CN103411941B - Parallel confocal micro imaging method based on senior secondary axes symmetrical polarized light and device - Google Patents

Abstract

The present invention proposes a kind of parallel confocal micro imaging method based on senior secondary axes symmetrical polarized light and device, utilizes the method can obtain the three-dimensional super-resolution micro-imaging of object.Described parallel confocal microscopic imaging device based on senior secondary axes symmetric polarized light beam includes: pinhole filter, collimating lens, polarization conversion system, iris filter, beam splitter, optical filter, pinhole array plate and sensor, wherein, sample is placed on a D translation platform, focal beam spot detecting location on detection sample can be changed, to realize the 3-D scanning imaging of sample by moving three dimension translation stage.

Description

Parallel confocal micro imaging method based on senior secondary axes symmetrical polarized light and device

Technical field

The present invention relates to parallel confocal micro-imaging technique, be specifically related to a kind of based on senior secondary axes symmetrical polarized light also Row confocal microscopic imaging method and device.

Background technology

In recent years, confocal microscopic imaging technology axially chromatographs into uniqueness due to its high accuracy, high-resolution, noncontact As ability and be easily achieved the abilities such as 3-D view reconstruct and obtain in the field such as micro-nano detection, accurate measurement and bioscience research Arrive extensive application, and a lot of major companies of European and American developed countries have also been proposed high-precision matured product.

Traditional confocal microscopy detection technique is all based on light source, tested object point and 3 principles being conjugated each other of detector Carrying out simple scan imaging, therefore image taking speed is relatively slow, and because the factors such as mechanical vibration limit measurement essence when scanning Degree, is difficulty with the most real-time three-dimensional measurement.But in order to disclose life process and the physics of material property from molecular level Essence, needs the dynamic changing process of Real Time Observation living cells and cell membrane.Therefore, optical means how is utilized to break through tradition light Learn microscopical resolution limit so that it is not only there is the optical resolution of nanoscale but also biomacromolecule can be monitored continuously With the evolution of organelle micro-structure, become a significant challenge and the opportunity of the development of confocal microscopic imaging technology.In order to solve The determination that confocal microscope image taking speed is slow, visual field is less, it is proposed that parallel confocal micro-imaging technique, becomes in recent years Individual study hotspot.The method uses micro-optical device to realize the segmentation to light beam, makes simple scan become multiple beam the most parallel Scanning, greatly improves measuring speed.By the end of current position, it is proposed that multiple parallel confocal micro imaging method, as Nipkow spining disk method, microlens array method, Darman raster method, method of astigmatism, integrated optical beam method, digital micro-mirror method and colored aberration Method etc..But on the whole, although these methods improve the speed of micro-imaging, but its resolution is the most relatively low, still can not expire Foot carries out the requirement of high resolution observations to subcellular fraction micro structure.

Summary of the invention

The present invention provides a kind of parallel confocal micro imaging method based on senior secondary axes symmetric polarized light beam and device, with Phase improve detection speed while, it is achieved high-resolution imaging, meet subcellular fraction micro-nano structure is carried out in real time, high-resolution The requirement of observation.

