CN102221327B - Phase shift interference microscopic device and method based on Zernike phase contrast imaging - Google Patents

Abstract

The invention relates to a phase shift interference microscopic device and method based on Zernike phase contrast imaging. The device comprises an illuminating unit, a microscopic amplifying unit and a phase contrast imaging unit which are arranged in sequence along the incident direction of light; the illuminating unit comprises a beam expanding system, a multi-beam illumination generating unit and a beam contraction collimating unit which are arranged in sequence along the light path direction; the beam expanding system comprises a laser, a light intensity controller and a beam expanding unit which are arranged in sequence along the light path direction; the multi-beam illumination generating unit comprises an axicon lens, a first lens, a rotary scatterer, a second lens and an amplitude mask plate which are arranged in sequence along a light path; a tested sample is arranged on a focal surface between a first objective lens and a second objective lens; and the tested sample and a CCD (Charge Coupled Device) camera meet an objective image relation. Due to the adoption of the device and the method, the vignetting effect of the conventional Zernike phase contrast imaging is avoided, quantitative measurement of a phase object is realized, and the light path has the advantages of low coherent noise, high vibration resistance, high transverse resolution and the like.

Description

Phase shift interference microscope equipment and method based on the Ze Nike phase contrast imaging

Technical field

The present invention relates to a kind of microscope equipment that is used to measure small items amplitude and PHASE DISTRIBUTION.

Background technology

General microscope can only be observed the intensity distributions of small items, and is powerless for the PHASE DISTRIBUTION of transparent substance.Yet the three-dimensional appearance of testee in the PHASE DISTRIBUTION direct correlation of testee, and the PHASE DISTRIBUTION of accurately measuring small items is significant.

The optical interferometry technology that is used to measure the small items PHASE DISTRIBUTION at present mainly comprises following several kinds:

One, optical interference microtechnic, it combines the optical interference method with microscope, for the PHASE DISTRIBUTION of accurately measuring small items provides powerful measure.Existing interference microscope spatially is divided into two bundles with laser beam, a branch ofly is used for shining sample, after amplifying collimation as thing light; Another restraints light as a reference, two-beam is combined at last again, utilizes interference between them to measure the PHASE DISTRIBUTION of small items.The digital hologram microscope DHM-1000 that Switzerland Lunceetec company develops just belongs to above-mentioned micro-light path.This technology can successfully be used to measure the PHASE DISTRIBUTION of small items, yet thing light has spatially been gone through different paths with reference light in the process of measuring, thereby ambient vibration is bigger to the measurement result influence, has reduced the stable and repeatable of device.

Two, phasecontrast microscope technology, this technology be through the phase place of the zero-frequency component of change thing light (containing sample information), thereby make the phase information of thing light become strength information.Zero-frequency and the high fdrequency component of thing light had been served as the reference light and the role of thing light in the general interference respectively during phase contrast was micro-, and they have passed through identical light path, so installed insensitive to the vibration of environment.For example the Popeseu of Massachusetts Institute Technology teaches the micro-light path of a kind of phase contrast that proposes: adopt the parallel light sample, through postponing to be positioned at the phase place of the central zero-frequency component of frequency spectrum, realized the quantitative measurment to the small items PHASE DISTRIBUTION.Though should technology insensitive to ambient vibration, yet because light path has adopted relevant fully parallel light, so this technology has shortcomings such as coherent noise is big, lateral resolution is low.

Holland scientist Ze Nike proposes the micro-method of other a kind of phase contrast: adopt the ring-shaped light sample that throws light on, adopt the ring-type phase mask plate to postpone the phase place of zero-frequency component simultaneously, thereby make the phase information of sample become strength information.The illumination of the annular beam of used partial coherence greatly reduces the coherent noise in the imaging in the phase contrast microscopic method, and has improved microscopical lateral resolution.Yet, because the phase place of the intensity of interference pattern and testee is not the linear transformation relation, so the Ze Nike phasecontrast microscope of this method can only be used for observing qualitatively.Simultaneously, the ring-type phase-plate has also postponed to be distributed in the high fdrequency component on this ring by error when postponing the zero-frequency component, and the halo effect that therefore produces has had a strong impact on the authenticity of phase contrast imaging.

Summary of the invention

The present invention combines Ze Nike phase contrast imaging and phase-shifting technique; The phase shift interference microscope equipment that a kind of freedom from vibration is good, lateral resolution is high, coherent noise is low has been proposed; This device can be used for the phase place of small items is measured; The invention solves existing Ze Nike phase contrast imaging and can only be used for the limitation of qualitative observation, overcome existing interference microscopy simultaneously ambient vibration responsive (poor stability), technical matters such as coherent noise is high, lateral resolution is low.

