CN102539381B - Refractive index chromatography device based on micro-off-axis microscopic interference projection - Google Patents
Refractive index chromatography device based on micro-off-axis microscopic interference projection Download PDFInfo
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- CN102539381B CN102539381B CN 201010604252 CN201010604252A CN102539381B CN 102539381 B CN102539381 B CN 102539381B CN 201010604252 CN201010604252 CN 201010604252 CN 201010604252 A CN201010604252 A CN 201010604252A CN 102539381 B CN102539381 B CN 102539381B
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Abstract
The invention discloses a refractive index chromatography device based on micro-off-axis microscopic interference projection; the refractive index chromatography device integrates technologies such as micro-off-axis interference, polarization phase shift, Hibert phase extraction, image high-speed collection and the like; the refractive index chromatography device is characterized in that two interference patterns are simultaneously obtained through the combination of the micro-off-axis interference technology and the polarization phase shift technology, then the obtaining speed of interference projection data can be increased by more than 3 times by extracting phase projection data carrying the space distribution information of reflective indexes of cell samples, on the basis, the multidirectional projection data of the space distribution of the refractive indexes of the cell samples is obtained through the two-dimensional scanning of an electronic control translation platform, and then the three-dimensional reconfiguration of the space distribution of the reflective indexes is realized; and the refractive index chromatography device has the prominent advantages of simpleness, fast speed, simple and convenient operation and the like.
Description
Technical field
The present invention relates to a kind of optical microphotograph interfering layer analysis apparatus, particularly a kind of micro-interference chromatographic apparatus that can be applicable to the quantitative measurment of biological cell inner refractive index space distribution.
Background technology
Refractive index and space distribution thereof are the Important Parameters of characterising biological tissue optical characteristics, and the space distribution of understanding refractive index in the biological sample in depth has great significance for the foundation of biological organism optical model and the research of relative photo transmission theory.Existing large quantity research has reported that the local anomaly of the interior refractive index of tissue is on impact and the application in development medical diagnosis on disease technology thereof of optical transmission process in the biological tissue.For example, have the difference of refractive index between the normal and malignant breast tissue, this has very important value for development optics without the wound diagnostic techniques.On the cell yardstick, refractive index can be to describe the Independent Parameters of biological sample optical characteristics, it is the basis of all kinds of optical phenomenas on the research and analysis cell yardstick, it is the basis of development disease optics Clinics equally, therefore on the cell yardstick, measuring the refractive index of biological sample and space distribution thereof has important value for the optical characteristics of deep understanding tissue sample, optical transmission process and development biological tissue in the biological tissue without the wound diagnostic techniques.Disclosed United States Patent (USP) on January 15th, 2009 (application number PCT/US2008/008447) has proposed time-based phase shift Mach Ceng Degan and has related to device, obtain the multi-direction refractive index projection of living cells by being rotated into shooting angle, as and if the three-dimensional chromatography that carries out the distribution of biological cell refractive index spatial has obtained the distributed in three dimensions of red blood cell inner refractive index with regard to.This method adopts coaxial interference technique, obtain the interference data for projection by the time phase shift technology, therefore in light path, must be with automatically controlled split-second precision phase changer, this is so that light path is comparatively complicated, poor reliability, also must gather simultaneously the interference data for projection that the above interferogram of three width of cloth could obtain a direction, so that interfere the acquisition process of data for projection to become comparatively complicated, particularly real-time is very poor, and this measurement for active somatic cell is very disadvantageous.
Summary of the invention
The object of the present invention is to provide a kind of optical microphotograph refractive index chromatography device simple, that real-time is good that installs, it relates to Mach Ceng Degan, littlely combine from technology such as axle micro-interference, polarization phase-shifting, high-speed image sampling, Hilbert phase extraction, effectively overcome the real time problems that the time phase shift brings, make simultaneously system have the advantages such as simple in structure, good stability and good reproducibility.