The present invention provides a kind of parallel confocal microscopic imaging device based on senior secondary axes symmetric polarized light beam, including Such as lower part: pinhole filter, the light beam that laser instrument sends filters through pinhole filter；Collimating lens, light after filtering The collimated collimated of bundle is collimated light beam；Polarization conversion system, this collimated light beam is converted to by polarization conversion system subsequently The senior secondary axes symmetric polarized light beam of predetermined form；Iris filter, described senior secondary axes symmetric polarized light beam is filtered through pupil Ripple device carries out amplitude and phase-modulation；Beam splitter, through the senior secondary axes symmetric polarized light beam of amplitude and phase-modulation by beam splitter Focus on after reflection on detection sample, obtain multiple focal beam spot, the information of the multiple position of sample can be detected simultaneously, and, from Optical signal transmission after beam splitter that the multiple detecting location of sample is reflected back；Optical filter, comprises original ripple in the light beam of transmission Long excitation signal and the fluorescence signal excited, utilize optical filter to be removed by excitation signal therein so that only allows fluorescence letter Number through and be focused on focal plane, obtain correspondence multiple focal beam spots；Pinhole array plate, is arranged on focal plane, should The pin hole position of pinhole array plate and the location matches of focal beam spot, make each focal beam spot pin hole the most from which saturating Penetrate, it is ensured that conjugate imaging relation；Sensor, is received by sensor from the fluorescence signal of pinhole array transmission, is sent to meter Counting carrying out follow-up data in process and image reconstruction, wherein, sample is placed on a D translation platform, passes through moving three dimension Translation stage can change focal beam spot detecting location on detection sample, to realize the 3-D scanning imaging of sample.

Optionally, obtaining multiple focal beam spot on focal plane, number of spots is that 2 × (P-1) is individual, and wherein P is light beam Polarization level time P.

Optionally, the polarization level time P of light beam is 3 or 4.

The present invention provides a kind of parallel confocal micro imaging method based on senior secondary axes symmetric polarized light beam, including Following steps:

The light beam that laser instrument sends filters through pinhole filter,；It is flat that light beam after filtering is collimated collimated Row light beam,；This collimated light beam is converted to the senior secondary axes symmetric polarized light beam of predetermined form by polarization conversion system subsequently,； Described senior secondary axes symmetric polarized light beam carries out amplitude and phase-modulation through iris filter,；Through amplitude and phase-modulation Senior secondary axes symmetric polarized light beam focuses on after being reflected by beam splitter on detection sample, obtains multiple focal beam spot, can visit simultaneously The information of the multiple position of test sample product, and, the optical signal transmission after beam splitter being reflected back from the multiple detecting location of sample；Thoroughly The excitation signal comprising original wavelength in the light beam penetrated and the fluorescence signal excited, utilize optical filter to be gone by excitation signal therein Remove so that only allow fluorescence signal to pass through and be focused on focal plane, obtain multiple focal beam spots of correspondence；Pinhole array plate Be arranged on focal plane, the pin hole position of this pinhole array plate and the location matches of focal beam spot, make each focal beam spot only from One of them pin hole transmission, it is ensured that conjugate imaging relation；Received by sensor from the fluorescence signal of pinhole array transmission, quilt It is sent on calculating carry out follow-up data process and image reconstruction；Wherein, sample is placed on a D translation platform, passes through Moving three dimension translation stage can change focal beam spot detecting location on detection sample, to realize the 3-D scanning imaging of sample.

Accompanying drawing explanation

Fig. 1 (a) is the spatial polarization distribution character of axially symmetry polarization light beam, and Fig. 1 (b)-Fig. 1 (d) is senior axial symmetry The spatial polarization distribution character of polarized beam, wherein, Fig. 1 (b) is the situation of polarization level time P=2, and Fig. 1 (c) is polarization level time P=3 Situation, Fig. 1 (d) is the situation of polarization level time P=4, and wherein arrow represents the orientation of correspondence position linear polarization.

Fig. 2 (a) is parallel confocal micro imaging system structural representation, and Fig. 2 (b) is differential confocal detecting system knot Structure schematic diagram.

Fig. 3 (a)-Fig. 3 (b) is the two kinds of typical methods generating senior secondary axes symmetric polarized light beam.

Fig. 4 (a)-Fig. 4 (b), Fig. 4 (c)-Fig. 4 (d) and Fig. 4 (e)-Fig. 4 (f) be respectively polarization level time be the three of 4,8 and 15 Plant senior secondary axes symmetric polarized light beam intensity distributions on focal plane.

Fig. 5 is the burnt spot size (FWHM) change with numerical aperture NA of the axially symmetry polarization light beam of high polarization level time, Wherein spot size is focal beam spot full width at half maximum degree radially.