Technical solution of the present invention is:

A kind of phase shift interference microscope equipment based on the Ze Nike phase contrast imaging is characterized in that: comprise the lighting unit, micro-amplifying unit and the phase contrast imaging unit that set gradually along the incident direction of light;

Said lighting unit comprises successively beam-expanding system, multiple beam illumination generation unit along the optical path direction setting, bundle collimation unit contracts;

Said beam-expanding system comprises laser instrument 1, light intensity control 2 and the expansion Shu Danyuan 3 that sets gradually along optical path direction;

Said multiple beam illumination generation unit comprises the axicon lens that sets gradually along light path 4, first lens 5, rotation scatterer 6, second lens 7 and vibration amplitude mask plate 8; Said rotation scatterer 6 is arranged on the back focal plane of first lens 5; Said vibration amplitude mask plate 8 is placed on the image planes after rotation scatterer 6 is formed images by second lens 7;

The said bundle collimation unit that contracts comprises the 3rd lens 9 and first object lens 10 successively along light path, and said vibration amplitude mask plate 8 is arranged on the front focal plane of the combination of lenses of being made up of the 3rd lens 9 and first object lens 10;

Said micro-amplifying unit comprises second object lens 12 and the 4th lens 13 successively along light path;

Said phase contrast imaging unit comprises phase mask plate 14 and CCD camera 15 successively along light path, and said phase mask plate 14 is arranged on the back focal plane of the combination of lenses of being made up of second object lens 12 and the 4th lens 13;

Said sample 11 is placed on the focal plane between first object lens 10 and second object lens 12, and said sample 11 satisfies object-image relation with CCD camera 15.

Above-mentioned multiple beam illumination generation unit also comprises the 5th lens (16), and said the 5th lens (16) are arranged on after second lens 7; Second lens 7 and the 5th lens 16 constitute first telescopic system; Said rotation scatterer 6 is arranged on the front focal plane of first telescopic system, and said vibration amplitude mask plate 8 is arranged on the first telescopic system back focal plane;

The above-mentioned bundle collimation unit that contracts comprises that also the 6th lens 17, the five lens 16 and the 6th lens 17 constitute second telescopic system, and said vibration amplitude mask plate 8 is arranged on the second telescopic system intermediate focal plane.

Above-mentioned micro-amplifying unit also comprises the 7th lens 18, and said the 7th lens 18 are arranged on after the 4th lens 13.

Above-mentioned phase contrast imaging unit comprises the 8th lens 19; The 5th telescopic system that said the 7th lens 18 and the 8th lens 19 constitute, said phase mask plate 14 is arranged on the intermediate focal plane of the 5th telescopic system, and said CCD camera 15 is arranged on the 8th lens 19 back focal planes.

Above-mentioned vibration amplitude mask plate 8 is an opaque flat board that is carved with a series of point-like transparent regions, and said point-like transparent region periodically is distributed on the annulus, and said annulus is with corresponding by the corresponding annulus of second lens, 7 imaging back lighting optical spectrums; Said point-like transparent region is round or rectangle, and the diameter of said round point-like transparent region is 10 μ m-50 μ m; The diameter of said rectangle point-like transparent region is 10 μ m-50 μ m;

Said phase mask plate (14) is the quartz glass that an etching a series of point-like phase slots; The diameter of said phase slots or the length of side are between the 10 μ m-50 μ m, and it periodically is distributed on the annulus; Said annulus with overlapped by the corresponding annulus of thing optical spectrum after the 4th lens (13) imagings; The periodicity of said phase slots is confirmed according to following mode: corresponding to the zero-frequency component of each thing light, the phase slots of different depth alternately is distributed in the one-period.

Phase shift interference microscopic method based on the Ze Nike phase contrast imaging is characterized in that: may further comprise the steps:

1] produce the pointolite of discrete and periodic distribution:

1.1] laser that sends of laser instrument 1 behind light intensity control 2, expanded Shu Danyuan 3 beam-expanding collimations and become directional light;

1.2] directional light is through becoming annular beam after being placed on the axicon lens 4 on the beam-expanding system back focal plane;

1.3] annular beam passes first lens 5, on the back focal plane of first lens 5, forms annulus;

1.4] place rotation scatterer 6 at the annulus place, make that every bit becomes mutual incoherent independent point light source on this annulus;

1.5] by second lens 7 this annulus is imaged onto on the vibration amplitude mask plate 8;

1.6] select the series of points light source of discrete and periodic distribution the picture of vibration amplitude mask plate 8 annulus from vibration amplitude mask plate;

1.7] the 3rd lens 9 with these point-sourcre imagings to the front focal plane of first object lens 10;

1.8] first object lens 10 are collimated into the multi beam directional light along different illumination directions with the picture of pointolite on the front focal plane, with different angles irradiation sample 11.

2] utilize 12 pairs of samples of second object lens to carry out micro-amplification;

3] PHASE DISTRIBUTION with sample 11 changes into intensity distributions:

3.1] after the illumination light of passing sample amplified by second object lens 12, the illumination light frequency spectrum was presented on the back focal plane of second object lens 12;

3.2] the illumination light frequency spectrum is imaged on the phase mask plate 14 by the 4th lens 13; Make the point-like phase slots of different depth cover the zero-frequency component of thing light under the different illumination directions successively through rotatable phase mask plate 14;

3.3] the thing light of frequency spectrum after by phase mask plate 14 modulation, its PHASE DISTRIBUTION is converted into visible intensity distributions, and is imaged onto on the CCD15.

Also comprise step 4] intensity distributions is calculated to be the PHASE DISTRIBUTION of quantification:

4.1] the rotatable phase mask plate selects the phase slots coverture light zero-frequency component of different depth, on the CCD face, obtains the interference pattern of the different phase-shift phases of three width of cloth successively;

4.2] utilize three width of cloth phase-shifted interference pattern to calculate the PHASE DISTRIBUTION of thing light, obtain the PHASE DISTRIBUTION of sample.

