CN110314714B

Movatterモバイル変換

Info

Publication number: CN110314714B
Application number: CN201910612791.6A
Authority: CN
Inventors: 王俊生; 成家乐
Original assignee: Dalian Maritime University
Current assignee: Dalian Maritime University
Priority date: 2019-07-09
Filing date: 2019-07-09
Publication date: 2021-06-25
Anticipated expiration: 2039-07-09
Also published as: CN110314714A

Abstract

Translated fromChinese

本发明提供一种基于三维图像特征的细胞活性状态表征监测装置及方法。本发明装置，包括PDMS基底层和电极层，电极层设置在PDMS基底层上，并与PDMS基底层上开设的卡槽键合，电极层由四块铜片电极组成，其中四块电极两两相对，从而产生非均匀电场。通过对电极施加交流电压，使得放置于旋转观察室中的细胞做匀速的旋转运动。在观察期间，滴入NaClO对细胞采取逐渐灭活操作。设置一定时间段为一个周期，通过显微镜拍摄细胞旋转一圈的过程中多个角度的图像，并且使用三维恢复技术恢复周期内细胞的三维全貌图像。连续观察多个周期，通过分析对比图像中所携带的生命体征信息，包括细胞椭球度、表面粗糙度、颜色矩阵、色素溢出率，来实时监测细胞各个周期的活性。

The present invention provides a device and method for characterizing and monitoring cell activity state based on three-dimensional image features. The device of the present invention includes a PDMS base layer and an electrode layer. The electrode layer is arranged on the PDMS base layer and is bonded to the slot opened on the PDMS base layer. The electrode layer is composed of four copper sheet electrodes, wherein two of the four electrodes are two In contrast, a non-uniform electric field is generated. By applying an alternating voltage to the electrodes, the cells placed in the rotating observation chamber are made to rotate at a uniform speed. During the observation period, the cells were gradually inactivated by dripping NaClO. A certain period of time is set as a cycle, and images of multiple angles during the rotation of the cells are captured through a microscope, and the three-dimensional overall image of the cells in the cycle is restored using a three-dimensional restoration technology. Continuously observe multiple cycles, and monitor the activity of cells in each cycle in real time by analyzing the vital signs information carried in the contrast images, including cell ellipsoid, surface roughness, color matrix, and pigment overflow rate.

Description

Cell activity state characterization monitoring device and method based on three-dimensional image characteristics

Technical Field

The invention relates to the technical field of cell manipulation and three-dimensional recovery, in particular to a cell activity state characterization monitoring device and method based on three-dimensional image characteristics.

Background

In recent years, microfluidic chips have gained more and more attention in various fields, particularly in the fields of chemical analysis, cell analysis, and the like. The micro-fluidic chip has the characteristics of simple processing, short analysis time, small detection capacity and easy integration, and is convenient for operating biological particles or cells based on the micro-fluidic chip. Therefore, the micro-fluidic chip has important influence and significance in the fields of biological diagnosis, bacteria detection and the like.

Methods for manipulating particles or cells by using the microfluidic chip generally include acoustic, magneto-mechanical, optical, and electro-mechanical methods. In the electric power, the Dielectrophoresis (DEP) technique is widely used for the manipulation of particles or cells such as capture, rotation, enrichment, separation, etc. Dielectrophoresis refers to the movement of particles under a non-uniform electric field based on the different properties of the particles and the solution.

At present, the shooting cost of the three-dimensional structure of the cell is extremely high, and the problems that the technology is not easy to master, the artificial training period is long, the material loss is serious and the like exist.

Disclosure of Invention

According to the technical problem, a cell activity state characterization monitoring device and a cell activity state characterization monitoring method based on three-dimensional image features are provided. The invention designs a non-uniform electric field, and utilizes a signal generator to generate the electric field, thereby generating dielectrophoresis force, and enabling sample cells to rotate at uniform angles so as to shoot the cells from different angles. Because the cell activity monitoring device can be used for rapidly shooting by using a microscope for monitoring, the cell activity can be monitored by extracting the characteristics of a three-dimensional cell image through a plurality of groups of photos.

