CN114088588B - Three-dimensional red blood cell size measuring method based on lens-free imaging - Google Patents
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- 210000003743 erythrocyte Anatomy 0.000 title claims abstract description 171
- 238000003384 imaging method Methods 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 17
- 238000005259 measurement Methods 0.000 claims abstract description 29
- 239000012472 biological sample Substances 0.000 claims abstract description 10
- 210000004027 cell Anatomy 0.000 claims description 27
- 230000000737 periodic effect Effects 0.000 claims description 7
- 238000000691 measurement method Methods 0.000 claims description 6
- 239000000523 sample Substances 0.000 claims description 6
- 238000013519 translation Methods 0.000 claims description 6
- 230000003822 cell turnover Effects 0.000 claims description 3
- 238000009792 diffusion process Methods 0.000 claims description 3
- 238000006073 displacement reaction Methods 0.000 claims description 3
- 230000010354 integration Effects 0.000 claims description 3
- 230000000877 morphologic effect Effects 0.000 claims description 3
- 230000007306 turnover Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
- 150000001875 compounds Chemical class 0.000 claims 3
- 210000004369 blood Anatomy 0.000 claims 1
- 239000008280 blood Substances 0.000 claims 1
- 230000001678 irradiating effect Effects 0.000 abstract 1
- 238000010586 diagram Methods 0.000 description 7
- 230000036544 posture Effects 0.000 description 6
- 210000000601 blood cell Anatomy 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000012123 point-of-care testing Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004159 blood analysis Methods 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/02—Investigating particle size or size distribution
- G01N15/0205—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging
- G01N15/0211—Investigating a scatter or diffraction pattern
Abstract
The invention discloses a three-dimensional dimension measuring method of red blood cells based on lens-free imaging, which comprises the steps of preparing biological sample solution; setting up a lens-free imaging image acquisition system; irradiating light on the microfluidic chip; collecting a diffraction image of the red blood cells; establishing a diffraction light intensity model; establishing an arc edge diffraction light intensity model; establishing a three-dimensional measurement model of red blood cells; and collecting diffraction patterns of the red blood cells to be detected, and identifying and estimating the sizes and the thicknesses of the red blood cells by using a three-dimensional dimension measurement model of the red blood cells. According to the invention, the three-dimensional size of the red blood cells is measured by utilizing the diffraction ring patterns corresponding to the red blood cells in a special posture in the motion process of the red blood cells in the micro-fluidic chip of the lens-free imaging system.
Description
Technical Field
The invention belongs to the technical field of medical diagnosis and image analysis, and relates to a three-dimensional erythrocyte dimension measuring method based on lens-free imaging.
Background
The Red Blood Cell (RBC) size is an important parameter for medical diagnosis. Whole blood cell count (CBC) is one of the common blood examination methods prescribed by doctors for clinical diagnosis and prognosis. In the international society for standardization of hematology (ICSH) 2014, blood cell analyzer evaluation guidelines, parameters related to red blood cell size, such as red blood cell distribution width (RDW), were also determined as part of CBC.
At present, blood cells are detected and measured mainly by two methods, one is an artificial method, and an optical microscope is used for cell detection; the other is cell detection by means of a flow cytometer. However, the detection devices of the two methods have the problems of large volume, high price, high requirement on professional knowledge of operators and the like. These problems limit the possibilities Of blood cell Testing in Point-Of-Care Testing (POCT).
Disclosure of Invention
The invention aims to provide a three-dimensional dimension measurement method of erythrocytes based on lens-free imaging, which utilizes diffraction ring patterns corresponding to erythrocytes in a special posture in the motion process of erythrocytes in a microfluidic chip of a lens-free imaging system to realize the measurement of the three-dimensional dimension of erythrocytes.
