CN106520535A - Label-free cell detection device and method based on light sheet illumination - Google Patents
Label-free cell detection device and method based on light sheet illumination Download PDFInfo
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Abstract
The invention discloses a label-free cell detection device and method based on light sheet illumination. The method includes the steps that a light sheet generation unit is used for shaping a laser beam into a uniform illumination light sheet with the micron-order thickness through a cylinder lens; the light sheet excites particles or a cell suspension placed in a sample micro-cavity chamber controlled by a precise displacement table to move; a form microscopic image and a two-dimensional light scattering pattern of a single particle or cell are recorded by a detector under the focusing mode and the defocusing mode through an objective lens; and a result is input into an imaging analysis system for image processing and recognizing and classifying. By means of the light sheet illumination method, a stimulation area can be effectively limited, background interference in light scattering imaging is inhibited, and effective stimulation of the single particle or cell and particle size discrimination of the sub-micron resolution ratio level are realized; and a light sheet illumination stimulation two-dimensional light scattering technology can avoid complex dyeing operation and a fluorescence signal detection process, and label-free detection and classification are carried out on aging cells. The label-free cell detection device and method based on light sheet illumination are high in applicability and capable of being popularized.
Description
Technical field
The present invention relates to a kind of label-free cell detection device and method illuminated based on mating plate, is capable of achieving the micro- of submicron
Particle size is recognized, and can be used for the classification of senile cell and normal cell, and cell ageing is related to various senile diseases such as
Malignant tumor and cardiovascular disease etc..
Background technology
Clinically the detection classification to cell depends on flow cytometer.Conventional flow cytometer is by using complexity
Laser beam shaping is limited excitation area into the elliptical beam close with cell volume by light path system, is reduced and is disturbed, and sternly
Lattice control sheath streaming system, keep laser beam orthogonal with sample flow, so as to reach the purpose of single sample signal detection and collection, control
Process processed is complicated, high cost.In terms of the detection to biological cell with classification, conventional cell instrument is generally needed using various fluorescence
Reagent or dyestuff carry out the dye marker of complexity, dying operation process very complicated, the knot that external force operation can be to cell to cell
Structure and function produce certain infringement, additionally, fluorescence signal is weaker, optical path is relative complex.Conventional flow cytometer passes through
Detection granular one dimension light scattering signal, i.e. forward light scattering intensity, are capable of achieving the size to granule and differentiate.It is this according to light intensity
Stability of the dimension measurement method of relative size to excitation 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, only excites the advantage of the fluorescence molecule on focal plane to improve imaging resolution using mating plate, be capable of achieving to biological tissue,
The high-resolution imaging of embryo, neutral net, cell mass etc..But, the application of current mating plate means of illumination depends on glimmering
There is certain limitation in signal technology, range of application.
Clinically to the detection of senile cell mainly by means of biomarker technology, i.e. SA- β-Gal dyeing detections.Aging
Cell due to beta galactosidase catalysis present navy blue, so as to pass through optical microscope microscopy can distinguish senile cell with not
Senile cell, the technology have been widely used in biology agings research.Another kind of method is by low cytometric analysis point
Select senile cell and non-senile cell.In flow cytomery, need to build table in the transcription regulating region of cell ageing
Up to the reporter gene of green fluorescent protein (GFP), so as to the non-senile cell of detection by quantitative and senile cell.Measured by flow cytometry
Method can more clearly distinguish senile cell, but the radom insertion of reporter gene occurs extra differential expression, causes
Reporter gene heterogeneity, affects Detection results.Sum it up, above method is easy to detection, accuracy is higher, but process steps are multiple
It is miscellaneous, and the used time is longer, it is relatively costly, while artificial diagosis is subjective and take time and effort, and cell marking, dyeing course
Cellularity may be changed, experimental result is affected.
