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CN106520535B - A kind of label-free cell detection device and method based on mating plate illumination - Google Patents

A kind of label-free cell detection device and method based on mating plate illumination Download PDF

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CN106520535B
CN106520535B CN201610890421.5A CN201610890421A CN106520535B CN 106520535 B CN106520535 B CN 106520535B CN 201610890421 A CN201610890421 A CN 201610890421A CN 106520535 B CN106520535 B CN 106520535B
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sample
light
light sheet
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illumination
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CN106520535A (en
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苏绚涛
林梅爱
刘巧
谯旭
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Shandong University
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    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434Optical arrangements
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Optical investigation techniques, e.g. flow cytometry
    • G01N15/149Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
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    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N2015/1006Investigating individual particles for cytology
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    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434Optical arrangements
    • G01N2015/144Imaging characterised by its optical setup
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1493Particle size

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Abstract

本发明公开一种基于光片照明的免标记细胞检测装置及方法,包括光片发生单元即通过柱面透镜将激光光束整形成微米级厚度的均匀照明光片,光片激发置于由精密位移台控制移动的样本微腔室中的微粒或细胞悬液,单个微粒或细胞的形态显微图像和二维光散射图样通过物镜分别在聚焦和去焦模式下被探测器记录,结果输入至成像分析系统进行图像处理和识别分类。本发明采用的光片照明方法能够有效限制激发区域,抑制光散射成像中的背景干扰,实现对单个微粒或细胞的有效激发以及在亚微米分辨率水平的微粒尺寸鉴别。光片照明激发二维光散射技术能够避免复杂的染色操作和荧光信号探测过程,对衰老细胞实现免标记检测和分类。本发明适用性强,具备推广性。

The invention discloses a label-free cell detection device and method based on light sheet illumination, comprising a light sheet generating unit, that is, a laser beam is shaped into a uniform illumination light sheet with a thickness of micrometers through a cylindrical lens, and the light sheet excitation is placed in a precise displacement The stage controls the particles or cell suspensions in the moving sample microchambers. The morphological microscopic images and two-dimensional light scattering patterns of individual particles or cells are recorded by the detector through the objective lens in focusing and defocusing modes, respectively, and the results are input to the imaging. The analysis system performs image processing and recognition classification. The light sheet illumination method adopted in the present invention can effectively limit the excitation area, suppress the background interference in the light scattering imaging, and realize the effective excitation of a single particle or cell and the particle size identification at the sub-micron resolution level. The two-dimensional light scattering technology excited by light sheet illumination can avoid complex staining operations and fluorescent signal detection processes, and realize label-free detection and classification of senescent cells. The invention has strong applicability and promotion.

Description

A kind of label-free cell detection device and method based on mating plate illumination
Technical field
The present invention relates to it is a kind of based on mating plate illumination label-free cell detection device and method, it can be achieved that sub-micron it is micro- Particle size identification, and can be used for the classification of senile cell and normal cell, and cell ageing is related to a variety of senile diseases such as Malignant tumour and cardiovascular disease etc..
Background technique
Flow cytometer clinically is depended on to the detection classification of cell.Conventional flow cytometer passes through using complicated Laser beam shaping at excitation area is limited with elliptical beam similar in cell volume, is reduced interference, and tight by light path system Lattice control sheath streaming system, keep laser beam orthogonal with sample flow, to achieve the purpose that single sample signal detection and acquisition, control Process processed is complicated, at high cost.In terms of the detection and classification to biological cell, conventional cell instrument is usually required using various fluorescence Reagent or dyestuff carry out complicated dye marker to cell, and dying operation process very complicated, external force operation can be to the knot of cell Structure and function generate certain damage, in addition, fluorescence signal is weaker, optical path is relative complex.Conventional flow cytometer passes through Granular one dimension light scattering signal, i.e. forward light scattering intensity are detected, it can be achieved that the size to particle identifies.It is this according to luminous intensity The dimension measurement method of relative size to the stability of excitation light source, detector sensitivity with the performance of signal adapter propose compared with High request.
Mating plate illumination is a kind of technology by beam modulation flakiness lighting source.The technology is mainly used in fluorescence microscopy Imaging, the advantages of fluorescent molecule on focal plane is only excited using mating plate improve imaging resolution, it can be achieved that biological tissue, The high-resolution imaging of embryo, neural network, cell mass etc..But at present mating plate means of illumination application depend on it is glimmering Signal technology, there are certain limitations for application range.
