CN112782059B - Method for measuring quasi-spherical cell height direction displacement in lens-free imaging system - Google Patents
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- 210000004027 cell Anatomy 0.000 claims description 152
- 239000000523 sample Substances 0.000 claims description 40
- 238000005259 measurement Methods 0.000 claims description 10
- 239000013079 quasicrystal Substances 0.000 claims description 9
- 239000012472 biological sample Substances 0.000 claims description 4
- 210000001090 spherocyte Anatomy 0.000 claims description 3
- 238000001514 detection method Methods 0.000 description 12
- 238000012123 point-of-care testing Methods 0.000 description 5
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- G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
- G01B11/00—Measuring arrangements characterised by the use of optical techniques
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- 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
- G01N15/10—Investigating individual particles
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- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
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Abstract
The invention discloses a method for measuring the height direction displacement of quasicpheric cells in a lens-free imaging system, which comprises the steps of establishing a Fresnel diffraction model according to the characteristics of the lens-free imaging system, establishing the height direction displacement measuring model of the quasicpheric cells of the lens-free imaging system, obtaining the distance from a diffraction ring of the quasicpheric cells to a real cell boundary through a diffraction image of the quasicpheric cells in a biological cell sample to be measured, substituting the distance into the measuring model, and obtaining the height direction displacement of the quasicpheric cells. The method can solve the problem that the displacement of the quasi-spherical cells in the height direction in the microfluidic chip cannot be accurately measured, is very suitable for POTC combined with the microfluidic chip and the lens-free imaging, is simple to operate and high in instantaneity, and has very important significance for detecting the biological cells combined with the lens-free imaging system and the microfluidic.
Description
Technical Field
The invention belongs to the technical field of medical image analysis, and relates to a quasi-spherical cell height direction displacement measurement method in a lens-free imaging system.
Background
Biological cell detection and analysis are indispensable means for medical research, disease diagnosis, etc. Development of cell detection devices and methods is an important force pushing advances in biomedical research and disease diagnosis techniques. Among them, the detection and analysis of quasimeric cells plays an important role.
The traditional biological cell detection mode is mainly divided into two types, 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. In recent years, point-Of-Care Testing (POCT), which is low cost, portable, user friendly and easy to operate, is becoming increasingly popular. The realization of biological cell detection under POCT conditions is becoming a research hotspot.
The microfluidic control can accurately control the quantity and the flow rate of the sample and the reagent, thereby realizing the separation and detection of the analyte with high precision and high sensitivity. The POCT technology based on micro-flow control has the advantages of simple preparation, low reagent consumption, short response time, continuous monitoring and analysis and the like, and provides a powerful platform for biological cell detection. The principles of imaging and the volume of the system of a lensless imaging system offer great advantages over a lensed microscope, while also providing a near infinite field of view and low cost. The development of lens-free imaging systems based on microfluidic technology has thus made it possible to detect biological cells based on POCT technology.
A typical lens-free imaging system based on microfluidics consists of a light source, a microfluidic chip and a CMOS image sensor (CMOS Image Sensor, CIS). The microfluidic chip containing the cells is directly arranged on the CIS, and as no lens exists between the cells and the CIS, a cell image cannot be truly formed on the CIS, and only diffraction fringes of the cells can be formed. Meanwhile, due to the introduction of the microfluidic chip, some technical difficulties are brought to lens-free imaging. Since the micro-channel of the microfluidic chip has a certain height, the cells will move in the height direction when the cells move within the micro-channel. The distance from the cell to the imaging surface varies and the variation is random. The uncertainty in the cell-to-imaging plane distance will seriously affect the imaging effect of a lens-less imaging system, and is detrimental to further analysis of the image. Therefore, how to use diffraction images to obtain the displacement information of cells in the height direction of the microfluidic chip has important significance in biological cell detection.
Disclosure of Invention
The invention aims to provide a method for measuring the height direction displacement of a quasicpheric cell in a lens-free imaging system, which utilizes quasicpheric cell diffraction fringes to realize the measurement of the height direction displacement of the quasicpheric cell.