Phase shift interference microscopic method based on the Ze Nike phase contrast imaging is characterized in that: may further comprise the steps:

1] produce the pointolite of discrete and periodic distribution:

1.1] laser that sends of laser instrument 1 behind light intensity control 2, expanded Shu Danyuan 3 beam-expanding collimations and become directional light;

1.2] directional light is through becoming annular beam after being placed on the axicon lens 4 on the beam-expanding system back focal plane;

1.3] annular beam passes first lens 5, on the back focal plane of first lens 5, forms annulus;

1.4] every bit becomes mutual incoherent independent point light source on annulus place placement rotation scatterer 6 makes this annulus;

1.5] utilize the telescopic system of forming by second lens 7 and the 5th lens 16 that this annulus is imaged onto on the vibration amplitude mask plate 8;

1.6] select the series of points light source of discrete and periodic distribution the annulus of vibration amplitude mask plate 8 on being imaged onto vibration amplitude mask plate;

1.7] behind the collimating effect of light that send of these pointolites, become directional light along different illumination directions through the 6th lens 17, shine sample 11 behind the bundle that contracts of these illumination light through the 3rd telescopic system formed by the 3rd lens 9 and first object lens 10;

2] under the irradiation of multiple beam, sample is carried out processing and amplifying:

Sample 11 is placed on the front focal plane of second object lens 12, through the output light of being made up of second object lens 12 and the 4th lens 13 after the amplification of system far away of looking around;

3] PHASE DISTRIBUTION with sample 11 changes into intensity distributions:

3.1] thing light after amplifying through Fourier transforms, occurs the spectrum distribution of thing light through the 7th lens 18 on the back focal plane of the 7th lens 18;

3.2] phase mask plate 14 is placed in the thing optical spectrum, the phase slots on the phase mask plate cut can coverture light the zero-frequency component and postpone the phase place of zero-frequency component, convert the PHASE DISTRIBUTION of thing light into intensity distributions.

Also comprise step 4] intensity distributions is calculated to be the quantification PHASE DISTRIBUTION:

4.1] the rotatable phase mask plate, the phase slots that makes different depth is the component of coverture light zero-frequency successively, thereby obtains the phase-shifted interference pattern of the different phase-shift phases of three width of cloth, and the CCD that is positioned on the 8th lens 19 back focal planes accepts;

4.2] utilize three width of cloth phase shift phase contrast interference patterns to calculate the PHASE DISTRIBUTION of thing light.

The present invention has the following advantages:

1, thing light and reference light are the high frequency and the zero-frequency component of thing light, spatially experience identical path, and this thing ginseng optical texture on road altogether makes that this device is insensitive to ambient vibration.

2, adopt incoherent each other N light beam along the different directions sample that throws light on simultaneously,

when making coherent noise in the interferometry reduce to single beam to throw light on (N=24)

3, the micro-light path of Ze Nike phase contrast after the improvement has adopted N light beam with the different directions synchronous lighting, " synthetic numerical aperture " SNA=NA of formation _Illum+ NA _MO, improved microscopical numerical aperture, thereby improved microscopical lateral resolution.

4, it is micro-to adopt the telescopic system of being made up of microcobjective 12 and long-focus lens 13 that sample is amplified in the light path, and thing light remains directional light after this system, has therefore eliminated the additive phase that object lens are introduced.

5, the micro-light path of Ze Nike phase contrast after the improvement combines with phase-shifting technique, can carry out quantitative measurment to the phase place of small items.Phase contrast after the improvement interferes micro-light path to overcome " halation " effect in traditional Ze Nike phase contrast imaging.Tradition Ze Nike phase contrast imaging adopts the ring-shaped light source lighting, and adopts the ring-type phase-plate to postpone the phase place of thing light zero-frequency component.The ring-type phase-plate has also postponed to be distributed in the high fdrequency component phase place on the ring when postponing the zero-frequency component phase, " halation " effect therefore occurred.Light path after the improvement adopts the point source of light illumination that separates, and the thing optical spectrum under the difference illumination light is spatially separated from one another, and point-like phase place step only changes the phase place of thing light zero-frequency component, has therefore overcome " halation " effect.

Description of drawings

Fig. 1 interferes the microstructure synoptic diagram for the Ze Nike phase contrast

Fig. 2 interferes micro-middle multiple beam lighting unit structure synoptic diagram for the Ze Nike phase contrast.

Fig. 3 interferes micro-middle phase contrast imaging cellular construction synoptic diagram for the Ze Nike phase contrast.

Fig. 4 (a) is illumination vibration amplitude mask plate synoptic diagram; (b) be the phase mask plate synoptic diagram.

Fig. 5 goes up the off-axis illumination synoptic diagram of difference for ring.

Fig. 6 is the phase shift phase contrast interference pattern and reproduction result of microlens array.(a-c) be that phase-shift phase is respectively 0 ,-pi/2, the interference pattern of the microlens array of pi/2; (d) be the three dimensional Phase distribution of the microlens array of reproduction.

Fig. 7 is apparatus of the present invention and the general comparison of interfering microscope equipment to same phase object measurement result.(a) be the PHASE DISTRIBUTION that adopts apparatus of the present invention to record; (b) be two kinds of measurement device results' comparison.