The technical scheme adopted by the invention is as follows: a three-dimensional measurement method of red blood cells based on lens-free imaging, the lens-free imaging system comprises a light source, a microfluidic chip and a CMOS image sensor, and is implemented according to the following steps:
step 1, preparing a biological sample solution;
step 2, constructing a lens-free imaging image acquisition system, fixing a microfluidic chip on a CMOS image sensor, and determining the distance between a monochromatic light source and the sensor;
step 3, introducing the biological sample into the microfluidic chip, and turning on a monochromatic light source to irradiate light on the microfluidic chip;
step 4, after the biological sample moves stably in the microfluidic chip, opening an image acquisition device, and acquiring a diffraction image of the red blood cells by using a CMOS image sensor;
step 5, according to the characteristics of a lens-free imaging system and the morphological characteristics of red blood cells, a diffraction light intensity model is established by adopting a Fresnel straight-edge diffraction theory;
step 6, establishing an arc edge diffraction light intensity model according to the Fresnel straight edge diffraction light intensity model;
step 7, taking diffraction superposition and light intensity attenuation into consideration to establish a three-dimensional measurement model of the red blood cells;
and 8, collecting diffraction patterns of the red blood cells to be detected, and identifying and estimating the sizes and the thicknesses of the red blood cells by using a three-dimensional dimension measurement model of the red blood cells.
The present invention is also characterized in that,
the monochromatic light source is a point light source or a parallel light source.
The step 5 is specifically as follows:
erythrocytes are quasi-spherical cells, and the diffraction edge is not a standard circle, so the diffraction at any point on the edge of erythrocytes can be regarded as diffraction on the tangent line of the point. The straight-edge Fresnel diffraction is met, diffraction occurs on a semi-infinite plane taking a sharp straight edge as a boundary, and the light intensity I on an imaging plane, namely, the straight-edge Fresnel diffraction model is expressed as:
in the formula (1), I 0 The average light intensity is that C (w) and S (w) are Fresnel integral;
c (w) and S (w) are expressed as:
in the formula (3), r 'is the distance from the light source to the biological cell sample, s' is the distance from the biological cell sample to the CMOS image sensor, x is the distance from the diffraction ring to the real red blood cell boundary in the red blood cell diffraction image, and λ is the wavelength of light.
The step 6 is specifically as follows:
for curved edges, the diffracted light has a larger diffusion space, and therefore the diffracted light intensity decays more than for straight edges. Since diffraction at any point on the arc is regarded as diffraction on a tangent line of the point, based on straight-edge fresnel diffraction, amplitude attenuation and periodic variation are considered, and the arc diffraction light intensity distribution, namely an arc diffraction model, is expressed as:
in the formula (4), I arc The light intensity is diffracted at the arc edge, and alpha is an attenuation coefficient;
obtained according to the difference between the arc edge diffraction light intensity integral area and the straight edge diffraction light intensity integral area
Wherein R is arc Is the radius of the arc edge,x is the distance from the arc edge to the arc center, and therefore the attenuation coefficient α is expressed as
The step 7 is specifically implemented according to the following steps:
step 7.1, according to the fresnel diffraction theory, the radius of the first bright ring in the diffraction image of the red blood cells is larger than the diameter of the red blood cells, so that the diffraction image of the red blood cells is a multi-position diffraction superposition result, the diffraction superposition influences the light intensity distribution of the diffraction image of the red blood cells, the center of the red blood cells is taken as a coordinate origin, and the superposition light intensity distribution of the diffraction image of the red blood cells, namely, a lens-free imaging light intensity model is expressed as follows:
in the formula (7), I cell Is the absolute light intensity distribution of the diffraction pattern of the quasicpheric cells;
step 7.2, constructing a three-dimensional measurement model of the red blood cells
The diffraction pattern is the result of integration, and for a moving point, the real and imaginary parts of the integrated function change sign multiple times, so the contributions of the elements generally cancel each other out, destructively interfere, but for an element at a stationary point, the stationary point is called a critical point or pole, the variation of the integrated function is slow, and the contributions of the elements do not cancel each other out;
