CN109839396B - Synchrotron radiation confocal fluorescence experiment method based on KB mirror focusing - Google Patents
Synchrotron radiation confocal fluorescence experiment method based on KB mirror focusing Download PDFInfo
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- CN109839396B CN109839396B CN201910062560.2A CN201910062560A CN109839396B CN 109839396 B CN109839396 B CN 109839396B CN 201910062560 A CN201910062560 A CN 201910062560A CN 109839396 B CN109839396 B CN 109839396B
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Abstract
The invention relates to a synchronous radiation confocal fluorescence experimental method based on KB mirror focusing, which comprises the following steps: the surface of a copper tie strip of the standard sample is vertical to the direction of a light path of the focused X-ray; obtaining a relation curve chart of the fluorescence signal intensity and the position of the first Y-direction motor; dividing the abscissa of the relation curve graph into n equal parts by taking the width or height of the confocal infinitesimal as a unit, and solving the area integral of the corresponding fluorescence signal intensity in each equal part; making the standard sample form an angle of 45 degrees with the direction of the light path of the focused X-ray; three-dimensional scanning is carried out on the standard sample, and the intensity of a fluorescence signal is recorded; calculating a normalized concentration for each portion of the standard; and drawing a three-dimensional relative concentration distribution graph of the standard sample according to the normalized concentration of each part of the standard sample. The confocal fluorescence experiment is realized by adopting X rays focused by a KB mirror; compared with the traditional confocal experiment, the confocal experiment device is designed aiming at small light spots, can realize smaller confocal infinitesimal and realize higher spatial resolution of the confocal experiment.
Description
Technical Field
The invention relates to a hard X-ray confocal experimental method, in particular to a synchrotron radiation confocal fluorescence experimental method based on KB mirror focusing.
Background
The X-ray fluorescence analysis method is a qualitative and quantitative method capable of determining element components in substances, is widely applied to the subject fields of biology, materials, geology, archaeology, environment and the like, and has the advantages of sensitivity, no damage, atmospheric environment and the like. However, in conventional fluorescence experiments, there is no depth spatial resolution capability. The three-dimensional spatial distribution information of the substance can be provided by an X-ray confocal experimental method.
X-ray confocal experimental method was proposed by Gibson and Kumakhov in 1992, and the first X-ray confocal experimental apparatus appeared in 2000. The existing confocal experimental method is mostly based on laboratory X-rays, and has low brightness and poor resolution capability. Although the brightness of the confocal method based on the synchrotron radiation light source is high, the spot size is large, the spatial resolution depends on the manufacturing process of the capillary, and great improvement is difficult.
Disclosure of Invention
In order to solve the problems of the prior art, the present invention provides a method for confocal fluorescence simultaneous radiation experiment based on KB mirror focusing, so as to greatly improve the spatial resolution of the confocal experiment.
The invention discloses a synchronous radiation confocal fluorescence experimental method based on KB mirror focusing, which comprises the following steps:
step 0, providing a synchronous radiation confocal fluorescence experiment device, which comprises:
a KB mirror for receiving incident unfocused hard X-rays and emitting said focused X-rays;
a sample control system, comprising: the device comprises a first X-direction motor, a first Y-direction motor, a first Z-direction motor and a sample rack for placing a sample, wherein the first X-direction motor, the first Y-direction motor, the first Z-direction motor and the sample rack are sequentially arranged together from bottom to top;
a microscope system, comprising: a microscope assembly; and
a detector system, comprising: a fluorescence detector and a capillary tube connected with the front end of the fluorescence detector;
wherein the coincidence part of the focal point of the KB mirror and the focal point of the capillary is a confocal micro element;
step 7, dividing the standard sample into a plurality of parts from the surface of the copper tie strip along the optical path direction of the focused X-ray, and setting the normalized concentration of each part as I1、I2、I3……ImThe following relationship is given:
I1＝a1/S1；
I2＝(a2-I1S2)/S1；
I3＝(a3-I1S3-I2S2)/S1；
……
Im＝(am-I1Sm-I2S(m-1)-···I(m-1)S2)/S1；
step 8, according to the normalized concentration I of each part of the standard sample1、I2、I3……ImAnd drawing to obtain a three-dimensional relative concentration distribution map of the standard sample.
Further, the step 6 comprises: firstly, adopting a first step length which is less than one tenth of the width or height of the confocal infinitesimal to enable the confocal infinitesimal to approach the standard sample along the optical path direction of the focused X-ray, and marking the position just contacting the standard sample as a standard sample boundary; then, with the width or height of the confocal micro-element as a second step length, scanning the inside of the standard sample along the optical path direction of the focused X-ray step by step, and recording the intensity a of the fluorescence signal1、a2、a3、……、am。
In the above KB mirror focusing based synchrotron confocal fluorescence experiment method, the preset thickness of the copper tie of the standard sample is 5-20nm, and the preset width of the copper tie of the standard sample is 5-10 μm.
In the above KB mirror focusing based synchrotron radiation confocal fluorescence experiment method, the sample control system further comprises:
a 45-degree X-direction motor mounted on the first Y-direction motor to move along the Y direction under the drive of the first Y-direction motor;
a 45-degree Y-direction motor which is arranged on the 45-degree X-direction motor and driven by the 45-degree X-direction motor to move along the directions forming 45 degrees with the X positive direction and the Y negative direction respectively;
the first Z-direction motor is arranged on the 45-degree Y-direction motor to move along the directions forming 45 degrees with the X positive direction and the Y positive direction under the driving of the first Z-direction motor;
a rotary motor mounted on the first Z-direction motor for movement in the Z-direction under the drive of the first Z-direction motor;
the sample holder is mounted on the rotary motor to rotate in a horizontal plane under the drive of the rotary motor.