The content of the invention
The present invention is in order to solve the above problems, it is proposed that a kind of label-free cell detection device illuminated based on mating plate and side
Method.The present invention uniformly excites single microparticle or cell using mating plate lighting engineering as mode of excitation, obtains the low back of the body of sample
The two-dimentional light scattering pattern 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 these goals, the present invention is adopted the following technical scheme that：
It is a kind of based on mating plate illuminate label-free cell detection device, including by laser beam shaping into thin photocatalytic film mating plate
Generating unit, the thin photocatalytic film uniformly excite what is moved by the three dimensions on vertically and horizontally face that sample control unit drives
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
Unit realizes classifying on the basis of image features are extracted automatically；
The mating plate generating unit, including LASER Light Source, reflecting optics, neutral density piece and cylindrical lenses, laser light
Cylindric light beam is launched in source, after reflecting mirror controls the direction of propagation and neutral density piece adjustment light intensity, projects cylinder saturating
Mirror, cylindrical lenses modulation width of light beam produce the illumination mating plate that thickness is several microns to tens microns.
The sample control unit, including the sample chip for carrying sample suspensions, for the holder 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 collecting unit includes microcobjective and cmos detector, and image acquisition units have focusing mode and go
Two kinds of mode of operations of burnt pattern；Under focusing mode, the sample to be tested after object lens alignment is accurately positioned is focused on, and is detected by CMOS
Device records the form micro-image of single sample；Under the pattern of defocusing, object lens are capturing sample away from sample to be tested certain distance
The scattered light in space is distributed in, the distance definition goes defocus distance, captured scattered light to project cmos detector and put down for forward direction
Face, detector record the two-dimentional light scattering diagram picture of the sample.
The imaging analysis unit includes Mie photon diffusion models analog modules, characteristic parameter extraction module and supporting vector
The automatic sort module of machine, the Mie photon diffusion models analog module, according to Mie light scattering theory models, realize two to microgranule
Dimension light scattering simulation；The characteristic parameter extraction module, extracts characteristic parameter, bag according to the sample two dimension light scattering pattern of capture
The light intensity scanning and Fourier methods for including pattern is analyzed；The automatic sort module of the support vector machine, by the sample knot of collection
Fruit is divided into training set and test set, and according to the eigenvalue of input, by finding optimized parameter, Automatic Optimal classification function is realized
The automatic classification of sample.
Based on the label-free cell detection method that mating plate is illuminated, comprise the following steps：
(1) build sample chip, from two panels Conventional glass thin slice as sample chamber upper and lower surface, two panels coverslip
As pad, two ends are respectively placed in, and are clamped between glass flake, composition can carry the micro chamber of sample liquor.
(2) sample suspensions are configured and is imported in sample micro chamber by pipet, by ready sample chip by certainly
Holder processed is fixed on precision three-dimensional electricity driving displacement platform；
(3) electricity driving displacement platform movement sample is controlled, sample to be imaged is determined, makes sample to be tested be located at object lens center, and it is right
Sample is focused on, and triggers the form micro-image that cmos detector records the sample；
(4) start mating plate generating meanss, calibrate light path, determine that laser beam is reflected in passing sequentially through mating plate generating unit
The optical axis of mirror, neutral density piece and cylindrical lenses, keeps beam level to propagate, and horizontal adjustment cylindrical lenses position makes illumination
The beam waist position of mating plate is in detection object lens center；
(5) illuminate mating plate and excite sample to be tested in suspension, from individual particle or individual cells scattering light distribution in
Three dimensions, adjustment microcobjective make object lens gather the scattering in the range of respective angles under the pattern of defocusing away from sample to be tested
Light, triggers the two-dimentional light scattering pattern that cmos detector records the sample；
(6) the two-dimentional light scattering pattern that cmos detector is captured is input into Image analysis system carries out image procossing and sample
This identification and classification.
In step (2), self-control holder be made up of intermediate plate and fixed screw, can stable holding sample chip, and and
It is dissolved in optics inverted microscope.
In step (6), using algorithm of support vector machine to the automatic classification of experimental result, to pretreated experiment knot
Fruit carries out study in groups training and prediction, there is provided classification results and classification assessed value.