The technical solution that realizes the object of the invention is: a kind of based on little refractive index chromatography device from the projection of axle micro-interference, comprise that polarization He-Ne laser instrument, beam-expanding collimation mirror, the first depolarization Amici prism, the second depolarization Amici prism, confocal microscopy objective lens are front microcobjective and rear microcobjective, the first five times regualting frame, the second five times regualting frame, automatically controlled D translation platform, quarter wave plate, the first total reflective mirror, the second total reflective mirror, polarization splitting prism, the first high-speed cmos digital image acquisition device and the second high-speed cmos digital image acquisition device; Place the beam-expanding collimation mirror after the linear polarization He-Ne laser instrument and carry out beam-expanding collimation, place thereafter a depolarization Amici prism and carry out polarization beam splitting; Place front microcobjective and rear microcobjective after first output face of depolarization Amici prism, the confocal some place of front microcobjective and rear microcobjective is placed with cell sample to be measured, cell sample to be measured is placed on the automatically controlled D translation platform, front microcobjective is placed on the first five times regualting frame, rear microcobjective is placed on the second five times regualting frame, places the depolarization Amici prism after the rear microcobjective; Place the first total reflective mirror after second output face of depolarization Amici prism light path is turn 90 degrees, place quarter wave plate after the first total reflective mirror and carry out the polarization state variation, place the second total reflective mirror after the quarter wave plate light path is turn 90 degrees again; Place the second depolarization Amici prism after the second total reflective mirror mutually orthogonal incident light is closed bundle, place polarization splitting prism after the second depolarization Amici prism, place the first high-speed cmos digital image acquisition device after the first output face of polarization splitting prism, place the second high-speed cmos digital image acquisition device after the second output face, the first high-speed cmos digital image acquisition device and the second high-speed cmos digital image acquisition device obtain respectively simultaneously phase shift and are
Interferogram, in order to the multi-direction interference data for projection of extract real-time.
The present invention compared with prior art, its remarkable advantage: 1. the present invention, can obtain simultaneously phase shift and is from technology such as axle interference, polarization phase-shifting, Hilbert phase extraction and High-speed Image Acquisitions in conjunction with little
Two width of cloth interferograms, then by Hilbert phase extraction method and phase unwrapping method, recovery carries the phase projection data of cell sample refractive index spatial distributed intelligence, can will interfere the data for projection acquisition speed to improve more than 3 times, compare simultaneously with from the axle interference, optimize spectral bandwidth, improved the later stage reconstruction precision.
2. replace time-phase displacement with polarization phase-shifting, simplified optical microphotograph interfering layer analysis apparatus, made that system architecture is more simple, operation is more easy, avoided the noise of time-phase displacement, reduced the cost of device.
3. replace traditional parallel light projection with the cone-shaped beam projection, can significantly improve the multiple that optical microphotograph amplifies, effectively improved optical microphotograph interfering layer analysis apparatus spatial resolution.
4. adopt the interferogram that obtains of high-speed cmos camera, can make the acquisition time of interference image foreshorten to a millisecond magnitude, the detection of dynamic ability of biological sample.
Description of drawings
Accompanying drawing is the structural representation that the present invention is based on little refractive index chromatography device from the projection of axle micro-interference.
Embodiment
Below in conjunction with accompanying drawing the present invention is described in further detail.
In order to overcome the problem in the background technology, the present invention is equally take Mach-Zehnder interferometer as the basis, proposed based on little refractive index chromatography technology from the projection of axle micro-interference, by combining from axle interference and polarization phase-shifting technology little, then synchronization gain two width of cloth interferograms obtain the interference data for projection of a direction by phase unwrapping; By sample is carried out two-dimensional scan, obtain the interference data for projection of a plurality of directions, carry out again the three-dimensional reconstruction that the testing sample refractive index spatial distributes.The method can interfere the data for projection acquisition speed to improve more than 3 times, Effective Raise measuring speed; Meanwhile with the phase shift of polarization phase-shifting takeover time, simplify the complicacy of experimental provision, the simplicity of operation, also reduced simultaneously the cost of device.
