CN103292690B - A kind of synthetic aperture microscope equipment selected based on light field - Google Patents
A kind of synthetic aperture microscope equipment selected based on light field Download PDFInfo
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- CN103292690B CN103292690B CN201310207072.9A CN201310207072A CN103292690B CN 103292690 B CN103292690 B CN 103292690B CN 201310207072 A CN201310207072 A CN 201310207072A CN 103292690 B CN103292690 B CN 103292690B
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Abstract
The invention discloses a kind of synthetic aperture microscopic method selected based on light field, comprise the following steps: 1) detect light and be irradiated on sample after expanding deviation process, utilize micro imaging system collection through the transmitted light of sample; 2) the transmission combiner that reference light is collected with micro imaging system after expanding deviation process also unifies polarization, then passes through the interference pattern of image recording sensor two-beam; 3) change the angle that detection illumination is mapped to sample, repeat step 1) and step 2), obtain multiple interference patterns corresponding with multiple angle; 4) utilize the method for Fourier transform often to be opened the PHASE DISTRIBUTION of interference pattern, then obtain micro-image by the reconstruct of randon algorithm.The invention also discloses a kind of synthetic aperture microscope equipment selected based on light field.The method that the present invention adds light field selection effectively adds microscopical synthetic aperture, and improves lateral resolution and longitudinal frame, achieves the effect of super-resolution.
Description
Technical field
The invention belongs to the micro-field of optical ultra-discrimination, particularly a kind of synthetic aperture microscopic method based on light field selection and device.
Background technology
21 century is period of biology information technology develop rapidly, and in the process of this development, the demand for the observation of the subcellular structure of living cells is also more and more urgent.First, in order to realize the use will avoiding coloring agent to the observation of living cells nature state as far as possible.Simultaneously a kind of method generally adopted at present because most of biological sample is the water white measurement by phase place, because microcobjective itself limits the low-frequency information that can only receive sample light, so the numerical aperture how expanding microcobjective carries high-resolution important channel.
WonshikChoi team in 2012 publishes thesis Three-dimensionaldifferentialinterferencecontrastmicrosc opyusingsyntheticapertureimaging.The mode scanned by 2-D vibration mirror is carried out light field simulation and is achieved the effect expanding microcobjective virtual aperture.Improve the resolution of transmission-type phase microscope, and apply for Patents.But problem be wherein two-dimensional scanning mirrors owing to not stablizing fixed point in scanning process, so certain systematic error will inevitably be introduced in the process of synthetic aperture.
YannCotte in 2013 publishes thesis Marker-freephasenanoscopy.Light will be inputted by using the method for prism wedge to rotate, achieving the effect expanding microcobjective virtual aperture.Improve resolution, and apply for Patents.Their scheme can introduce certain angle of inclination when prism wedge rotates principle, and due to the angle of wedge of prism wedge fix thus synthetic aperture process in lack adjustability.
Summary of the invention
In order to solve present optical ultra-discrimination field about the deficiency existed in synthetic aperture method, enrich the associative operation mode in synthetic aperture field simultaneously, the invention provides a kind of synthetic aperture microscopic method based on light field selection and device, the method selected by adding light field effectively adds microscopical synthetic aperture.Structure of the present invention is simple, and lateral resolution and longitudinal frame are obtained for and significantly improve, and can be used for the field such as optical microphotograph field and biological subcellular structure acquisition of information, high precision test.
Based on the synthetic aperture microscopic method that light field is selected, comprise the following steps:
1) detect light is irradiated on sample after expanding deviation process, utilizes micro imaging system collection through the transmitted light of sample;
2) the transmission combiner that reference light is collected with micro imaging system after expanding deviation process also unifies polarization, then passes through the interference pattern of image recording sensor two-beam;
3) change the angle that detection illumination is mapped to sample, repeat step 1) and step 2), obtain multiple interference patterns corresponding with multiple angle;
4) utilize the method for Fourier transform often to be opened the PHASE DISTRIBUTION of interference pattern, then obtain micro-image by the reconstruct of randon algorithm.
Described detection light and reference light are obtained by same laser beam light splitting, make that reference light is identical with the frequency of detection light, phasic difference is constant and direction of vibration is consistent, and the two-beam after conjunction bundle just can be made to produce interference.
Detection light is different with the multiple that reference light expands, and the multiple that described detection light expands is 7 ~ 8 times, and the multiple that described reference light expands is 4 ~ 6 times.
Through expand deviation process-detection light is irradiated on sample after light field is selected, described light field is selected to be used for focusing on detection light center uniform parts.Light beam focuses on back focus and drops on the back focal plane of convergent lens, becomes directional light be like this irradiated on sample by the laser of convergent lens.