A kind of Dimensions recognition system to submicron particle, using the above-mentioned label-free cell detection dress illuminated based on mating plate
Put.
A kind of categorizing system to senile cell and normal cell, using the above-mentioned label-free cell inspection illuminated based on mating plate
Survey device.
Beneficial effects of the present invention are：
(1) present invention realizes the sensitive of the different scale of granule pattern of sub-micron level using the lateral two dimension light scattering pattern of detection
Detection, it is relatively more stable according to the method for one-dimensional forward direction light intensity signal than in conventional flow cytometer；
(2) present invention adopts quiescence cells suspension scanning imagery mode, has broken away from the liquid of complexity in conventional flow cytometer
Flow control system, easy to operate, relative inexpensiveness, possesses broad applicability；
(3) in traditional microscopy, generally require when preparing biological specimen smear by drying, fixation and freezing etc.
Reason, can cause certain destruction to the structure of sample, affect 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 can allow sample closer to its initial condition
Under measure, be particularly suited for living cells imaging, it is ensured that the verity and effectiveness of DATA REASONING；
(4) present invention adopts mating plate lighting system as excitation source, there is provided high-quality to excite mating plate to limit lasing region
Domain, it is to avoid excite other cells to cause interference simultaneously, there is provided be preferably imaged signal to noise ratio, flexible mating plate thickness and position control
System is easy to cell is quickly positioned and scanned, and efficient capacity usage ratio can reduce being imaged required exciting power, reduce light
Impact of the toxicity to cytoactive；
(5) harvester of the present invention possesses two mode of operations, can capture the morphological image and two of individual cells simultaneously
Dimension light scattering pattern, contributes to setting up the corresponding relation of cellularity and optical property, realizes the imaging to individual cells behavior
And analysis, the deficiency of light intensity signal is only provided when compensate for conventional flow cytometer to individual particle or cell analysis；
(6) present invention is detected to senile cell using label-free method, can be realized quick with normal cell to aging
Automatically classify, traditional method has been broken away to cell dyeing and the complicated processes of artificial diagosis；
(7) present invention is applied to the imaging identification and classification of other biological cell, possesses popularity.
Description of the drawings
Structure and principle schematic of the Fig. 1 for apparatus of the present invention；
Different mating plate thickness mating plates and its measurement result that Fig. 2 (a)-Fig. 2 (c) is provided for present example；
The micro-image of the different-grain diameter polystyrene microsphere that Fig. 3 (a)-Fig. 3 (f) is provided for present example and two-dimentional light
Scattering pattern；
Fig. 4 is fast Fourier (FFT) the analysis result schematic diagram for testing and simulating polystyrene microsphere light scattering；
Fig. 5 (a)-Fig. 5 (f) is dissipated with the micro-image of senile cell and two-dimentional light for the normal cell that present example is provided
Penetrate pattern；
Fig. 6 is human body into fiber aging and the characteristic parameter distribution schematic diagram of normal cell light scattering pattern.
Wherein, 1, laser instrument, 2, reflecting mirror, 3, neutral-density filter, 4, cylindrical lenses, 5, sample chamber, 6, sample
Room holder, 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 below in conjunction with the accompanying drawings.
A kind of label-free cell detection device and method illuminated based on mating plate, the mating plate illumination that the present invention is adopted can have
Imitate and limit excitation area, suppress the ambient interferences in light scattering imaging, control process is simple and flexible.Two-dimentional light scattering technique is carried
For the light scattering on sample to be tested polar angle and two, azimuth angular range, the letter abundanter than one-dimensional light scattering can be obtained
Breath, realizes differentiating in the microparticle size of submicron resolution level, and can avoid the dying operation and fluorescence signal of complexity
Detection process, realizes label-free identification and classification to senile cell.In the image acquisition process of the present invention, sample to be tested nature
It is placed under suspension state, does not both need the sheath flow control system of flow cytometer, processes in also having broken away from optical microscope microscopy
Drying that smear is related to, fixed equivalent damage operation, it is ensured that the verity and effectiveness of measurement.
