CN104296685A - Device and method for measuring smooth free-form surface sample based on differential STED - Google Patents

Abstract

The invention provides a device and method for measuring a smooth free-form surface sample based on differential STED, and belongs to the field of optical microscopic measurement. The surface of the smooth free-form surface sample is plated with a fluorescent film, and by the utilization of the characteristic that the fluorescent film radiates fluorescence in various directions under laser radiation, the problem that signal light is difficult to collect due to specular reflection of light beams on a smooth free-form surface is solved; meanwhile, by the adoption of the STED microscopy principle, light spots formed by collecting two pulse laser beams of different wavelengths coincide with each other, the portions, located in the center regions of the light spots, of the fluorescent film on the sample excite fluorescence, the portions, located in the light spots but not located in the center regions of the light spots, of the fluorescent film generate back excitation and do not emit fluorescence, and therefore super-resolution imaging of the sample is achieved; by the adoption of a differential detection structure, differential operation is conducted on two paths of collected signals, a differential response curve is acquired, the surface position of the sample is determined through a zero point of the differential response curve, and finally the purpose of measuring the smooth free-form surface sample shape with the large curvature at high accuracy is achieved.

Description

The apparatus and method of smooth free form surface sample are measured based on differential STED

Technical field

The apparatus and method measuring smooth free form surface sample based on differential STED belong to optical microphotograph fields of measurement.

Background technology

For smooth free form surface surface sample, in normal and the large region of optical axis direction angle, because illumination cannot be collected to the light of sample surfaces completely because of mirror-reflection, or almost cannot be collected, so its surface topography cannot high-acruracy survey, even cannot measure.Thus the sample that how high-acruracy survey has a smooth free form surface of larger slope has become a difficult problem of field of optical measurements.

In sample surface morphology fields of measurement, laser scanning co-focusing microscopy (Laser Scanning Confocal Microscopy; LSCM) be conventional method.The method adopts precise pinhole filtering technique, the information be only in focal plane position can be detected, inhibit the parasitic light of non-focusing plane to greatest extent, have higher signal to noise ratio (S/N ratio); Simultaneity factor can realize harmless optical tomography along Z-direction, thus realizes the three-dimensional imaging to thicker sample.But when using the measurement of laser scanning co-focusing microscopy to have the sample on smooth free form surface surface, at sample surfaces slope comparatively large regions, because light beam generation mirror-reflection causes illumination light almost cannot collect, so the pattern in this region cannot be measured.Simultaneously because the resolving power of laser scanning co-focusing microscopy affects by optical diffraction limit, super-resolution cannot be realized and measure.

Differential confocal scanning survey is one of typical modified confocal measurement method.Differential confocal scanning survey comprises the confocal interference microscope (publication number CN1614457A) with high-space resolution imaging ability, there is shaping circular light bundle formula differential confocal sensor (publication number CN1529123A) of high spatial resolution, three differential confocal microscopic imaging method and device (publication number CN1587898A), three-differential focasing micro-three-dimensional super-resolution imaging method (publication number CN1609590A), three-dimensional super-resolution confocal array scanning micro-detecting method and device (publication number CN1632448A), shaping circular light three differential confocal microscope (publication number CN1588157A), there is the differential confocal scanning detection method (publication number CN1527026A) etc. of high spatial resolution, it is equidistant away from burnt and nearly focal plane that two confocal point probes are placed in image space by differential confocal measurement system respectively, bipolarity response characteristic is obtained by the differential calculating of intensity response, produce and follow the tracks of zero point, overcome the deficiency that conventional confocal can only carry out relative position measurement.The relative conventional confocal microtechnic of this technology significantly improves signal to noise ratio (S/N ratio), and axial resolution and measurement range are two times of conventional confocal microtechnic, and it is more accurate especially to measure at null position.But the reflected signal that traditional differential confocal measurement technology is still difficult to by fully collecting smooth free curved surface sample improves signal to noise ratio (S/N ratio), thus realize the object of the smooth free curved surface sample topography of high-acruracy survey.