Present invention also offers a kind of synthetic aperture microscope equipment selected based on light field, comprising:
The first laser instrument arranged successively along reference light light path and beam expanding lens;
Along second laser, light field selector switch, sample stage and micro imaging system that detection light light path is arranged successively;
For the first polarization splitting prism of the transmission combiner by described reference light and micro imaging system collection;
The analyzer of interfering is produced for making the reference light after conjunction bundle and transmitted light;
For gathering the image acquisition device of interference pattern;
And for controlling the computing machine of described image acquisition device and light field selector switch.
The first described laser instrument and second laser are same laser instrument, and the laser beam that described laser instrument sends is divided into described detection light and reference light through the second polarization splitting prism.Single laser instrument makes that the structure of whole microscope equipment is relatively simple, energy consumption is low, realize interfering for making reference light and transmitted light, need that the frequency of two-beam is identical, phasic difference is constant and direction of vibration is consistent, and the light beam that two laser instruments send is difficult to meet this requirement, but the reference light obtained by single laser instrument light splitting and transmitted light more easily produce interference.
Described beam expanding lens is 4f system, this 4f system comprise along light path arrange successively first before beam expanding lens, the first catoptron and first after beam expanding lens.4f system is used for expanding reference light, makes the clear aperature expanded to CCD with a tight waist of reference light close.
Light field selector switch has two kinds of selectable schemes, the first is: described light field selector switch is made up of the beam-expanding system arranged successively along light path and light field option board, described beam-expanding system comprise along light path arrange successively second before beam expanding lens, the second catoptron and second after beam expanding lens, described light field option board comprises rotating disk and the eccentric lens be inlaid on rotating disk, and described rotating disk is controlled by described computing machine; The second is: described light field selector switch comprises fiber coupler, polarization maintaining optical fibre, the fiber exit device arranged successively along light path, and described fiber exit device bias is arranged on a rotating disc, and described rotating disc is controlled by described computing machine.
In the light field selector switch of the first scheme, beam-expanding system is for being extended to scope of activities when can cover lens rotation on light field selector switch by detecting light beam, and lens only carry out selective focus to the relatively more uniform part of beam center; Light field selector switch in first scheme, detection couples light in polarization maintaining optical fibre by fiber coupler, the other end of polarization maintaining optical fibre is connected with fiber exit device, fiber exit device port also has micro lens again can focus on beam projecting light, this fiber exit device rotates with a rotating disc, is mapped to angle on sample for changing detection illumination.
As preferably, described image acquisition device is CCD.
Principle of work of the present invention is as follows:
Laser is irradiated on the first polarization spectroscope and is divided into detection light and reference light, detection light focuses on the back focal plane of convergent lens after light field selector switch, lens in light field selector switch form the 4f system from axle with convergent lens, when the lens eccentric position adjustment of light field selector switch can enable light focus on the different point assembling back focal plane in time following and rotate together with rotating disk, the light like this through convergent lens can with different angular illumination on sample.Due to the restriction in the aperture of microcobjective own, during single angular illumination sample, microcobjective can only receive the information of sample low frequency part, and when multi-angle irradiation time, the sample message that angle of inclination incidence obtains belongs to high-frequency information for straight incidence.Transmitted light and reference light overlap on polarization splitting prism, and the perfection being realized two-beam by the effect of analyzer is interfered.Change by adjustment the angle that detection illumination is mapped to sample, and record the interferogram of respective angles illumination with high-speed CCD, use
represent, wherein
for irradiating the wave vector of light.
The first step, to the data obtained
carry out Fourier transform; Second step, adopts shift frequency principle the one-level frequency spectrum obtained after Fourier transform to be moved on to the position of zero level frequency spectrum, then adopts Hamming window to carry out filtering, retain the spectrum information of reflection object phase; 3rd step, the information after the shift frequency obtain second step is carried out inverse Fourier transform and is obtained PHASE DISTRIBUTION information Z
θ 4th step, passes through formula
calculate the PHASE DISTRIBUTION under this wave vector angle; 5th step, due to each
under all correspond to some angles
pHASE DISTRIBUTION, adopt randon conversion method realize the three-dimensional microscopic image that three-dimensionalreconstruction obtains super-resolution.
The present invention is based on the three-dimensional microtechnic of synthetic aperture, by expanding the effect of numerical aperture to the selection of light field and simulated implementation, achieve the effect of super-resolution, and due to system more succinct, control convenient, have good economic advantages.
Compared with prior art, the present invention has following useful technique effect:
(1) error ratio of bringing in light field simulation is less than existing system;
(2) lateral resolution comparatively simple microscope significantly improve, lateral resolution can reach below 200nm;
(3) apparatus structure is succinct, and fast and easy high precision adjusts, and economy is strong.
Accompanying drawing explanation
Fig. 1 the present invention is based on the structural representation that light field selects the microscope equipment of synthetic aperture.
Fig. 2 is a kind of embodiment of light field selector switch.