The method comprises the steps of performing three-dimensional recovery on images continuously shot in a specified time period by using a three-dimensional recovery technology to obtain recovered three-dimensional images; periodically repeating the operation to obtain an image of the target cell carrying vital sign information in an observation period, and observing the change; the vital sign information is divided into an ellipsoid representing the whole size and the outline of the periphery of the cell, surface roughness representing the small distance of the cell surface and the unevenness of tiny peaks and valleys, and a color symbiotic matrix representing the color and texture information through the integration of information in images such as the size, the gray value and the like, and the images of part of cells may also carry a pigment overflow rate representing the overflow degree of the pigment in the cell.

The invention analyzes the vital sign information of the three-dimensional recovery image of the target cell in the whole period, compares the change of the ellipsoid degree of the cell in the whole monitoring process by analyzing the change of the whole size and the outline of the periphery of the cell, and the larger the ellipsoid degree is, the lower the cell activity is.

The method analyzes the vital sign information of the three-dimensional recovery image of the target cell in the whole period, compares the change of the surface roughness of the cell in the whole monitoring process by comparing the change of the small distance and the tiny peak valley of the cell surface, and the higher the surface roughness of the cell is, the lower the activity of the cell is.

The invention analyzes the vital sign information of the three-dimensional recovery image of the target cell in the whole period, compares the change of the color matrix of the cell in the whole monitoring process by analyzing the color and texture information of the cell, and the closer the color matrix is, the lower the cell activity is.

The invention analyzes the vital sign information of the three-dimensional recovery image of the target cell in the whole period, compares the change of the pigment overflow rate of the cell in the whole monitoring process by analyzing the pigment overflow degree in the cell, and the higher the pigment overflow rate is, the lower the cell activity is.

The cell activity state characterization monitoring device and method based on the three-dimensional image features are visually compared, and the change of cells in the whole monitoring process is judged, so that the activity of the cells is effectively monitored.

The technical means adopted by the invention are as follows:

a cell activity state characterization monitoring device based on three-dimensional image features comprises: a PDMS substrate layer and an electrode layer; the electrode layer is arranged on the PDMS substrate layer and is bonded with the clamping groove formed in the PDMS substrate layer.

Further, the card slot includes:

the PDMS substrate layer is provided with a square groove arranged in the center of the PDMS substrate layer, a first clamping groove and a third clamping groove which are arranged on the left side and the right side of the square groove in a facing manner, and a second clamping groove and a fourth clamping groove which are arranged on the upper side and the lower side of the square groove in a facing manner;

the electrode layer is composed of a first copper sheet electrode, a second copper sheet electrode, a third copper sheet electrode and a fourth copper sheet electrode, the bottom of the first copper sheet electrode is inserted into the first clamping groove, the bottom of the second copper sheet electrode is inserted into the second clamping groove, the bottom of the third copper sheet electrode is inserted into the third clamping groove, and the bottom of the fourth copper sheet electrode is inserted into the fourth clamping groove.

Furthermore, the first copper sheet electrode, the second copper sheet electrode, the third copper sheet electrode and the fourth copper sheet electrode in the electrode layer are all cuboid sheets with the same size, and are respectively connected with the multichannel phase generator.

Further, the relative distance between the first copper sheet electrode and the third copper sheet electrode is D1, the relative distance between the second copper sheet electrode and the fourth copper sheet electrode is D2, and D1 is equal to D2; the square groove is a rotary observation chamber, the depth is D3, the length is D4, and the width is D5; the first clamping groove, the second clamping groove, the third clamping groove and the fourth clamping groove are consistent in specification, the length is D6, the width is D7, and the depth is D8.

The invention also provides a cell activity state characterization and monitoring method based on the three-dimensional image characteristics, which comprises the following steps:

step S1: placing a small amount of target cell sample solution to be observed in a centrifuge, performing centrifugal treatment, placing buffer solution, and vibrating to uniformly mix; repeating the operation, and adding a proper amount of buffer solution to dilute to obtain a cell solution with a specified concentration;

step S2: sucking a solution containing a cell by a pipette, and transferring the solution into a square groove arranged at the center of the PDMS substrate layer;

step S3: sucking a proper amount of low-concentration NaClO solution through a pipette gun, and dropwise adding the NaClO solution into a square groove arranged in the center of the PDMS substrate layer to gradually kill cells;