Fig. 6 is the structural representation of pinhole array plate.

Detailed description of the invention

Axially symmetry polarization light beam (Axially Symmetric Polarized Beams, ASPBs) is that one has axle pair Claiming the polarized beam of characteristic, axis of symmetry is the propagation axis of light beam.As shown in Fig. 1 (a), light beam on cross section any point (in Except heart point) polarization state be linear polarization, polaried orientation is in cross section.Assume the cross section that x-y plane is light beam, z-axis generation The propagation axis of mass color bundle, S (r, φ) is the certain point (except central point) on beam cross-section, and its polaried orientation meets such as ShiShimonoseki System,

Φ(r,φ)=P×φ+φ₀(P≠0) (1)

Wherein, P is referred to as polarization level time, represents the periodicity of polaried orientation change when light beam along the circumferential direction changes 360 °； φ 0 is the initial polarization azimuth corresponding when φ=0, and its value is relevant with choosing of x-axis；The polaried orientation of S point is corresponding with this point Attitude relevant.When polarizing level time P more than 1, the most senior secondary axes symmetric polarized light beam, as Fig. 1 (b)- Fig. 1 (d).

The confocal micro imaging system as shown in Fig. 2 (a) is set up based on senior secondary axes symmetric polarized light beam.Wherein, laser The light beam that device 201 sends collimated lens 203 collimation after pinhole filter 202 filters is collimated light beam, this collimated light beam Be converted to the senior secondary axes symmetric polarized light beam of particular form by polarization conversion system 204 subsequently, be then passed through pupil filtering Device 205 carries out amplitude and phase-modulation, is focused on the spy on D translation platform 209 by condenser lens 207 after beam splitter 206 reflects On test sample product 208, obtain multiple focal beam spot, the information of the multiple position of sample can be detected simultaneously；From the multiple detecting location of sample The optical signal being reflected back transmission after beam splitter 206, the excitation signal comprising original wavelength in the light beam of transmission and exciting Fluorescence signal, utilizes optical filter 210 to be removed by excitation signal therein and only allows fluorescence signal to pass through, and this fluorescence signal is by optically focused Mirror 211 focuses on, and obtains multiple focal beam spots of correspondence on focal plane.Focal plane is placed a pinhole array plate 212, this pin The pin hole position of hole array board and the location matches of focal beam spot, make the pin hole transmission the most from which of each focal beam spot, Ensure that conjugate imaging relation.Received by area array CCD below or sensor 213 from the fluorescence signal of pinhole array transmission, quilt It is sent on calculating carry out follow-up data process and image reconstruction.Wherein, sample is placed on a D translation platform 209, Focal beam spot detecting location on detection sample can be changed by moving three dimension translation stage 209, sweep realizing the three-dimensional of sample Retouch imaging.In the present invention, owing to using senior cylindrical polarized beam to obtain the focal beam spot of multiple super-resolution, D translation Sample Scan the most just can be measured the information of multiple position by platform simultaneously, therefore, it is possible to realize " parallel " micro-imaging, relatively In simple scan imaging, the present invention can significantly improve image taking speed and meet the performance requirement of the micro-detection of super-resolution.

In Fig. 2 (a), collimated beam is converted to senior secondary axes symmetric polarized light beam by available multiple method, arranges here Lift 2 kinds of typical methods:

(a) based on 4f system and spatial light modulator from Coherent decomposition be synthetically generated method, specifically refer to Xilin The document of Wang et al. " utilizes spatial light modulator and interference with common path to measure and arranges any vector beam of generation (Generation of arbitrary vector beams with a spatial light modulator and a Common path interferometric arrangement) .Opt.Lett., 32:3549,2007 ".

(b) generation based on spatial polarization converter method.Design a kind of piecemeal optics device being made up of multiple half-wave plates Part, making each half-wave plate fast axle circumferentially is certain rule change, incident line polarized light can be converted to vibration Director space change line polarized light, specifically refer to Chinese patent application CN201210562648.9 " polarization converter, Vector beam generates system and method ".