The present invention is based on little refractive index chromatography device from the projection of axle micro-interference, its principle of work is: the laser beam that linear polarization He-Ne laser instrument sends is after the beam-expanding collimation of beam-expanding collimation mirror, by through the depolarization Amici prism it being divided into thing light and the reference light of mutual vertical transmission, and the linear polarization state when keeping incident; The first object lens in the confocal objective group are focused at thing light on the cell sample, and the second object lens in the confocal objective group will from the object lens collimation of cell sample outgoing, then incide on the depolarization light-combining prism; Reference light is through the reflection of completely reflecting mirror, and the direction of propagation changes 90 degree, then through a quarter wave plate, polarization state becomes circularly polarized light (or elliptically polarized light), by a completely reflecting mirror its direction of propagation is changed 90 degree more afterwards, incide on the depolarization light-combining prism, a branch of with the thing light compositing; Then, by polarization splitting prism it is carried out polarization spectro, the interferogram that the P polarized light component forms is by one road high-speed cmos collected by camera, and sends into Computer Processing, the interferogram that the S polarized component forms is by another road high-speed cmos collected by camera, and sends into Computer Processing.Little from Spindle Status for whole optical interference circuit is in, the deflection angle of regulating first level crossing makes and has a little θ angle between reference light and the thing light, to guarantee interference spectrum figure's
Mutually not overlapping between the level, but all overlap with zero order fringe, namely be in little from Spindle Status.On this basis, by Hilbert phase extraction method and phase unwrapping method, from the carrier frequency interferogram, recover the phase projection data that carry the distributed intelligence of cell sample refractive index spatial.Accurate control by electronic control translation stage, can realize the two-dimensional scan of cell sample, thereby obtain the multi-direction phase projection data that the cell sample refractive index spatial distributes, then quote the three-dimensionalreconstruction algorithm space distribution of cell sample refractive index is carried out three-dimensionalreconstruction, obtain the distributed in three dimensions data of refractive index.
In conjunction with Fig. 1, the present invention is based on little refractive index chromatography device from the projection of axle micro-interference, comprise polarization He-Ne laser instrument 1, beam-expanding collimation mirror 2, the first depolarization Amici prism 3, the second depolarization Amici prism 7, confocal microscopy objective lens i.e. front microcobjective 4 and rear microcobjective 6, the first five times regualting frame 15, the second five times regualting frame 16, automatically controlled D translation platform 5, quarter wave plate 9, the first total reflective mirror 8, the second total reflective mirror 10, polarization splitting prism 11, the first high-speed cmos digital image acquisition device 12 and the second high-speed cmos digital image acquisition device 13; Place beam-expanding collimation mirror 2 after the linear polarization He-Ne laser instrument 1 and carry out beam-expanding collimation, place thereafter a depolarization Amici prism 3 and carry out polarization beam splitting; Place front microcobjective 4 and rear microcobjective 6 after first output face of depolarization Amici prism 3, the confocal some place of front microcobjective 4 and rear microcobjective 6 is placed with cell sample to be measured, cell sample to be measured is placed on the automatically controlled D translation platform 5, front microcobjective 4 is placed on the first five times regualting frame 15, rear microcobjective 6 is placed on the second five times regualting frame 16, places depolarization Amici prism 7 after the rear microcobjective 6; Place the first total reflective mirror 8 after second output face of depolarization Amici prism 3 light path is turn 90 degrees, place quarter wave plate 9 after the first total reflective mirror 8 and carry out polarization state and change, place the second total reflective mirror 10 after the quarter wave plate 9 light path is turn 90 degrees again; Place 7 pairs of mutually orthogonal incident lights of the second depolarization Amici prism after the second total reflective mirror 10 and close bundle, place polarization splitting prism 11 after the second depolarization Amici prism 7, place the first high-speed cmos digital image acquisition device 12 after the first output face of polarization splitting prism 11, placing the second high-speed cmos digital image acquisition device 13, the first high-speed cmos digital image acquisition devices 12 and the second high-speed cmos digital image acquisition device 13 after the second output face obtains respectively simultaneously phase shift and is
Interferogram, in order to the multi-direction interference data for projection of extract real-time.
The present invention is based on little refractive index chromatography device from the projection of axle micro-interference, realize by quarter wave plate 9 and polarization splitting prism 11
Polarization phase-shifting, can obtain simultaneously phase shift in conjunction with the first high-speed cmos digital image acquisition device 12 and the second high-speed cmos digital image acquisition device 13 and be
Two width of cloth interferograms.