In the above KB mirror focusing based synchrotron radiation confocal fluorescence experiment method, the microscope system further comprises:
a second Y-direction motor;
a second X-direction motor mounted on the second Y-direction motor for movement in the Y-direction under the drive of the second Y-direction motor;
a second Z-direction motor mounted on the second X-direction motor for moving along the X direction under the driving of the second X-direction motor;
the microscope adapter is arranged on the second Z-direction motor and driven by the second Z-direction motor to move along the Z direction;
the microscope assembly is mounted on the microscope adaptor.
In the above KB mirror focusing based synchrotron radiation confocal fluorescence experiment method, the detector system further includes:
a third Y-direction motor;
a third X-direction motor mounted on the third Y-direction motor for movement in the Y-direction under the drive of the third Y-direction motor;
a third Z-direction motor mounted on the third X-direction motor for movement in the X-direction under the drive of the third X-direction motor;
a swing angle motor mounted on the third Z-direction motor to move along the Z direction under the driving of the third Z-direction motor;
a pitch motor mounted on said yaw motor;
the detector cover is sleeved at the signal acquisition end of the fluorescent detector at one end, and the capillary tube is inserted at the other end of the detector cover;
the fluorescence detector is arranged on the pitching motor, and is driven by the swing angle motor to adjust the swing angle and is driven by the pitching motor to adjust the pitching angle.
Due to the adoption of the technical scheme, the confocal fluorescence experiment is realized by using the characteristic that the KB mirror can focus the X-ray into a small point and the X-ray focused by the KB mirror; compared with the traditional confocal experiment, the confocal experiment device is designed aiming at small light spots, and can realize smaller confocal infinitesimal, thereby realizing higher spatial resolution of the confocal experiment.
Drawings
FIG. 1 is a schematic structural diagram of a confocal fluorescence synchronous radiation experimental apparatus involved in a confocal fluorescence synchronous radiation experimental method based on KB mirror focusing according to the present invention;
FIG. 2 is an exploded top view of the confocal fluorescence experimental apparatus shown in FIG. 1;
FIG. 3 is a schematic diagram of a sample control system in the confocal fluorescence synchronous radiation experiment apparatus shown in FIG. 1;
FIG. 4 is a schematic structural diagram of a microscope system in the confocal fluorescence testing apparatus of FIG. 1;
FIG. 5 is a schematic diagram of a detector system of the confocal fluorescence synchronous radiation experimental apparatus shown in FIG. 1;
FIG. 6 is a schematic structural diagram of the confocal fluorescence synchronous radiation experimental apparatus shown in FIG. 1 after step S101 is performed in the preparation process of the standard sample;
FIG. 7 is a schematic structural diagram of the confocal fluorescence synchronous radiation experimental apparatus shown in FIG. 1 after step S102 is performed on the standard sample during the preparation process;
FIG. 8 is a schematic structural diagram of the confocal fluorescence synchronous radiation experimental apparatus shown in FIG. 1 after step S103 is performed on the standard sample during the preparation process;
FIG. 9 is a schematic structural diagram of the confocal fluorescence synchrotron radiation experiment apparatus of FIG. 1 when the standard sample is used in the confocal fluorescence synchrotron radiation experiment;
FIG. 10 is a schematic diagram of a KB mirror focusing based synchrotron radiation confocal fluorescence experiment method of the present invention;
FIG. 11 is a schematic diagram of step S204 executed when calibrating the apparatus in the confocal fluorescence synchronous radiation experiment apparatus in FIG. 1;
FIGS. 12a and b are schematic diagrams illustrating a positional relationship between the capillary and the third Y-direction motor after steps S206 and S207 are executed to calibrate the apparatus in the CCFL experimental apparatus in FIG. 1;
FIG. 13 is a schematic structural diagram of a confocal micro-element in the confocal fluorescence experimental apparatus of FIG. 1;
FIG. 14 is a schematic diagram of the confocal infinitesimal size measurement method in the confocal fluorescence experimental apparatus of FIG. 1 when step S301 is performed;
FIG. 15 is a schematic diagram of the confocal infinitesimal size measurement method in the confocal fluorescence experimental apparatus of FIG. 1 when step S304 is performed;
FIG. 16 is a schematic diagram of the confocal infinitesimal size measurement method in the confocal fluorescence experimental apparatus of FIG. 1 when step S308 is executed;
FIG. 17 is a graph illustrating the relationship between the fluorescence signal intensity obtained after step 403 is performed and the position of the first Y-direction motor in the KB mirror focusing-based confocal fluorescence experiment method according to the present invention;
FIG. 18 is a graph illustrating the relationship between the fluorescence signal intensity obtained after step 404 is executed and the position of the first Y-direction motor in the KB mirror focusing-based confocal fluorescence experiment method according to the present invention.
Detailed Description
The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
First, a description will be given of a confocal fluorescence synchrotron radiation experimental apparatus according to the present invention, that is, a confocal fluorescence synchrotron radiation experimental method based on KB mirror focusing.
Herein, the various directions involved in the above-mentioned confocal fluorescence experiments are defined as follows:
when the experimental device is overlooked, the incident direction along the X-ray is the Y direction; the direction perpendicular to the incident direction of the X ray in the horizontal plane is the X direction; the vertical upward direction perpendicular to the horizontal plane is the Z direction.