Clinically to the detection of senile cell mainly by means of biomarker technology, i.e. SA- β-Gal dyeing detection.Aging Cell due to beta galactosidase catalysis present navy blue, thus by optical microscopy microscopy can distinguish senile cell with not Senile cell, the technology have been widely used in biology agings research.Another method is by low cytometric analysis point Select senile cell and non-senile cell.In flow cytomery, the transcription regulating region building table in cell ageing is needed Up to the reporter gene of green fluorescent protein (GFP), thus the non-senile cell of quantitative detection and senile cell.Measured by flow cytometry Method can more clearly distinguish senile cell, but the radom insertion of reporter gene will appear additional differential expression, cause Reporter gene is inhomogenous, influences detection effect.To sum up, above method is easy to detect, accuracy is higher, but process steps are multiple It is miscellaneous, and the used time is longer, higher cost, while artificial diagosis is subjective and takes time and effort, and cell marking, dyeing course Eucaryotic cell structure may be changed, influence experimental result.
Summary of the invention
The present invention to solve the above-mentioned problems, propose it is a kind of based on mating plate illumination label-free cell detection device and side Method.The present invention uniformly excites single microparticle or cell as mode of excitation using mating plate lighting engineering, obtains the low back of sample The two-dimentional light scattering diagram sample of scape noise, and it is applied to the rapid dimensional identification and the label-free inspection of senile cell of single microparticle Survey classification.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of label-free cell detection device based on mating plate illumination, including the mating plate by laser beam shaping at thin photocatalytic film Generating unit, the thin photocatalytic film uniformly excite the three-dimensional space movement on vertically and horizontally face driven by sample control unit Sample to be tested, the form micro-image of sample and two-dimentional light scattering diagram picture are captured by image acquisition units, transfer to imaging analysis list Member realizes automatic classification on the basis of extracting image features;
The mating plate generating unit, including laser light source, reflecting optics, neutral density piece and cylindrical lens, laser light Source emits cylindric light beam, and after reflecting mirror controls the direction of propagation and neutral density piece adjustment light intensity, it is saturating to project cylinder Mirror, cylindrical lens modulate width of light beam generation with a thickness of several microns to tens microns of illumination mating plate.
The sample control unit, the sample chip including carrying sample suspensions, for the fixator of fixed sample chip, With the electricity driving displacement platform for driving sample to move along x-axis, y-axis and z-axis.
Described image acquisition unit includes microcobjective and cmos detector, and image acquisition units have focusing mode and go Two kinds of operating modes of burnt mode;Under focusing mode, the sample to be tested after object lens alignment is accurately positioned is focused, and is detected by CMOS The form micro-image of device record single sample;Under the mode of defocusing, object lens are capturing sample far from sample to be tested certain distance It is distributed in the scattering light in space, which is that forward direction defocuses distance, and captured scattering light projection to cmos detector is put down Face, detector record the two-dimentional light scattering diagram picture of the sample.
The imaging analysis unit includes Mie photon diffusion models analog module, characteristic parameter extraction module and supporting vector The automatic categorization module of machine, the Mie photon diffusion models analog module are realized according to Mie light scattering theory model to the two of particle Tie up light scattering analogue;The characteristic parameter extraction module extracts characteristic parameter, packet according to the sample two dimension light scattering diagram sample of capture Include the luminous intensity scanning and Fourier methods analysis of pattern;The automatic categorization module of support vector machines, by the sample knot of acquisition Fruit is divided into training set and test set, and according to the characteristic value of input, by finding optimized parameter, Automatic Optimal classification function is realized The automatic classification of sample.
Label-free cell detection method based on mating plate illumination, comprising the following steps:
(1) sample chip is constructed, upper and lower surface of the two panels Conventional glass thin slice as sample chamber, two panels coverslip are selected As gasket, both ends are respectively placed in, and are clamped between glass flake, the micro chamber that can carry sample liquor is constituted.
(2) sample suspensions are configured and are imported in sample micro chamber by pipette, ready sample chip is passed through certainly Fixator processed is fixed on precision three-dimensional electricity driving displacement platform;
(3) the mobile sample of control electricity driving displacement platform, determines sample to be imaged, so that sample to be tested is located at object lens center, and right Sample focuses, and triggering cmos detector records the form micro-image of the sample;
(4) start mating plate generating device, calibrate optical path, determine that laser beam passes sequentially through and reflected in mating plate generating unit The optical axis of mirror, neutral density piece and cylindrical lens keeps beam level to propagate, and horizontal adjustment cylindrical lens position makes to illuminate The beam waist position of mating plate is in detection object lens center;
(5) illumination mating plate excitation suspension in sample to be tested, the scattering light distribution from individual particle or individual cells in Three-dimensional space adjusts microcobjective far from sample to be tested, object lens is made to acquire the scattering within the scope of respective angles under the mode of defocusing Light, triggering cmos detector record the two-dimentional light scattering diagram sample of the sample;
(6) the two-dimentional light scattering diagram sample that cmos detector captures is input to Image analysis system and carries out image procossing and sample This identification and classification.