the cells in the microfluidic chip have two moving modes, namely translation and overturning, the translation does not change the relative position of the points on the edge where diffraction occurs, the light intensity distribution of the diffraction pattern is not influenced, the overturning changes the relative position of the points on the edge where diffraction occurs, the light intensity distribution of the diffraction pattern shows a periodic change rule, and the red blood cells are identified according to the light intensity distribution characteristics of the diffraction pattern;
determining saddle points A, A ', B, B' of red blood cells as rest points, wherein saddle points A, A ', B, B' are positioned at the edge of a disk of the red blood cells, the connecting line of AA 'and the connecting line of BB' are both disk diameters and are mutually perpendicular, when the red blood cells are overturned by taking BB 'as axes, the red blood cells have larger relative displacement at points B and B', so that the light intensity of a diffraction image is smaller at points B and B ', the light intensity of the diffraction image corresponds to the minimum light intensity point of a first bright ring of the red blood cell diffraction image, the relative position of the diffraction image is unchanged at points A and A', the light intensity point of the first bright ring of the diffraction image corresponds to the maximum light intensity point of the diffraction image, and using the characteristics as a three-dimensional measurement model of the red blood cells for identifying the red blood cells, namely when two light intensity minimum points exist in the BB 'direction and two light intensity maximum points exist in the AA' direction, the red blood cells are;
since the shape of the red blood cells is a biconcave disk, when the red blood cells rotate around BB 'as an axis, and the red blood cells turn over to the surface of the cell disk and are vertical to the direction of the light source, the diffraction patterns at A, A' and B, B 'have the same diffraction ring positions, and the radius R of the first bright ring of the diffraction pattern at the point A, A' is according to the three-dimensional dimension measurement model of the red blood cells 1 I.e. radius R of red blood cells, R is measured 1 Obtaining the radius r of the red blood cells; when the red blood cells are turned over to make the surface of the biconcave disk parallel to the direction of the light source, the light intensity of the diffraction pattern reaches the minimum value at the point B, B' and the radius R of the first bright ring of the diffraction pattern at the point A, A 2 I.e. the thickness h of the red blood cells, R is measured 2 Obtaining the thickness h of the red blood cells, wherein under the gesture, the radius of the arc B and the arc B 'is smaller and is the rotation axis of the red blood cells, the diffraction patterns cannot be effectively overlapped, and the radius R of the first bright ring of the diffraction pattern passes through the B, B' point 3 The relationship with cell radius measures the radius r of the red blood cells.
The step 8 is specifically as follows:
the diffraction images of red blood cell turnover at different times in the micro-fluidic chip are acquired through the image acquisition device, the light intensity of a first bright ring of the diffraction images is calculated, and according to the constructed three-dimensional measurement model of the red blood cells, when two light intensity minimum points exist in the BB 'direction and two light intensity maximum points exist in the AA' direction, the red blood cells can be judged. When the diffraction patterns at four points A, A ', B, B ' have the same light intensity, the radius of the red blood cells is measured according to the three-dimensional measurement model of the red blood cells, and when the diffraction patterns at points A and A ' have the maximum light intensity and the diffraction patterns at points B and B ' have the minimum light intensity, the thickness of the red blood cells is measured according to the three-dimensional measurement model of the red blood cells by using the radius of the first bright ring of the diffraction patterns at points A and A '.
The beneficial effects of the invention are as follows:
according to the three-dimensional dimension measuring method of the erythrocytes based on the lens-free imaging, when the erythrocytes move in the microfluidic chip, the postures of the erythrocytes in the microfluidic chip are determined through periodic characteristics, and then the three-dimensional dimension of the erythrocytes is obtained through diffraction images under special postures; the method is very suitable for POTC combined with micro-fluidic chip and lens-free imaging, can solve the problem that the three-dimensional size of red blood cells cannot be determined in micro-fluidic, is simple to operate and high in real-time, and has important significance for medical diagnosis.
