CN103411560A - Device and method for measuring microstructure through angular spectrum scanning illumination fluorescent follow-up pinhole detection - Google Patents

Abstract

The microstructure measurement device and method for angle-spectrum scanning illumination fluorescence follow-up pinhole detection belong to the field of ultra-precise three-dimensional microstructure surface topography measurement, and mainly relate to a microstructure measurement device and method based on angle-spectrum scanning illumination fluorescence follow-up pinhole detection ; The present invention is provided with an angular spectrum scanning illumination optical path and a fluorescent follow-up pinhole detection optical path, and proposes a three-dimensional microstructure sample measurement method; the present invention can not only avoid some areas that cannot be illuminated or complicated by the existing converging beam illumination technology. The problem of reflection can effectively solve the problem that the detection signal strength is attenuated and the background noise is enhanced, which causes the measurement accuracy to decrease or even fail to measure. Moreover, it can be realized that there is a corresponding fluorescent pinhole in front of each CCD camera pixel, so that there is no need for precise adjustment between the fluorescent follow-up pinhole and the CCD camera pixel.

Description

Microstructure measurement device and method for angular spectrum scanning illumination fluorescence follow-up pinhole detection

technical field

The microstructure measurement device and method for angle-spectrum scanning illumination fluorescence follow-up pinhole detection belong to the field of ultra-precise three-dimensional microstructure surface topography measurement, and mainly relate to a microstructure measurement device and method based on angle-spectrum scanning illumination fluorescence follow-up pinhole detection .

Background technique

The processing and application of microstructures are mainly reflected in three aspects: microelectronics technology, microsystem technology and micro-optical technology, such as typical applications such as computer chips, biochips and microlens arrays. The common feature of the above-mentioned technologies is that they have a three-dimensional structure, and the size of the functional structure is on the order of micron, submicron or nanometer. development of related industries. With the rapid development of micromachining technology, instruments capable of rapid and non-destructive three-dimensional detection of such samples will have great application prospects.

U.S. Patent US3013467 discloses a confocal imaging technology for the first time. This invention obtains the axial detection ability of the sample contour by introducing the confocal imaging technology of the three-point optical conjugation of point light source, point illumination and point detection. Cooperate with the movement of the stage in the horizontal direction to realize three-dimensional measurement. Chinese patent CN1395127A discloses a confocal microscopic measurement system. The invention utilizes confocal technology and introduces an interference optical path into the confocal optical path to obtain a highly sensitive interferometric signal and realize high-precision measurement of the axial direction of the sample. US Patent US6282020B1 discloses a confocal microscope system based on a scanning galvanometer. The invention utilizes the confocal principle and introduces the galvanometer scanning technology to obtain the ability of converging the illumination spot to move at high speed on the sample surface, realize fast confocal detection, and improve the measurement speed. However, the above three methods use the microscope objective lens to focus the light beam to the surface of the sample with ups and downs for illumination. In this way, there will be problems that some areas cannot be illuminated or complex reflections, which will cause the attenuation of the detection signal intensity and the background noise. Enhanced, the measurement accuracy is reduced, or even impossible to measure.

Chinese Patent Publication No. CN1971333A, the name of the invention is a confocal microscopic imaging system using virtual pinhole technology, which discloses a confocal microscopic imaging system based on virtual pinhole technology. The invention utilizes the corresponding position on the two-dimensional digital image collected by the CCD A virtual pinhole is set, and the light intensity information in the virtual pinhole is obtained through computer processing, thereby realizing a confocal microscopic imaging system without a physical pinhole, which has the characteristics of controllable pinhole position and size, and convenient calibration. However, this invention is based on the basic principle of confocal point-to-point imaging. One detection can only image a point on the sample. If you want to image a certain area of the sample under test, you need to add a mechanical three-dimensional scanning mechanism, which makes it difficult to increase the measurement speed. Chinese patent CN1632448A, the title of the invention is a three-dimensional super-resolution confocal array scanning microscopic detection method and device, which discloses an array pinhole technology, which realizes parallel confocal measurement by introducing a pinhole array in front of the CCD. However, this method requires precise alignment between the array pinhole position and the CCD pixel position, which leads to difficulties in assembly and adjustment.