In the step (2), self-control fixator is made of intermediate plate and fixed screw, can stable holding sample chip, and it is simultaneous It is dissolved in optics inverted microscope.
In the step (6), classified automatically using algorithm of support vector machine to experimental result, pretreated experiment is tied Fruit is grouped learning training and prediction, provides classification results and classification assessed value.
The Dimensions recognition system of a kind of pair of submicron particle is filled using the above-mentioned label-free cell detection based on mating plate illumination It sets.
The categorizing system of a kind of pair of senile cell and normal cell is examined using the above-mentioned label-free cell based on mating plate illumination Survey device.
The invention has the benefit that
(1) different scale of the invention using the lateral two-dimentional light scattering diagram sample realization particle pattern of sub-micron level of detection is sensitive Detection, it is relatively more stable than the method in conventional flow cytometer according to one-dimensional forward direction light intensity signal;
(2) present invention uses quiescence cells suspension scanning imagery mode, gets rid of liquid complicated in conventional flow cytometer Flow control system, easy to operate, relative inexpensiveness has broad applicability;
(3) it in traditional microscopy, is generally required when preparing biological sample smear through drying, fixation and freezing etc. Reason, will cause certain destruction to the structure of sample, influences observing effect.In the present invention, sample to be tested is placed under suspension state Observation and imaging, provide preferable structure and function and protecting for sample, and sample can be allowed in its closer reset condition Under measure, be particularly suitable for living cells imaging, ensure that the authenticity and validity of DATA REASONING;
(4) present invention provides the excitation mating plate limitation lasing region of high quality using mating plate lighting method as excitation light source Domain avoids while other cells being excited to cause to interfere, and provides preferable imaging signal to noise ratio, and flexible mating plate thickness and position are controlled System is easy to that cell is quickly positioned and scanned, and exciting power needed for efficient capacity usage ratio can reduce imaging reduces light Influence of the toxicity to cell activity;
(5) acquisition device of the present invention has two operating modes, can capture the morphological image and two of individual cells simultaneously Light scattering diagram sample is tieed up, the corresponding relationship for establishing eucaryotic cell structure and optical property is facilitated, realizes the imaging to individual cells behavior And analysis, the deficiency of light intensity signal is only provided when compensating for conventional flow cytometer to individual particle or cell analysis;
(6) present invention detects senile cell using label-free method, can be realized to the quick of aging and normal cell Automatic classification, gets rid of conventional method to the complicated processes of cell dyeing and artificial diagosis;
(7) present invention is suitable for the imaging identification and classification of other biological cell, has popularity.
Detailed description of the invention
Fig. 1 is the structure and schematic illustration of apparatus of the present invention;
Fig. 2 (a)-Fig. 2 (c) is the different mating plate thickness mating plates and its measurement result that present example provides;
Fig. 3 (a)-Fig. 3 (f) is the micro-image and two-dimentional light for the different-grain diameter polystyrene microsphere that present example provides Scattering pattern;
Fig. 4 is fast Fourier (FFT) the analysis result schematic diagram of experiment and simulation polystyrene microsphere light scattering;
Fig. 5 (a)-Fig. 5 (f) is that the micro-image of normal cell and senile cell that present example provides and two-dimentional light dissipate Penetrate pattern;
Fig. 6 is characteristic parameter distribution schematic diagram of the human body at fiber aging and normal cell light scattering diagram sample.
Wherein, 1, laser, 2, reflecting mirror, 3, neutral-density filter, 4, cylindrical lens, 5, sample chamber, 6, sample Room fixator, 7, three-D electric displacement platform, 8, microcobjective, 9, cmos detector, 10, data analysis system.
Specific embodiment:
The invention will be further described with example with reference to the accompanying drawing.
