CN104122228B - A kind of Integrated Light interferes the micro imaging system and method analyzed with scattered information - Google Patents
A kind of Integrated Light interferes the micro imaging system and method analyzed with scattered information Download PDFInfo
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Abstract
The invention discloses the micro imaging system that a kind of Integrated Light is interfered and scattered information is analyzed, it is based on coaxial Michelson interference light path and meter Lao interference device, sample is positioned on object stage, object stage is a semi-transparent semi-reflecting lens, reflection light beam produces to interfere by rice labor interference device in Michelson interference light path, the interference image being amplified by microscope, can extract the phase information of sample from image. Meanwhile, by the forward direction transmitted light beam of sample after semi-transparent semi-reflecting minute surface, producing the little angle of forward direction and large-angle scatter, this dispersion image can be received by forward direction CCD, therefrom can obtain forward scattering amplitude distribution, can the size of destructing sample by scattering theory. Apparatus of the present invention can detect the morphosis of sample more exactly by integrated phase and scattered information, has important practical value, particularly in the detection application of biological cell substructure form in transparent substance microscopic morphology measurement.
Description
Technical field
The invention belongs to biological cell ortho states micro-imaging technique field, be specifically related to a kind of Integrated Receiver and analyze the micro imaging system of interference information and scattered information.
Background technology
Although most cells is often thought of as water white, but strictly speaking, it is unlikely to be fully transparent, namely pure-phase object it is unlikely to be, therefore when after light transmission cell, the amplitude of light and phase place all can change, and all contain the information of cell through the amplitude of light of cell and phase place. Phase imaging detection technique goes to obtain the information of cell from the cell amount of phase modulation to incident light wave, and light scattering testing technology is then start with from cell to the amount of amplitude modulation of incident illumination with obtaining the information of cell. Therefore in theory, two aspects of the optical information being through cell that phase imaging detection technique is studied with light scattering testing technology, not only separate but also connect each other, complement one another.
Existing major part phase imaging detection technique is all based on interferometric method, the whole audience imaging of object can be realized, not only its transverse spatial resolution is high, general only limited by diffraction limit, and its axial resolution is high, up to sub-wavelength magnitude, it is adaptable to without the detection of scatterer or weak scattering object. According to two interference lights (Object light wave and reference light wave) whether record plane exists angle, it is possible to be divided into coaxial-type and off-axis formula. Typical coaxial quantitative phase imaging technology includes GabrielPopescu and teaches the Fourier phase microscopy (FPM) that research group proposes and grows up for 2004, the parallel coaxial holographic phase microscopy that research group proposes is taught by Duke University AdamWax, etc., coaxial quantitative phase imaging technology has good stability, can suppress certain phase noise, but it is generally required to repetitive measurement; Typical off-axis quantitative imaging technique includes Hilbert phase microscopy (HPM), diffraction phase microscopy (DPM), two step phase shift dual pathways interference etc., and this type of imaging technique image taking speed is fast, however it is necessary that big broadband, CCD space.In recent years, professor AdamWax proposes slight off-axis and interferes (SlightlyOff-AxisInterference), this technology is interfered between coaxial the interference between off-axis, compare off-axis and interfere the broadband, CCD space that needs are less, compare the coaxial measurement interfering needs less, optimize spatial resolution. GabrielPopescu in 2006 teaches research group and proposes diffraction phase microscopy (DPM), it combines off-axis light path and the advantage of coaxial light path: have good stability and high acquisition rate, but the reason due to light source, there is speckle in the image of diffraction phase microtechnique (DPM), this will reduce the sensitivity of space phase, and limit its application in ultrastructure is studied, within 2011, it is taught research group proposition spatial light interference technique (SLIM) by GabrielPopescu to adopt white light to overcome speckle effect as light source equally, but this technology needs to gather four width intensity images for each quantitative phase figure. afterwards, GabrielPopescu research group proposition white light in 2012 makes the diffraction phase microtechnique (wDPM) of light source, and its patented technology US2014/0085715A1 delivered for 2014 (Diffractionphasemicroscopywithwhitelight: the diffraction phase microtechnique of white light source) achieves single shot and high time and susceptiveness spatially. but above-mentioned technology is all the interference information obtaining sample, only can detect transparent phase body sample, remain the substructure of sample in detection difficult.
Light scattering testing technology generally need not be interfered, measured directly is scatter intensity distribution, the decay etc. of scattared energy distribution or light intensity, the information that it provides is to measure the meansigma methods in volume, the equalization single composition contribution to scattering, sample substructure form is bigger on its scattering impact, so there being important application in cell size measurement, such as patented technology WO2014/084957A1 (Opticalsystem, apparatusandmethodforflow-cytometry: the optical system of flow cytometer, apparatus and method), but, the quantitative measurement that this type of technology changes for cellular morphology, simply analyze from scattering aspect, it is not related to interference information.