Contents of the invention

In order to solve the above problems, the present invention designs a microstructure measurement device and method for angular spectrum scanning illumination fluorescence follow-up pinhole detection; the device and method can not only avoid some areas that cannot be illuminated or complicated The problem of reflection can effectively solve the problem that the detection signal intensity attenuation and the background noise increase cause the measurement accuracy to decrease or even fail to measure, and it can realize that there is a corresponding fluorescent pinhole in front of each CCD camera pixel, so that the fluorescence can follow There is no need for precise alignment between the pinhole and the CCD camera pixels.

The purpose of the present invention is achieved like this:

Angle-spectrum scanning illumination fluorescence follow-up pinhole detection microstructure measurement device, including laser, first scanning galvanometer, second scanning galvanometer, scanning lens, first aperture, first imaging lens, beam splitter, second aperture , microscope objective lens, scanning stage, tube mirror, fluorescence follow-up pinhole, second imaging lens, narrow-band filter and CCD camera; the beam emitted from the laser is reflected by the first scanning galvanometer and the second scanning galvanometer Afterwards, through the scanning lens, the first aperture, the first imaging lens, the beam splitter, the second aperture, and the microscope objective lens, it irradiates the surface of the measured microstructure sample that moves axially with the scanning stage to form an angular spectrum scan. Illumination light path: the light beam diffusely reflected from the surface of the microstructure sample to be tested passes through the microscope objective lens and the second aperture again, and is reflected by the beam splitter, and converges to the fluorescent follow-up pinhole through the tube lens, and the excited fluorescence is captured by the second imaging lens and the narrow-band filter are imaged to the CCD camera to form a fluorescence follow-up pinhole detection optical path; the rotation axes of the first scanning galvanometer and the second scanning galvanometer are perpendicular to each other, and the back focal plane of the scanning lens coincides with the object plane of the first imaging lens On the plane where the first diaphragm is located; the image plane of the first imaging lens coincides with the rear focal plane of the microscope objective lens on the plane where the second diaphragm is located; the front focal plane of the tube mirror coincides with the object plane of the second imaging lens on the fluorescence follower The plane where the moving pinhole is; the CCD camera is positioned at the image plane of the second imaging lens, and the filter is placed between the fluorescent moving pinhole and the CCD camera; the fluorescent moving pinhole is evenly coated with Transparent thin base material for fluorescent substances.

The transparent thin base material is glass with a thickness of no more than 0.17mm, and the upper and lower surfaces are parallel and subjected to light-splitting treatment.

Angular spectrum scanning illumination fluorescence follower pinhole detection microstructure measurement method, the method includes the following steps:

Step a, set the number of step rotations of the first scanning galvanometer as N _x , the number of step rotations of the second scanning galvanometer as N _y , and the number of step movements of the scanning stage along the optical axis as N _z , The number of pixels in the CCD camera is M;

Step b. Arranging and combining the stepping movement position of the scanning stage set in step a, the stepping rotation position of the first scanning galvanometer and the stepping rotation position of the second scanning galvanometer to obtain N _x × N _y × N _z different spatial positions, at each spatial position, perform angular spectrum illumination on the microstructure sample to be tested, and then form N _x ×N _y ×N _z images on the surface of the fluorescent pinhole;

In step c, use M fluorescence point detector arrays composed of fluorescence tracking pinholes and CCD cameras to detect N _x ×N _y ×N _z images of the microstructure to be measured, and then obtain N _x ×N _y ×N _z ×M light intensity data;

Step d, use the computer to process the N _x ×N _y ×N _z ×M light intensity data obtained in step c, and obtain the axial axis under the illumination angles of M points and N _x ×N _y angular spectra on the microstructure sample to be tested Response curve, according to the principle of confocal, first judge the axial response curve of each point that best matches the square curve of the theoretical sinc function, that is, the optimal angular spectrum illumination angle, and then calculate the axial coordinates of each point;

Step e, obtaining the corresponding axial coordinates according to the plane position of each point and step d, and reconstructing the three-dimensional structure of the microstructure sample to be measured.

In the method for measuring the microstructure of the above-mentioned angle-spectrum scanning illumination fluorescence follow-up pinhole detection, in the step a, the angle between two adjacent step rotation positions of the first scanning galvanometer is the same or different.