Drawings
FIG. 1 is a schematic diagram of a lens-free imaging system used in a three-dimensional measurement method of red blood cells based on lens-free imaging according to the present invention;
fig. 2 is a schematic diagram corresponding to a side view and a top view of a red blood cell turning model in step 7 of the three-dimensional dimension measurement method of red blood cells based on lens-free imaging, in which fig. 2 (a) is a schematic diagram corresponding to a side view and a top view when red blood cells are turned over to a cell disk surface and are perpendicular to a light source direction, and fig. 2 (b) is a schematic diagram corresponding to a side view and a top view when red blood cells are turned over to make the biconcave disk surface parallel to the light source direction;
fig. 3 is a schematic diagram of the positions of the first bright ring at different positions in two typical postures in step 7 of the three-dimensional measurement method for red blood cells based on lens-free imaging according to the present invention, wherein fig. 3 (a) is a schematic diagram of the positions of the first bright ring when red blood cells are turned over to the cell disc surface perpendicular to the light source direction, and fig. 3 (b) is a schematic diagram of the positions of the first bright ring when red blood cells are turned over to make the biconcave disc surface parallel to the light source direction.
In the figure, 9. A monochromatic light source, 10. A micropore, 11. A convex lens, 12. A light shielding plate, 13. A microfluidic chip and 14. A CMOS image sensor.
Detailed Description
The invention will be described in detail below with reference to the drawings and the detailed description.
The invention relates to a three-dimensional dimension measuring method of red blood cells based on lens-free imaging, as shown in figure 1, wherein a lens-free imaging system comprises a light source, a microfluidic chip and a CMOS image sensor, and is implemented according to the following steps:
and step 1, preparing biological sample solution with specific solubility.
And 2, constructing a lens-free imaging image acquisition system, fixing the microfluidic chip on the CMOS image sensor, and determining the distance between the monochromatic light source and the sensor.
And step 3, introducing the biological sample into the microfluidic chip, and turning on a monochromatic light source, wherein the monochromatic light source is a point light source or a parallel light source, so that light irradiates the microfluidic chip.
And 4, after the biological sample moves stably in the microfluidic chip, opening an image acquisition device, and acquiring a diffraction image of the red blood cells by using a CMOS image sensor.
Step 5, according to the characteristics of a lens-free imaging system and the morphological characteristics of red blood cells, a diffraction light intensity model is established by adopting a Fresnel straight-edge diffraction theory;
the step 5 specifically comprises the following steps:
erythrocytes are quasi-spherical cells, and the diffraction edge is not a standard circle, so the diffraction at any point on the edge of erythrocytes can be regarded as diffraction on the tangent line of the point. The straight-edge Fresnel diffraction is met, diffraction occurs on a semi-infinite plane taking a sharp straight edge as a boundary, and the light intensity I on an imaging plane, namely, the straight-edge Fresnel diffraction model is expressed as:
in the formula (1), I 0 The average light intensity is that C (w) and S (w) are Fresnel integral;
c (w) and S (w) are expressed as:
in the formula (3), r 'is the distance from the light source to the biological cell sample, s' is the distance from the biological cell sample to the CMOS image sensor, x is the distance from the diffraction ring to the real red blood cell boundary in the red blood cell diffraction image, and λ is the wavelength of light.
Step 6, establishing an arc edge diffraction light intensity model according to the Fresnel straight edge diffraction light intensity model;
the step 6 specifically comprises the following steps:
for curved edges, the diffracted light has a larger diffusion space, and therefore the diffracted light intensity decays more than for straight edges. Since diffraction at any point on the arc is regarded as diffraction on a tangent line of the point, based on straight-edge fresnel diffraction, amplitude attenuation and periodic variation are considered, and the arc diffraction light intensity distribution, namely an arc diffraction model, is expressed as:
in the formula (4), I arc The light intensity is diffracted at the arc edge, and alpha is an attenuation coefficient;
obtained according to the difference between the arc edge diffraction light intensity integral area and the straight edge diffraction light intensity integral area
Wherein R is arc Is the radius of the arc edge, x is the distance from the arc edge to the arc center, and therefore the attenuation coefficient α is expressed as
Step 7, taking diffraction superposition and light intensity attenuation into consideration to establish a three-dimensional measurement model of the red blood cells;
step 7.1, according to the fresnel diffraction theory, the radius of the first bright ring in the diffraction image of the red blood cells is larger than the diameter of the red blood cells, so that the diffraction image of the red blood cells is a multi-position diffraction superposition result, the diffraction superposition influences the light intensity distribution of the diffraction image of the red blood cells, the center of the red blood cells is taken as a coordinate origin, and the superposition light intensity distribution of the diffraction image of the red blood cells, namely, a lens-free imaging light intensity model is expressed as follows:
in the formula (7), I cell Is the absolute light intensity distribution of the diffraction pattern of the quasicpheric cells;
step 7.2, constructing a three-dimensional measurement model of the red blood cells
The diffraction pattern is the result of integration, and for a moving point, the real and imaginary parts of the integrated function change sign multiple times, so the contributions of the elements generally cancel each other out, destructively interfere, but for an element at a stationary point, the stationary point is called a critical point or pole, the variation of the integrated function is slow, and the contributions of the elements do not cancel each other out;
the cells in the microfluidic chip have two moving modes, namely translation and overturning, the translation does not change the relative position of the points on the edge where diffraction occurs, the light intensity distribution of the diffraction pattern is not influenced, the overturning changes the relative position of the points on the edge where diffraction occurs, the light intensity distribution of the diffraction pattern shows a periodic change rule, and the red blood cells are identified according to the light intensity distribution characteristics of the diffraction pattern;
as shown in fig. 2, saddle points A, A ', B, B' of the red blood cells are determined to be rest points, saddle points A, A ', B, B' are all located at the disc edges of the red blood cells, the connecting line of AA 'and the connecting line of BB' are all disc diameters and are perpendicular to each other, when the red blood cells are turned around by the BB 'as axes, the red blood cells have larger relative displacement at points B and B', so that the light intensity of the diffraction image is smaller at points B and B ', which correspond to the minimum light intensity point of the first bright ring of the diffraction image of the red blood cells, and the relative positions of the diffraction image are unchanged at points a and a', which correspond to the maximum light intensity point of the first bright ring of the diffraction image, the characteristics are utilized as a three-dimensional measurement model of the red blood cells for red blood cell identification, namely, when there are two light intensity minimum points in the direction of BB ', and two light intensity maximum points in the direction AA', the red blood cells are obtained.
Since the shape of the red blood cells is a biconcave disk, when the red blood cells are pivoted about BB ', and the red blood cells are turned over to the surface of the cell disk to be perpendicular to the direction of the light source, as shown in FIG. 2 (a), in this posture, the diffraction patterns at A, A', B, B 'have the same diffraction ring positions, the diffraction light intensity distribution of which is shown in FIG. 3 (a), the radius R of the first bright ring of the diffraction pattern at A, A' is calculated from the three-dimensional measurement model of red blood cells 1 I.e. radius R of red blood cells, R is measured 1 Obtaining the radius r of the red blood cells; when the red blood cells are turned over to make the biconcave disk surface parallel to the light source direction, as shown in FIG. 2 (b), in this posture, the diffraction pattern light intensity at B, B' reaches a minimum value, and the diffraction light intensity distribution thereof is as shown in FIG. 3 (b), radius R of the first bright ring of the diffraction pattern at A, A 2 I.e. the thickness h of the red blood cells, R is measured 2 Obtaining the thickness h of the red blood cells, wherein under the gesture, the radius of the arc B and the arc B 'is smaller and is the rotation axis of the red blood cells, the diffraction patterns cannot be effectively overlapped, and the radius R of the first bright ring of the diffraction pattern passes through the B, B' point 3 The relationship with cell radius measures the radius r of the red blood cells.
And 8, collecting diffraction patterns of the red blood cells to be detected, and identifying and estimating the sizes and the thicknesses of the red blood cells by using a three-dimensional dimension measurement model of the red blood cells.
The diffraction images of red blood cell turnover at different times in the micro-fluidic chip are acquired through the image acquisition device, the light intensity of a first bright ring of the diffraction images is calculated, and according to the constructed three-dimensional measurement model of the red blood cells, when two light intensity minimum points exist in the BB 'direction and two light intensity maximum points exist in the AA' direction, the red blood cells can be judged. When the diffraction patterns at four points A, A ', B, B ' have the same light intensity, the radius of the red blood cells is measured according to the three-dimensional measurement model of the red blood cells, and when the diffraction patterns at points A and A ' have the maximum light intensity and the diffraction patterns at points B and B ' have the minimum light intensity, the thickness of the red blood cells is measured according to the three-dimensional measurement model of the red blood cells by using the radius of the first bright ring of the diffraction patterns at points A and A '.