step S4: the phase generator is respectively connected with a first copper sheet electrode, a second copper sheet electrode, a third copper sheet electrode and a fourth copper sheet electrode in the electrode layer, and cells move to the center of the rotary observation chamber by adjusting the phases of the direct/alternating voltage and the alternating voltage;

step S5: the phase generator is respectively connected with a first copper sheet electrode, a second copper sheet electrode, a third copper sheet electrode and a fourth copper sheet electrode in the electrode layer, and the cells start to slowly rotate in the center of the rotary observation chamber by adjusting the phases of the direct/alternating voltage and the alternating voltage;

step S6: observing the rotating cell in the rotating observation chamber through a microscope, and continuously taking images of the cell at a plurality of angles in a time period T; the three-dimensional image of the cells in the time period T is restored in real time through three-dimensional restoration to obtain the image of the target cells in the observation period, so that the vital sign information carried in the image is analyzed; the activity status of the cells is monitored in real time.

Further, the vital sign information comprises an ellipsoid representing the overall size and contour of the periphery of the cell, surface roughness representing the small intervals and the unevenness of tiny peaks and valleys on the surface of the cell, and a color symbiotic matrix representing the color and texture information of the cell, and the image of part of the cells also carries a pigment overflow rate representing the degree of pigment overflow in the cell.

Further, the monitoring method also comprises the steps of analyzing vital sign information of the three-dimensional recovery image of the target cell in the whole period, and comparing the change of the ellipsoid degree of the cell in the whole monitoring process by analyzing the change of the peripheral overall dimension and contour of the cell.

Further, the monitoring method also comprises the steps of analyzing vital sign information of the three-dimensional recovery image of the target cell in the whole period, and comparing the change of the surface roughness of the cell in the whole monitoring process by comparing the change of the small space and the tiny peak-valley of the cell surface.

Further, the monitoring method also comprises the steps of analyzing vital sign information of a three-dimensional recovery image of the target cells in the whole period, and comparing the change of the color matrix of the cells in the whole monitoring process by analyzing cell color and texture information.

Furthermore, the monitoring method also comprises the steps of analyzing the vital sign information of the three-dimensional recovery image of the target cell in the whole period, and comparing the change of the pigment overflow rate of the cell in the whole monitoring process by analyzing the pigment overflow degree in the cell.

Compared with the prior art, the invention has the following advantages:

1. the device provided by the invention generates a non-uniform electric field by applying voltage and phase change to the electrodes, utilizes dielectrophoresis force to enable target particles to rotate at a uniform angle under the action of the dielectrophoresis force, and the rotating angle and the rotating speed are controlled by the change of the electrodes.

2. The device provided by the invention has high mechanical shooting efficiency and can carry out continuous shooting in a longer time period; and extracting features related to the activity condition of the cells in the image through the recovered three-dimensional image, and analyzing vital sign information carried in the image.

3. According to the method provided by the invention, the mechanical shooting has the advantages that the defects of high manual error, high manual training cost, high material consumption and the like are avoided when a microscope is used for shooting;

for the reasons, the invention can be widely popularized in the fields of cell manipulation, three-dimensional recovery and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a schematic view of the overall structure of the apparatus of the present invention.

FIG. 2 is a schematic structural diagram of a PDMS substrate layer of the device of the present invention.

FIG. 3 is a schematic diagram of the structure of an electrode layer of the device of the present invention.

In the figure: 1. a PDMS base layer; 2. an electrode layer; 3. a first card slot; 4. a second card slot; 5. a third card slot; 6. a fourth card slot; 7. a square groove; 8. a first copper sheet electrode; 9. a second copper sheet electrode; 10. a third copper sheet electrode; 11. and a fourth copper sheet electrode.

Detailed Description

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise. Meanwhile, it should be understood that the sizes of the respective portions shown in the drawings are not drawn in an actual proportional relationship for the convenience of description. Techniques, methods, and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate. Any specific values in all examples shown and discussed herein are to be construed as exemplary only and not as limiting. Thus, other examples of the exemplary embodiments may have different values. It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, further discussion thereof is not required in subsequent figures.