In the method for measuring the microstructure of the above-mentioned angle-spectrum scanning illumination fluorescence follow-up pinhole detection, in the step a, the angle between two adjacent step rotation positions of the second scanning galvanometer is the same or different.

In the method for measuring the microstructure of the above-mentioned angle-spectrum scanning illumination fluorescence follow-up pinhole detection, in the step a, the distance between two adjacent step-translation positions of the scanning stage is the same or different.

The present invention introduces the angular spectrum scanning illumination light path, realizes parallel light beams illuminating the tested microstructure sample at different incident angles, and then enables each point of the tested microstructure sample to find the corresponding optimal lighting angle, avoiding the existing converging light beam illumination The problem that certain areas cannot be illuminated or complex reflection caused by technology can effectively solve the problem of attenuation of detection signal strength and enhancement of background noise, resulting in reduced measurement accuracy or even impossible measurement.

The present invention also introduces the detection light path of the fluorescence servo pinhole, and uses the fluorescent material film with the Stokes property to realize the detection of the array pinhole points, thereby greatly improving the measurement speed; Due to the high density of fluorescent substances, there is a corresponding fluorescent pinhole in front of each CCD camera pixel, so that there is no need for precise adjustment between the fluorescent follower pinhole and the CCD camera pixel.

Description of drawings

Fig. 1 is a structural schematic diagram of the microstructure measuring device for angular spectrum scanning fluorescence follow-up pinhole detection of the present invention.

Fig. 2 is a schematic diagram of the angular spectrum scanning illumination light path in the angular spectrum scanning fluorescence follow-up pinhole detection microstructure measuring device of the present invention.

Fig. 3 is a schematic diagram of the optical path of fluorescence following pinhole detection in the angular spectrum scanning fluorescence following pinhole detection microstructure measuring device of the present invention.

Fig. 4 Schematic diagram of beam incident fluorescence follower pinhole.

Fig. 5 is a flow chart of the microstructure measurement method for angular spectrum scanning fluorescence following pinhole detection of the present invention.

In the figure: 1 laser, 2 first scanning galvanometer, 3 second scanning galvanometer, 4 scanning lens, 5 first aperture, 6 first imaging lens, 7 beam splitter, 8 second aperture, 9 microscope objective lens , 10 scanning stage, 11 tube mirror, 12 fluorescent follow-up pinhole, 13 second imaging lens, 14 narrow-band filter, 15CCD camera.

Detailed ways

The specific embodiments of the present invention will be further described in detail below in conjunction with the accompanying drawings.

Specific embodiment one

The structural diagram of the angular spectrum scanning illumination fluorescence follow-up pinhole detection microstructure measurement device of this embodiment is shown in Figure 1, the device includes a laser 1, a first scanning galvanometer 2, a second scanning galvanometer 3, a scanning lens 4, The first diaphragm 5, the first imaging lens 6, the beam splitter 7, the second diaphragm 8, the microscope objective lens 9, the scanning stage 10, the tube mirror 11, the fluorescent follow-up pinhole 12, the second imaging lens 13, Narrow-band filter 14 and CCD camera 15; the beam emitted from laser 1 is reflected by first scanning galvanometer 2 and second scanning galvanometer 3, then passes through scanning lens 4, first diaphragm 5, and first imaging lens 6 in sequence , the beam splitter 7, the second aperture 8, and the microscopic objective lens 9 irradiate the surface of the microstructure sample to be measured with the axial movement of the scanning stage 10 to form an angular spectrum scanning illumination optical path, as shown in Figure 2; from the measured The light beam diffusely reflected on the surface of the microstructure sample passes through the microscope objective lens 9 and the second aperture 8 again, and is reflected by the beam splitter 7, and then converges to the fluorescent follow-up pinhole 12 through the tube lens 11, and the excited fluorescence is captured by the second imaging lens 13 and the narrow-band filter 14 are imaged to the CCD camera 15 to form a fluorescence follow-up pinhole detection optical path, as shown in Figure 3; The focal plane coincides with the object plane of the first imaging lens 6 on the plane where the first aperture 5 is located; the image plane of the first imaging lens 6 coincides with the plane of the second aperture 8 where the image plane of the first imaging lens 9 is located; The front focal plane of 11 coincides with the object plane of the second imaging lens 13 on the plane where the fluorescence follow-up pinhole 12 is; Between the CCD cameras 15; the fluorescent follow-up pinhole 12 is a transparent thin base material uniformly coated with a fluorescent substance having a Stokes property. The transparent thin base material is glass with a thickness of no more than 0.17mm, and the upper and lower surfaces are parallel and subjected to light-splitting treatment. Choosing a glass of no more than 0.17mm can not only reduce the aberration, but also directly use the cover glass as the raw material of the transparent thin substrate material, saving cost.