Fig. 8 is the comparison of lateral resolution measurement result under single beam coaxial-illuminating and the multiple beam off-axis illumination.(a) be single beam coaxial-illuminating result; (b) be multiple beam off-axis illumination result.

Wherein Reference numeral is following: 1-laser instrument, 2-light intensity control, 3-beam-expanding system, 4-axicon lens, 5-first lens; 6-rotates scatterer, 7-second lens, 8-vibration amplitude mask plate, 9-the 3rd lens, 10-first object lens; The 11-sample, 12-second object lens, 13-the 4th lens, 14-phase mask plate, 15-CCD camera; 16-the 5th lens, 17-the 6th lens, 18-the 7th lens, 19-the 8th lens.

Embodiment

Modular construction and function that the present invention comprised are following:

1, laser instrument 1, and wavelength is at visible-range, and the output laser power stability is used to produce illumination light.

2, light intensity control 2, can be continuously adjustable attenuator, also can be the polaroid (second polaroid is used for fixing the polarization direction of illumination light, rotates first polaroid and regulates the illumination light intensity) of two parallel placements.

3, first lens 5, second lens 7, the 3rd lens 9, the 4th lens 13, the 5th lens 16, the 6th lens 17, the 7th lens 18, the eight lens 19 require to be aplanat.

4, rotation scatterer 6, the scatterer that promptly rotates, can be one by driven by motor and the frosted glass plate of center fixation on motor shaft, through rotating, make that every bit is irrelevant mutually on the illumination annulus.Require the rotation alignment good, the position of frosted glass plate is positioned on the back focal plane of first lens.

4, axicon lens 4, can be the refractive axicon lens, also can be the diffraction type axicon lens.Require the annular beam dispersion angle of generation moderate.

6, vibration amplitude mask plate 8, are the opaque flat board that is carved with a series of point-like transparent regions above.These point-like transparent regions periodically are distributed on the annulus.This annulus is with corresponding by the corresponding annulus of second lens, 7 imaging back lighting optical spectrums.The point-like transparent region can also can be rectangle for round, and the regional diameter or the length of side are advisable at 10 μ m-50 μ m.The point that vibration amplitude mask plate is used on illumination light frequency spectrum (being circular), choosing series of discrete is as new lighting source.

7, phase mask plate 14, are the quartz glass that an etching a series of point-like phase slots.The diameter of phase slots or the length of side are between the 10 μ m-50 μ m, and they periodically are distributed on this annulus.This annulus with overlapped by the corresponding annulus of thing optical spectrum after the imaging of the 4th lens 13.Corresponding to the zero-frequency component of each thing light, the phase slots of three different depths (corresponding different phase is poor, as 0 ,-pi/2, pi/2) alternately be distributed in the corresponding cycle.In addition, should keep identical spacing between the phase slots of different depth.Phase mask plate is used for postponing the phase place of the zero-frequency component of different directions illumination light, but does not change the phase place of thing light high fdrequency component.

8, the CCD camera 15, are generally black-white CCD (or CMOS) camera, have appropriate gray shade rank, Pixel Dimensions and pixel quantity.

Ze Nike phase contrast proposed by the invention interferes micro-light path can be divided into multiple beam lighting unit (as shown in Figure 2) and phase contrast imaging unit (as shown in Figure 3).

In the multiple beam lighting unit, the laser that laser instrument 1 sends is expanded Shu Danyuan 3 beam-expanding collimations and is become directional light through behind the light intensity control 2.This directional light becomes annular beam through after being placed on the axicon lens 4 on the beam-expanding system back focal plane.The frequency spectrum of this annular beam appears on the back focal plane of first lens 5, and this frequency spectrum is an annulus that glitters.The scatterer 6 of one rotation is placed on this annulus position, makes that every bit becomes mutual incoherent independent point light source on this annulus.These pointolites are imaged onto this annulus on the vibration amplitude mask plate 8 by second lens 7.The detailed structure of vibration amplitude mask plate is shown in Fig. 4 (a): a series of point-like transparent regions that on opaque flat board, distributing, and these dotted region periodically are distributed on the corresponding annulus of annular beam.Vibration amplitude mask plate is selected series of points light source separation and periodic distribution on the annulus of illumination light.These pointolites to the front focal plane of first object lens 10, are collimated into directional light along different directions irradiation sample 11 by first object lens 10 by the 3rd lens imaging again, and are as shown in Figure 5.

In the phase contrast imaging unit, sample is placed on the front focal plane of second object lens 12, by the combination of lenses amplification imaging of being made up of object lens 12 and the 4th lens 13.The frequency spectrum of thing light appears on the back focal plane of this combination of lenses.For thereby the phase place that postpones thing light zero-frequency component realizes phase contrast imaging, a phase mask plate 14 is placed on the frequency plane of thing light.The structure of this phase mask plate is shown in Fig. 4 (b): the point-like phase slots of different depth alternately is distributed on the annulus.For the zero-frequency component of each thing light, there is the phase slots of three different depths corresponding with it respectively.Select the phase slots coverture light zero-frequency component of different depth through the rotatable phase mask plate, thereby can give of the delay of thing light zero-frequency component, therefore between thing light zero-frequency component and high fdrequency component, realize different phase shifts with out of phase.Thing light after frequency spectrum is modulated, its PHASE DISTRIBUTION have become intensity distributions and have been imaged on the CCD face.