A kind of label-free cell detection device and method based on mating plate illumination, the mating plate illumination that the present invention uses can have Effect limits excitation area, inhibits the background interference in light scattering imaging, and control process is simple and flexible.Two-dimentional light scattering technique mentions For the light scattering on sample to be tested polar angle and two, azimuth angular range, it can get and scatter letter more abundant than one-dimensional light Breath is realized and is identified in the microparticle size of submicron resolution level, and can be avoided complicated dying operation and fluorescence signal Detection process realizes label-free identification and classification to senile cell.In image acquisition process of the invention, sample to be tested is natural It is placed under suspension state, has not both needed the sheath flow control system of flow cytometer, also got rid of in optical microscopy microscopy and handle Drying that smear is related to, fixed equivalent damage operation, ensure that the authenticity and validity of measurement.
A kind of label-free cell detection device and method based on mating plate illumination, including mating plate generating unit, sample control Unit, image acquisition units and data processing unit.Mating plate generating unit is by laser beam shaping at the thin of micron order thickness Mating plate, the mating plate of generation enter sample control unit excitation sample to be tested, and sample control unit is being hung down for controlling sample to be tested Three-dimensional space on straight and horizontal plane is mobile, and image acquisition units capture the form micro-image and two-dimentional light scattering diagram of sample Picture, and be transmitted to imaging analysis unit and carry out image procossing, feature extraction and data analysis.
Mating plate generating unit includes laser light source, reflecting optics, neutral density piece and cylindrical lens.Laser light source transmitting Cylindric light beam projects cylindrical lens after reflecting mirror and neutral density piece, cylindrical lens squeezed light in one-dimensional direction Beam width forms the illumination mating plate with a thickness of several microns to tens microns.Laser light source selected type is that diode semiconductor is solid Body laser, wavelength are 532nm, and the laser beam spot sizes of generation are 1.052mm.Neutral density piece is for adjusting laser light beam intensity Degree, optional transmitance are 50%, 32%, 10%, 1%, 0.1%.
Sample control unit includes special sample chip, fixator and electricity driving displacement platform.Sample chip is by four 170 μm Thick glass flake is composed, wherein two panels glass flake is as sample chip upper and lower surface, and in addition two panels is put as gasket It sets at both ends, clamping between the upper and lower surfaces, forms the chamber that volume is about 25.4mm x 10.0mm x 0.17mm, is used for Sample loading suspension.Sample chip intermediate plate is clamped and is fixed on electricity driving displacement platform.The electricity driving displacement platform is three-axis accurate Displacement platform can be controlled separately sample in x-axis, and y-axis and z-axis are mobile, and displacement resolution is up to 20nm or more.
Image acquisition units include microcobjective and cmos detector.Image acquisition units have focusing mode and the mould that defocuses Two kinds of operating modes of formula.Under focusing mode, the sample to be tested after object lens alignment is accurately positioned is focused, and is remembered by cmos detector Record the form micro-image of single sample;Under the mode of defocusing, object lens work far from sample to be tested certain distance, and the distance is fixed Justice is that forward direction defocuses distance, and the two-dimentional light scattering diagram picture of sample to be tested is obtained in the case where forward direction removes defocus distance.In the present invention, it defocuses Distance is 200 μm.Object lens collect the two-dimensional scattering light of the sample in corresponding angular range, pass through cmos detector record The two-dimentional light scattering diagram sample of a sample.
Imaging analysis unit includes the simulation of Mie photon diffusion models, and the feature extraction of image and support vector machines (SVM) are certainly Dynamic sorting algorithm.In algorithm of support vector machine, the sample results of acquisition are divided into training set and test set, by finding optimal ginseng Number, Automatic Optimal classification function obtain classification results and assessment result.
As shown in Figure 1, a kind of label-free cell detection device based on mating plate illumination, including laser 1, reflecting mirror 2, in Property density filters 3, cylindrical lens 4, sample chamber 5, sample room's fixator 6, three-D electric displacement platform 7, microcobjective 8, Cmos detector 9, data analysis system 10.
Specifically include following procedure:
The laser beam that laser 1 issues adjusts the direction of propagation by reflecting mirror 2, and is controlled by neutral-density filter 3 Laser beam after laser intensity processed, adjustment direction and energy projects cylindrical lens 4,4 vertical direction modulation light of cylindrical lens Beam compresses light beam in one-dimensional direction, is shaped to the thin photocatalytic film of micron order thickness, and the size of mating plate thickness depends on light Beam projects the effective numerical aperture size of cylindrical lens;
Mating plate is illuminated from 5 side illumination sample suspensions of sample chamber, to excite the sample to be tested in suspension.Fixator 6 Sample room 5 is fixed on three-D electric displacement platform 7, three-D electric displacement platform 7 controls sample in vertical direction and horizontal plane On accurate movement;
After sample is excited by laser light-piece, the scattering light for being distributed in three-dimensional space is detected the detection of object lens 8 and collects, CMOS Detector 9 captures two-dimentional light scattering diagram sample and records;
The image data of record is transmitted to analysis system 10 and carries out data processing and analysis.