A kind of label-free cell detection device and method illuminated based on mating plate, including mating plate generating unit, sample control
Unit, image acquisition units and data processing unit.Mating plate generating unit is by laser beam shaping into the thin of micron order thickness
Mating plate, the mating plate of generation excite sample to be tested, sample control unit to be used for controlling sample to be tested vertical into sample control unit
Three dimensions movement on straight and horizontal plane, the form micro-image and two-dimentional light scattering diagram of image acquisition units capture sample
Picture, and be sent to imaging analysis unit and carry out image procossing, feature extraction and data analysiss.
Mating plate generating unit includes LASER Light Source, reflecting optics, neutral density piece and cylindrical lenses.LASER Light Source is launched
Cylindric light beam, after reflecting mirror and neutral density piece, projects cylindrical lenses, cylindrical lenses squeezed light in one-dimensional square
Beam width, forms the illumination mating plate that thickness is 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 used for adjusting laser light beam intensity
Degree, optional transmitance are 50%, 32%, 10%, 1%, 0.1%.
Sample control unit includes special sample chip, holder and electricity driving displacement platform.Sample chip is by four 170 μm
Thick glass flake is combined, and wherein, two panels glass flake is put as pad as sample chip upper and lower surface, in addition two panels
Put at two 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 control sample respectively in x-axis, y-axis and z-axis movement, and displacement resolution is up to more than 20nm.
Image acquisition units include microcobjective and cmos detector.Image acquisition units have focusing mode and the mould that defocuses
Two kinds of mode of operations of formula.Under focusing mode, the sample to be tested after object lens alignment is accurately positioned is focused on, and is remembered by cmos detector
The form micro-image of record single sample；Under the pattern of defocusing, object lens are working away from sample to be tested certain distance, and the distance is fixed
Justice removes defocus distance for forward direction, obtains the two-dimentional light scattering diagram picture of sample to be tested in the case where forward direction removes defocus distance.In the present invention, defocus
Distance is 200 μm.Object lens collect the two-dimensional scattering light of the sample in corresponding angular range, by cmos detector label
The two-dimentional light scattering pattern of individual sample.
Imaging analysis unit includes that Mie photon diffusion models are simulated, and the feature extraction of image and support vector machine (SVM) are certainly
Dynamic sorting algorithm.In algorithm of support vector machine, the sample results of collection are divided into into training set and test set, by finding optimum 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 illuminated based on mating plate, including laser instrument 1, reflecting mirror 2, in
Property density filters 3, cylindrical lenses 4, sample chamber 5, sample room's holder 6, three-D electric displacement platform 7, microcobjective 8,
Cmos detector 9, data analysis system 10.
Specifically include procedure below：
The laser beam that laser instrument 1 sends adjusts the direction of propagation through reflecting mirror 2, and is controlled by neutral-density filter 3
Laser beam after laser intensity processed, adjustment direction and energy projects cylindrical lenses 4,4 vertical direction of cylindrical lenses modulation light
Beam, makes light beam be compressed in one-dimensional square, 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 lenses；
Mating plate is illuminated from 5 side illumination sample suspensions of sample chamber, so as to excite the sample to be tested in suspension.Holder 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, it is distributed in the detected object lens 8 of three-dimensional scattered light and detects and collect, CMOS
Detector 9 captures two-dimentional light scattering pattern and records；
The view data of record is sent 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.Light scattering imaging in, be reduce imaging process in background noise, and while ensure sample to be tested be subject to Uniform Illumination,
It is for the mating plate that the sample of different size yardstick provides different-thickness is excited in the present invention, thick with mating plate is pointedly controlled
Degree, limits excitation area, it is to avoid come from the interference of other sample scatter light.By control light beam expand multiple and cylinder is saturating
The focal length of mirror produces the mating plate of desired thickness.The light of different-thickness is realized in this example by changing different focal cylindrical lenses
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 for building；
(2) LASER Light Source is opened, calibrates light path, making laser beam pass sequentially through optical element includes that reflecting mirror, neutrality are close
The optical axis of degree piece and cylindrical lenses, keeps horizontal transmission.90 ° of Rotating cylindrical surface lens, make light beam be compressed in the horizontal direction,
Adjustment cylinder lens position, makes illumination mating plate beam waist position in detection object lens center；
(3) increase wavelength filter before cmos detector, filter excitation wavelength (532nm), it is allowed to rhodamine solution
Launch wavelength (575nm) pass through, trigger 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) to portray parameter as thickness；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 present invention for sensitivity and the accuracy of the discriminating of micron level particle size, using polystyrene mark
Quasi- microsphere carries out experimental verification and device calibration.In the present invention, selection particle diameter is that 3.87 μm and 4.19 μm of standard microsphere is
Imaged samples, obtain its micro-image and two-dimensional scattering figure, and the result by experimental result with Mie simulations carry out FFT contrasts
Analysis.In the present invention, selected microcobjective is 20 times of object lens that numerical aperture is 0.4, its corresponding detectable angular range
It it is 72.5 ° -107.5 °, so in Mie simulations, scattering polar angle and azimuth value are this scope.Mating plate is adjusted in this example
Thickness is 13 μm or so.
