating step two and step 3, until reconstructed results reaches stable, no longer further convergence; Finally obtaining high-quality size is 150nm, appear face for the three-dimensional reconstruction structure of the trioctahedron gold-monocrystal nano particle of 552}, as shown in Figure 4.
Embodiment 2:
The present embodiment selects the high symmetry regular dodecahedron mouse herpesvirus particle to having 60 symmetry operations to implement three-dimensional imaging, and step is as follows:
1), sample preparation:
In the present embodiment, the symmetry sample is selected has the symmetric deactivation regular dodecahedron of height space mouse herpesvirus particle; This viral sample profile symmetry belongs to 532 crystallographic point groups, comprises 6 five axis of symmetry, 10 axises of trigonal symmetry, 15 diadaxis;
By mouse herpesvirus particle ultraviolet inactivation, utilize 3% glutaraldehyde solution to fix, utilize hydro-extractor to select 3000 rev/mins to carry out centrifugally, filter out the uniform virion of particle diameter; Utilize methanol solution by the virion solution dilution to about 10000/μ L, utilize the vortex oscillator that dilute sample shake to processing 5 minutes, virion is disperseed fully; Drawing 3 μ L with pipettor, to drop to thickness be 100nm, is of a size of the Si of 2mm * 2mm
4n
3on film, virion is uniformly distributed on film, concentration is about 0.01/μ m
2;
2), with relevant X-ray diffraction microscopie unit, use X ray free-electron laser monopulse to irradiate the sample of preparation, directly obtain the relevant X ray diffracting data of the two dimension of sample in Fourier space:
Described relevant X-ray diffraction imaging device is in the same manner as in Example 1, and wherein the distance between CCD and sample stage is selected 1800mm, and the X ray energy is selected 5.5KeV, and other parameters of imaging device are identical with embodiment 1; Data acquisition step is with step 2 in embodiment 1) described method is identical;
3), data screening, determine the normal incidence two dimension diffraction data of individual particle sample:
In the data screening process, choose the zone of diffraction data center 100 pixels * 100 pixels, threshold value setting is maximum intensity 80%, and other settings are identical with the described method of step 3) and parameter in embodiment 1;
4), two-dimentional diffraction data is processed:
Data handling procedure is identical with step 4) in embodiment 1, wherein in the present embodiment, 15 pixels of diffraction data * 15 pixels is superposed to a pixel;
5) dimensional orientation of virion:
The spatial orientation method of virion is identical with the described crystal orientation method of step 5) in embodiment 1;
6), three dimensional diffraction data acquisition:
Because virus is regular dodecahedron, belong to 532 point groups, have 60 symmetry operations, in rectangular coordinate system, the XYZ axle is all by the diadaxis of regular dodecahedron, centre coordinate (x, y, z) be the center of virion, other methods of obtaining the three dimensional diffraction data are identical with embodiment 1 step 6);
7), three-dimensional reconstruction:
Virion three-dimensional reconstruction process is identical with the described three-dimensional reconstruction process of step 7) in embodiment 1.