A schematic diagram of the light beam incident on the fluorescent follower pinhole 12 is shown in FIG. 4 . Among them, the circle represents the fluorescent molecule, the diameter is generally tens to hundreds of nanometers, the rectangle below the fluorescent molecule is a transparent thin substrate material, and the black rectangle at the bottom is the effective imaging area, and its diameter is generally about five microns, much larger than the fluorescent molecule diameter. The corresponding black fluorescent molecules are effective follow-up pinholes; the white rectangles are invalid imaging regions, and the corresponding white fluorescent molecules are invalid follow-up pinholes. Therefore, when a small displacement occurs in the effective imaging area, there is still a corresponding fluorescent follower pinhole to match it.

The Stokes property in this embodiment means that after the incident light beam is absorbed by the fluorescent substance, a light beam with a longer wavelength than the incident light beam is emitted.

The flow chart of the microstructure measurement method for angular spectrum scanning illumination fluorescence following pinhole detection in this embodiment is shown in Figure 5, and the method includes the following steps:

Step a, set the number of step rotations of the first scanning galvanometer 2 as N _x , the number of step rotations of the second scanning galvanometer 3 as N _y , and the number of step movements of the scanning stage 10 along the optical axis as N _z , the number of pixels in the CCD camera 15 is M;

Step b, arrange and combine the stepping movement position of the scanning stage 10 set in step a, the stepping rotation position of the first scanning galvanometer 2 and the stepping rotation position of the second scanning galvanometer 3, to obtain N _x × N _y ×N _z different spatial positions, and perform angular spectrum illumination on the microstructure sample to be tested at each spatial position, and then form N _x ×N _y ×N _z images on the surface of the fluorescent follower pinhole 12;

In step c, use M fluorescence point detector arrays composed of fluorescence follow-up pinhole 12 and CCD camera 15 to detect N _x ×N _y ×N _z images of the microstructure to be measured, and then obtain N _x ×N _y × N _z ×M light intensity data;

Step d, use the computer to process the N _x ×N _y ×N _z ×M light intensity data obtained in step c, and obtain the axial axis under the illumination angles of M points and N _x ×N _y angular spectra on the microstructure sample to be tested Response curve, according to the principle of confocal, first judge the axial response curve of each point that best matches the square curve of the theoretical sinc function, that is, the optimal angular spectrum illumination angle, and then calculate the axial coordinates of each point;

Step e, obtaining the corresponding axial coordinates according to the plane position of each point and step d, and reconstructing the three-dimensional structure of the microstructure sample to be measured.

In the method for measuring the microstructure of the above-mentioned angular spectrum scanning illumination fluorescence follow-up pinhole detection, in the step a, the angle between the two adjacent stepping rotation positions of the first scanning galvanometer 2 is the same; the angle between the two adjacent scanning galvanometers 3 The angles between the two stepping rotation positions are the same; the distance between two adjacent stepping translational positions of the scanning stage 10 is the same. The setting of the same angle between the stepping and rotating positions and the same distance between the stepping and translational positions is convenient for adjustment.

Specific embodiment two

The difference between this embodiment and the specific embodiment 1 is that in the step a, the first scanning vibrating mirror 2 is adjacent to two stepping rotation positions; the second scanning vibrating mirror 3 is adjacent to two stepping rotation positions; At least one of the two adjacent step-translation positions of the stage 10 is different. The setting of different angles between the step rotation positions or the step translation position spacing can achieve local fine adjustment.