Stimulated radiation loss (Stimulated Emission Depletion, STED) microscopy proposes (Hell by Stefan W.Hell, S.W.and Wichmann, J. (1994), ' Breaking the diffraction resolution limit by stimulated emission:Stimulated-emission-depletion fluorescence microscopy ', Optics Letters, 19 (11): 780 – 782), its basic thought uses a branch of picosecond pulse laser light beam to be converged to the first focal beam spot at sample surfaces, the picosecond pulse laser light beam using another Shu Bochang different is converged to the second focal beam spot of donut-like at sample surfaces, two spot center are overlapped, the fluorescent material being wherein positioned at spot center region inspires fluorescence, be arranged in hot spot but do not send out at the fluorescent material generation de excitation in spot center region, do not send fluorescence, thus realize super-resolution imaging.Because the measuring object of field of industrial measurement cannot send fluorescence, so STED microscopy device is mainly used in biology microscope imaging field, not yet there is the precedent being applied in production piece topography measurement at present.Even if STED microscopy to be transplanted to simply production piece topography measurement field, also cannot to overcome light beam and produce the extremely weak problem of the collection signal light intensity that causes of mirror-reflection at the sample surfaces with smooth free form surface.

Summary of the invention

Object of the present invention is exactly the problem being difficult to measure the smooth free form surface sample topography with larger slope for above-mentioned classic method, propose a kind of apparatus and method measuring smooth free form surface sample based on differential STED, one deck fluorescent film is plated at smooth free form surface sample surfaces, utilize fluorescent film under laser irradiates, give off the characteristic of fluorescence to all directions, avoid light beam and cause flashlight to be difficult to the problem of collecting at smooth free form surface generation mirror-reflection; Simultaneously, adopt STED microscopy principle, the hot spot that two bundle different wave length pulsed laser beams are converged to is overlapped, the fluorescent film be positioned on the sample in spot center region inspires fluorescence, be arranged in hot spot but do not send out at the fluorescent film generation de excitation in spot center region, do not send fluorescence, thus realize the super-resolution imaging to sample; Adopt differential detection structure, the two paths of signals collected is carried out calculus of differences and obtains differential response curve, determine specimen surface positions zero point by differential response curve, final solution high-acruracy survey has the problem of the smooth free form surface sample topography of larger slope.

The object of the present invention is achieved like this:

Measure the device of smooth free form surface sample based on differential STED, comprising: excitation module, loss module, differential image-forming module and plated film sample;

Described excitation module comprises: wavelength is λ ₁the first picosecond pulse laser, first conduction optical fiber, the first collimator objective, the first plane mirror; Be λ at wavelength ₁the first picosecond pulse laser after configure the first conduction optical fiber, the first collimator objective and the first plane mirror successively;

Described loss module comprises: wavelength is λ ₂the second picosecond pulse laser, second conduction optical fiber, the second collimator objective, the second plane mirror, vortex shape phase-modulation plate and 1/2nd wave plates; Be λ at wavelength ₂the second picosecond pulse laser after configure the second conduction optical fiber, the second collimator objective and the second plane mirror successively, the second plane mirror reflected light path configures vortex shape phase-modulation plate and 1/2nd wave plates successively;

Described differential image-forming module comprises: the first dichroic mirror, the second dichroic mirror, quarter-wave plate, focusing objective len, three-dimensional micrometric displacement objective table, optical filter, Amici prism, first collect object lens, the first pin hole, the first photodetector, the second collection object lens, the second pin hole and the second photodetector; The reflected light path of the first dichroic mirror configures the second dichroic mirror, quarter-wave plate, focusing objective len and three-dimensional micrometric displacement objective table successively, the reverse transmitted light path of first dichroic mirror configures optical filter, Amici prism, first collects object lens, the first pin hole and the first photodetector, the transmitted light path of Amici prism configures second and collects object lens, the second pin hole and the second photodetector;

Described plated film sample is the testing sample of plated surface fluorescent material film;

Wavelength in described excitation module is λ ₁the first picosecond pulse laser send exciting light, directional light is formed after the first conduction optical fiber and the first collimator objective, parallel beam forms the first focal beam spot after the first plane mirror, the first dichroic mirror reflects and the second dichroic mirror, quarter-wave plate, focusing objective len transmission on plated film sample, the fluorescent film on the first described focal beam spot excited sample surface thus send fluorescence;