Concrete operation step：
(1) appropriate polystyrene microsphere stock solution is drawn, is diluted with ultra-pure water, and microsphere suspensions are imported into sample microcavity
Room；
(2) sample chip for being loaded with microsphere suspensions for preparing is fixed on three-D displacement platform by holder, is controlled
Displacement platform, focusing objective len determine the imageable target in visual field, finely tune displacement platform, make targeted microspheres be located at object lens centre position；
(3) cmos detector is triggered, records the form micro-image under focusing mode of microsphere；
(4) LASER Light Source is opened, adjusts mating plate generating unit device, make laser beam holding level and in optical element
On central optical axis, mating plate and the 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 be operated in collection of scattered light under the pattern of defocusing, triggering CMOS is visited
Device is surveyed, the two-dimentional light scattering pattern of the microsphere is recorded；
(6) according to Mie light scattering theory models, simulate the two-dimentional light scattering result of microsphere.Simulated conditions are optical source wavelength
532nm, microsphere refractive index 1.59, solution medium refractive index 1.334 residing for microsphere, scattering polar angle and azimuthal angular range are
72.5°-107.5°；
(7) the two-dimentional light scattering diagram picture respectively to testing and simulate carries out horizontal sweep, obtains the gray scale of light scatter intensity
Value curve.The quantitative analysiss of one-dimensional FFT are carried out by Fourier transformation, the frequency main peak value in FFT collection of illustrative plates can quantify light scattering
The fringe distribution of pattern.The difference of the frequency main peak value produced according to the light scattering fringe distribution of various sizes of microsphere, realizes
Size identification and classification to microsphere.In this example, the frequency step of FFT samplings is set to 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 microsphere of m is obtained under 20 times of object lens focusing task patterns.Fig. 3 (b) and Fig. 3 (e) is under the pattern of defocusing respectively
The two-dimentional light scattering pattern of the two kinds of beads for obtaining.The size difference of two kinds of microspheres is 320nm, focuses on mould in ordinary optical object lens
More difficult differentiation two kinds of particles directly perceived under formula.By contrast, the two-dimentional light scattering pattern of two kinds of beads is in striped quantity and distribution
Notable difference is presented, 7 bright fringes occurs in the light scattering pattern of 3.87 μm of microspheres, occurs 8 bright fringes in 4.19 μm of microspheres,
Therefore, it can the striped quantity by judging light scattering pattern and directly quickly distinguish two kinds of microspheres.Fig. 3 (c) and Fig. 3 (f) is root
According to the light scattering pattern result of two kinds of beads of Mie modelings, the result is presented than more consistent scattering strip with experimental result
Stricture of vagina is distributed.Difference and experiment and analog result further with fast Fourier FFT methods two kinds of beads of quantitative analysiss
Identical property, as a result as shown in Figure 4.For 4.19 μm of microspheres, simulation is 1.432 (1/ with the FFT frequency main peak values of experimental result
degree)；For 3.87 μm of microspheres, the frequency main peak value of experimental result is 1.2963 (1/degree), and analog result is
1.2825 (1/degree), the only difference of a sampling step length, also than more consistent.This example demonstrates the present invention for sub-micro
Feasibility and accuracy that rice resolution particle size differentiates.