Present invention is generally directed to the deficiency that above-mentioned two class technology exist, invented the device that can simultaneously receive sample light scattering and phase place microscopy information, applying this device can calculate according to different models and algorithm, may finally obtain the ultrastructure form result of pinpoint accuracy.
Summary of the invention
It is an object of the invention to provide a kind of Integrated Light is interfered and scattered information is analyzed micro imaging system and method, enable interference information and the scattered information of comprehensive object analysis, more accurately obtain the substructure form of object.
In order to solve above technical problem, the concrete technical scheme that the present invention adopts is as follows:
The micro imaging system that a kind of Integrated Light is interfered and scattered information is analyzed, it is characterised in that including: coaxial Michelson interference light path and meter Lao interference device, particularly as follows:
Described rice labor interference device is made up of the 3rd lens (7), illuminator (8), the second spectroscope (9); Through described rice labor interference device image through microscope (14) amplify after, interference image receptor and CCD-2 (16) be received as digit phase image and carried out numerical computations and process by computer (17);
Described Michelson interference optical routing light source (1), the first spectroscope (6), illuminator (8), the second spectroscope (9), object stage (10) composition;The beams extended by lens colimated light system that also includes being made up of the 4th lens (11) and the 5th lens (12), scattering strength receptor and CCD-1 (13), interference image receptor and CCD-2 (16), microscope (14), the 6th lens (15), for scattered information and phase information being acquired storage and the computer (17) processed;
Described object stage (10) is the semi-transparent semi-reflecting lens of an adjustable angle, adjustable with illuminator (8) angle, it may be achieved the optical path adjusting that coaxial, off-axis and slight off-axis are interfered;
Described object stage (10) is semi-transparent semi-reflecting lens, to ensure that can pass through object stage through the scattering light of sample transmits downwards;
First spectroscope (6) is with horizontal plane in angle of 45 degrees; The putting position of the second spectroscope (9) ensures that the reflection light making object stage (10) is equal with the distance reflecting light passed of illuminator (8); Illuminator (8) is placed on the 3rd lens (7) center below, its size can guarantee that occur reflection basis on the smaller the better.
The micro imaging method of the micro imaging system analyzed with scattered information is interfered, it is characterised in that comprise the following steps according to described a kind of Integrated Light:
Step one, after making the beam shaping system that the light beam of light source (1) forms via the first lens (2), field stop (3), the second lens (4), aperture diaphragm (5), transmit to the first spectroscope (6); The reflection light of described first spectroscope (6) is transferred to the second spectroscope (9) by the 3rd lens (7), light beam is divided into two parts by the second spectroscope (9): a part reflexes on illuminator (8), and a part is transmitted on sample; It is transmitted to the light beam on sample when object stage (10), a part reflects to obtain reflection light, described reflection light meets again by after sample produce to interfere with the reflection light of illuminator (8), through the 3rd lens (7), the first spectroscope (6), microscope (14) and the 6th lens (15) after elder generation, at CCD-2 (16) upper formation micro-interference image; Described micro-interference image obtains the PHASE DISTRIBUTION information of reflection sample morphology structure after computer (17) processes;
Step 2, utilizes light a part of light transmission object stage (10) when object stage (10) that the second spectroscope (9) is transmitted on sample to obtain transmission light; Namely described transmission light comprise the scattered information of sample, through the beams extended by lens colimated light system that the 4th lens (11), the 5th lens (12) form, described scattered information is received as digital picture by CCD-1 (13) and is carried out numerical computations by computer (17) and obtain scattering strength and process to obtain the Scattering Amplitude Distribution value information of reflection sample size;
The scattered information that step 3, interference information that described step one is obtained by computer (17) and step 2 obtain, carries out calculating based on scattering theory and phase imaging theory, may finally obtain phase place body sample morphology, structure and size information.
The operation principle of the present invention is as follows:
The light beam sent by light source 1, onwards transmission is via the first lens 2, field stop 3, second lens 4, after the beam shaping system of aperture diaphragm 5 composition, transmit to the first spectroscope 6, the reflection light of described first spectroscope 6 continues travel downward, transmit to the second spectroscope 9 through the 3rd lens 7, light beam is divided into two parts by described second spectroscope 9, a part reflexes on illuminator 8, a part is transmitted on sample: the light part being transmitted on sample reflects, the reflection light of this partially reflective light and illuminator 8 meets at the second spectroscope 9 place and interferes, after through the 3rd lens 7, first spectroscope 6, microscope 14, 6th lens 15, micro-interference pattern is received by CCD-216,Be transmitted to the light on sample some through the object stage of semi-transparent semi-reflecting lens, through the beams extended by lens colimated light system of the 4th lens the 11, the 5th lens 12 composition, CCD-113 collect sample scattering strength information. The last computer 17 interference information to obtaining and scattered information, carry out calculating based on scattering theory and phase imaging theory, may finally obtain the phase place body sample morphology of pinpoint accuracy, structure and size information.