Referring to fig. 1-5, the experimental apparatus comprises: KB mirror 1, sample control system 2, microscope system 3, and detector system 4, wherein,
a KB mirror 1(Kirkpatrick-Baez mirror) for receiving the unfocused hard X-rays (as indicated by arrow A in FIG. 2) generated by the synchrotron radiation device (not shown) and focusing the hard X-rays to generate focused X-rays;
the sample control system 2 is used for adjusting the posture of the standard sample 5 placed on the sample control system, and adjusting the standard sample 5 to the focus of the focused X-ray under the assistance of the microscope system 3, so that the standard sample 5 generates a fluorescence effect under the irradiation of the focused X-ray and emits fluorescence signals in all directions;
the microscope system 3 is used for assisting the positioning of the sample 5, and the angle between the microscope system 3 and the optical path of the focused X-ray (i.e. Y direction) is 45 degrees (in order to make the fluorescence signal of the sample 5 reach the detector system 4, and the signal-to-noise ratio is best, the sample 5 needs to be at 45 degrees with the optical path of the focused X-ray (i.e. Y direction), and the microscope system 3 is placed perpendicular to the surface of the sample 5 so as to observe and position the sample 5, so that the angle between the microscope system 3 and the optical path of the focused X-ray (i.e. Y direction) is 45 degrees;
the detector system 4 is placed in a direction at an angle of 90 degrees to the optical path of the focused X-rays (i.e. the X-direction) (the detector system 4 is at an angle of 45 degrees to the microscope system 3) for collecting the fluorescence signal in that direction (as indicated by arrow B in fig. 2) because the scattered signal in that direction is minimal, thereby maximizing the signal-to-noise ratio of the fluorescence signal.
As shown in fig. 3, the sample control system 2 specifically includes:
a first X-direction motor 201;
a first Y-direction motor 202 mounted on the first X-direction motor 201 to move in the X-direction by being driven by the first X-direction motor;
a 45-degree X-direction motor 203 mounted on the first Y-direction motor 202 to move in the Y direction by being driven by the motor;
a 45-degree Y-direction motor 204 mounted on the 45-degree X-direction motor 203 to move in directions at 45 degrees to each of the X-positive direction and the Y-negative direction (as shown in fig. 2) under the drive of the motor;
a first Z-direction motor 205 mounted on the 45-degree Y-direction motor 204 to move in a direction forming an angle of 45 degrees with each of the positive X-direction and positive Y-direction (as shown in fig. 2);
a rotary motor 206 mounted on the first Z-direction motor 205 to be moved in the Z-direction by being driven by the same;
a sample holder installed on the rotating motor 206 to rotate in a horizontal plane under the driving of the rotating motor, which is used for placing the standard sample 5 thereon, and under the adjustment of the rotating motor 206, the surface of the standard sample 5 can be made to be right opposite to the focused X-ray or the microscope system 3.
In this embodiment, the sample holder is made of teflon, and since the teflon does not contain metal atoms, the fluorescence interference experiment result is not generated; in addition, the sample holder specifically includes: a base 271 fixedly connected with the rotating motor 206 and a conical member 272 vertically installed on the top surface of the base 271, wherein the conical member 272 reduces scattering from the sample holder as much as possible while fixing the standard 5.
In the adjustment process of the sample control system 2, when the surface of the sample 5 is opposite to the focused X-ray, the first Z-direction motor 205, the first X-direction motor 201, and the first Y-direction motor 202 form a set of scanning system to provide three-dimensional scanning drive for the sample 5; when the surface of the standard sample 5 forms an angle of 45 degrees with the focused X-ray, the first Z-direction motor 205, the 45-degree X-direction motor 203, and the 45-degree Y-direction motor 204 form a set of scanning system, providing a three-dimensional scanning drive for the standard sample 5.
As shown in fig. 4, the microscope system 3 specifically includes:
a second Y-direction motor 301;
a second X-direction motor 302 mounted on the second Y-direction motor 301 to move in the Y-direction by being driven by the second Y-direction motor;
a second Z-direction motor 303 mounted on the second X-direction motor 302 to move in the X-direction by being driven by the second X-direction motor;
a microscope adaptor 304 mounted on the second Z-direction motor 303 to move in the Z-direction under the drive of the second Z-direction motor;
a microscope assembly 305, comprised of an optical microscope and a camera, mounted on the microscope adapter 304, can be used to remotely view the standard 5.
Before the confocal fluorescence experiment, the focus of the microscope assembly 305 can be positioned to coincide with the focus of the focused X-ray by the second Y-direction motor 301, the second X-direction motor 302 and the second Z-direction motor 303, and the sample 5 can be moved to the focus of the microscope assembly 305 by the sample control system 2 to realize the auxiliary positioning of the sample 5.
As shown in fig. 5, the detector system 4 specifically includes:
a third Y-direction motor 401;
a third X-direction motor 402 mounted on the third Y-direction motor 401 to move in the Y-direction by being driven by the third Y-direction motor;
a third Z-direction motor 403 mounted on the third X-direction motor 402 to move in the X-direction by being driven;
a swing angle motor 404 installed on the third Z-direction motor 403 to be moved in the Z-direction by being driven, which may be implemented by a commercially available rotating motor;
a tilt motor 405 mounted on the swing angle motor 404;
a fluorescence detector 406 mounted on the tilt motor 405, and driven by the tilt motor 404 to adjust the swing angle (the general adjustment range is ± 2 degrees), and driven by the tilt motor 405 to adjust the tilt angle (the general adjustment range is ± 2 degrees), wherein the fluorescence detector 406 is used for detecting the fluorescence signal of the focused X-ray, and has energy resolution;
a detector cover 407, one end of which is sleeved on the signal acquisition end of the fluorescence detector 406, and the other end of which is inserted with the capillary tube 408, wherein the detector cover 407 is used for placing the capillary tube 408 at a proper position in front of the fluorescence detector 406, shielding stray light in the environment and improving the signal-to-noise ratio of acquired data, is made of an aluminum alloy material and has a hollow cylindrical structure; the capillary tube 408 is used to turn the focused X-rays into parallel light.