Embodiment 1
Using the label-free cell detection device and method illuminated based on mating plate, the Uniform Illumination light of different-thickness is realized Piece.In light scattering imaging, to reduce the ambient noise in imaging process, and guarantee simultaneously sample to be tested by Uniform Illumination, The mating plate for providing different-thickness for the sample of different size scale in the present invention excites, and has pointedly control mating plate thickness Degree limits excitation area, avoids the interference from other sample scatter light.By control light beam expand multiple and cylinder is saturating The focal length of mirror generates the mating plate of required thickness.The light of different-thickness is realized in this example by conversion different focal length cylindrical lens Piece.Different thickness is visualized by rhodamine solution, and measures mating plate thickness by vertical scanning image pixel.
Concrete operation step:
(1) rhodamine 6G solution is configured, and solution is imported in the sample chamber built;
(2) open laser light source, calibrate optical path, make laser beam pass sequentially through optical element include reflecting mirror, it is neutral close The optical axis of piece and cylindrical lens is spent, horizontal transmission is kept.90 ° of Rotating cylindrical surface lens, compress light beam in the horizontal direction, Cylinder lens position is adjusted, makes to illuminate mating plate beam waist position in detection object lens center;
(3) increase wavelength filter before cmos detector, filter out excitation wavelength (532nm), allow rhodamine solution Launch wavelength (575nm) pass through, triggering cmos detector record mating plate visualization after image;
(4) by vertical scanning image pixel, gray value is obtained, determines the with a tight waist of mating plate, and measure its mating plate thickness, this Place uses halfwidth (FWHM) as thickness and portrays parameter;As a result as shown in Fig. 2 (a), Fig. 2 (b), Fig. 2 (c), mating plate thickness Respectively 5.6 μm, 13.7 μm and 53.3 μm.
Embodiment 2
In order to verify the sensitivity and accuracy that the present invention identifies micron level particle size, polystyrene mark is used Quasi- microballoon carries out experimental verification and device calibration.In the present invention, the partial size is selected to be for the standard microballoon of 3.87 μm and 4.19 μm Imaged samples obtain its micro-image and two-dimensional scattering figure, and the result of experimental result and Mie simulation are carried out FFT comparison Analysis.Selected microcobjective is 20 times of object lens that numerical aperture is 0.4, corresponding detectable angular range in the present invention It is 72.5 ° -107.5 °, so in Mie simulation, scatters polar angle and azimuth value range thus.Mating plate is adjusted in this example With a thickness of 13 μm or so.
Concrete operation step:
(1) appropriate polystyrene microsphere stoste is drawn, is diluted with ultrapure water, and microsphere suspensions are imported into sample microcavity Room;
(2) sample chip for being loaded with microsphere suspensions prepared is fixed on three-D displacement platform by fixator, is controlled Displacement platform, focusing objective len determine the imageable target in visual field, finely tune displacement platform, targeted microspheres is made to be located at object lens middle position;
(3) cmos detector is triggered, the form micro-image under focusing mode of microballoon is recorded;
(4) laser light source is opened, mating plate generating unit device is adjusted, keeps laser beam holding horizontal and in optical element On central optical axis, the mating plate and targeted microspheres of formation are on sustained height, and mating plate beam waist position is maintained at the object lens visual field Center;
(5) object lens are finely tuned, forward direction defocuses 200 μm, makes object lens work collection of scattered light under the mode of defocusing, and triggering CMOS is visited Device is surveyed, the two-dimentional light scattering diagram sample of the microballoon is recorded;
(6) according to Mie light scattering theory model, the two-dimentional light scattering result of microballoon is simulated.Simulated conditions are optical source wavelength 532nm, microballoon refractive index 1.59, solution medium refractive index 1.334 locating for microballoon, scatters polar angle and azimuthal angular range is 72.5°-107.5°;
(7) horizontal sweep is carried out to the two-dimentional light scattering diagram picture of experiment and simulation respectively, obtains the gray scale of light scatter intensity It is worth curve.The quantitative analysis of one-dimensional FFT is carried out by Fourier transformation, the frequency main peak value in FFT map can quantify light scattering The fringe distribution of pattern.According to the difference for the frequency main peak value that the distribution of the light scattered striation of various sizes of microballoon generates, realize Size identification and classification to microballoon.In this example, the frequency step of FFT sampling is set as 0.0138 (1/degree).