Fig. 3 is the structural representation of light field option board.
Fig. 4 is the another embodiment of light field selector switch.
Embodiment
Describe the present invention in detail below in conjunction with embodiment and accompanying drawing, but the present invention is not limited to this.
As shown in Figure 1, based on the synthetic aperture microscope equipment that light field is selected, comprising: laser instrument 1, focus lamp 2, aperture 3, collimation lens 4, reflective mirror 5, polarization splitting prism 6, light field selector switch 7, convergent lens 8, sample 9, microcobjective 10, field lens 11, front beam expanding lens 12, reflective mirror 13, rear beam expanding lens 14, polarization splitting prism 15, polarization analyzer 16, high-speed CCD 17 and main control computer 18.
Light field selector switch 7 has two kinds of different structures.The first structure is shown in Fig. 2, comprises and arranges front beam expanding lens 19, catoptron 20, rear beam expanding lens 21 and light field option board 22 successively along light path; As shown in Figure 3, light field option board is made up of rotating disk 24 and the eccentric micro objective 23 be inlaid on rotating disk, and rotating disk is by driving stepper motor and be controlled by main control computer 18.
As shown in Figure 4, the light field selector switch 7 of the second structure comprises the coupling light device 25, polarization maintaining optical fibre 26, fiber exit device 27 and the rotating disc 28 that connect successively, fiber exit device 27 to be positioned on rotating disc 28 and to rotate with it, and rotating disc 28 is controlled by main control computer 18.
The course of work of said apparatus is as follows:
Laser instrument 1 sends laser beam, focus lamp 2, aperture 3 and collimation lens 4 form small filter, for carrying out filtering and collimation to laser beam, laser is divided into detection light and reference light by polarization spectroscope 6, reference light is through front beam expanding lens 12, catoptron 13 and rear beam expanding lens 14 carry out expanding deviation, detection light is incident in light field selector switch 7, and focus on difference on convergent lens 8 front focal plane after being undertaken expanding deviation by light field selector switch 7, convergent lens 8 converts the detection light focusing on front focal plane to directional light and is irradiated on sample 9, microcobjective 10 receives the transmitted light through sample and coordinates with field lens 11 and forms micro imaging system, polarization splitting prism 15 is by transmitted light with reference to combiner, and the polarization of the unified two-beam of analyzer 16 also realizes perfect the interference, records the interference pattern information of reference light and transmitted light with high-speed CCD 17.Main control computer 18 is connected with high-speed CCD 17 with light field selector switch 7, for the shooting of the rotation and high-speed CCD 17 of coordinating light field selector switch, and processes data and obtains high-resolution sample image.
Wherein, in two kinds of structures of light field selector switch, if the light field selector switch in use Fig. 2, detection light passes through front beam expanding lens 19, catoptron 20, and rear beam expanding lens 21 realizes expanding and turning to, detection illumination after expanding is mapped on light field option board 22, the rotating disk 24 of light field option board rotates under the drive of stepper motor, and the part light irradiated thereon focuses on by micro objective 23 again, focuses on the back focal plane of convergent lens 8; If the light field selector switch in use Fig. 4, detection illumination is mapped on fiber coupler 25, the at utmost coupling of light beam is realized by the focus lamp and angle adjusting fiber coupler, because light can be adjusted to the angle wanted by polarization maintaining optical fibre 26 pliability easily, the other end of polarization maintaining optical fibre is connected with fiber exit device 27, and the light that polarization maintaining optical fibre 26 passes over can focus on the back focal plane of convergent lens 8 by the embedded micro lens of fiber exit device 27.Fiber exit device 27 can be followed dish 23 of walking around and be rotated together, realizes the difference of Laser Focusing on convergent lens 8 back focal plane.