A detecting pinhole can be placed before the detectors, utilize co-focusing imaging principle to improve the resolution of fluorescence signal detection Rate, it is possible on this basis, introduces differential confocal detecting system, improves sensitivity and the resolution of signal axial detection, such as Fig. 2 Shown in (b).

Fig. 3 (a)-Fig. 3 (b) is the two kinds of typical methods generating senior secondary axes symmetric polarized light beam, and wherein SLM represents sky Between photomodulator, P1 is polaroid 1, and L1 is Fourier transform lens 1, and L2 is Fourier transform lens 2, and F is spatial filter, G is Ronchi grating, and PS1 is polarization converter.

Under normal circumstances, senior secondary axes symmetric polarized light beam has following light field COMPLEX AMPLITUDE,

${\stackrel{\→}{E}}_{\mathrm{in}}(r,\mathrm{\φ},z)=\mathrm{AP}\left(r\right)\left\{\cos \right[(P-1)\mathrm{\φ}+{\mathrm{\φ}}_0]{\stackrel{\→}{e}}_r+\sin [(P-1)\mathrm{\φ}+{\mathrm{\φ}}_0\left]{\stackrel{\→}{e}}_{\mathrm{\φ}}\right\}---\left(2\right)$

Wherein, A is a constant, represents the mean amplitude of tide size of light field；P (r) is the pupil function of light beam, characterizes light beam Relative amplitude and PHASE DISTRIBUTION；P is the polarization level time of light beam；It is respectively along unit vector radially and tangentially.

Based on Vector Diffraction Theory, the high NA focus field distribution of senior secondary axes symmetric polarized light beam can be derived,

$\stackrel{\→}{E}(r_S,{\mathrm{\φ}}_S,z_S)=\left[\begin{array}{c}{E}_r^{\left(S\right)}\\ E_{\mathrm{\φ}}^{\left(S\right)}\\ E_z^{\left(S\right)}\end{array}\right]$

$=-i^{(3P+1)}A\underset{0}{\overset{\mathrm{\α}}{\∫}}P\left(\mathrm{\θ}\right)A\left(\mathrm{\θ}\right)T\left(\mathrm{\θ}\right)\sin \mathrm{\θexp}\left({\mathrm{ikz}}_S\cos \mathrm{\θ}\right)$

$\×\left[\begin{array}{c}\cos [(P-1){\mathrm{\φ}}_S+{\mathrm{\φ}}_0]\left\{\cos \mathrm{\θ}\right[J_P\left({\mathrm{kr}}_S\sin \mathrm{\θ}\right)-J_{P-2}\left({\mathrm{kr}}_S\sin \mathrm{\θ}\right)]+J_P\left({\mathrm{kr}}_S\sin \mathrm{\θ}\right)+J_{P-2}\left({\mathrm{kr}}_S\sin \mathrm{\θ}\right)\}\\ \sin [(P-1){\mathrm{\φ}}_S+{\mathrm{\φ}}_0]\left\{\cos \mathrm{\θ}\right[J_P\left({\mathrm{kr}}_S\sin \mathrm{\θ}\right)+J_{P-2}\left({\mathrm{kr}}_S\sin \mathrm{\θ}\right)]+J_P\left({\mathrm{kr}}_S\sin \mathrm{\θ}\right)-J_{P-2}\left({\mathrm{kr}}_S\sin \mathrm{\θ}\right)\}\\ 2i\cos [(P-1){\mathrm{\φ}}_S+{\mathrm{\φ}}_0]\sin \mathrm{\θ}{J}_{P-1}\left({\mathrm{kr}}_S\sin \mathrm{\θ}\right)\end{array}\right]\mathrm{d\θ}---\left(3\right)$