Wavelength in described loss module is λ ₂the second picosecond pulse laser send loss light, directional light is formed after the second conduction optical fiber and the second collimator objective, through the second plane mirror reflection, vortex shape phase-modulation plate, 1/2nd wave plate transmissions, after second dichroic mirror reflects and quarter-wave plate, focusing objective len transmission, form the second focal beam spot of donut-like at plated film sample surfaces;

The fluorescence that described plated film sample surfaces the inspires prism that is split after focusing objective len, quarter-wave plate, the second dichroic mirror, the first dichroic mirror, filter transmission is divided into two-beam, light beam is collected object lens by first and is assembled, collected by the first photodetector after the first pin hole, another light beam is collected object lens by second and is assembled, and is collected after the second pin hole by the second photodetector.

The above-mentioned device measuring smooth free form surface sample based on differential STED, described plated film sample surfaces plates one deck organic fluorescence film by the method for evaporation, and the soluble in water or organic solvent of described fluorescent film, thickness is no more than 1 μm.

The above-mentioned device measuring smooth free form surface sample based on differential STED, the first described pin hole be positioned at the first collection object lens focal plane after z ₀place, the second pin hole be positioned at the second collection object lens focal plane before z ₀place.

The method measuring smooth free form surface sample based on differential STED that the above-mentioned device measuring smooth free form surface sample based on differential STED realizes, comprises the following steps:

A () forms at sample surfaces the fluorescent film that a layer thickness is no more than 1 μm by the method for evaporation, described fluorescent film and sample profile fit tightly, and forms plated film sample;

B () wavelength is λ ₁the first picosecond pulse laser send exciting light, directional light is formed after the first conduction optical fiber and the first collimator objective, parallel beam forms the first focal beam spot after the first plane mirror, the first dichroic mirror reflects and the second dichroic mirror, quarter-wave plate, focusing objective len transmission on plated film sample, and the fluorescent film on the first described focal beam spot excited sample surface sends fluorescence;

C () wavelength is λ ₂the second picosecond pulse laser send loss light, directional light is formed after the second conduction optical fiber and the second collimator objective, through the second plane mirror reflection, vortex shape phase-modulation plate, 1/2nd wave plate transmissions, after second dichroic mirror reflects and quarter-wave plate, focusing objective len transmission, the second focal beam spot of donut-like is formed, the first focal beam spot and the second focal beam spot center superposition at plated film sample surfaces;

D fluorescence that () fluorescent film inspires is after the first photodetector and the second photodetector are collected, and two paths of signals carries out calculus of differences and obtains differential response curve, determines plated film specimen surface positions zero point by differential response curve;

Two kinds of computings can be carried out for obtained two-way differential wave:

1. differential calculating is carried out to two paths of signals:

$\begin{array}{c}{I}_{D1}(x_s,z_s)=({\left|U_D(x_s,z_s\mathrm{\Δz})\right|}^2+c_0)-({\left|u_D(x_s,z_s,-\mathrm{\Δz})\right|}^2+c_0)\\ ={\left|U_D(x_s,z_s,\mathrm{\Δz})\right|}^2-{\left|U_S(x_s,z_s,\mathrm{\Δz})\right|}^2\end{array}$

Wherein I _d1represent the intensity response function of system during differential calculating, U _drepresent the amplitude response function of the lower system of bilateral illumination, x _sand z _srepresent position coordinates, △ z represents detector defocusing amount, c ₀represent the common-mode noise in signal;

2. resistance to crosstalk calculating is carried out to two paths of signals:

$\begin{array}{c}{I}_{D1}(x_s,z_s)=\frac{({\left|U_D(x_s,z_s,\mathrm{\Δz})\right|}^2+c_0)-({\left|U_D(x_s,z_s,\mathrm{z\Δ})\right|}^2+c_0)}{({\left|U_D(x_s,z_s,\mathrm{\Δz})\right|}^2+c_0)+({\left|U_D(x_s,z_s,\mathrm{\Δz})\right|}^2+c_0)}\\ =\frac{|U_D(x_s,z_s,\mathrm{\Δz}){|}^2-|U_D(x_s,z_s,\mathrm{\Δz}){|}^2}{|U_D(x_s,z_s,\mathrm{\Δz}){|}^2+{|U}_D(x_s,z_s,\mathrm{\Δz}){|}^2+2c_0}\end{array}$