Shown in the experimental result of this example such as Fig. 3 (a)-Fig. 3 (f).Fig. 3 (a) and Fig. 3 (d) is 3.87 μm and 4.19 μ respectively The micro-image that the microballoon of m obtains under 20 times of object lens focusing task modes.Fig. 3 (b) and Fig. 3 (e) is under the mode of defocusing respectively The two-dimentional light scattering diagram sample of the two kinds of beads obtained.The size difference of two kinds of microballoons is 320nm, focuses mould in ordinary optical object lens More difficult intuitive differentiation two kinds of particles under formula.In contrast, the two-dimentional light scattering diagram sample of two kinds of beads is in striped quantity and distribution Notable difference is presented, there are 7 bright fringes, 8 bright fringes occurs in 4.19 μm of microballoons in the light scattering diagram sample of 3.87 μm of microballoons, Therefore, two kinds of microballoons directly can quickly be distinguished by judging the striped quantity of light scattering diagram sample.Fig. 3 (c) and Fig. 3 (f) is root According to the light scattering diagram sample of two kinds of beads of Mie modeling as a result, the result consistent scattering strip compared with experimental result is presented Line distribution.Further utilize the difference of two kinds of beads of fast Fourier FFT method quantitative analysis and experiment and analog result Identical property, as a result as shown in Figure 4.For 4.19 μm of microballoons, simulation and the FFT frequency main peak value of experimental result are 1.432 (1/ degree);For 3.87 μm of microballoons, the frequency main peak value of experimental result is 1.2963 (1/degree), and analog result is 1.2825 (1/degree), the difference of only one sampling step length, also than more consistent.This example demonstrates the present invention for sub-micro The feasibility and accuracy that rice resolution ratio particle size identifies.
Embodiment 3
Using the label-free cell detection device and method illuminated based on mating plate, to human body at fiber senile cell and normally Cell carries out cell recognition and automatic classification.It is about 50 μm that mating plate thickness is adjusted in this example.Aging is induced to by hydrogen peroxide Human body carry out cell morphology image at fiber senile cell and normal cell and its two-dimentional light scattering diagram sample acquisition, every class are random Acquire 55 experimental results.Characteristic parameter extraction is carried out to 110 light scattering diagram pictures of acquisition, and inputs support vector machines (SVM) algorithm classifies automatically to two class cells as characteristic parameter.
Specific implementation:
(1) prepare the processed human body of hydrogen peroxide into fiber senile cell and normal cell, be configured to carefully with PBS buffer solution Born of the same parents' suspension imports sample micro chamber, control bit moving stage and imaging unit device, and the micrograph of cell is obtained under focusing mode Picture;Such as Fig. 5 (a), Fig. 5 (b).
(2) starting mating plate lighting device obtains the two-dimentional light scattering diagram of two class cells under the 200 μm of modes that defocus respectively Sample, such as Fig. 5 (c), Fig. 5 (d).
(3) algorithm detects and extracts the characteristic parameter of speckle distribution.Local intensity maxima point is detected first, determines spot Position and center.In the present invention, pixel more than intensity 6 units bigger than surrounding pixel is defined as maximum intensity value point. After determining spot centers, the pixel region being less than within the scope of 6 strength differences of the central point is defined as with the local maxima Speckle area centered on hot spot.110 experimental results are scanned, the numerical value ginseng of corresponding speckle count and speckle area is extracted Number characterizes different cells according to speckle count and the distribution relation of speckle area, and realization is to senile cell and normal cell Classification, as shown in Figure 6.
(4) using the numerical value of speckle count and speckle area as the characteristic of division value of support vector machines, when executing SVM algorithm Linear kernel function is chosen, and classifier is constructed using 5- folding cross-validation method.110 data of two class cell results are divided at random For 5 data sets, each data set has 22 samples, in turn using 4 data sets therein as training set, a remaining number After being used as test set, repeated overlapping to verify 5 times according to collection, using the average value of the accuracy of 5 prediction results as finally accurate Degree.Classification results are as shown in table 1.Wherein, the cell quantity that accuracy rate is defined as correctly being classified accounts for whole cell quantities Percentage;Sensitivity definition is the ratio of the quantity that senile cell can correctly be classified and senile cell total quantity;Specificity It is defined as the ratio of quantity and normal cell total quantity that normal cell is correctly classified.AUC parameter is for assessing classifier Performance is worth closer to 1, and presentation class device performance is better.