Before the synchrotron radiation confocal fluorescence experiment is performed, the main optical axis of the capillary 208 is adjusted to be completely parallel to the movement direction of the third Y-direction motor 401 by using the pitch angle motor 406 and the swing angle motor 404, and the focus of the capillary 408 is calibrated to be completely overlapped with the focus of the focused X-ray by using the third Y-direction motor 401, the third X-direction motor 402 and the third Z-direction motor 403, so that the experiment can be started.
The preparation method of the standard sample 5 for the synchrotron radiation confocal fluorescence experimental device comprises the following steps:
step S101, depositing a copper thin film 52 with the thickness of 5-20nm (preferably 5nm) on the whole top surface of the silicon substrate 51 by magnetron sputtering, molecular beam epitaxy and other methods, and cutting the silicon substrate 51 with the copper thin film 52 into a length of 3-7mm (preferably 5mm) and a width of 0.8-1.2mm (preferably 1 mm); (as shown in FIG. 6)
Step S102, etching the copper film 52 and the silicon substrate 51 with partial thickness adjacent to the copper film 52 by an ion beam etching method to reduce their widths, forming a copper strip 53 with a width of 50-200 μm (preferably 100 μm) and a first substrate portion 54; (as shown in FIG. 7)
In step S103, the copper strip 53 and the first substrate portion 54 are etched by a focused ion beam etching method, so that two opposite side surfaces of the copper strip 53 and the first substrate portion 54 extending in the extension direction are recessed inward at a middle position thereof, thereby forming a copper-based strip 55 and a second substrate portion 56 (as shown in fig. 8), wherein the copper-based strip 55 has a width of 5-10 μm (preferably 5 μm) and a length of 20-50 μm (preferably 20 μm), and preferably, the copper-based strip 55 has a width of 5 μm, a length of 20 μm, and a thickness of 5nm, which is an effective area of the standard sample 5.
When the standard sample 5 is used in a synchrotron radiation confocal fluorescence experiment, the standard sample 5 is vertically placed, one end face of the standard sample extending along the width direction faces downwards, and the copper plating layer faces the incidence direction of synchrotron radiation X-rays; specifically, as shown in fig. 9, in use, hard X-rays are incident perpendicularly to the surface of the copper strip 53 of the standard sample 5 (as indicated by an arrow a in fig. 9), then the position of the standard sample 5 is adjusted so that the focus of the focused X-rays falls at the middle position O of the copper strip 55, and then the detector system is placed perpendicularly to the direction of the focused X-rays to detect the fluorescence signal of copper emitted from the standard sample 5 (as indicated by an arrow B in fig. 9); in this process, the absorption of the fluorescence signal of copper is negligible, because the width of the copper tie 55 is narrow. Therefore, the standard sample 5 can be placed perpendicular to the incident direction of the synchrotron radiation X-ray, the fluorescence signal can still reach the fluorescence detector 406 without being affected, and at this time, the intensity of the fluorescence signal emitted by the standard sample 5 is the same as the intensity of the fluorescence signal emitted by the standard sample 5 placed at an angle of 45 degrees with respect to the incident direction of the synchrotron radiation X-ray. Therefore, the dimension measurement result of the confocal infinitesimal can be directly obtained when scanning the confocal infinitesimal in the synchrotron radiation confocal fluorescence experiment, an inclined plane is not needed for scanning, the result is calculated (the inclined plane scanning can bring errors, the errors have little influence on the confocal infinitesimal with large volume, but have great influence on the measurement of the confocal infinitesimal with small volume), and the thickness error can not be introduced into the instrument calibration because the copper belt system has very thin thickness. In addition, it should be noted that the above parameters are derived from the investigation of many experiments, and are the result of the balance of the parameters when the standard sample is extremely small.
As shown in fig. 10, according to the schematic diagram of the synchrotron radiation confocal fluorescence experiment implemented by the present invention, hard X-rays generate focused X-rays through a KB mirror 1, then the focus of the focused X-rays is made to fall on a standard sample 5, so as to excite the fluorescence signals of the standard sample 5, and finally the fluorescence signals are observed by using a detector system 4 forming an angle of 90 degrees with the optical path of the focused X-rays, wherein a capillary 408 for limiting the beam is placed in front of the fluorescence detector 406, so that only the fluorescence signals in the field of view of the capillary 408 can be transmitted to the fluorescence detector 406.
The smallest cross-section (shown as line a-a in fig. 10) of the field of view of the capillary tube 408 (i.e., the optical path of the capillary tube 408) is the focal point of the capillary tube 408. By adjusting the device, the focal point of the KB mirror 1 (indicated by the line b-b in FIG. 10) can be made to coincide with the focal point of the capillary 408. The coincidence of the focal point of the KB mirror 1 and the focal point of the capillary 408 is the confocal infinitesimal (as shown by the portion filled by oblique lines in fig. 10), and the minimum size of the confocal infinitesimal determined by the field of view of the capillary 408 is the highest spatial resolution of the confocal device.