Wherein I _d2the intensity response function of system when representing that resistance to crosstalk calculates;

E () three-dimensional micrometric displacement objective table drives plated film sample three-dimensional mobile, form 3-D scanning imaging;

F () uses water or organic solvent to wash the fluorescent film of sample surfaces.

Good result of the present invention is:

1) by plating the mode of fluorescent film to testing sample, overcome the optical measuring techniques such as conventional confocal method by the restriction of light beam at smooth free form surface sample surfaces generation mirror-reflection, the apparatus and method measuring smooth free form surface sample based on differential STED can realize the topography measurement to the smooth free form surface sample with larger slope, realize the collection of the flashlight to sample side reflection.

2) STED microtechnic is applied to field of industrial measurement, the super-resolution imaging to sample can be realized.

3) adopt the equidistant out of focus detection of image space, after measurement result process, can effectively suppress common-mode noise or series mode noise.

Accompanying drawing explanation

Fig. 1 is the apparatus structure schematic diagram measuring smooth free form surface sample based on differential STED.

In figure, piece number illustrates: 1, wavelength is λ ₁the first picosecond pulse laser, 2, the first conduction optical fiber, 3, the first collimator objective, 4, the first plane mirror, 5, wavelength is λ ₂the second picosecond pulse laser, 6, second conduction optical fiber, 7, second collimator objective, 8, second plane mirror, 9, vortex shape phase-modulation plate, 10, / 2nd wave plates, 11, first dichroic mirror, 12, second dichroic mirror, 13, quarter-wave plate, 14, focusing objective len, 15, plated film sample, 16, three-dimensional micrometric displacement objective table, 17, optical filter, 18, Amici prism, 19, first collects object lens, 20, first pin hole, 21, first photodetector, 22, second collects object lens, 23, second pin hole, 24, second photodetector.

Embodiment

Below in conjunction with accompanying drawing, the embodiment of the present invention is described in detail.

Specific embodiment one

The present embodiment is device embodiment.

The device measuring smooth free form surface sample based on differential STED of the present embodiment, structural representation as shown in Figure 1.This device comprises: excitation module, loss module, differential image-forming module and plated film sample 15;

Described excitation module comprises: wavelength is λ ₁the first picosecond pulse laser 1, first conduct optical fiber 2, first collimator objective 3, first plane mirror 4;

Described loss module comprises: wavelength is λ ₂the second picosecond pulse laser 5, second conduct optical fiber 6, second collimator objective 7, second plane mirror 8, vortex shape phase-modulation plate 9 and 1/2nd wave plates 10;

Described differential image-forming module comprises: the first dichroic mirror 11, second dichroic mirror 12, quarter-wave plate 13, focusing objective len 14, three-dimensional micrometric displacement objective table 16, optical filter 17, Amici prism 18, first are collected object lens 19, first pin hole 20, first photodetector 21, second and collected object lens 22, second pin hole 23 and the second photodetector 24; The first described pin hole 20 be positioned at the first collection object lens 19 focal plane after z ₀place, the second pin hole 23 be positioned at the second collection object lens 22 focal plane before z ₀place;

Described plated film sample 15 plates one deck fluorescent film by the method for evaporation, the soluble in water or organic solvent of described fluorescent film, and thickness is no more than 1 μm;

When measuring use:

Wavelength in excitation module is λ ₁the first picosecond pulse laser 1 send exciting light, after the first conduction optical fiber 2 and the first collimator objective 3, form directional light, parallel beam reflects through the first plane mirror 4, first dichroic mirror 11 and the second dichroic mirror 12, quarter-wave plate 13, on plated film sample 15, forms the first focal beam spot after focusing objective len 14 transmission;