Claims (4)
1. a method that realizes the micro-nano sample three-dimensional imaging of symmetry with the X ray single measurement, step is:
1) will there is nanocrystal or the viral sample of symmetric profile, utilize ultrasonic or concussion mode dispersion treatment 1～10 minute, then be prepared in the Si that thickness is 20～200nm
3n
4on film, and make the lip-deep sample rate of film not higher than 0.1/μ m
2, make single dispersed sample;
2) utilize relevant X-ray diffraction microscopie unit, use X ray free-electron laser monopulse to irradiate the sample of preparation, directly obtain the relevant X ray diffracting data of the two dimension of sample in Fourier space; Wherein said microscopie unit internal vacuum is not less than 10
-6torr, be arranged in order and be provided with the X ray free-electron laser light source that pulse width is 6～30fs along optical path direction, response time is not less than the X ray shutter of 0.03s, first group of X ray slit that slit sizes is 0.5～20 μ m, second group of X ray slit that slit sizes is 1～40 μ m, XY direction moving range be 20 ± 1mm and the mobile accuracy sample stage that is 0.3 μ m and be placed in sample stage after 20～8000mm and the single Pixel Dimensions ccd detector that is 50 μ m * 50 μ m;
The part of diffraction data central area 100 pixels of 3) selection step 2) obtaining * 100 pixels～500 pixels * 500 pixels, calculate the average diffracted intensity in this zone, 50%～80% of the maximum intensity of take is intensity threshold, and Van Gogh can be defined as the normal incidence two dimension diffraction data of individual particle sample in the diffraction data of this threshold value;
4) for the two-dimentional diffraction data of the normal incidence of the individual particle sample of selecting, first deduct background and the CCD dark noise of diffraction data, remove discontinuous line and the discontinuous point of diffraction data, then by diffraction data, adjacent 3 pixels * 3 pixels～25 pixels * 25 pixels are merged into a pixel, and diffraction data is done to the Central Symmetry operation, obtain complete diffractogram;
5) determine the line at a Tiao Guoqi center on the diffractogram obtained in step 4), by at interval of 0.1～2 degree, making the line that the central rotation mode is set another Tiao Guo center, calculate respectively the intensity difference of every two lines, the intensity threshold that is less than 5-10% according to intensity difference is determined self equivalent lines, record the corresponding angle occurred of equivalent lines and the quantity of self equivalent lines, for determining the spatial orientation of described sample simultaneously;
6) spatial symmetry of definite sample space orientation and this sample according to step 5), in rectangular coordinate system, described sample two dimension diffraction data being carried out to the symmetric rotation of crystallographic point groups and Central Symmetry under the symmetry sample processes, obtain the three dimensional diffraction data of described sample, wherein in rectangular coordinate system, the interpolation without numerical value lattice point position corresponding with diffraction data selected the method for linear interpolation;
7) the three dimensional diffraction data of utilizing oversampling smoothness reconstruction algorithm to obtain step 6) are carried out phase bit recovery and three-dimensional reconstruction, the three dimensional diffraction data are carried out to iterative computation; First in iterative process, the initial phase information of three dimensional diffraction data is selected random phase information, in each iterative process, the phase information of three dimensional diffraction data is selected a front resulting phase place of iterative computation afterwards, and its strength information is selected the three dimensional diffraction data of obtaining by step 6); Set the square constraint condition identical with sample size in the real space in iterative process, the negatron density in constraint condition and the electron density outside constraint condition are by being multiplied by coefficient 0.05～0.3 cancellation gradually; Select the three-dimensional gauss low frequency filter of gradual change to carry out the gradient noise reduction process to the three dimensional diffraction data, its high frequency percent of pass threshold interval is 0.1～0.8; Through 1000-10000 iteration, every through an iteration, phase place is all upgraded, and finally obtains the three-dimensional structure image of the micro-nano sample of symmetry.
2. realize as claimed in claim 1 the method for the micro-nano sample three-dimensional imaging of symmetry with the X ray single measurement, it is characterized in that: the described nanocrystal with symmetric profile is that tetrahexahedron is nanocrystalline, trapezohedron is nanocrystalline, and trioctahedron is nanocrystalline or hexoctahedron is nanocrystalline; The described virus with symmetric profile is the icosahedron viruses particles.
3. realize as claimed in claim 1 the method for the micro-nano sample three-dimensional imaging of symmetry with the X ray single measurement, it is characterized in that: in relevant X-ray diffraction microscopie unit, the pulse width of described X ray free-electron laser is 6～20fs, frequency is 10-60Hz, first group of X ray slit is of a size of 2～10 μ m, second group of X ray slit is of a size of 5～20 μ m, and described ccd detector is placed in 500～4000mm place after sample stage.
4. realize as claimed in claim 1 the method for the micro-nano sample three-dimensional imaging of symmetry with the X ray single measurement, it is characterized in that: use X ray free-electron laser monopulse to irradiate sample, the method of directly obtaining the relevant X ray diffracting data of the two dimension of sample in Fourier space is: it is 10Hz that X ray free-electron laser pulsed frequency is set, the X ray shutter time shutter is 0.1s, stepping rate mobile example platform according to 5～40 μ m/ steps, and often move the sample stage that moves a step, the fast exposure gate of X ray once, to being prepared in Si
3n
4single dispersed sample on film is carried out the monopulse irradiation and is obtained the relevant X ray diffracting data of the two dimension of sample in Fourier space, and realizes record and the output of ccd detector to diffracted signal.
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