In sum, the phase contrast after the improvement interferes micro-light path to overcome " halation " effect in traditional Ze Nike phase contrast imaging.Tradition Ze Nike phase contrast imaging adopts the ring-shaped light source lighting, and adopts the ring-type phase-plate to postpone the phase place of thing light zero-frequency component.The ring-type phase-plate has also postponed to be distributed in the high fdrequency component phase place on the ring when postponing the zero-frequency component phase, " halation " effect therefore occurred.Light path after the improvement adopts the point source of light illumination that separates, and the thing optical spectrum under the difference illumination light is spatially separated from one another, and point-like phase place step only changes the phase place of thing light zero-frequency component, has therefore overcome " halation " effect.

Principle of the present invention:

Ze Nike phase contrast after the improvement interferes micro-light path to adopt on the illumination annulus N pointolite with the different directions sample that throws light on.As shown in Figure 5, on first object lens, 10 front focal planes, N pointolite periodically is distributed on the circumference.The light that these pointolites send is collimated into directional light with different directions illumination sample 11 through object lens 10.These illumination light can be used identical off-axis angle α _IllumWith different azimuth angle theta _iCharacterize:

$E_{\mathrm{illum}}({\mathrm{\θ}}_i,x,y)=\frac{2\mathrm{\π}}{\mathrm{\λ}}\sin {\mathrm{\α}}_{\mathrm{illum}}({\cos \mathrm{\θ}}_{i}x+\sin {\mathrm{\θ}}_{i}y).---\left(1\right)$

Wherein, x and y represent the volume coordinate on the sample plane.The multiple transmitance of sample can be designated as the illumination light of

different directions through forming different thing light behind the sample.The frequency spectrum of these thing light is positioned on the back focal plane of object lens, they spatially be separated from each other and periodic distribution on a circumference.The frequency spectrum of each thing light is all in the form of annular discs, and wherein the zero-frequency component is positioned at disc centre, high fdrequency component be centered around the zero-frequency component around.Phase mask plate with point-like phase slots is placed on this frequency plane, is used for the spectrum distribution of modulator light.On phase mask plate, the phase slots of three kinds of different depths is with different orientations θ i distribute periodically (corresponding one by one with the zero-frequency component of thing light), the zero-frequency component of coverture light just on the circumference.The position angle is θ _iThe transmitance of phase slots can use Γ (θ _i, represent that δ) wherein δ representes phase-delay quantity.Deflection is θ _iThing light zero-frequency component by phase place step Γ (θ _i, δ) modulation can be expressed as in (on the sample image planes) thing recovery distribution of amplitudes on the CCD face:

O _r(θ _i，x′，y′，δ)＝FT ^-1{FT[O(x，y)·E _illum(θ _i，x，y)]·Γ(θ _i，δ)}.(2)

Here FT and FT ^-1Represent Fourier transform and inverse Fourier transform respectively, the volume coordinate on x ' and y ' expression CCD plane.Because the thing light of different illumination directions is irrelevant mutually each other, so the light distribution on the CCD plane is each thing light light intensity sum.

$I(x^{\′},y^{\′},\mathrm{\δ})=\underset{i=1}{\overset{N}{\mathrm{\Σ}}}{\left|O_r({\mathrm{\θ}}_i,x^{\′},y^{\′},\mathrm{\δ})\right|}^2.---\left(3\right)$

In fact, if neglect the different directions incidental phase distortion of throwing light on, the zero-frequency component of the thing light under the different illumination directions is modulated by corresponding point-like phase slots, and the thing light after the modulation will have identical intensity distributions.Formula (2) can be rewritten into O _r(x ', y ')=O ₀(x ', y ') exp (i δ)+O _d(x ', y '), O here ₀(x ', y ') and O _d(x ', y ') representes the zero-frequency and the high fdrequency component of thing light respectively.Through the rotatable phase mask plate, the phase place step (corresponding different phase postpones δ) of choosing different depth comes the zero-frequency component of coverture light, thereby can obtain the interference pattern of different phase-shift phases.If three corresponding phase-shift phase δ of phase place steps are respectively 0, when-pi/2 and pi/2, then the last intensity distributions of CCD is expressed as respectively:

$\left\{\begin{array}{c}{I}_1(x^{\&prime;},y^{\&prime;})={|{\mathrm{<figure-callout\; id="0"\; label="O"\; filenames="HDA0000058544150000041.png,HDA0000058544150000111.png"\; state="\{\{state\}\}">O</figure-callout>}}_0+O_d|}^2,\\ I_2(x^{\&prime;},y^{\&prime;})={|{\mathrm{<figure-callout\; id="0"\; label="O"\; filenames="HDA0000058544150000041.png,HDA0000058544150000111.png"\; state="\{\{state\}\}">O</figure-callout>}}_0+\exp (-\mathrm{i\&pi;}/2)O_d|}^2,\\ I_3(x^{\&prime;},y^{\&prime;})={|{\mathrm{<figure-callout\; id="0"\; label="O"\; filenames="HDA0000058544150000041.png,HDA0000058544150000111.png"\; state="\{\{state\}\}">O</figure-callout>}}_0+\exp (\mathrm{i\&pi;}/2)O_d|}^2.\end{array}\right.---\left(4\right)$