Embodiment 3
Using the label-free cell detection device and method illuminated based on mating plate, to human body into fiber senile cell and normal
Cell carries out cell recognition and classification automatically.About 50 μm of mating plate thickness is adjusted in this example.To inducing aging through hydrogen peroxide
Human body carry out cell morphology image and its two-dimentional light scattering pattern collection into fiber senile cell and normal cell, it is random per class
55 experimental results of collection.110 light scattering diagram pictures to obtaining carry out characteristic parameter extraction, and are input into support vector machine
(SVM) algorithm is classified to two class cells automatically as characteristic parameter.
Implement：
(1) prepare the water-treated human body of dioxygen into fiber senile cell and normal cell, be configured to carefully with PBS
Born of the same parents' suspension, imports sample micro chamber, control bit moving stage and image-generating unit device, obtains the microgram of cell under focusing mode
Picture；Such as Fig. 5 (a), Fig. 5 (b).
(2) start mating plate illuminator, under the 200 μm of patterns that defocus, obtain the two-dimentional light scattering diagram of two class cells respectively
Sample, such as Fig. 5 (c), Fig. 5 (d).
(3) algorithm is detected and extracts the characteristic parameter of speckle distribution.Local intensity maxima point is detected first, determines speckle
Position and center.In the present invention, pixel more than big 6 units of strength ratio surrounding pixel is defined as maximum intensity value point.
After determining spot centers, it will be defined as less than the pixel region in the range of 6 strength differences of the central point with the local maxima
Speckle area centered on hot spot.110 experimental results of scanning, extract the numerical value ginseng of corresponding speckle count and speckle area
Number, according to the speckle count cell different from the distribution relation of speckle area sign, 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 support vector machine characteristic of division value, perform SVM algorithm when
Linear kernel function is chosen, and grader is built using 5- folding cross-validation methods.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
According to collection as test set, after repeated overlapping verifies 5 times, the meansigma methodss of the accuracy that 5 times are predicted the outcome are used as final 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 ratio；Sensitivity definition is the ratio of the quantity that senile cell correctly can be classified and senile cell total quantity；Specificity
It is defined as the ratio of quantity that normal cell correctly classified and normal cell total quantity.AUC parameters are used for assessing grader
Performance, value are closer to 1, and presentation class device performance is better.
Svm classifier result of 1 human body of table into fiber aging and normal cell
Although the above-mentioned accompanying drawing that combines is described to the specific embodiment of the present invention, not to present invention protection model
The restriction enclosed, one of ordinary skill in the art should be understood that on the basis of technical scheme those skilled in the art are not
The various modifications made by needing to pay creative work or deformation are still within protection scope of the present invention.
Claims (9)
1. a kind of label-free cell detection device illuminated based on mating plate, is characterized in that：Including by laser beam shaping into glimmer
The mating plate generating unit of piece, the thin photocatalytic film uniformly excite the three-dimensional space on vertically and horizontally face driven by sample control unit
Between the sample to be tested that moves, the form micro-image of sample and two-dimentional light scattering diagram picture are captured by image acquisition units, are transferred to into
As analytic unit is realized classifying on the basis of image features are extracted automatically；
The mating plate generating unit, including LASER Light Source, reflecting optics, neutral density piece and cylindrical lenses, LASER Light Source is sent out
Cylindric light beam is penetrated, after reflecting mirror controls the direction of propagation and neutral density piece adjustment light intensity, cylindrical lenses, post is projected
Face lens modulation width of light beam produces illumination mating plate.