The present invention has beneficial effect. Light source of the present invention adopts Low coherence white light source, improves resolution, reduces noise; The present invention adopts coaxial Michelson interference light path, improves the stability of experiment; Object stage of the present invention is the semi-transparent semi-reflecting lens of an adjustable angle, can guarantee that the scattering light comprising sample scattered information can pass through object stage and continue transmission, and with illuminator adjustable angle, it may be achieved the optical path adjusting that coaxial, off-axis and slight off-axis are interfered. This utilizes CCD-113 collected specimens scattering strength, CCD-216 to receive micro-interference image, makes computer can comprehensively analyze scattered information and interference information, obtain having the phase place body sample morphology of pinpoint accuracy, structure and size information; After the microcobjective of the present invention is positioned at interference system, phase noise can be reduced. Therefore, present system wide application, there is very strong using value.
Accompanying drawing explanation
Fig. 1 is that Integrated Light of the present invention interferes the light path schematic diagram corresponding with the micro imaging system of scattered information analysis.
In figure: 1. light source; 2. the first lens; 3. field stop; 4. the second lens; 5. aperture diaphragm; 6. the first spectroscope; 7. the 3rd lens; 8. illuminator; 9. the second spectroscope; 10. object stage; 11. the 4th lens; 12. the 5th lens; 13.CCD-1; 14. microscope; 15. the 6th lens; 16.CCD-2; 17. computer.
Detailed description of the invention
Below in conjunction with accompanying drawing, technical scheme is described in further details.
The micro imaging system that the Integrated Light of the present invention is interfered and scattered information is analyzed, is realized by coaxial Michelson's light path and meter Lao interferometer, as shown in Figure 1.
The light beam sent by light source 1, onwards transmission is through the first lens 2, field stop 3, second lens 4, the beam shaping system of aperture diaphragm 5 composition, arrive the first spectroscope 6, the reflection light of described first spectroscope 6 transmits to the second illuminator 9 through the 3rd lens 7, light beam is divided into two parts by the second spectroscope 9, a part reflexes on illuminator 8, a part is transmitted on sample: the light part being transmitted on sample reflects, the reflection light of this partially reflective light and illuminator 8 meets at the second spectroscope 9 place and interferes, after through the 3rd lens 7, first spectroscope 6, microscope 14, 6th lens 15, micro-interference image is received by CCD-216, and it is stored in computer 17, being transmitted to the light on sample also has part to continue downward transmission through the object stage 10 of semi-transparent semi-reflecting lens, through the beams extended by lens colimated light system of the 4th lens the 11, the 5th lens 12 composition, gathers scattering strength, and be stored in computer 17 on CCD-113. last computer 17, according to the sample micro-interference image obtained and scattering strength information, carries out calculating based on scattering theory and phase imaging theory, may finally obtain phase place body sample morphology, structure and size information.
Claims (2)
1. an Integrated Light interferes the micro imaging system analyzed with scattered information, it is characterised in that including: coaxial Michelson interference light path and meter Lao interference device, particularly as follows:
Described rice labor interference device is made up of the 3rd lens (7), illuminator (8), the second spectroscope (9);Through described rice labor interference device image through microscope (14) amplify after, interference image receptor and CCD-2 (16) be received as digit phase image and carried out numerical computations and process by computer (17);
Described Michelson interference light path includes light source (1), the first spectroscope (6), illuminator (8), the second spectroscope (9), object stage (10); The beams extended by lens colimated light system that also includes being made up of the 4th lens (11) and the 5th lens (12), scattering strength receptor and CCD-1 (13), interference image receptor and CCD-2 (16), microscope (14), the 6th lens (15), for scattered information and phase information being acquired storage and the computer (17) processed;
Described object stage (10) is the semi-transparent semi-reflecting lens of an adjustable angle, adjustable with illuminator (8) angle, it is possible to realize coaxial, off-axis and the optical path adjusting of slight off-axis interference;
Described object stage (10) is semi-transparent semi-reflecting lens, to ensure that can pass through object stage through the scattering light of sample transmits downwards;
First spectroscope (6) is with horizontal plane in angle of 45 degrees; The putting position of the second spectroscope (9) ensures that the reflection light making object stage (10) is equal with the distance reflecting light passed of illuminator (8); Illuminator (8) is placed on the 3rd lens (7) center below, its size can guarantee that occur reflection basis on the smaller the better.