In the above embodiment, the mentioned step d is specifically:

The light field distribution of the plane where the fluorescent follower pinhole 12 is located is:

$\mathrm{<span\; class="notranslate">\; u</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; x</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; ,</span>{\mathrm{<span\; class="notranslate">\; the\; y</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; =</span><span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; u</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; ,</span>\mathrm{<span\; class="notranslate">\; the\; y</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; T</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; ,</span>\mathrm{<span\; class="notranslate">\; the\; y</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; ]</span><span\; class="notranslate">\; \&CircleTimes;</span>\mathrm{<span\; class="notranslate">\; h</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; ,</span>\mathrm{<span\; class="notranslate">\; the\; y</span>}<span\; class="notranslate">\; ;</span>{\mathrm{<span\; class="notranslate">\; x</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; ,</span>{\mathrm{<span\; class="notranslate">\; the\; y</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>$

$<span\; class="notranslate">\; =</span><span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; A</span>}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; exp</span>}<span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; jk</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; x</span>}\mathrm{<span\; class="notranslate">\; cos</span>}\mathrm{<span\; class="notranslate">\; \&alpha;</span>}<span\; class="notranslate">\; +</span>\mathrm{<span\; class="notranslate">\; the\; y</span>}\mathrm{<span\; class="notranslate">\; cos</span>}\mathrm{<span\; class="notranslate">\; \&beta;</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; ]</span><span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; T</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; ,</span>\mathrm{<span\; class="notranslate">\; the\; y</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; ]</span><span\; class="notranslate">\; \&CircleTimes;</span>\mathrm{<span\; class="notranslate">\; h</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; x</span>}<span\; class="notranslate">\; ,</span>\mathrm{<span\; class="notranslate">\; the\; y</span>}<span\; class="notranslate">\; ;</span>{\mathrm{<span\; class="notranslate">\; x</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; ,</span>{\mathrm{<span\; class="notranslate">\; the\; y</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>$

Where U(x',y') is the image square light field distribution, U(x,y) is the illumination light field, T(x,y) is the microstructure sample object function, h(x,y;x',y ') is the point spread function of the microscope objective. The incident angle of the plane wave is continuously modulated by angular spectrum scanning illumination, that is, the phase value corresponding to exp[jk(xcosα+ycosβ)]. Fluorescent follow-up pinhole 12 is coated with a fluorescent substance with Stokes properties, and each fluorescent substance with Stokes properties is equivalent to a detector with a very small size. If the scanning stage 10 moves at this time, the When the microstructure sample passes through the object plane of the microscopic objective lens 9 along the optical axis direction, it satisfies the confocal measurement principle, and the detected normalized light intensity distribution should be:

$\mathrm{<span\; class="notranslate">\; I</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; u</span>}<span\; class="notranslate">\; ,</span>\mathrm{<span\; class="notranslate">\; 0</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; =</span>{\mathrm{<span\; class="notranslate">\; N</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}{<span\; class="notranslate">\; [</span>\frac{\mathrm{<span\; class="notranslate">\; sin</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; u</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; 4</span>}<span\; class="notranslate">\; )</span>}{\mathrm{<span\; class="notranslate">\; u</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; 4</span>}}<span\; class="notranslate">\; ]</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}$

Where u≈kzNA ² is the optical dimensionless coordinate along the optical axis, and N=πa ² /λf is the Fresnel number. And when the measured point moves to the object plane of the microscope objective lens 9, it is conjugated with the fluorescent molecule on the corresponding fluorescent follower pinhole 12, and the light field energy detected by the fluorescent molecule is the highest at this time. Further, the light intensity of the illuminating plane wave is adjusted so that the highest light energy collected by the fluorescent molecules is just above its fluorescence excitation threshold, and then the fluorescence phenomenon of another wavelength is excited, which passes through the second imaging lens 13 and the filter behind it. The light sheet 14 is imaged to the CCD camera 15, so the axial coordinates of the measured point conjugated with the fluorescent molecule are completed.

Claims (6)