Claims (4)
1. The three-dimensional red blood cell size measuring method based on lens-free imaging, the lens-free imaging system comprises a light source, a microfluidic chip and a CMOS image sensor, and is characterized by comprising the following steps:
step 1, preparing a biological sample solution;
step 2, constructing a lens-free imaging image acquisition system, fixing a microfluidic chip on a CMOS image sensor, and determining the distance between a monochromatic light source and the sensor;
step 3, introducing the biological sample into the microfluidic chip, and turning on a monochromatic light source to irradiate light on the microfluidic chip;
step 4, after the biological sample moves stably in the microfluidic chip, opening an image acquisition device, and acquiring a diffraction image of the red blood cells by using a CMOS image sensor;
step 5, according to the characteristics of a lens-free imaging system and the morphological characteristics of red blood cells, a diffraction light intensity model is established by adopting a Fresnel straight-edge diffraction theory;
step 6, establishing an arc edge diffraction light intensity model according to the Fresnel straight edge diffraction light intensity model;
step 7, taking diffraction superposition and light intensity attenuation into consideration to establish a three-dimensional measurement model of the red blood cells;
the step 7 is specifically implemented according to the following steps:
step 7.1, according to the fresnel diffraction theory, the radius of the first bright ring in the diffraction image of the red blood cells is larger than the diameter of the red blood cells, so that the diffraction image of the red blood cells is a multi-position diffraction superposition result, the diffraction superposition influences the light intensity distribution of the diffraction image of the red blood cells, the center of the red blood cells is taken as a coordinate origin, and the superposition light intensity distribution of the diffraction image of the red blood cells, namely, a lens-free imaging light intensity model is expressed as follows:
（7）
in the formula (7), the amino acid sequence of the compound,is the absolute light intensity distribution of the diffraction pattern of the quasicpheric cells, ">Is the average light intensity +.>Is the intensity of arc diffraction->Is the radius of the arc edge, < >>Is the distance from the arc edge to the arc center;
step 7.2, constructing a three-dimensional measurement model of the red blood cells
The diffraction pattern is the result of integration, and for a moving point, the real and imaginary parts of the integrated function change sign multiple times, so the contributions of the elements generally cancel each other out, destructively interfere, but for an element at a stationary point, the stationary point is called a critical point or pole, the variation of the integrated function is slow, and the contributions of the elements do not cancel each other out;
the cells in the microfluidic chip have two moving modes, namely translation and overturning, the translation does not change the relative position of the points on the edge where diffraction occurs, the light intensity distribution of the diffraction pattern is not influenced, the overturning changes the relative position of the points on the edge where diffraction occurs, the light intensity distribution of the diffraction pattern shows a periodic change rule, and the red blood cells are identified according to the light intensity distribution characteristics of the diffraction pattern;
determining saddle points A, A ', B, B' of red blood cells as rest points, wherein saddle points A, A ', B, B' are positioned at the edge of a disk of the red blood cells, the connecting line of AA 'and the connecting line of BB' are both disk diameters and are mutually perpendicular, when the red blood cells are overturned by taking BB 'as axes, the red blood cells have larger relative displacement at points B and B', so that the light intensity of a diffraction image is smaller at points B and B ', the light intensity of the diffraction image corresponds to the minimum light intensity point of a first bright ring of the red blood cell diffraction image, the relative position of the diffraction image is unchanged at points A and A', the light intensity point of the first bright ring of the diffraction image corresponds to the maximum light intensity point of the diffraction image, and using the characteristics as a three-dimensional measurement model of the red blood cells for identifying the red blood cells, namely when two light intensity minimum points exist in the BB 'direction and two light intensity maximum points exist in the AA' direction, the red blood cells are;