In conjunction with Fig. 1 the present invention is done more detailed explanation, based on little refractive index chromatography device from the projection of axle micro-interference, it is comprised of polarization He-Ne laser instrument, beam-expanding collimation mirror, depolarization Amici prism, confocal microscopy objective lens, five times regualting frame, cell sample to be measured, motorized precision translation stage, level crossing, depolarization Amici prism and high-speed image sampling device etc., wherein the output wavelength of linear polarization He-Ne laser instrument 1 is 632.8nm, power 4mW, the about 7mm of spot size, stability less than
Beam-expanding collimation mirror 2 by plano-concave lens (
,
) and plano-convex lens (
,
) form, expand than being 10 times; Depolarization Amici prism 3 and 7 splitting ratio are 1:1, and can guarantee that the polarization state of emergent light is consistent with incident light; Confocal objective is 25 * (25/1.48,160/0.17) to 4 and 6 magnification, and numerical aperture is 0.4, is positioned on the five times regualting frame in order to carry out focal adjustment; Cell sample to be measured is that erythrocyte can carry out accurately movement of three-dimensional, step pitch 0.078 μ m by electronic control translation stage 5; The quick shaft direction of quarter wave plate becomes miter angle with the polarization direction of incident light; Polarization splitting prism 11 can separate P and S polarized component, from orthogonal both direction outgoing; High- speed cmos camera 12 and 13 models are Mintron 1310, and pixel count is
, per second can gather 500 width of cloth images.Its course of work is: the laser beam that linear polarization He-Ne laser instrument 1 sends is divided into thing light and the reference light of mutual vertical transmission by depolarization Amici prism 3 with it behind the beam-expanding collimation of beam-expanding collimation mirror 2, and the linear polarization state when keeping incident; The first object lens 4 in the confocal objective group are focused at thing light on the cell sample, and the second object lens 6 in the confocal objective group will from the object lens collimation of cell sample outgoing, then incide on the depolarization Amici prism 7; Reference light is through the reflection of completely reflecting mirror 8, the direction of propagation changes 90 degree, then through a quarter wave plate 9, polarization state becomes circularly polarized light (or elliptically polarized light), by a completely reflecting mirror 10 its direction of propagation is changed 90 degree more afterwards, incide on the depolarization Amici prism 7, a branch of with the thing light compositing; Then, by polarization splitting prism 11 it is carried out polarization spectro, the interferogram that the P polarized light component forms is gathered by one road high-speed cmos camera 12, and sends into computing machine 14 and process, the interferogram that the S polarized component forms is gathered by another road high-speed cmos camera 13, and sends into computing machine 14 and process.Little from Spindle Status for whole optical interference circuit is in, the deflection angle of regulating first level crossing 8 makes and has a little θ angle between reference light and the thing light, to guarantee interference spectrum figure's
Mutually not overlapping between the level, but all overlap with zero order fringe, namely be in little from Spindle Status.On this basis, by Hilbert phase extraction method and phase unwrapping method, from the carrier frequency interferogram, recover the phase projection data that carry the distributed intelligence of cell sample refractive index spatial.Accurate control by electronic control translation stage, can realize the two-dimensional scan of cell sample, thereby obtain the multi-direction phase projection data that the cell sample refractive index spatial distributes, then quote the three-dimensionalreconstruction algorithm space distribution of cell sample refractive index is carried out three-dimensionalreconstruction, obtain the distributed in three dimensions data of refractive index.
Hilbert phase extraction of the present invention and unpacking method can be described as: when the intensity distributions of interference fringe is
（1）
In the formula,
With
Represent respectively the distribution of reference light and thing light,
That the phase place that sample causes changes,
Be the spatial frequency of striped, determined by the angle between reference light and the sample light.
,
Expression adds
The phase place of reference path changes behind the wave plate.
If
(2)
Then
(3)
Wherein, HT is Hilbert transform.
Interferogram is after pre-service, distribute by the field of behaviour that can obtain wrapping up after the Hilbert transform, namely real phase value distribution is through the modular arithmetic of 2 π, therefore, also must phase unwrapping (Phase Unwrapping) computing, recover real PHASE DISTRIBUTION.The cardinal rule of phase unwrapping is along certain path the modulation phase data to be carried out " integration ".If the path determines, then certain any phase gradient is:
, wherein
Be the picture point sequence number.If
Greater than a certain threshold value, for example
, then think the phase place stripe edge, phase value has
Discontinuous.This phase place is discontinuous can be revised, and the method for correction is basis
positive and negatively add or deduct at phase value accordingly
, then write down the phase place striped ordinal number N of present picture element point, repeat this process, when instantly being not the phase place stripe edge, then the phase place striped ordinal number of picture element still is N, and when running into phase place stripe edge point, then the phase place striped ordinal number N of picture element is corresponding adds or deduct 1.After the phase value of all picture elements all passed through rectification calculating, demodulation phase can be expressed as:
Adoption Network stream method of the present invention is carried out phase unwrapping, first Phase unwrapping is converted into the extreme-value problem of finding the solution minimum norm in the mathematics, can effectively avoid propagation of error, and the phase place of launching is twined rear and original interferometric phase high conformity again, can effectively solve the Phase unwrapping of biological tissue's interferogram, and still keep higher phase unwrapping precision with respect to Local treatment.