Wavelength in loss module is λ ₂the second picosecond pulse laser 5 send loss light, directional light is formed after the second conduction optical fiber 6 and the second collimator objective 7, reflect through the second plane mirror 8, the transmission of vortex shape phase-modulation plate 9,1/2nd wave plate 10, second dichroic mirror 12 reflects and quarter-wave plate 13, after focusing objective len 14 transmission, at the second focal beam spot of plated film sample 15 surface formation donut-like;

The first described focal beam spot and the second focal beam spot center superposition, effect is only have fluorescence molecule on the fluorescent film of the first focal beam spot central area can normal fluorescence excitation, the fluorescence molecule in other regions is sent out by the second focal beam spot de excitation of donut-like, cannot produce fluorescence;

The fluorescence that described plated film sample 15 surface excitation the goes out prism 18 that is split after focusing objective len 14, quarter-wave plate 13, second dichroic mirror 12, first dichroic mirror 11, optical filter 17 transmission is divided into two-beam, light beam is collected object lens 19 by first and is assembled, collected by the first photodetector 21 after the first pin hole 20, another light beam is collected object lens 22 by second and is assembled, and is collected after the second pin hole 23 by the second photodetector 24.

Specific embodiment two

The embodiment of the method for the present embodiment for realizing on device described in specific embodiment one.

The method measuring smooth free form surface sample based on differential STED of the present embodiment, comprises the following steps:

A fluorescent material rhodamine B soluble in water is plated to sample surfaces by the method for evaporation by (), form the fluorescent film that a layer thickness is no more than 1 μm, described fluorescent film and sample profile fit tightly, and form plated film sample 15;

B () wavelength is λ ₁the first picosecond pulse laser 1 send exciting light, directional light is formed after the first conduction optical fiber 2 and the first collimator objective 3, parallel beam reflects through the first plane mirror 4, first dichroic mirror 11 and the second dichroic mirror 12, quarter-wave plate 13, on plated film sample 15, forms the first focal beam spot after focusing objective len 14 transmission, and the fluorescent film on the first described focal beam spot excited sample surface sends fluorescence;

C () wavelength is λ ₂the second picosecond pulse laser 5 send loss light, directional light is formed after the second conduction optical fiber 6 and the second collimator objective 7, reflect through the second plane mirror 8, vortex shape phase-modulation plate 9, / 2nd wave plate 10 transmissions, second dichroic mirror 12 reflects and quarter-wave plate 13, after focusing objective len 14 transmission, the second focal beam spot of donut-like is formed on plated film sample 15 surface, first focal beam spot and the second focal beam spot center superposition, effect is only have fluorescence molecule on the fluorescent film of the first focal beam spot central area can normal fluorescence excitation, the fluorescence molecule in other regions is sent out by the second focal beam spot de excitation of donut-like, fluorescence cannot be produced

D fluorescence that () fluorescent film inspires is after the first photodetector 21 and the second photodetector 24 are collected, and two paths of signals carries out calculus of differences and obtains differential response curve, determines plated film specimen surface positions zero point by differential response curve;

Two kinds of computings can be carried out for obtained two-way differential wave:

1. differential calculating is carried out to two paths of signals:

$\begin{array}{c}{I}_{D1}(x_s,z_s)=({\left|U_D(x_s,z_s\mathrm{\Δz})\right|}^2+c_0)-({\left|u_D(x_s,z_s,-\mathrm{\Δz})\right|}^2+c_0)\\ ={\left|U_D(x_s,z_s,\mathrm{\Δz})\right|}^2-{\left|U_S(x_s,z_s,\mathrm{\Δz})\right|}^2\end{array}$

Wherein I _d1represent the intensity response function of system during differential calculating, U _drepresent the amplitude response function of the lower system of bilateral illumination, x _sand z _srepresent position coordinates, △ z represents detector defocusing amount, c ₀represent the common-mode noise in signal;

2. resistance to crosstalk calculating is carried out to two paths of signals:

$\begin{array}{c}{I}_{D1}(x_s,z_s)=\frac{({\left|U_D(x_s,z_s,\mathrm{\Δz})\right|}^2+c_0)-({\left|U_D(x_s,z_s,\mathrm{z\Δ})\right|}^2+c_0)}{({\left|U_D(x_s,z_s,\mathrm{\Δz})\right|}^2+c_0)+({\left|U_D(x_s,z_s,\mathrm{\Δz})\right|}^2+c_0)}\\ =\frac{|U_D(x_s,z_s,\mathrm{\Δz}){|}^2-|U_D(x_s,z_s,\mathrm{\Δz}){|}^2}{|U_D(x_s,z_s,\mathrm{\Δz}){|}^2+{|U}_D(x_s,z_s,\mathrm{\Δz}){|}^2+2c_0}\end{array}$

Wherein I _d2the intensity response function of system when representing that resistance to crosstalk calculates;

E () three-dimensional micrometric displacement objective table 16 drives plated film sample 15 three-dimensional mobile, form 3-D scanning imaging;

F () uses water or organic solvent to wash the rhodamine B fluorescent film of sample surfaces.

Claims (5)

1. measure the device of smooth free form surface sample based on differential STED, it is characterized in that, comprising: excitation module, loss module, differential image-forming module and plated film sample (15);

Described excitation module comprises: wavelength is λ ₁the first picosecond pulse laser (1), first conduction optical fiber (2), the first collimator objective (3), the first plane mirror (4); Be λ at wavelength ₁the first picosecond pulse laser (1) after configure the first conduction optical fiber (2), the first collimator objective (3) and the first plane mirror (4) successively;

Described loss module comprises: wavelength is λ ₂the second picosecond pulse laser (5), the second conduction optical fiber (6), the second collimator objective (7), the second plane mirror (8), vortex shape phase-modulation plate (9) and 1/2nd wave plates (10); Be λ at wavelength ₂the second picosecond pulse laser (5) after configure the second conduction optical fiber (6), the second collimator objective (7) and the second plane mirror (8) successively, the second plane mirror (8) reflected light path configures vortex shape phase-modulation plate (9) and 1/2nd wave plates (10) successively;

Described differential image-forming module comprises: the first dichroic mirror (11), the second dichroic mirror (12), quarter-wave plate (13), focusing objective len (14), three-dimensional micrometric displacement objective table (16), optical filter (17), Amici prism (18), first collect object lens (19), the first pin hole (20), the first photodetector (21), the second collection object lens (22), the second pin hole (23) and the second photodetector (24), the reflected light path of the first dichroic mirror (11) configures the second dichroic mirror (12) successively, quarter-wave plate (13), focusing objective len (14) and three-dimensional micrometric displacement objective table (16), first dichroic mirror (11) oppositely transmitted light path configure optical filter (17), Amici prism (18), first collects object lens (19), first pin hole (20) and the first photodetector (21), the transmitted light path of Amici prism (18) configures second and collects object lens (22), second pin hole (23) and the second photodetector (24),

The testing sample of described plated film sample (15) the fluorescent material film that has been plated surface;

Wavelength in described excitation module is λ ₁the first picosecond pulse laser (1) send exciting light, after the first conduction optical fiber (2) and the first collimator objective (3), form directional light, parallel beam through the first plane mirror (4), the first dichroic mirror (11) reflects and the second dichroic mirror (12), quarter-wave plate (13), go up formation first focal beam spot at plated film sample (15) after focusing objective len (14) transmission;

Wavelength in described loss module is λ ₂the second picosecond pulse laser (5) send loss light, directional light is formed after the second conduction optical fiber (6) and the second collimator objective (7), reflect through the second plane mirror (8), vortex shape phase-modulation plate (9), 1/2nd wave plates (10) transmission, second dichroic mirror (12) reflection and quarter-wave plate (13), after focusing objective len (14) transmission, at the second focal beam spot of plated film sample (15) surface formation donut-like;

The fluorescence that described plated film sample (15) surface excitation goes out is through focusing objective len (14), quarter-wave plate (13), second dichroic mirror (12), first dichroic mirror (11), the prism (18) that is split after optical filter (17) transmission is divided into two-beam, light beam is collected object lens (19) by first and is assembled, collected by the first photodetector (21) after the first pin hole (20), another light beam is collected object lens (22) by second and is assembled, collected by the second photodetector (24) after the second pin hole (23).