since the shape of the red blood cells is biconcave disk, when the red blood cells rotate around BB 'as the axis, the red blood cells turn over to the surface of the cell disk and are vertical to the direction of the light source, the diffraction patterns at A, A' and B, B 'have the same diffraction ring positions, and the radius of the first bright ring of the diffraction pattern at A, A' is calculated according to the three-dimensional dimension measurement model of the red blood cellsI.e.the radius r of the erythrocytes, measuring +.>Obtaining the radius r of the red blood cells; when the red blood cells are turned over to make the surface of the biconcave disk parallel to the direction of the light source, the light intensity of the diffraction pattern reaches the minimum value at the point B, B' and the radius of the first bright ring of the diffraction pattern at the point A, A +.>Namely the thickness h of the red blood cells, measuring +.>Obtaining the thickness h of the red blood cells, in this attitude, the radii of the B and B 'circular arcs are small and are the axes of rotation of the red blood cells, the diffraction patterns of which cannot be effectively superimposed, the radius of the first bright ring of the diffraction pattern passing through the B, B' point +.>The relationship between the cell radius and the radius r of the red blood cells is measured;
step 8, collecting diffraction patterns of the red blood cells to be detected, and identifying and estimating the size and thickness of the red blood cells by using a three-dimensional dimension measurement model of the red blood cells;
the step 8 specifically comprises the following steps:
the diffraction images of red blood cell turnover at different times in the micro-fluidic chip are collected through the image collecting device, the light intensity of a first bright ring of the diffraction images is calculated, and according to the constructed three-dimensional measurement model of the red blood cells, when two light intensity minimum points exist in the BB 'direction and two light intensity maximum points exist in the AA' direction, the red blood cells can be judged; when the diffraction patterns at four points A, A ', B, B ' have the same light intensity, the radius of the red blood cells is measured according to the three-dimensional measurement model of the red blood cells, and when the diffraction patterns at points A and A ' have the maximum light intensity and the diffraction patterns at points B and B ' have the minimum light intensity, the thickness of the red blood cells is measured according to the three-dimensional measurement model of the red blood cells by using the radius of the first bright ring of the diffraction patterns at points A and A '.
2. A three-dimensional measurement method of red blood cells based on lens-free imaging according to claim 1, wherein the monochromatic light source is a point light source or a parallel light source.
3. The method for three-dimensional measurement of red blood cells based on lens-free imaging according to claim 2, wherein said step 5 is specifically:
the red blood cells are quasi-spherical cells, and the diffraction edge of the red blood cells is not a standard circle, so that the diffraction at any point of the red blood cells edge can be regarded as the diffraction on the tangent line of the point, the diffraction accords with the straight-edge Fresnel diffraction, the diffraction occurs on a semi-infinite plane with a sharp straight edge as a boundary, and the light intensity on an imaging planeI.e. straight-sided fresnel diffraction model is expressed as:
（1）
in the formula (1), the components are as follows,is the average light intensity +.>、/>Is a fresnel integral;
、/>expressed as:
（2）
（3）
in the formula (3), the amino acid sequence of the compound,is the distance of the light source to the biological cell sample, +.>Is the distance of the biological cell sample to the CMOS image sensor,/->Is the distance of the diffraction ring from the true red blood cell boundary in the diffraction image of red blood cells, +.>Is the wavelength of light.
4. A method for three-dimensional measurement of red blood cells based on lens-free imaging according to claim 3, wherein said step 6 is specifically:
for an arc edge, the diffracted light has a larger diffusion space, so the attenuation of the diffracted light intensity is larger than that of a straight line edge, and because the diffraction of any point on the arc edge is regarded as diffraction on a tangent line of the point, the straight-edge Fresnel diffraction is based on the consideration of amplitude attenuation and periodic variation, and the arc edge diffraction light intensity distribution, namely an arc edge diffraction model is expressed as:
（4）
in the formula (4), the amino acid sequence of the compound,is the intensity of arc diffraction->Is the attenuation coefficient;
obtained according to the difference between the arc edge diffraction light intensity integral area and the straight edge diffraction light intensity integral area
（5）
Wherein the method comprises the steps ofIs the radius of the arc edge, < >>Is the distance from the arc edge to the arc center, thus the attenuation coefficient +.>Represented as
（6）。
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