2. a kind of label-free cell detection device illuminated based on mating plate as claimed in claim 1, is characterized in that：The imaging
Analytic unit includes Mie photon diffusion models analog modules, characteristic parameter extraction module and the automatic sort module of support vector machine；Institute
Mie photon diffusion models analog modules are stated, according to Mie light scattering theory models, the two-dimentional light scattering simulation to microgranule is realized；It is described
Characteristic parameter extraction module, extracts characteristic parameter according to the sample two dimension light scattering pattern of capture, sweeps including the light intensity of pattern
Retouch and analyze with Fourier methods；The sample results of collection are divided into training set and survey by the automatic sort module of the support vector machine
Examination collection, according to the eigenvalue of input, by finding optimized parameter, Automatic Optimal classification function realizes the automatic classification of sample.
3. a kind of label-free cell detection device illuminated based on mating plate as claimed in claim 1, is characterized in that：The sample
Control unit, including the sample chip for carrying sample suspensions, for the holder of fixed sample chip, and drives sample along x-axis,
Y-axis and the electricity driving displacement platform of z-axis movement.
4. a kind of label-free cell detection device illuminated based on mating plate as claimed in claim 1, is characterized in that：Described image
Collecting unit includes microcobjective and cmos detector, and image acquisition units have focusing mode and the two kinds of Working moulds of pattern that defocus
Formula, under focusing mode, the sample to be tested after object lens alignment is accurately positioned is focused on, and records single sample by cmos detector
Form micro-image, under the pattern of defocusing, object lens are capturing sample distribution in the scattering in space away from sample to be tested certain distance
Light, the distance definition go defocus distance, captured scattered light to project cmos detector plane for forward direction, and detector records the sample
This two-dimentional light scattering diagram picture.
5. a kind of Dimensions recognition system to submicron particle, is characterized in that：Including as any one of claim 1-4
Based on the label-free cell detection device that mating plate is illuminated.
6. a kind of categorizing system to senile cell and normal cell, is characterized in that：Including such as any one of claim 1-4 institute
The label-free cell detection device illuminated based on mating plate stated.
7. the label-free cell detection method for being illuminated based on mating plate, is characterized in that, comprise the following steps：
(1) build sample chip, from two panels Conventional glass thin slice as sample chamber upper and lower surface, two panels coverslip conduct
Pad, is respectively placed in two ends, and is clamped between glass flake, and composition can carry the micro chamber of sample liquor；
(2) sample suspensions are configured and is imported in sample micro chamber by pipet, ready sample chip is solid by self-control
Determine device to be fixed on precision three-dimensional electricity driving displacement platform；
(3) electricity driving displacement platform movement sample is controlled, sample to be imaged is determined, makes sample to be tested be located at object lens center, and to sample
Focus on, trigger the form micro-image that cmos detector records the sample；
(4) start mating plate generating meanss, calibrate light path, determine laser beam pass sequentially through reflecting mirror in mating plate generating unit, in
Property density sheet and cylindrical lenses optical axis, keep beam level propagate, horizontal adjustment cylindrical lenses position makes illumination mating plate
Beam waist position in detection object lens center；
(5) illumination mating plate excites the sample to be tested in suspension, and the scattering light distribution from individual particle or individual cells is in three-dimensional
Space, adjustment microcobjective make object lens gather the scattered light in the range of respective angles under the pattern of defocusing away from sample to be tested, touch
Send out the two-dimentional light scattering pattern that cmos detector records the sample；
(6) the two-dimentional light scattering pattern that cmos detector is captured is input into Image analysis system carries out image procossing and sample knowledge
Other and classification.
8. the label-free cell detection method for being illuminated based on mating plate as described in claim 7, is characterized in that：The step
(2) in, it is described self-control holder be made up of intermediate plate and fixed screw, can stable holding sample chamber, and be compatible with optics inversion
Microscope.
9. the label-free cell detection method for being illuminated based on mating plate as described in claim 7, is characterized in that：The step
(6), in, using algorithm of support vector machine to the automatic classification of experimental result, the instruction that studies in groups is carried out to pretreated experimental result
Practice and predict, there is provided classification results and classification assessed value.
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