1 human body of table at fiber aging and normal cell svm classifier result
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not protects model to the present invention The limitation enclosed, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not Need to make the creative labor the various modifications or changes that can be made still within protection scope of the present invention.

Claims (9)

1.一种基于光片照明的免标记细胞检测装置,其特征是:包括将激光光束整形成薄光片的光片发生单元,该薄光片均匀激发由样本控制单元带动的在垂直和水平面上的三维空间移动的待测样本,样本的形态显微图像和二维光散射图像由图像采集单元捕获,交由成像分析单元在提取图像特征参数的基础上实现自动分类;1. A label-free cell detection device based on light sheet illumination, characterized in that: it comprises a light sheet generating unit that shapes a laser beam into a thin light sheet, and the thin light sheet uniformly excites the vertical and horizontal planes driven by the sample control unit. On the three-dimensional space moving sample to be tested, the morphological microscopic image and two-dimensional light scattering image of the sample are captured by the image acquisition unit, and then handed over to the imaging analysis unit to realize automatic classification based on the extraction of image feature parameters; 所述光片发生单元,包括激光光源、反射镜片、中性密度片以及柱面透镜,激光光源发射圆柱状光束,经过反射镜控制传播方向以及中性密度片调整光强后,投射到柱面透镜,柱面透镜调制光束宽度产生照明光片。The light sheet generating unit includes a laser light source, a reflecting lens, a neutral density sheet and a cylindrical lens. The laser light source emits a cylindrical light beam, which is projected onto the cylindrical surface after the reflector controls the propagation direction and the neutral density sheet adjusts the light intensity. A lens, a cylindrical lens modulates the beam width to produce an illuminating light sheet. 2.如权利要求1所述的一种基于光片照明的免标记细胞检测装置,其特征是:所述成像分析单元包括Mie光散射模型模拟模块、特征参数提取模块和支持向量机自动分类模块;所述Mie光散射模型模拟模块,根据Mie光散射理论模型,实现对微粒的二维光散射模拟;所述特征参数提取模块,根据捕获的样本二维光散射图样提取特征参数,包括图样的光强度扫描和傅里叶方法分析;所述支持向量机自动分类模块,将采集的样本结果分为训练集和测试集,根据输入的特征值,通过寻找最优参数,自动优化分类函数,实现样本的自动分类。2. A label-free cell detection device based on light sheet illumination according to claim 1, wherein the imaging analysis unit comprises a Mie light scattering model simulation module, a feature parameter extraction module and a support vector machine automatic classification module The Mie light scattering model simulation module, according to the Mie light scattering theoretical model, realizes the two-dimensional light scattering simulation of particles; the feature parameter extraction module extracts the feature parameters according to the captured sample two-dimensional light scattering pattern, including the pattern Light intensity scanning and Fourier method analysis; the support vector machine automatic classification module divides the collected sample results into a training set and a test set, and automatically optimizes the classification function by finding the optimal parameters according to the input eigenvalues. Automatic classification of samples. 3.如权利要求1所述的一种基于光片照明的免标记细胞检测装置,其特征是:所述样本控制单元,包括承载样本悬液的样本芯片,用于固定样本芯片的固定器,和带动样本沿x轴,y轴以及z轴移动的电动位移台。3 . The label-free cell detection device based on light sheet illumination according to claim 1 , wherein the sample control unit comprises a sample chip carrying the sample suspension, a holder for fixing the sample chip, 4 . And a motorized stage that moves the sample along the x-axis, y-axis and z-axis. 4.如权利要求1所述的一种基于光片照明的免标记细胞检测装置,其特征是:所述图像采集单元包括显微物镜和CMOS探测器,图像采集单元具有聚焦模式和去焦模式两种工作模式,在聚焦模式下,物镜对准精确定位后的待测样本聚焦,通过CMOS探测器记录单个样本的形态显微图像,在去焦模式下,物镜在远离待测样本一定距离捕获样本分布于空间的散射光,该距离定义为正向去焦距离,被捕获的散射光投射到CMOS探测器平面,探测器记录该样本的二维光散射图像。4 . The label-free cell detection device based on light sheet illumination according to claim 1 , wherein the image acquisition unit comprises a microscope objective lens and a CMOS detector, and the image acquisition unit has a focusing mode and a defocusing mode. 5 . There are two working modes. In the focusing mode, the objective lens is focused on the precisely positioned sample to be tested, and the morphological microscopic image of a single sample is recorded by the CMOS detector. In the defocusing mode, the objective lens is captured at a certain distance away from the sample to be tested. The scattered light distributed by the sample in space, this distance is defined as the forward defocusing distance, the captured scattered light is projected onto the CMOS detector plane, and the detector records the two-dimensional light scattering image of the sample. 