2. the micro imaging method of the micro imaging system that a kind of Integrated Light according to claim 1 is interfered and scattered information is analyzed, it is characterised in that comprise the following steps:
Step one, after making the beam shaping system that the light beam of light source (1) forms via the first lens (2), field stop (3), the second lens (4), aperture diaphragm (5), transmit to the first spectroscope (6); The reflection light of described first spectroscope (6) is transferred to the second spectroscope (9) by the 3rd lens (7), light beam is divided into two parts by the second spectroscope (9): a part reflexes on illuminator (8), and a part is transmitted on sample; It is transmitted to the light beam on sample when object stage (10), a part reflects to obtain reflection light, described reflection light meets again by after sample produce to interfere with the reflection light of illuminator (8), through the 3rd lens (7), the first spectroscope (6), microscope (14) and the 6th lens (15) after elder generation, at CCD-2 (16) upper formation micro-interference image; Described micro-interference image obtains the PHASE DISTRIBUTION information of reflection sample morphology structure after computer (17) processes;
Step 2, utilizes light a part of light transmission object stage (10) when object stage (10) that the second spectroscope (9) is transmitted on sample to obtain transmission light; Namely described transmission light comprise the scattered information of sample, through the beams extended by lens colimated light system that the 4th lens (11), the 5th lens (12) form, described scattered information is received as digital picture by CCD-1 (13) and is carried out numerical computations by computer (17) and obtain scattering strength and process to obtain the Scattering Amplitude Distribution value information of reflection sample size;
The scattered information that step 3, interference information that described step one is obtained by computer (17) and step 2 obtain, carries out calculating based on scattering theory and phase imaging theory, may finally obtain phase place body sample morphology, structure and size information.
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| CN105651733B (en) * | 2014-12-07 | 2019-01-18 | 上海微电子装备（集团）股份有限公司 | Material scattering characteristic measuring device and method |
| CN104534979B (en) * | 2014-12-10 | 2016-10-19 | 佛山市南海区欧谱曼迪科技有限责任公司 | A kind of multi-wavelength phase shift micro imaging system and method |
| CN104535572B (en) * | 2015-01-06 | 2017-04-05 | 江苏大学 | Based on the hemocyte combined detection system under light scattering and phase imaging |
| CN107688014B (en) * | 2016-09-21 | 2020-06-30 | 北京大学 | Cell imaging method |
| CN110530783B (en) * | 2018-05-24 | 2023-12-15 | 深圳市帝迈生物技术有限公司 | Lateral beam collection method and device for flow cytometer and flow cytometer |
| EP3575773A1 (en) * | 2018-05-28 | 2019-12-04 | Universität für Bodenkultur Wien | A method for determining a three-dimensional particle distribution in a medium |
| CN109596647B (en) * | 2018-12-01 | 2023-08-29 | 金华职业技术学院 | Method for neutron scattering experiment |
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| CN111122509B (en) * | 2019-11-08 | 2023-11-24 | 桂林电子科技大学 | F-P interferometer-based reflection transmission type phase microscopic imaging measurement system |
| CN113092346B (en) * | 2021-04-06 | 2022-02-11 | 中国水利水电科学研究院 | Algae cell counting detection system and detection method thereof |
| CN116321647B (en) * | 2023-03-29 | 2024-05-03 | 中国科学技术大学 | Flexible adjustable interferometer and method applied to plasma phase measurement |
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| CN100590421C (en) * | 2003-06-19 | 2010-02-17 | 麻省理工学院 | System and method for measuring phase |
| US8027032B2 (en) * | 2008-08-22 | 2011-09-27 | President & Fellows Of Harvard College | Microscopy imaging system and method employing stimulated raman spectroscopy as a contrast mechanism |
| JP4916573B2 (en) * | 2010-01-28 | 2012-04-11 | パナソニック株式会社 | Optical interference measurement method and optical interference measurement apparatus |
| CZ2010288A3 (en) * | 2010-04-14 | 2011-06-15 | Vysoké ucení technické v Brne | Interferometric system with spatial carrier frequency displaying in polychromatic radiation |
| CN102981261B (en) * | 2012-11-30 | 2015-03-11 | 山东大学 | Laser coherence diffraction microscopic imaging device and application thereof |
| CN103336419B (en) * | 2013-07-01 | 2016-05-25 | 江苏大学 | The micro-Polaroid system and method for a kind of two step phase shift interference phase place |
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