Claims (2)
1. one kind based on little refractive index chromatography device from the projection of axle micro-interference, it is characterized in that: comprise polarization He-Ne laser instrument [1], beam-expanding collimation mirror [2], the first depolarization Amici prism [3], the second depolarization Amici prism [7], the confocal microscopy objective lens i.e. front microcobjective [4] and rear microcobjective [6], the first five times regualting frame [15], the second five times regualting frame [16], automatically controlled D translation platform [5], quarter wave plate [9], the first total reflective mirror [8], the second total reflective mirror [10], polarization splitting prism [11], the first high-speed cmos digital image acquisition device [12] and the second high-speed cmos digital image acquisition device [13]; Polarization He-Ne laser instrument [1] is placed afterwards beam-expanding collimation mirror [2] and is carried out beam-expanding collimation, places thereafter the first depolarization Amici prism [3] and carries out polarization beam splitting; Place front microcobjective [4] and rear microcobjective [6] after first output face of the first depolarization Amici prism [3], the confocal some place of front microcobjective [4] and rear microcobjective [6] is placed with cell sample to be measured, cell sample to be measured is placed on the automatically controlled D translation platform [5], front microcobjective [4] is placed on the first five times regualting frame [15], rear microcobjective [6] is placed on the second five times regualting frame [16], and rear microcobjective [6] is placed the second depolarization Amici prism [7] afterwards; Placing the first total reflective mirror [8] after second output face of the first depolarization Amici prism [3] turn 90 degrees light path, the first total reflective mirror [8] is placed afterwards quarter wave plate [9] and is carried out the polarization state variation, and quarter wave plate [9] is placed afterwards the second total reflective mirror [10] light path is turn 90 degrees again; The second total reflective mirror [10] is placed afterwards the second depolarization Amici prism [7] mutually orthogonal incident light is closed bundle, the second depolarization Amici prism [7] is placed polarization splitting prism [11] afterwards, place the first high-speed cmos digital image acquisition device [12] after the first output face of polarization splitting prism [11], place the second high-speed cmos digital image acquisition device [13] after the second output face, the first high-speed cmos digital image acquisition device [12] and the second high-speed cmos digital image acquisition device [13] obtain respectively simultaneously phase shift and are
Interferogram, in order to the multi-direction interference data for projection of extract real-time.
2. according to claim 1 based on little refractive index chromatography device from the projection of axle micro-interference, it is characterized in that: realize by quarter wave plate [9] and polarization splitting prism [11]
Polarization phase-shifting, can obtain simultaneously phase shift in conjunction with the first high-speed cmos digital image acquisition device [12] and the second high-speed cmos digital image acquisition device [13] and be
Two width of cloth interferograms.
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| CN106872469B (en) * | 2017-03-16 | 2019-11-19 | 浙江大学 | A kind of chromatography phase microscopic method and device based on corner-sharing interference |
| US11125686B2 (en) * | 2017-07-06 | 2021-09-21 | Ramot At Tel-Aviv University Ltd. | System and method for three-dimensional label-free optical imaging of a biological cell sample in an environmental chamber |
| CN107482432B (en) * | 2017-08-16 | 2019-06-21 | 中国科学院上海光学精密机械研究所 | Annular multi-pass laser amplification device |
| CN110411333A (en) * | 2019-06-28 | 2019-11-05 | 北方工业大学 | Novel laser polarization phase shift interference chromatography measuring device and method |
| CN111198169A (en) * | 2019-11-08 | 2020-05-26 | 桂林电子科技大学 | Microstructure optical fiber high resolution three-dimensional refractive index testing method |
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