2. the device measuring smooth free form surface sample based on differential STED according to claim 1, it is characterized in that, described plated film sample (15) surface plates one deck organic fluorescence film by the method for evaporation, soluble in water or the organic solvent of described fluorescent film, thickness is no more than 1 μm.

3. the device measuring smooth free form surface sample based on differential STED according to claims 1, is characterized in that, the second focal beam spot center superposition of the first described focal beam spot and donut-like.

4. the device measuring smooth free form surface sample based on differential STED according to claims 1, is characterized in that, described the first pin hole (20) be positioned at the first collection object lens (19) focal plane after z ₀place, the second pin hole (23) be positioned at the second collection object lens (22) focal plane before z ₀place.

5. measure the method for smooth free form surface sample based on STED, it is characterized in that, comprise the following steps:

A () forms at sample surfaces the organic fluorescence film that a layer thickness is no more than 1 μm by the method for evaporation, described fluorescent film and sample profile fit tightly, and forms plated film sample (15);

B () wavelength is λ ₁the first picosecond pulse laser (1) send exciting light, after the first conduction optical fiber (2) and the first collimator objective (3), form directional light, parallel beam through the first plane mirror (4), the first dichroic mirror (11) reflects and the second dichroic mirror (12), quarter-wave plate (13), go up formation first focal beam spot at plated film sample (15) after focusing objective len (14) transmission;

C () wavelength is λ ₂the second picosecond pulse laser (5) send loss light, directional light is formed after the second conduction optical fiber (6) and the second collimator objective (7), reflect through the second plane mirror (8), vortex shape phase-modulation plate (9), 1/2nd wave plates (10) transmission, second dichroic mirror (12) reflection and quarter-wave plate (13), after focusing objective len (14) transmission, the second focal beam spot of donut-like is formed, the first focal beam spot and the second focal beam spot center superposition on plated film sample (15) surface;

D fluorescence that () fluorescent film inspires is after the first photodetector (21) and the second photodetector (24) are collected, two paths of signals carries out calculus of differences and obtains differential response curve, determines plated film specimen surface positions zero point by differential response curve;

Two kinds of computings can be carried out for obtained two-way differential wave:

1. differential calculating is carried out to two paths of signals:

I _D1(x _s,z _s)＝(|U _D(x _s,z _s,△z)| ²+c ₀)-(|U _D(x _s,z _s,-△z)| ²+c ₀)

＝|U _D(x _s,z _s,△z)| ²-|U _D(x _s,z _s,-△z)| ²

Wherein I _d1represent the intensity response function of system during differential calculating, U _drepresent the amplitude response function of the lower system of bilateral illumination, x _sand z _srepresent position coordinates, △ z represents detector defocusing amount, c ₀represent the common-mode noise in signal;

2. resistance to crosstalk calculating is carried out to two paths of signals:

$\begin{array}{c}{I}_{D1}(x_s,z_s)=\frac{({\left|U_D(x_s,z_s,\mathrm{\Δz})\right|}^2+c_0)-({\left|U_D(x_s,z_s,-\mathrm{\Δz})\right|}^2+c_0)}{({\left|U_D(x_s,z_s,\mathrm{\Δz})\right|}^2+c_0)+({\left|U_D(x_s,z_s,-\mathrm{\Δz})\right|}^2+c_0)}\\ =\frac{{\left|U_D(x_s,z_s,\mathrm{\Δz})\right|}^2-{\left|U_D(x_s,z_s,-\mathrm{\Δz})\right|}^2}{{\left|U_D(x_s,z_s,\mathrm{\Δz})\right|}^2+{\left|U_D(x_s,z_s,-\mathrm{\Δz})\right|}^2+2c_0}\end{array}$

Wherein I _d2the intensity response function of system when representing that resistance to crosstalk calculates;

E () three-dimensional micrometric displacement objective table (16) drives plated film sample (15) three-dimensional mobile, form 3-D scanning imaging;

F () uses water or organic solvent to wash the fluorescent film of sample surfaces.