Due to limitations in manufacturing processes and the like, the focal point of the capillary 408 is difficult to be made small, that is, the focal point size of the KB mirror 1 is much smaller than that of the capillary 408. As can be seen from fig. 10, the light path shape near the focal point of the KB mirror 1 and the capillary 408 is saddle-shaped, the light path cross section at the focal point is the smallest, and the cross section at a distance from the focal point is sharply enlarged. Since the size of the cross section of the optical path near the focal point is not very different, the cross sections of the optical path within the distance are considered to be equal, and the distance is called the depth of field. Because the focus of the KB mirror 1 is small, the depth of field L is only a plurality of micrometers, and the size of a light spot outside the range of the depth of field L is increased sharply; the field of view of the capillary 408 is larger, so the edge of its intersection with the KB mirror 1 optical path inevitably falls outside the KB mirror 1 focal depth L; once this edge deviates from the KB mirror 1 focus, it causes the size of the confocal infinitesimal to increase dramatically. Therefore, in order to obtain the minimum size of confocal infinitesimal, the capillary 408 and the focus of the KB mirror 1 are required to be exactly coincident, and the error needs to be controlled within one micron. This requires a highly accurate method of calibrating the instrument to exactly coincide the focal points of the two.
Based on the above situation, the calibration method for each instrument in the confocal fluorescence synchronous radiation experimental apparatus includes the following steps:
step S201, adjusting the focus of the microscope assembly 305 to be coincident with the focus of the KB mirror 1;
step S202, placing the above-mentioned standard sample 5 on the sample control system 2, making the surface of the copper tie 55 (i.e., the effective area) of the standard sample 5 perpendicular to the focused X-ray, and adjusting the standard sample 5 to the focus of the KB mirror 1 by the auxiliary observation of the microscope assembly 305;
step S203, directly aligning the fluorescence detector 406 with the standard sample 5 (the capillary 408 is not arranged at the front end of the fluorescence detector 406);
step S204, by scanning the first Y-direction motor 202 and the first Z-direction motor 205, the sample 5 is two-dimensionally imaged, and the focused X-ray finally falls on one side edge of the copper tie 55 of the sample 5 close to the fluorescence detector 406 (as shown in fig. 11) (because the spot size of the focused X-ray is small, and the copper tie 55 of the sample 5 is very thin, the copper generating the fluorescence signal can be used as a point signal source), (considering that only a part of the width of the copper tie 55 is narrow, the focused X-ray must be irradiated on the narrowest part of the copper tie 55 to ensure that the fluorescence signal enters the fluorescence detector 406 without being affected, and otherwise, the focused X-ray is absorbed by the copper film 52. therefore, the purpose of this step S204 is to make the focused X-ray finally fall on a specific position of the copper tie 55 of the sample 5, at this time, the volume of the copper generating the fluorescence signal is very small, can be considered a point where the resulting fluorescence signal can be received by fluorescence detector 406 with little copper self-absorption, thereby further reducing the effect of copper on the fluorescence signal absorption);
step S205, mounting the capillary tube 408 at the front end of the fluorescence detector 406;
step S206, scanning the third X-direction motor 402 at different positions of the third Y-direction motor 401, and correcting the position of the swing angle motor 404 according to the peak position change of the fluorescence signal intensity detected by the fluorescence detector 406; specifically, the method comprises the following steps: when the third Y-direction motor 401 is at the Y1 position, the third X-direction motor 402 is scanned to obtain the peak position of the fluorescence signal intensity variation curve by the fluorescence detector 406 as X1, and when the third Y-direction motor 401 is at the Y2 position, the third X-direction motor 402 is scanned to obtain the peak position of the fluorescence signal intensity variation curve by the fluorescence detector 406 as X2, and then the horizontal included angle α between the main optical axis of the capillary 408 and the movement direction of the third Y-direction motor 401 (the movement direction of the third Y-direction motor 401 is the direction in which the fluorescence approaches or leaves the detector 406 to or away from the standard sample 5) is tan-1[(X1-X2)/(Y1-Y2)](as shown in the top view of FIG. 12 a);
step S207, scanning the third Z-direction motor 403 at different positions of the third Y-direction motor 401, respectively, and correcting the position of the tilt motor 405 according to the change in the peak position of the fluorescence signal intensity detected by the fluorescence detector 406; specifically, the method comprises the following steps: when the third Y-direction motor 401 is at the Y1 position, the third Z-direction motor 403 is scanned to obtain the peak position of the fluorescence signal intensity variation curve by the fluorescence detector 406 as Z1, and when the third Y-direction motor 401 is at the Y2 position, the third Z-direction motor 403 is scanned to obtain the peak position of the fluorescence signal intensity variation curve by the fluorescence detector 406 as Z2, and then the angle β between the main optical axis of the capillary 408 and the vertical plane of the movement direction of the third Y-direction motor 401 is tan-1[(Z1-Z2)/(Y1-Y2)](as shown in the elevation view of FIG. 12 b);
step S208, repeatedly executing the above steps S206 and S207 until the main optical axis of the capillary 408 is completely parallel to the moving direction of the third Y-direction motor 401, at which time the included angles α and β are zero (i.e., the main optical axis of the capillary 408 cannot be made more parallel to the moving direction of the third Y-direction motor 401 within the moving accuracy of the motors), and it should be noted that after step S208 is executed, the positions of the tilt angle motor 404 and the tilt motor 405 are kept unchanged;
step S209, the third X-direction motor 402 and the third Z-direction motor 403 are moved to the positions of the peak intensity values of the fluorescence signals (i.e. the fluorescence signals are received by the fluorescence detector 406, the intensity of the fluorescence signals is counted, and the maximum value counted by the fluorescence detector 406 is the peak intensity value of the fluorescence signals) (when the step S209 is executed, the third X-direction motor 402 and the third Z-direction motor 403 are moved respectively and have no sequence, i.e. one of the motors is moved first and the other is moved later)
Step S210 is to scan the third Y-direction motor 401, obtain a relationship curve between the fluorescence signal intensity and the position of the third Y-direction motor 401 through the fluorescence detector 406, and move the third Y-direction motor 401 to the position of the fluorescence signal intensity peak according to the relationship curve, so that the main optical axis of the capillary 408 can be calibrated to coincide with the focus of the focused X-ray.