S(r_s,φ_s,z_s) it is a certain point of observation near focal plane, in above formula, E_r、E_φAnd E_zBe radially, tangential and axial three The light field complex amplitude of individual quadrature component；A is a constant；P (θ) is the pupil function of light beam, characterizes relative the shaking of incident beam Width and PHASE DISTRIBUTION；A (θ) is the apodizing function of condenser lens, relevant with lens type, such as, meet sine condition when lens Time, A (θ)=cos^1/2θ, when lens meet He Muhuosi condition, A (θ)=cos^-3/2θ；T (θ) is the filtering letter of iris filter Number；K is wave number；θ is beam convergence angle, the most a certain light beam wave vector and the angle of optical axis, and therefore maximum convergent angle α is with focusing thoroughly The relation of mirror numerical aperture is α=sin^-1(NA/n), wherein n is the refractive index of surrounding media；J_m() be progression be the first kind of m Bessel function.Based on above formula, the amplitude of focousing field, phase place and intensity distributions under different situations can be calculated.

The pupil function assuming incident beam is

$P\left(\mathrm{\θ}\right)=\exp \left[{-\mathrm{\β}}^2{\left(\frac{\sin \mathrm{\θ}}{\sin \mathrm{\α}}\right)}^2\right]{\left(\sqrt{2}\mathrm{\β}\frac{\sin \mathrm{\θ}}{\sin \mathrm{\α}}\right)}^P{L}_p^l\left({2\mathrm{\β}}^2\frac{{\sin }^2\mathrm{\θ}}{{\sin }^2\mathrm{\α}}\right)---\left(4\right)$

Wherein,Representing that radially coefficient is p, tangential coefficient is the Legnedre polynomial of l, β is lens fill factor, It is assumed herein that β=1, p=0, φ₀=0,A=1,n=1.Meanwhile, assume that the condenser lens selected meets sine condition simultaneously.The most false Determining condenser lens is a kind of high-NA oil immersion objective, and the numerical aperture of lens is 1.40, and wherein the refractive index of oil is 1.518。

The intensity distributions of focousing field on focal plane when Fig. 4 (a)-Fig. 4 (f) gives polarization level time respectively 4,8 and 15. From result of calculation, obtaining multiple focal beam spot on focal plane, number of spots is relevant with the polarization level of light beam time, be 2 × (P-1) individual；The size of focal beam spot is relevant with the numerical aperture of lens, and numerical aperture is the biggest, then spot size is the least, micro- When being used for detecting sample in system, then resolution is the highest.

Fig. 5 is the change with numerical aperture NA of high polarization level secondary axes symmetric polarized light beam Jiao's spot size (FWHM), wherein Spot size is focal beam spot full width at half maximum degree radially.

Seeing accompanying drawing 4 (a)-Fig. 4 (f) and Fig. 5, here, when lens numerical aperture is 1.40, polarization level time is 4 Time, hot spot full width at half maximum degree is 0.53 λ, and focal beam spot number is 6；When polarization level time is 8, hot spot full width at half maximum degree is 0.65 λ, Focal beam spot number is 14；When polarizing level time and being 15, hot spot full width at half maximum degree is 0.86 λ, and focal beam spot number is 28.Along with The increase of polarization level time, number of focal spots increases, and hot spot full width at half maximum degree also increased.High polarization level secondary axes symmetric polarized light beam The size of focal beam spot is also closely related with numerical aperture NA, and when numerical aperture increases, the size of each focal beam spot is gradually Reduce.As it is shown in figure 5, in the case of high-NA, the axially symmetry polarization light beam of P=3,4 and 5 can obtain the least gathering Burnt hot spot.And when numerical aperture NA is bigger, the size of the focal beam spot of P=3 and P=4 polarized beam is less than diffraction limit, There is super-resolution focus characteristic.Therefore, when selecting suitably polarization level time, the full width at half maximum degree of hot spot can be at sub-wavelength amount Level, and when it is for parallel confocal micro-imaging, improve imaging resolution and image taking speed the most simultaneously.