Above-mentioned device realizes the micro-method of synthetic aperture, and its process is as follows:
(1) laser instrument 1 launches light beam, and line focus lens 2, aperture 3 and collimation lens obtain collimated light beam, and this collimated light beam is divided into detection light and reference light through polarizing beam splitter 5;
(2) light field selector switch is as shown in Figure 2 adopted, detection illumination is mapped on light field selector switch 7, premenstrual beam expanding lens 19, catoptron 20, then beam expander is irradiated on light field option board 22 by rear beam expanding lens 21, the position adjusting front beam expanding lens 19 and rear beam expanding lens 21 ensures that the beam center homogeneous area expanded can cover the scope of micro objective 23 activity, the part light be radiated at above it focuses on by micro objective 23 again, distance between adjustment light field option board 22 and convergent lens 8 ensures that its focus is on the back focal plane of convergent lens 8, detection light becomes directional light through convergent lens 8 and is radiated on sample 9, transmitted light through sample is received by microcobjective 10, being coordinated by microcobjective field lens 11 supporting is with it irradiated on polarization splitting prism 15,
(3) reference light through front beam expanding lens 12, catoptron 13 and after beam expanding lens 14 carry out expanding turning to, before adjustment, the position of beam expanding lens 12 and catoptron 13 makes reflecting surface be positioned on the back focal plane of front beam expanding lens 12, after adjustment, the position of beam expanding lens 14 ensures that its front focus is at reflecting surface place, the face shape of catoptron 13 can being reduced like this on the impact on reference arm light wave corrugated as far as possible, being irradiated on polarization splitting prism 15 through expanding the reference light turned to;
(4) adjust the angle of polarization splitting prism 15 and the angle of catoptron 13, ensure that transmitted light and reference light overlap as far as possible, the illumination after coincidence is mapped on analyzer 16, the angle of adjustment analyzer 16, and transmitted light and reference light perfection are interfered;
(5) with the interference pattern of the lower two-beam of quick CCD17 shooting, be transferred to master control computer 18, master control computer 18 controls light field selector switch 7 and carries out can realizing carrying out sample the transillumination of different angles when light field is selected, and record detection illumination is mapped to angle on sample and the interference pattern corresponding with this angle.Light field selector switch rotates a circle, and completes the data acquisition needed for a three-dimensionalreconstruction, obtains the interference pattern of multiple and angle automatching;
(6) main control computer 18 utilizes the method for Fourier transform to obtain PHASE DISTRIBUTION to often opening interference pattern, then obtains high-resolution micro-image by randon conversion.
Claims (6)
1., based on the synthetic aperture microscope equipment that light field is selected, it is characterized in that, comprising:
The first laser instrument arranged successively along reference light light path and beam expanding lens;
Along second laser, light field selector switch, sample stage and micro imaging system that detection light light path is arranged successively;
For the first polarization splitting prism of the transmission combiner by described reference light and micro imaging system collection;
The analyzer of interfering is produced for making the reference light after conjunction bundle and transmitted light;
For gathering the image acquisition device of interference pattern;
And for controlling the computing machine of described image acquisition device and light field selector switch.
2. as claimed in claim 1 based on the synthetic aperture microscope equipment that light field is selected, it is characterized in that, the first described laser instrument and second laser are same laser instrument, and the laser beam that described laser instrument sends is divided into described detection light and reference light through the second polarization splitting prism.
3., as claimed in claim 1 based on the synthetic aperture microscope equipment that light field is selected, it is characterized in that, described beam expanding lens is 4f system, this 4f system comprise along light path arrange successively first before beam expanding lens, beam expanding lens after the first catoptron and first.
4. as claimed in claim 1 based on the synthetic aperture microscope equipment that light field is selected, it is characterized in that, described light field selector switch is made up of the beam-expanding system arranged successively along light path and light field option board, described beam-expanding system comprise along light path arrange successively second before beam expanding lens, the second catoptron and second after beam expanding lens, described light field option board comprises rotating disk and the eccentric lens be inlaid on rotating disk, and described rotating disk is controlled by described computing machine.
5. as claimed in claim 1 based on the synthetic aperture microscope equipment that light field is selected, it is characterized in that, described light field selector switch comprises fiber coupler, polarization maintaining optical fibre, the fiber exit device arranged successively along light path, beam projecting device can realize again focusing on beam projecting light beam, described fiber exit device bias is arranged on a rotating disc, and described rotating disc is controlled by described computing machine.
6., as claimed in claim 1 based on the synthetic aperture microscope equipment that light field is selected, it is characterized in that, described image acquisition device is CCD.
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| CN103900681A (en) * | 2014-04-09 | 2014-07-02 | 西安电子科技大学 | Scanning laser vibration measurement system |
| CN105300934B (en) * | 2014-05-26 | 2018-09-25 | 中国科学院上海高等研究院 | A kind of single channel optical super-resolution imaging instrument |
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| CN105511066A (en) * | 2016-01-29 | 2016-04-20 | 福州大学 | Microscopic polarization imaging device based on microwave sheet array and implement method thereof |
| US11209586B2 (en) * | 2016-12-15 | 2021-12-28 | Fusao Ishii | Ghost image elimination of doe using fourier optics method |
| EP3396430B1 (en) * | 2017-04-27 | 2023-08-16 | Euroimmun Medizinische Labordiagnostika AG | Optical scanning arrangement and method |
| CN111819485B (en) * | 2018-02-14 | 2022-07-29 | 国立研究开发法人理化学研究所 | Autofocus device, optical device provided with same, and microscope |
| CN109745008A (en) * | 2019-01-31 | 2019-05-14 | 北京超维景生物科技有限公司 | Adsorbable formula microscope detection device and laser scanning microscope |
| US11561389B2 (en) * | 2019-12-27 | 2023-01-24 | The Chinese University Of Hong Kong | High spatial and temporal resolution synthetic aperture phase microscopy (HISTR-SAPM) |
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