Further, since the capillary 408 cannot reduce the field of view to this level due to manufacturing process, etc., when the X-rays are focused to the micrometer or smaller scale using the synchrotron radiation light source in conjunction with optical devices such as KB mirrors, the depth dimension of the confocal micro-element is much larger than the other two dimensions, and the confocal micro-element 6 looks like a long bar (as shown in fig. 13).
In order to accurately measure the size of the confocal infinitesimal, the method for measuring the size of the confocal infinitesimal in the synchrotron radiation confocal fluorescence experimental device comprises the following steps:
step S301, placing the above-mentioned standard sample 5 on the sample control system 2, and making the surface of the copper tie 55 (i.e., the effective area) of the standard sample 5 perpendicular to the focused X-ray (as shown in fig. 14), and adjusting the effective area of the standard sample 5 to the focus of the focused X-ray spot by the auxiliary observation of the microscope assembly 305;
step S302, moving the first X-direction motor 201 and the first Z-direction motor 205 to move the focus of the focused X-ray to the center position of the effective area of the standard sample 5;
step S303, scanning the first Y-direction motor 202 (as shown in fig. 14) along the Y direction (i.e., the optical path direction of the focused X-ray), and recording by the fluorescence detector 406 to obtain a first relation curve between the fluorescence signal intensity and the position of the standard sample 5 (i.e., the position of the first Y-direction motor 202), where the full width at half maximum of the first relation curve is the depth resolution fwhm (Y) of the confocal infinitesimal 6 because the fluorescence signal intensity changes in a gaussian distribution;
step S304, the surface of the copper tie 55 of the sample 5 is directed to the fluorescence detector 406, so that the surface of the copper tie 55 is parallel to the focused X-ray (as shown in FIG. 15), and the microscope assembly 305 is used for assisting observation to adjust the effective area of the sample 5 to the focus of the focused X-ray spot;
step S305, moving the first Z-direction motor 205 to move the focus of the focused X-ray to the center position of the effective area of the standard sample 5;
step S306, scanning the first Y-direction motor 202 (i.e., scanning along the Y-direction), and observing the sample by the fluorescence detector 406 to place the sample at the position where the fluorescence signal is strongest (the fluorescence detector 406 counts and counts the received fluorescence signal in unit time, and the maximum value of the count is the strongest value of the fluorescence signal);
step S307, scanning the first X-direction motor 201 (as shown in fig. 15) along the X direction (i.e., the placement direction of the fluorescence detector 406), and recording by the fluorescence detector 406 to obtain a second relationship curve between the fluorescence signal intensity and the position of the standard sample 5 (i.e., the position of the first X-direction motor 201), where the full width at half maximum of the second relationship curve is the width resolution fwhm (X) of the confocal infinitesimal 6 because the fluorescence signal intensity changes in a gaussian distribution;
step S308, horizontally placing the sample 5, making the surface of the copper tie 55 face upward, rotating the sample 5 by 1 ° toward the capillary 408 with the optical path direction of the focused X-ray as an axis, so that the fluorescence signal enters the capillary 408 in a glancing manner (as shown in "rotation direction" in fig. 16), and adjusting the effective area of the sample 5 to the focus of the focused X-ray spot through the auxiliary observation of the microscope assembly 305;
step S309, moving the first X-direction motor 201 to move the focus of the focused X-ray to the center position of the effective area of the standard sample 5;
step S310, scanning the first Y-direction motor 202 (i.e., scanning along the Y-direction), and observing the sample by the fluorescence detector 406 to place the sample at the position where the fluorescence signal is strongest (the fluorescence detector 406 counts and counts the received fluorescence signal in unit time, and the maximum value of the count is the strongest value of the fluorescence signal);
step S311, scanning the first Z-direction motor 205 along the Z direction (i.e., the vertical upward direction) (as shown in fig. 16), and recording a third relation curve between the fluorescence signal intensity and the position of the standard sample 5 (i.e., the position of the first Z-direction motor 205) by the fluorescence detector 406, wherein the full width at half maximum of the third relation curve is the height resolution fwhm (Z) of the confocal micro-element 6 because the fluorescence signal intensity changes in a gaussian distribution;
it should be noted that, in step S311, since the surface of the standard sample 5 and the capillary 408 have a grazing exit angle of 1 ° (as shown in fig. 16), the full width at half maximum FWHM of the scanned third relation curve2(measurement) ═ FWHM2(X)*tan2(1°)+FWHM2(Z)＝0.0003*FWHM2(X)+FWHM2(Z)≈FWHM2(Z) due to 0.0003 FWHM2The value of (X) is negligible, so the full width at half maximum of the third relation curve is defined as the height resolution FWHM (Z) of the confocal micro-element 6.
The standard sample can be regarded as a point fluorescent signal source because the copper thickness of the fluorescent light generated by the standard sample is known and accurately controllable (the thickness of a copper film is 5nm and is far smaller than the dimension of a confocal infinitesimal). Thus, the full width at half maximum of the directly measured fluorescence signal intensity variation is the full width at half maximum of the confocal infinitesimal. The testing method is simple and direct, and based on a special standard sample, the fluorescent signal can be directly scanned by using the motor in the first X, Y, Z direction, and the scanning direction is consistent with the boundary direction of the confocal infinitesimal, so that the size of the obtained confocal infinitesimal is more accurate.