The technical scheme adopted by the invention is that the method for measuring the quasi-spherical cell height direction displacement in the lens-free imaging system is implemented according to the following steps:
step 1, preparing a standard biological cell sample containing standard spherocytes;
step 2, placing the microfluidic chip on a CMOS image sensor of a lens-free imaging system, and introducing a biological cell sample into the microfluidic chip;
step 3, turning on a monochromatic light source of the lens-free imaging system to enable light to irradiate on a microfluidic chip with a standard biological sample;
step 4, opening an image acquisition device, and acquiring diffraction images of the quasimeric cells in the standard biological cell sample by using a CMOS image sensor;
step 5, establishing a Fresnel diffraction model according to the imaging principle and characteristics of the lens-free imaging system;
step 6, calculating the diffraction light intensity amplitude of Fresnel diffraction to obtain a relation between the position of a diffraction ring and the distance from a quasicpheric cell to an imaging surface, and establishing a quasicpheric cell height direction displacement measurement model of the lens-free imaging system, wherein the distance from the quasicpheric cell to the imaging surface is the distance from a standard biological cell sample to a CMOS image sensor;
and 7, collecting diffraction images of the quasicpheric cells in the biological cell sample to be detected, obtaining the distance from the diffraction ring of the quasicpheric cells to the real cell boundary according to the diffraction images, and calculating the distance from the quasicpheric cells to the imaging surface in the biological cell sample to be detected through a height direction displacement measurement model, wherein the distance from the quasicpheric cells to the imaging surface is the height direction displacement of the quasicpheric 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:
since the distance from the light source to the imaging surface in the lens-free system is a finite distance, the diffraction principle accords with the Fresnel diffraction, and since the diffraction edge of the quasi-spherical cell is not a standard circle, the diffraction accords with the straight-edge Fresnel diffraction, the diffraction occurs on a semi-infinite plane taking a sharp straight edge as a boundary, and the light intensity I on the 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 standard biological cell sample, s' is the distance from the standard biological cell sample to the CMOS image sensor, x is the distance from the diffraction ring to the real cell boundary in the diffraction image of the standard biological cell sample, λ is the wavelength of light, r ', λ are both known amounts, and s', x are both unknown amounts.
Step 6 is specifically implemented according to the following steps:
the following formula is obtained from the formula (1),
is obtained by the following formulas (2) and (3):
from formulas (4) and (5):
at the positions of the bright stripes and the dark stripes of the fresnel diffraction pattern of the quasimeric cells in the standard biological cell sample, C (w) =s (w) is satisfied, and the diffraction intensity amplitude obtained by substituting C (w) =s (w) into formula (6) is:
substituting the formula (3) into the formula (7) to obtain a relational expression of the distance from the diffraction ring of the quasicrystal cell to the real cell boundary and the distance from the quasicrystal cell to the imaging surface in the standard biological cell sample, wherein the relational expression is the quasicrystal cell height direction displacement measurement model of the lens-free imaging system.
The step 7 is specifically implemented according to the following steps:
collecting diffraction images of the quasicpheric cells in the biological cell sample to be detected through an image collecting device, obtaining the distance from the diffraction ring of the quasicpheric cells to the real cell boundary according to the diffraction images, substituting the distance from the diffraction ring to the real cell boundary into a measuring model of the quasicpheric cell height direction displacement, obtaining the distance from the quasicpheric cells to the imaging surface, wherein the distance from the quasicpheric cells to the imaging surface is the height direction displacement of the quasicpheric cells, and measuring the height direction displacement of the quasicpheric cells in the biological cell sample to be detected.
The beneficial effects of the invention are as follows:
the invention relates to a method for measuring the height direction displacement of quasicpheric cells in a lens-free imaging system, which comprises the steps of acquiring diffraction images of quasicpheric cells in micro-flow control by using the lens-free imaging system, and obtaining the relationship between the positions of quasicpheric cell diffraction rings and the distance from cells to an imaging surface; the method can accurately measure the displacement of the quasimmons in the height direction, is very suitable for POTC combined with micro-fluidic chip and lens-free imaging, can solve the problem that the displacement of the quasimmons in the height direction in the micro-fluidic chip cannot be accurately measured, is simple to operate and high in instantaneity, and has very important significance for biological cell detection under a lens-free imaging system.
Drawings
Fig. 1 is a schematic diagram of a lens-less imaging system to which the present invention is applied.