In fact, the amplitude of the zero-frequency component of thing light and phase place spatially slowly change, and its PHASE DISTRIBUTION can be ignored.From above three phase-shifted interference pattern, can solve the corresponding distribution of amplitudes of thing light zero-frequency and high fdrequency component respectively: | O ₀(x ', y ') and | O _d(x ', y ') |.Simultaneously, the COMPLEX AMPLITUDE of thing light high fdrequency component also can solve from following formula:

$O_d(x^{\&prime;},y^{\&prime;})=\frac{(1-i)(I_1-I_2)+(1+i)(I_1-I_3)}{4\left|{\mathrm{<figure-callout\; id="0"\; label="O"\; filenames="HDA0000058544150000041.png,HDA0000058544150000111.png"\; state="\{\{state\}\}">O</figure-callout>}}_0\right|}.---\left(5\right)$

The PHASE DISTRIBUTION of thing light zero-frequency component has been left in the basket in the following formula.At last, with the complex amplitude O of thing light zero-frequency that solves and high fdrequency component ₀(x ', y ') and O _d(x ', y ') addition, thus the COMPLEX AMPLITUDE of thing light can be obtained.

$O(x^{\&prime;},y^{\&prime;})={\mathrm{<figure-callout\; id="0"\; label="O"\; filenames="HDA0000058544150000041.png,HDA0000058544150000111.png"\; state="\{\{state\}\}">O</figure-callout>}}_0+O_d\frac{(1-i)(I_1-I_2)+(1+i)(I_1-I_3)}{4\left|{\mathrm{<figure-callout\; id="0"\; label="O"\; filenames="HDA0000058544150000041.png,HDA0000058544150000111.png"\; state="\{\{state\}\}">O</figure-callout>}}_0\right|}+O_0---\left(6\right)$

From following formula, can obtain the PHASE DISTRIBUTION of sample:

Imag (O) and Real (O) represent the imaginary part and the real part of thing recovery amplitude respectively here.

Case study on implementation and performance are demarcated:

We use following parameter that the feasibility of this patent is described as example.Experimentally, the He-Ne laser instrument with wavelength 633nm is used as lighting source.The focal length of first lens, 5 to the 8th lens 18 is followed successively by along light path: 30mm, 100mm, 100mm, 80mm, 80mm, 80mm, 80mm, 400mm.First object lens 10 are two identical Zeiss object lens with second object lens 12, and magnification is 10X, numerical aperture NA=0.3.The total magnification of whole experiment device is M=25.6 (the magnification M of the telescopic system of being made up of object lens 12 and the 4th lens 13 ₁Magnification M with last telescopic system ₂Common decision).The cycle P=8 μ m of axicon lens DOE-Axicon, the off-axis angle of the annular beam that diffraction produces is α=λ/P=0.08rad.Magnification from axicon lens to the sample plane optical system is M _Relay=0.5, the off-axis angle of illumination light is α on sample plane so _Illum=α/M _Relay=0.16rad.On the back focal plane of first lens 5, the frequency spectrum of this annular beam is a dazzling annulus, and its diameter is 4.75mm.After the scattering of rotation scatterer 6, this annulus is imaged onto on the back focal plane of lens 16 by the telescopic system of being made up of lens 7 and lens 16.Place a vibration amplitude mask plate 8 on this plane, formed new lighting source.This vibration amplitude mask plate is an opaque flat board, is carved with a series of point-like transparent regions above.These transparent regions are the rectangle of 20 μ m * 70 μ m, are that to be evenly distributed on diameter the cycle be on the 4.75mm annulus with π/12rad.Therefore, this vibration amplitude mask plate is chosen N=24 point as new lighting source.The light that these pointolites send is behind the collimation and the bundle that contracts, with identical off-axis angle (sin α _Illum=0.16) with different position angle irradiation sample.Corresponding thing optical spectrum is imaged onto on the phase mask plate 14 of lens 18 back focal planes.This phase mask plate is the quartz glass that an etching a series of phase slots.The size of phase slots is 20 μ m * 70 μ m, and they are that to be evenly distributed on diameter the cycle be on the 4.75mm annulus with π/12rad still.In each cycle, the phase slots of three different depths (corresponding phase differs from 0 respectively ,-pi/2 and pi/2) alternately distributes, and the idle interval of double-width is arranged between the phase slots.This phase mask plate is fixed on the automatically controlled universal stage.In order to realize phase shift, 5 ° of each rotatable phase mask plates make the phase slots of different depth come the zero-frequency component of coverture light successively.

As laboratory sample, 5 ° of each rotatable phase mask plates obtain three width of cloth phase-shifted interference pattern, shown in Fig. 6 (a-c) with microlens array.Through adopting formula (4)-(6) described reproducting method that interference pattern is reproduced, the three dimensional Phase that obtains tested microlens array distributes, shown in Fig. 6 (d).

In order to demarcate this measuring precision of experimental apparatus, a part that adopts a phase place annulus adopts apparatus of the present invention and general interference microscope equipment to measure respectively as laboratory sample.The measurement result that adopts apparatus of the present invention is shown in Fig. 7 (a); The comparison of the measurement result that two kinds of methods obtain is shown in Fig. 7 (b).Need to prove: two lines among the figure are that the same position in two measurement results is taken out.From comparison, be not difficult to find out, two kinds of basically identicals as a result that method obtains, but the PHASE DISTRIBUTION that apparatus of the present invention obtain has lower noise.In addition, we measure the stability of apparatus of the present invention: when in experimental provision, not having the setting-out article, and the repeatedly measurement of having carried out, the mean value of each point gained phase place root mean square (RMS) in repeatedly measuring is λ/45 in the CCD visual field.