As described above, although a better focusing device is employed, the capillary 408 limits the improvement of the resolution of the entire apparatus; in order to reduce the volume of the confocal micro-element 6, effectively reduce the depth of the confocal micro-element 6 to the same magnitude as the width and the height thereof and improve the resolution of the device, the synchronous radiation confocal fluorescence experimental method based on KB mirror focusing comprises the following steps:
step S401, placing the above-mentioned standard sample 5 on the sample control system 2, and making the surface of the copper tie 55 (i.e., the effective area) of the standard sample 5 perpendicular to the focused X-ray (as shown in fig. 14), and adjusting the effective area of the standard sample 5 to the focus of the focused X-ray spot through the microscope assembly 305 for auxiliary observation;
step S402, moving the first X-direction motor 201 and the first Z-direction motor 205 to move the focus of the focused X-ray to the center position of the effective area of the standard sample 5;
step S403, scanning the first Y-direction motor 202 along the Y-direction (i.e., the optical path direction of the focused X-ray) (as shown in fig. 14), and recording by the fluorescence detector 406 a relationship curve between the fluorescence signal intensity and the position of the standard sample 5 (i.e., the position of the first Y-direction motor 202), and drawing a relationship curve graph (as shown in fig. 17) with the fluorescence signal intensity as the ordinate and the position of the first Y-direction motor 202 as the abscissa; since the moving step of the first Y-direction motor 202 is much smaller than the width of the confocal micro-element 6, and the intensity variation of the fluorescence signal is gaussian distributed (e.g. a gaussian fit curve drawn by a smooth line in fig. 17), the full width at half maximum of the relation curve (e.g. a curve drawn by a non-smooth line in fig. 17) is the depth resolution fwhm (Y) of the confocal micro-element 6 (based on the above-mentioned measurement method of the dimension of the confocal micro-element 6, the full width at half maximum of the width and height of the confocal micro-element 6 obtained in this embodiment is about 2 micrometers, but the depth is about 30 micrometers, which is much larger than the width and height);
in step S404, the abscissa of the graph is divided into n equal parts based on the width of the confocal micro-element 6 (generally, the width and the height of the confocal micro-element 6 are equal), and the area integral of the fluorescence signal intensity corresponding to each equal part is obtained and is sequentially marked as S1、S2、……、Sn(as shown in fig. 18);
step S405, rotating the sample 5 towards the position of the fluorescence detector so that the angle between the sample and the optical path direction of the focused X-ray is 45 degrees, and determining the region of interest of the surface of the copper tie 55 of the sample 5 (i.e. the sample region to be detected, because the sample may be very large and the laboratory does not necessarily measure all the samples, so that only the region concerned by the experimenter needs to be measured, which is called the region of interest) by the aid of the microscope assembly 305;
step S406, the sample 5 is scanned three-dimensionally by the first X-direction motor 201, the first Y-direction motor 202, and the first Z-direction motor 205 step by step, and the intensity a of the fluorescent signal is recorded1、a2、a3、……、amM is any positive integer (the value of m has no special requirement and is determined according to the experimental requirement, when a standard sample with a larger volume needs to be scanned, the value of m is larger, otherwise, the value of m is smaller);
specifically, in each scanning cycle, scanning is performed from an area without the standard sample 5 to the standard sample 5, firstly, the confocal micro-element 6 is close to the standard sample 5 along the Y direction by adopting a first step length which is less than one tenth of the width of the confocal micro-element 6, and the position just contacting the standard sample 5 is marked as a standard sample boundary;
then, the width of the confocal micro-element 6 is taken as the second step length, the scanning is performed gradually towards the interior of the standard sample 5 along the Y direction, and the intensity a of the fluorescence signal is recorded1、a2、a3、……、am；
Therefore, the confocal micro-element 6 is divided into a plurality of right cubes with the same length, width and height, and the side length of each right cube is equal to the width and height of the original confocal micro-element;
in step S407, the standard sample 5 is divided into a plurality of portions in the Y direction from the surface of the copper tie 55 by the plurality of orthocubes, and the normalized concentration of each portion is defined as I1、I2、I3……ImThe following relationship is given:
I1＝a1/S1；
I2＝(a2-I1S2)/S1；
I3＝(a3-I1S3-I2S2)/S1；
……
Im＝(am-I1Sm-I2S(m-1)-···I(m-1)S2)/S1；
step S408, according to the normalized concentration I of each part of the standard sample 51、I2、I3……Im(these normalized concentration values can be plotted on a graph according to coordinates), a three-dimensional relative concentration profile of the standard 5 is plotted.
According to the experimental method, the depth size of the confocal micro element can be reduced to be the same as the width size and the height size by recursive calculation and combining with instrument parameters measured by a standard sample, so that the advantage of synchrotron radiation micro focusing light spots is fully utilized, and compared with a common confocal fluorescence experiment, the spatial resolution is improved by one order of magnitude.
The above embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present patent application. The invention has not been described in detail in order to avoid obscuring the invention.