In the figure, a light source 8, a micropore 9, a convex lens 10, parallel rays 11, a light shielding plate 12, a microfluidic chip 13 and a CMOS image sensor 14.
Detailed Description
The invention will be described in detail below with reference to the drawings and the detailed description.
The invention relates to a method for measuring the height direction displacement of quasicpheric cells in a lens-free imaging system. The lens-free imaging system, as shown in fig. 1, comprises a light shielding plate 12, a light source 8, a convex lens 10, a micro-fluidic chip 13 and a CMOS image sensor 14, wherein the light source 8 is a point light source, light rays of the light source 8 penetrate through micropores 9 and are refracted through the convex lens 10 to form parallel light rays 11, the parallel light rays 11 irradiate on the micro-fluidic chip 13, a standard biological sample is introduced into the micro-fluidic chip 13 and is placed on the CMOS image sensor 14, and the CMOS image sensor 14 collects diffraction images of biological cells to be detected.
The invention discloses a method for measuring the height direction displacement of quasimerical cells in a lens-free imaging system, which is implemented according to the following steps:
step 1, preparing a standard biological cell sample containing standard spherocytes.
Step 2, placing the microfluidic chip 13 on a CMOS image sensor 14 of a lens-free imaging system, and introducing a biological cell sample into the microfluidic chip 13.
And 3, turning on a monochromatic light source 8 of the lens-free imaging system to enable light to irradiate on a microfluidic chip 13 which is communicated with a standard biological sample.
And 4, turning on an image acquisition device, and acquiring diffraction images of the quasimeric cells in the standard biological cell sample by using the CMOS image sensor 14.
Step 5, establishing a Fresnel diffraction model according to the imaging principle and characteristics of the lens-free imaging system;
the step 5 specifically comprises the following steps:
since the distance from the light source to the imaging surface in the lens-free system is a finite distance, the diffraction principle accords with the Fresnel diffraction, and since the diffraction edge of the quasi-spherical cell is not a standard circle, the diffraction accords with the straight-edge Fresnel diffraction, the diffraction occurs on a semi-infinite plane taking a sharp straight edge as a boundary, and the light intensity I on the 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 standard biological cell sample, s' is the distance from the standard biological cell sample to the CMOS image sensor, x is the distance from the diffraction ring to the real cell boundary in the diffraction image of the standard biological cell sample, λ is the wavelength of light, r ', λ are both known amounts, and s', x are both unknown amounts.
Step 6, calculating the diffraction light intensity amplitude of Fresnel diffraction to obtain a relation between the position of a diffraction ring and the distance from a quasicpheric cell to an imaging surface, and establishing a quasicpheric cell height direction displacement measurement model of the lens-free imaging system, wherein the distance from the quasicpheric cell to the imaging surface is the distance from a standard biological cell sample to the CMOS image sensor 14;
the step 6 is specifically implemented according to the following steps:
the following formula is obtained from the formula (1),
is obtained by the following formulas (2) and (3):
from formulas (4) and (5):
at the positions of the bright stripes and the dark stripes of the fresnel diffraction pattern of the quasimeric cells in the standard biological cell sample, C (w) =s (w) is satisfied, and the diffraction intensity amplitude obtained by substituting C (w) =s (w) into formula (6) is:
substituting the formula (3) into the formula (7) to obtain a relational expression of the distance from the diffraction ring of the quasicrystal cell to the real cell boundary and the distance from the quasicrystal cell to the imaging surface in the standard biological cell sample, wherein the relational expression is the quasicrystal cell height direction displacement measurement model of the lens-free imaging system.
And 7, collecting diffraction images of the quasicpheric cells in the biological cell sample to be detected, obtaining the distance from the diffraction ring of the quasicpheric cells to the real cell boundary according to the diffraction images, and calculating the distance from the quasicpheric cells to the imaging surface in the biological cell sample to be detected through a height direction displacement measurement model, wherein the distance from the quasicpheric cells to the imaging surface is the height direction displacement of the quasicpheric cells.