At last, the lateral resolution of apparatus of the present invention is measured.In the experiment, utilize one-period to be P=2 μ m, dutycycle is that 1/2 amplitude grating is as sample.Adopt the synchronous off-axis illumination of single beam coaxial-illuminating and multiple beam (lighting systems of apparatus of the present invention) that sample is carried out micro-imaging respectively.Under the single beam coaxial-illuminating, the picture of the sample of CCD record is shown in Fig. 8 (a); The image that adopts record under the synchronous off-axis illumination of multiple beam is shown in Fig. 8 (b).In Fig. 8 (a), the information of sample almost can not be differentiated.This is because the cycle of grating is too little, differentiates L less than the limit of object lens _Limit=2.57 μ m.When adopting multiple beam with the different directions synchronous lighting, illumination light makes object lens have " synthetic numerical aperture ", i.e. NA _Syn=NA _Illum+ NA _MOIllumination light has improved NA with the numerical aperture of object lens _Illum=0.16, the corresponding limiting resolution of device this moment is L _Limit=1.67 μ m are so can be the grating of 2 μ m in the resolution cycle still.In addition, we also can find out from the comparison of Fig. 8, adopt apparatus of the present invention can reduce coherent noise effectively.

Claims (10)

1. the phase shift interference microscope equipment based on the Ze Nike phase contrast imaging is characterized in that: comprise the lighting unit, micro-amplifying unit and the phase contrast imaging unit that set gradually along the incident direction of light;

Said lighting unit comprises successively beam-expanding system, multiple beam illumination generation unit along the optical path direction setting, bundle collimation unit contracts;

Said beam-expanding system comprises laser instrument (1), light intensity control (2) and the expansion Shu Danyuan (3) that sets gradually along optical path direction;

Said multiple beam illumination generation unit comprises axicon lens (4), first lens (5), rotation scatterer (6), second lens (7) and the vibration amplitude mask plate (8) that sets gradually along light path; Said rotation scatterer (6) is arranged on the back focal plane of first lens (5); Said vibration amplitude mask plate (8) is placed on the image planes after rotation scatterer (6) is formed images by second lens (7);

The said bundle collimation unit that contracts comprises the 3rd lens (9) and first object lens (10) successively along light path, and said vibration amplitude mask plate (8) is arranged on the front focal plane of the combination of lenses of being made up of the 3rd lens (9) and first object lens (10);

Said micro-amplifying unit comprises second object lens (12) and the 4th lens (13) successively along light path;

Said phase contrast imaging unit comprises phase mask plate (14) and CCD camera (15) successively along light path, and said phase mask plate (14) is arranged on the back focal plane of the combination of lenses of being made up of second object lens (12) and the 4th lens (13);

Said sample (11) is placed on the focal plane between first object lens (10) and second object lens (12), and said sample (11) and CCD camera (15) satisfy object-image relation.

2. the phase shift interference microscope equipment based on the Ze Nike phase contrast imaging according to claim 1 is characterized in that:

Said multiple beam illumination generation unit also comprises the 5th lens (16), and said the 5th lens (16) are arranged on second lens (7) afterwards; Second lens (7) and the 5th lens (16) constitute first telescopic system; Said rotation scatterer (6) is arranged on the front focal plane of first telescopic system, and said vibration amplitude mask plate (8) is arranged on the first telescopic system back focal plane.

3. the phase shift interference microscope equipment based on the Ze Nike phase contrast imaging according to claim 1 and 2; It is characterized in that: the said bundle collimation unit that contracts also comprises the 6th lens (17); The 5th lens (16) and the 6th lens (17) constitute second telescopic system, and said vibration amplitude mask plate (8) is arranged on the second telescopic system intermediate focal plane.

4. the phase shift interference microscope equipment based on the Ze Nike phase contrast imaging according to claim 3 is characterized in that: said micro-amplifying unit also comprises the 7th lens (18), and said the 7th lens (18) are arranged on the 4th lens (13) afterwards.

5. the phase shift interference microscope equipment based on the Ze Nike phase contrast imaging according to claim 4 is characterized in that: said phase contrast imaging unit comprises the 8th lens (19); The 5th telescopic system that said the 7th lens (18) and the 8th lens (19) constitute, said phase mask plate (14) is arranged on the intermediate focal plane of the 5th telescopic system, and said CCD camera (15) is arranged on the 8th lens (19) back focal plane.

6. the phase shift interference microscope equipment based on the Ze Nike phase contrast imaging according to claim 4; It is characterized in that: said vibration amplitude mask plate (8) is an opaque flat board that is carved with a series of point-like transparent regions; Said point-like transparent region periodically is distributed on the annulus, and said annulus is with corresponding by the corresponding annulus of second lens (7) imaging back lighting optical spectrum; Said point-like transparent region is round or rectangle, and the diameter of said round point-like transparent region is 10 μ m-50 μ m; The length of side of said rectangle point-like transparent region is 10 μ m-50 μ m;

Said phase mask plate (14) is the quartz glass that an etching a series of point-like phase slots; The diameter of said phase slots or the length of side are between the 10 μ m-50 μ m, and it periodically is distributed on the annulus; Said annulus with overlapped by the corresponding annulus of thing optical spectrum after the 4th lens (13) imagings; The periodicity of said phase slots is confirmed according to following mode: corresponding to the zero-frequency component of each thing light, the phase slots of different depth alternately is distributed in the one-period.