It is true that utilize iris filter that senior secondary axes symmetric polarized light beam is carried out amplitude and phase-modulation, it is also possible to Reduce the size of focal beam spot further, improve the resolution of observation.

Especially, in order to realize confocal microscopic imaging, need to design specific pinhole array plate to mate focal beam spot, as Shown in Fig. 6, a flat board is uniformly arranged multiple pin hole along annular direction, the radius of annulus and the annular radii of focal beam spot Identical, pin hole quantity and size determine according to focal beam spot quantity and size.

Below the embodiment of the present invention is described.It will be understood by those skilled in the art, however, that without departing substantially from by right In the case of the true scope and spirit of the invention determined by requirement, these embodiments can be modified and modification.

Claims (6)

1. a parallel confocal microscopic imaging device based on senior secondary axes symmetrical polarized light, including such as lower part:

Pinhole filter, the light beam that laser instrument sends filters through pinhole filter,

Collimating lens, it is collimated light beam that light beam after filtering is collimated collimated,

Polarization conversion system, senior the axial symmetry that this collimated light beam is converted to predetermined form by polarization conversion system subsequently is inclined Shake light beam, and described senior secondary axes symmetric polarized light beam has a following light field COMPLEX AMPLITUDE:

${\stackrel{\→}{E}}_{in}(r,\mathrm{\φ},z)=AP\left(r\right)\{\cos \[(P-1)\mathrm{\φ}+{\mathrm{\φ}}_0\]{\stackrel{\→}{e}}_r+\sin \[(P-1)\mathrm{\φ}+{\mathrm{\φ}}_0\]{\stackrel{\→}{e}}_{\mathrm{\φ}}\}$

Wherein, A is a constant, represents the mean amplitude of tide size of light field, and P (r) is the pupil function of light beam, characterizes the phase of light beam To amplitude and PHASE DISTRIBUTION, P is the polarization level time of light beam,For along unit vector radially,For Unit Vector tangentially Amount, r is the lens aperture that light beam is corresponding, and φ is the polarization azimuth of light beam, φ₀For the initial polarization azimuth of light beam,

Pupil function is: $P\left(\mathrm{\θ}\right)=\exp \[-{\mathrm{\β}}^2{\left(\frac{sin\mathrm{\θ}}{\sin \mathrm{\α}}\right)}^2\]{\left(\sqrt{2}\mathrm{\β}\frac{sin\mathrm{\θ}}{sin\mathrm{\α}}\right)}^P{L}_p^1\left(2{\mathrm{\β}}^2\frac{{\sin }^2\mathrm{\θ}}{{\sin }^2\mathrm{\α}}\right),$

Wherein, θ is beam convergence angle, and α is maximum convergent angle, and β is lens fill factor,Represent that radially coefficient is p, tangential Coefficient is the Legnedre polynomial of l,

Iris filter, described senior secondary axes symmetric polarized light beam carries out amplitude and phase-modulation through iris filter,

Beam splitter, focuses on detection sample after the senior secondary axes symmetric polarized light beam of amplitude and phase-modulation is reflected by beam splitter On, obtain multiple focal beam spot, the information of the multiple position of sample can be detected simultaneously, and, reflect from the multiple detecting location of sample The optical signal transmission after beam splitter returned,

Optical filter, the excitation signal comprising original wavelength in the light beam of transmission and the fluorescence signal excited, utilize optical filter by it In excitation signal remove so that only allow a fluorescence signal pass through and be focused on focal plane, obtain corresponding multiple focusing Hot spot,

Pinhole array plate, is arranged on focal plane, the pin hole position of this pinhole array plate and the location matches of focal beam spot, makes every An individual focal beam spot pin hole transmission the most from which, it is ensured that conjugate imaging relation,

Sensor, is received by sensor from the fluorescence signal of pinhole array transmission, is sent on computer carry out follow-up number According to processing and image reconstruction,

Wherein, sample is placed on a D translation platform, can change focal beam spot at detection sample by moving three dimension translation stage Detecting location on product, to realize the 3-D scanning imaging of sample.