The step 7 is specifically implemented according to the following steps:
collecting diffraction images of the quasicpheric cells in the biological cell sample to be detected through an image collecting device, obtaining the distance from the diffraction ring of the quasicpheric cells to the real cell boundary according to the diffraction images, substituting the distance from the diffraction ring to the real cell boundary into a measuring model of the quasicpheric cell height direction displacement, obtaining the distance from the quasicpheric cells to the imaging surface, wherein the distance from the quasicpheric cells to the imaging surface is the height direction displacement of the quasicpheric cells, and measuring the height direction displacement of the quasicpheric cells in the biological cell sample to be detected.
Claims (3)
1. The method for measuring the quasi-spherical cell height direction displacement in the lens-free imaging system is characterized by comprising the following steps of:
step 1, preparing a standard biological cell sample containing standard spherocytes;
step 2, placing the microfluidic chip on a CMOS image sensor of a lens-free imaging system, and introducing a biological cell sample into the microfluidic chip;
step 3, turning on a monochromatic light source of the lens-free imaging system to enable light to irradiate on a microfluidic chip with a standard biological sample;
step 4, opening an image acquisition device, and acquiring diffraction images of the quasimeric cells in the standard biological cell sample by using a CMOS image sensor;
step 5, establishing a Fresnel diffraction model according to the imaging principle and characteristics of the lens-free imaging system;
the step 5 specifically comprises the following steps:
since the distance from the light source to the imaging surface in the lens-free system is a finite distance, the diffraction principle accords with the Fresnel diffraction, and since the diffraction edge of the quasi-spherical cell is not a standard circle, the diffraction accords with the straight-edge Fresnel diffraction, the diffraction occurs on a semi-infinite plane taking a sharp straight edge as a boundary, and the light intensity I on the 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 standard biological cell sample, s' is the distance from the standard biological cell sample to the CMOS image sensor, x is the distance from the diffraction ring to the real cell boundary in the diffraction image of the standard biological cell sample, lambda is the wavelength of light, r ', lambda are both known quantities, and s', x are both unknown quantities;
step 6, calculating the diffraction light intensity amplitude of Fresnel diffraction to obtain a relation between the position of a diffraction ring and the distance from a quasicpheric cell to an imaging surface, and establishing a quasicpheric cell height direction displacement measurement model of the lens-free imaging system, wherein the distance from the quasicpheric cell to the imaging surface is the distance from a standard biological cell sample to a CMOS image sensor;
the step 6 is specifically implemented according to the following steps:
the following formula is obtained from the formula (1),
is obtained by the following formulas (2) and (3):
from formulas (4) and (5):
at the positions of the bright stripes and the dark stripes of the fresnel diffraction pattern of the quasimeric cells in the standard biological cell sample, C (w) =s (w) is satisfied, and the diffraction intensity amplitude obtained by substituting C (w) =s (w) into formula (6) is:
substituting the formula (3) into the formula (7) to obtain a relational expression of the distance from the diffraction ring of the quasicrystal cell to the real cell boundary and the distance from the quasicrystal cell to the imaging surface in the standard biological cell sample, wherein the relational expression is a quasicrystal cell height direction displacement measurement model of the lens-free imaging system;
and 7, collecting diffraction images of the quasicpheric cells in the biological cell sample to be detected, obtaining the distance from the diffraction ring of the quasicpheric cells to the real cell boundary according to the diffraction images, and calculating the distance from the quasicpheric cells to the imaging surface in the biological cell sample to be detected through a height direction displacement measurement model, wherein the distance from the quasicpheric cells to the imaging surface is the height direction displacement of the quasicpheric cells.
2. The method for measuring the height direction displacement of the quasimlobal cells in the lens-free imaging system according to claim 1, wherein the monochromatic light source is a point light source or a parallel light source.
3. The method for measuring the height direction displacement of the quasimlobular cells in the lens-free imaging system according to claim 1, wherein the step 7 is specifically performed according to the following steps:
collecting diffraction images of the quasicpheric cells in the biological cell sample to be detected through an image collecting device, obtaining the distance from the diffraction ring of the quasicpheric cells to the real cell boundary according to the diffraction images, substituting the distance from the diffraction ring to the real cell boundary into a measuring model of the quasicpheric cell height direction displacement, obtaining the distance from the quasicpheric cells to the imaging surface, wherein the distance from the quasicpheric cells to the imaging surface is the height direction displacement of the quasicpheric cells, and measuring the height direction displacement of the quasicpheric cells in the biological cell sample to be detected.
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