5.一种对亚微米微粒的尺寸识别系统,其特征是:包括如权利要求1-4中任一项所述的基于光片照明的免标记细胞检测装置。5. A size identification system for submicron particles, characterized by comprising the label-free cell detection device based on light sheet illumination according to any one of claims 1-4. 6.一种对衰老细胞与正常细胞的分类系统,其特征是:包括如权利要求1-4中任一项所述的基于光片照明的免标记细胞检测装置。6. A classification system for senescent cells and normal cells, characterized by comprising the label-free cell detection device based on light sheet illumination as claimed in any one of claims 1-4. 7.基于光片照明的免标记细胞检测方法,其特征是,包括以下步骤:7. A label-free cell detection method based on light sheet illumination, characterized in that it comprises the following steps: (1)构建样本芯片,选用两片常规玻璃薄片作为样本腔室的上下表面,两片盖玻片作为垫片,分别置于两端,并被夹持在玻璃薄片之间,构成可承载样本液溶液的微腔室;(1) To construct a sample chip, two conventional glass sheets are used as the upper and lower surfaces of the sample chamber, and two cover glass sheets are used as spacers, which are placed at both ends and sandwiched between the glass sheets to form a load-bearing sample. Microchambers for liquid solutions; (2)配置样本悬液并通过移液管导入样本微腔室中,将准备好的样本芯片通过自制固定器固定在精密三维电动位移台上;(2) Configure the sample suspension and introduce it into the sample microchamber through a pipette, and fix the prepared sample chip on a precision three-dimensional electric displacement stage through a self-made holder; (3)控制电动位移台移动样本,确定待成像样本,使待测样本位于物镜中心,并对样本聚焦,触发CMOS探测器记录该样本的形态显微图像;(3) Control the electric displacement stage to move the sample, determine the sample to be imaged, make the sample to be tested at the center of the objective lens, focus the sample, and trigger the CMOS detector to record the morphological microscopic image of the sample; (4)启动光片发生装置,校准光路,确定激光光束依次通过光片发生单元中反射镜、中性密度片、以及柱面透镜的光轴,保持光束水平传播,水平调整柱面透镜位置,使照明光片的束腰位置处于探测物镜中心;(4) Start the light sheet generating device, calibrate the optical path, determine that the laser beam passes through the reflector, the neutral density sheet, and the optical axis of the cylindrical lens in the light sheet generating unit in turn, keep the beam horizontally propagate, and adjust the position of the cylindrical lens horizontally, Make the beam waist position of the illumination light sheet in the center of the detection objective lens; (5)照明光片激发悬液中的待测样本,来自单个颗粒或单个细胞的散射光分布于三维空间,调整显微物镜远离待测样本,使物镜在去焦模式下采集相应角度范围内的散射光,触发CMOS探测器记录该样本的二维光散射图样;(5) The illumination light sheet excites the sample to be tested in the suspension, the scattered light from a single particle or a single cell is distributed in three-dimensional space, adjust the microscope objective lens away from the sample to be tested, and make the objective lens collect in the corresponding angle range in defocusing mode The scattered light of the sample is triggered, and the CMOS detector is triggered to record the two-dimensional light scattering pattern of the sample; (6)将CMOS探测器捕获的二维光散射图样输入至成像分析系统进行图像处理和样本识别及分类。(6) Input the two-dimensional light scattering pattern captured by the CMOS detector to the imaging analysis system for image processing and sample identification and classification. 8.如权利要求7中所述的基于光片照明的免标记细胞检测方法,其特征是:所述步骤(2)中,所述自制固定器由夹片和固定螺丝组成,可稳定夹持样本腔室,并兼容于光学倒置显微镜。8. The label-free cell detection method based on light sheet illumination as claimed in claim 7, wherein in the step (2), the self-made fixator is composed of a clip and a fixing screw, which can be stably clamped Sample chamber and compatible with optical inverted microscopes. 9.如权利要求7中所述的基于光片照明的免标记细胞检测方法,其特征是:所述步骤(6)中,采用支持向量机算法对实验结果自动分类,对预处理后的实验结果进行分组学习训练和预测,提供分类结果以及分类评估值。9. The label-free cell detection method based on light sheet illumination as claimed in claim 7, wherein in the step (6), a support vector machine algorithm is used to automatically classify the experimental results, and the preprocessed experiments are The results are subjected to group learning training and prediction, providing classification results and classification evaluation values.
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