7. based on the phase shift interference microscopic method of Ze Nike phase contrast imaging, it is characterized in that: may further comprise the steps:

1] produce the pointolite of discrete and periodic distribution:

1.1] laser that sends of laser instrument (1) behind light intensity control (2), expanded Shu Danyuan (3) beam-expanding collimation and become directional light;

1.2] directional light is through becoming annular beam after being placed on the axicon lens (4) on the beam-expanding system back focal plane;

1.3] annular beam passes first lens (5), on the back focal plane of first lens (5), forms annulus;

1.4] place rotation scatterer (6) at the annulus place, make that every bit becomes mutual incoherent independent point light source on this annulus;

1.5] by second lens (7) this annulus is imaged onto on the vibration amplitude mask plate (8);

1.6] select the series of points light source of discrete and periodic distribution the picture of vibration amplitude mask plate (8) annulus from vibration amplitude mask plate;

1.7] the 3rd lens (9) with these point-sourcre imagings to the front focal plane of first object lens (10);

1.8] first object lens (10) are collimated into the multi beam directional light along different illumination directions with the picture of pointolite on the front focal plane, with different angles irradiation sample (11),

2] utilize second object lens (12) that sample is carried out micro-amplification;

3] PHASE DISTRIBUTION with sample (11) changes into intensity distributions:

3.1] after the illumination light of passing sample amplified by second object lens (12), the illumination light frequency spectrum was presented on the back focal plane of second object lens (12);

3.2] the illumination light frequency spectrum is imaged on the phase mask plate (14) by the 4th lens (13); Make the point-like phase slots of different depth cover the zero-frequency component of thing light under the different illumination directions successively through rotatable phase mask plate (14);

3.3] frequency spectrum is by the thing light after phase mask plate (14) modulation, its PHASE DISTRIBUTION is converted into visible intensity distributions, and is imaged onto on the CCD (15).

8. the phase shift interference microscopic method based on the Ze Nike phase contrast imaging according to claim 7 is characterized in that: also comprise step 4] intensity distributions is calculated to be the PHASE DISTRIBUTION of quantification:

4.1] the rotatable phase mask plate selects the phase slots coverture light zero-frequency component of different depth, on the CCD face, obtains the interference pattern of the different phase-shift phases of three width of cloth successively;

4.2] utilize three width of cloth phase-shifted interference pattern to calculate the PHASE DISTRIBUTION of thing light, obtain the PHASE DISTRIBUTION of sample.

9. based on the phase shift interference microscopic method of Ze Nike phase contrast imaging, it is characterized in that: may further comprise the steps:

1] produce the pointolite of discrete and periodic distribution:

1.1] laser that sends of laser instrument (1) behind light intensity control (2), expanded Shu Danyuan (3) beam-expanding collimation and become directional light;

1.2] directional light is through becoming annular beam after being placed on the axicon lens (4) on the beam-expanding system back focal plane;

1.3] annular beam passes first lens (5), on the back focal plane of first lens (5), forms annulus;

1.4] every bit becomes mutual incoherent independent point light source on annulus place placement rotation scatterer (6) makes this annulus;

1.5] utilize the telescopic system of forming by second lens (7) and the 5th lens (16) that this annulus is imaged onto on the vibration amplitude mask plate (8);

1.6] select the series of points light source of discrete and periodic distribution the annulus of vibration amplitude mask plate (8) on being imaged onto vibration amplitude mask plate;

1.7] behind the collimating effect of light through the 6th lens (17) that send of these pointolites; Become the directional light along different illumination directions, these illumination light are restrainted back irradiation sample (11) through the contracting of being made up of the 3rd lens (9) and first object lens (10) of the 3rd telescopic system;

2] under the irradiation of multiple beam, sample is carried out processing and amplifying:

Sample (11) is placed on the front focal plane of second object lens (12), through the output light of being made up of second object lens (12) and the 4th lens (13) after the amplification of system far away of looking around;

3] PHASE DISTRIBUTION with sample (11) changes into intensity distributions:

3.1] thing light after amplifying through Fourier transform, occurs the spectrum distribution of thing light through the 7th lens (18) on the back focal plane of the 7th lens (18);

3.2] phase mask plate (14) is placed in the thing optical spectrum, the phase slots on the phase mask plate cut can coverture light the zero-frequency component and postpone the phase place of zero-frequency component, convert the PHASE DISTRIBUTION of thing light into intensity distributions.

10. the phase shift interference microscopic method based on the Ze Nike phase contrast imaging according to claim 9 is characterized in that: also comprise step 4] intensity distributions is calculated to be the quantification PHASE DISTRIBUTION:

4.1] the rotatable phase mask plate, the phase slots that makes different depth is the component of coverture light zero-frequency successively, thereby obtains the phase-shifted interference pattern of the different phase-shift phases of three width of cloth, and the CCD that is positioned on the 8th lens (19) back focal plane accepts;

4.2] utilize three width of cloth phase shift phase contrast interference patterns to calculate the PHASE DISTRIBUTION of thing light.

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