1. the servo-actuated pin hole of angular spectrum scanning lighting fluorescent is surveyed microstructure measuring device, it is characterized in that: comprise laser instrument (1), the first scanning galvanometer (2), the second scanning galvanometer (3), scanning lens (4), the first diaphragm (5), the first imaging len (6), spectroscope (7), the second diaphragm (8), microcobjective (9), scanning objective table (10), Guan Jing (11), the servo-actuated pin hole of fluorescence (12), the second imaging len (13), narrow band pass filter (14) and CCD camera (15); The light beam sent from laser instrument (1) is after the first scanning galvanometer (2) and the second scanning galvanometer (3) reflection, pass through successively scanning lens (4), the first diaphragm (5), the first imaging len (6), spectroscope (7), the second diaphragm (8), microcobjective (9) shine with the axially-movable of scanning objective table (10) by the micro-measuring structure sample surfaces, form angular spectrum scanning illumination path; From by the irreflexive light beam of micro-measuring structure sample surfaces, again being passed through microcobjective (9), the second diaphragm (8), and reflected by spectroscope (7), through Guan Jing (11), converge to the servo-actuated pin hole of fluorescence (12), the fluorescence inspired is imaged onto CCD camera (15) by the second imaging len (13) and narrow band pass filter (14), forms the servo-actuated pin hole of fluorescence and surveys light path; The first scanning galvanometer (2) is mutually vertical with the rotating shaft of the second scanning galvanometer (3), and the object plane of the back focal plane of scanning lens (4) and the first imaging len (6) coincides with the first diaphragm (5) plane, place; The picture plane of the first imaging len (6) and the back focal plane of microcobjective (9) coincide with the second diaphragm (8) plane, place; The object plane of the front focal plane of Guan Jing (11) and the second imaging len (13) coincides with the servo-actuated pin hole of fluorescence (12) plane, place; CCD camera (15) is positioned at the second imaging len (13) as plane, and optical filter (14) is positioned between the servo-actuated pin hole of fluorescence (12) and CCD camera (15); The servo-actuated pin hole of described fluorescence (12) is for evenly being coated with the thin transparent base material of the fluorescent material with Stokes character.

2. the servo-actuated pin hole of angular spectrum according to claim 1 scanning lighting fluorescent is surveyed microstructure measuring device, and it is characterized in that: described thin transparent base material is the glass that thickness is no more than 0.17mm, and upper and lower surface is parallel and cut open light and process.

3. the servo-actuated pin hole of angular spectrum scanning lighting fluorescent is surveyed the microstructure measuring method, it is characterized in that: said method comprising the steps of:

The stepping number of revolutions of step a, setting the first scanning galvanometer (2) is N _x, the second scanning galvanometer (3) the stepping number of revolutions be N _y, along the stepping of optical axis direction, to move number of times be N to scanning objective table (10) _z, the number of pixels in CCD camera (15) is M;

Stepping shift position, the stepping turned position of the first scanning galvanometer (2) and the stepping turned position permutation and combination of the second scanning galvanometer (3) of step b, scanning objective table (10) that step a is set, obtain N _x* N _y* N _zIndividual different spatial, to by the micro-measuring structure sample, being carried out the angular spectrum illumination, and then form N in each locus on the servo-actuated pin hole of fluorescence (12) surface _x* N _y* N _zIndividual picture;

M the phosphor dot detector array that step c utilizes the servo-actuated pin hole of fluorescence (12) and CCD camera (15) to form, to the N by micro-measuring structure _x* N _y* N _zIndividual picture is surveyed, and then obtains N _x* N _y* N _z* M light intensity data;

Steps d, the N that utilizes computing machine treatment step c to obtain _x* N _y* N _z* M light intensity data, obtain by M point, N on the micro-measuring structure sample _x* N _yAxial response curve under individual angular spectrum light angle, at first judge every bit and the theoretical sinc function square axial response curve that curve mates most according to confocal principle, i.e. best angular spectrum light angle, and then calculate the axial coordinate of every bit;

Step e, obtain corresponding axial coordinate according to planimetric position and the steps d of every bit, reconstruct by the three-dimensional structure of micro-measuring structure sample.

4. the servo-actuated pin hole of angular spectrum scanning lighting fluorescent according to claim 3 is surveyed the microstructure measuring method, and it is characterized in that: in described step a, adjacent two the stepping turned position angles of the first scanning galvanometer (2) are identical or different.

5. the servo-actuated pin hole of angular spectrum scanning lighting fluorescent according to claim 3 is surveyed the microstructure measuring method, and it is characterized in that: in described step a, adjacent two the stepping turned position angles of the second scanning galvanometer (3) are identical or different.

6. the servo-actuated pin hole of angular spectrum scanning lighting fluorescent according to claim 3 is surveyed the microstructure measuring method, it is characterized in that: in described step a, adjacent two the stepping translation location gap of scanning objective table (10) are identical or different.

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