2. parallel confocal microscopic imaging device as claimed in claim 1, wherein obtains multiple focal beam spot, light on focal plane Speckle quantity is that 2 × (P-1) is individual, and wherein P is the polarization level time P of light beam.

3. parallel confocal microscopic imaging device as claimed in claim 1, wherein the polarization level time P of light beam is 3 or 4.

4. a parallel confocal micro imaging method based on senior secondary axes symmetrical polarized light, including following steps:

The light beam that laser instrument sends filters through pinhole filter,

It is collimated light beam that light beam after filtering is collimated collimated,

This collimated light beam is converted to the senior secondary axes symmetric polarized light beam of predetermined form, described height by polarization conversion system subsequently Level secondary axes symmetric polarized light beam has a following light field COMPLEX AMPLITUDE:

${\stackrel{\→}{E}}_{in}(r,\mathrm{\φ},z)=AP\left(r\right)\{\cos \[(P-1)\mathrm{\φ}+{\mathrm{\φ}}_0\]{\stackrel{\→}{e}}_r+\sin \[(P-1)\mathrm{\φ}+{\mathrm{\φ}}_0\]{\stackrel{\→}{e}}_{\mathrm{\φ}}\}$

Wherein, A is a constant, represents the mean amplitude of tide size of light field, and P (r) is the pupil function of light beam, characterizes the phase of light beam To amplitude and PHASE DISTRIBUTION, P is the polarization level time of light beam,For along unit vector radially,For unit tangentially Vector, r is the lens aperture that light beam is corresponding, and φ is the polarization azimuth of light beam, φ₀For the initial polarization azimuth of light beam,

Pupil function is: $P\left(\mathrm{\θ}\right)=\exp \[-{\mathrm{\β}}^2{\left(\frac{sin\mathrm{\θ}}{\sin \mathrm{\α}}\right)}^2\]{\left(\sqrt{2}\mathrm{\β}\frac{sin\mathrm{\θ}}{sin\mathrm{\α}}\right)}^P{L}_p^1\left(2{\mathrm{\β}}^2\frac{{\sin }^2\mathrm{\θ}}{{\sin }^2\mathrm{\α}}\right),$

Wherein, θ is beam convergence angle, and α is maximum convergent angle, and β is lens fill factor,Represent that radially coefficient is p, tangential Coefficient is the Legnedre polynomial of l,

Described senior secondary axes symmetric polarized light beam carries out amplitude and phase-modulation through iris filter,

Focus on after the senior secondary axes symmetric polarized light beam of amplitude and phase-modulation is reflected by beam splitter on detection sample, obtain Multiple focal beam spots, can detect the information of the multiple position of sample simultaneously, and, the light letter being reflected back from the multiple detecting location of sample Number transmission after beam splitter,

The excitation signal comprising original wavelength in the light beam of transmission and the fluorescence signal excited, utilize optical filter to excite therein Signal is removed so that only allows fluorescence signal to pass through and be focused on focal plane, obtains multiple focal beam spots of correspondence,

Pinhole array plate is arranged on focal plane, the pin hole position of this pinhole array plate and the location matches of focal beam spot, makes every An individual focal beam spot pin hole transmission the most from which, it is ensured that conjugate imaging relation,

Received by sensor from the fluorescence signal of pinhole array transmission, be sent on computer to carry out follow-up data process and Image reconstruction,

Wherein, sample is placed on a D translation platform, can change focal beam spot at detection sample by moving three dimension translation stage Detecting location on product, to realize the 3-D scanning imaging of sample.

5. parallel confocal micro imaging method as claimed in claim 4, wherein obtains multiple focal beam spot, light on focal plane Speckle quantity is that 2 × (P-1) is individual, and wherein P is the polarization level time P of light beam.

6. parallel confocal micro imaging method as claimed in claim 4, wherein the polarization level time P of light beam is 3 or 4.