Claims (6)
1. A synchrotron radiation confocal fluorescence experiment method based on KB mirror focusing is characterized by comprising the following steps:
step 0, providing a synchronous radiation confocal fluorescence experiment device, which comprises:
a KB mirror for receiving incident unfocused hard X-rays and emitting focused X-rays;
a sample control system, comprising: the device comprises a first X-direction motor, a first Y-direction motor, a first Z-direction motor and a sample rack for placing a sample, wherein the first X-direction motor, the first Y-direction motor, the first Z-direction motor and the sample rack are sequentially arranged together from bottom to top;
a microscope system, comprising: a microscope assembly; and
a detector system, comprising: a fluorescence detector and a capillary tube connected with the front end of the fluorescence detector;
wherein the coincidence part of the focal point of the KB mirror and the focal point of the capillary is a confocal micro element;
step 1, placing the standard sample on the sample holder, enabling the surface of a copper tie of the standard sample to be vertical to the light path direction of the focused X-ray, and then adjusting the copper tie of the standard sample to the focus of the focused X-ray under the auxiliary observation of a microscope component;
step 2, moving the first X-direction motor and the first Z-direction motor to move the focus of the focused X-ray to the central position of the copper tie of the standard sample;
step 3, scanning the first Y-direction motor along the light path direction of the focused X-ray, recording by the fluorescence detector to obtain a first relation curve of the fluorescence signal intensity and the position of the first Y-direction motor, and drawing a relation curve graph taking the fluorescence signal intensity as a vertical coordinate and the position of the first Y-direction motor as a horizontal coordinate;
step 4, dividing the abscissa of the relation curve chart into n equal parts by taking the width or the height of the confocal infinitesimal as a unit, and solving the area integral of the corresponding fluorescence signal intensity in each equal part, which is sequentially marked as S1、S2、……、Sn；
Step 5, rotating the standard sample towards the position of the fluorescence detector so that the standard sample forms an angle of 45 degrees with the light path direction of the focused X-ray, and determining the region of interest of the surface of the copper tie of the standard sample by the aid of the microscope component;
step 6, gradually carrying out three-dimensional scanning on the standard sample through the first X-direction motor, the first Y-direction motor and the first Z-direction motor, and recording the intensity a of the fluorescence signal1、a2、a3、……、amM is any positive integer;
step 7, dividing the standard sample into a plurality of parts from the surface of the copper tie strip along the optical path direction of the focused X-ray, and setting the normalized concentration of each part as I1、I2、I3……ImThe following relationship is given:
I1＝a1/S1；
I2＝(a2-I1S2)/S1；
I3＝(a3-I1S3-I2S2)/S1；
……
Im＝(am-I1Sm-I2S(m-1)-···I(m-1)S2)/S1；
step 8, according to the normalized concentration I of each part of the standard sample1、I2、I3……ImAnd drawing to obtain a three-dimensional relative concentration distribution map of the standard sample.
2. The KB mirror focusing-based synchrotron confocal fluorescence experimental method of claim 1, wherein the step 6 comprises: firstly, adopting a first step length which is less than one tenth of the width or height of the confocal infinitesimal to enable the confocal infinitesimal to approach the standard sample along the optical path direction of the focused X-ray, and marking the position just contacting the standard sample as a standard sample boundary; then, with the width or height of the confocal micro-element as a second step length, scanning the inside of the standard sample along the optical path direction of the focused X-ray step by step, and recording the intensity a of the fluorescence signal1、a2、a3、……、am。
3. The KB mirror focusing-based synchrotron confocal fluorescence experimental method of claim 1, wherein the copper ties of the standard sample have a predetermined thickness of 5-20nm and a predetermined width of 5-10 μm.
4. The KB mirror focusing-based synchrotron confocal fluorescence experimental method of claim 1, wherein the sample control system further comprises:
a 45-degree X-direction motor mounted on the first Y-direction motor to move along the Y direction under the drive of the first Y-direction motor;
a 45-degree Y-direction motor which is arranged on the 45-degree X-direction motor and driven by the 45-degree X-direction motor to move along the directions forming 45 degrees with the X positive direction and the Y negative direction respectively;
the first Z-direction motor is arranged on the 45-degree Y-direction motor to move along the directions forming 45 degrees with the X positive direction and the Y positive direction under the driving of the first Z-direction motor;
a rotary motor mounted on the first Z-direction motor for movement in the Z-direction under the drive of the first Z-direction motor;
the sample holder is mounted on the rotary motor to rotate in a horizontal plane under the drive of the rotary motor.
5. The KB mirror focusing-based synchrotron confocal fluorescence experimental method of claim 1, wherein the microscope system further comprises:
a second Y-direction motor;
a second X-direction motor mounted on the second Y-direction motor for movement in the Y-direction under the drive of the second Y-direction motor;
a second Z-direction motor mounted on the second X-direction motor for moving along the X direction under the driving of the second X-direction motor;
the microscope adapter is arranged on the second Z-direction motor and driven by the second Z-direction motor to move along the Z direction;
the microscope assembly is mounted on the microscope adaptor.
6. The KB mirror focusing-based synchrotron confocal fluorescence experimental method of claim 1, wherein the detector system further comprises:
a third Y-direction motor;
a third X-direction motor mounted on the third Y-direction motor for movement in the Y-direction under the drive of the third Y-direction motor;
a third Z-direction motor mounted on the third X-direction motor for movement in the X-direction under the drive of the third X-direction motor;
a swing angle motor mounted on the third Z-direction motor to move along the Z direction under the driving of the third Z-direction motor;
a pitch motor mounted on said yaw motor;
the detector cover is sleeved at the signal acquisition end of the fluorescent detector at one end, and the capillary tube is inserted at the other end of the detector cover;
the fluorescence detector is arranged on the pitching motor, and is driven by the swing angle motor to adjust the swing angle and is driven by the pitching motor to adjust the pitching angle.
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