In the description of the present invention, it is to be understood that the orientation or positional relationship indicated by the directional terms such as "front, rear, upper, lower, left, right", "lateral, vertical, horizontal" and "top, bottom", etc., are generally based on the orientation or positional relationship shown in the drawings, and are used for convenience of description and simplicity of description only, and in the absence of any contrary indication, these directional terms are not intended to indicate and imply that the device or element so referred to must have a particular orientation or be constructed and operated in a particular orientation, and therefore should not be considered as limiting the scope of the present invention: the terms "inner and outer" refer to the inner and outer relative to the profile of the respective component itself.

Spatially relative terms, such as "above … …," "above … …," "above … …," "above," and the like, may be used herein for ease of description to describe one device or feature's spatial relationship to another device or feature as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is turned over, devices described as "above" or "on" other devices or configurations would then be oriented "below" or "under" the other devices or configurations. Thus, the exemplary term "above … …" can include both an orientation of "above … …" and "below … …". The device may be otherwise variously oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

It should be noted that the terms "first", "second", and the like are used to define the components, and are only used for convenience of distinguishing the corresponding components, and the terms have no special meanings unless otherwise stated, and therefore, the scope of the present invention should not be construed as being limited.

Example 1

As shown in fig. 1, the present invention provides a cell activity state characterization and monitoring device based on three-dimensional image features, comprising: a PDMS substrate layer and an electrode layer; the electrode layer is arranged on the PDMS substrate layer and is bonded with the clamping groove formed in the PDMS substrate layer. The clamping grooves comprise a square groove arranged in the center of the PDMS substrate layer, a first clamping groove and a third clamping groove which are arranged on the left side and the right side of the square groove in a facing manner, and a second clamping groove and a fourth clamping groove which are arranged on the upper side and the lower side of the square groove in a facing manner; the square groove is a rotary observation chamber, the depth is D3, the length is D4, and the width is D5; the first clamping groove, the second clamping groove, the third clamping groove and the fourth clamping groove are consistent in specification, the length is D6, the width is D7, and the depth is D8.

As shown in fig. 2, the electrode layer is composed of a first copper sheet electrode, a second copper sheet electrode, a third copper sheet electrode and a fourth copper sheet electrode, the bottom of the first copper sheet electrode is inserted into the first card slot, the bottom of the second copper sheet electrode is inserted into the second card slot, the bottom of the third copper sheet electrode is inserted into the third card slot, and the bottom of the fourth copper sheet electrode is inserted into the fourth card slot. The first copper sheet electrode, the second copper sheet electrode, the third copper sheet electrode and the fourth copper sheet electrode in the electrode layer are all cuboid slices with the same size and are respectively connected with the multichannel phase generator. The relative distance between the first copper sheet electrode and the third copper sheet electrode is D1, the relative distance between the second copper sheet electrode and the fourth copper sheet electrode is D2, and D1 is equal to D2;

example 2

On the basis ofembodiment 1, the invention also provides a cell activity state characterization and monitoring method based on three-dimensional image characteristics, which comprises the following steps:

in this example, the target cell sample to be observed is an animal cell sample.

Step S1: placing a small amount of animal cell sample solution to be observed in a centrifuge, performing centrifugal treatment, placing buffer solution, and vibrating to uniformly mix; repeating the operation, and adding a proper amount of buffer solution to dilute to obtain a cell solution with a specified concentration;

step S2: sucking a solution containing an animal cell by a pipette gun, and transferring the solution into a square groove arranged at the center of the PDMS substrate layer;

step S6: observing the animal cells rotating in the rotating observation chamber through a microscope, and continuously taking images of the cells at a plurality of angles in a time period T; the three-dimensional image of the cells in the time period T is restored in real time through three-dimensional restoration to obtain the image of the target cells in the observation period, so that the vital sign information carried in the image is analyzed; the above experiment was repeated to monitor the activity status of the cells in real time.

As a preferred embodiment of the invention, the vital sign information comprises an ellipsoid representing the overall size and contour of the cell periphery, a surface roughness representing the unevenness of small pitches and tiny peaks and valleys on the cell surface, a color symbiotic matrix representing the color and texture information of the cell, and a pigment overflow rate representing the degree of pigment overflow in the cell is also carried in the image of part of the cell.

As a preferred embodiment of the invention, the vital sign information of the three-dimensional recovery image of the target cell in the whole period is analyzed, and the change of the ellipsoid degree of the cell in the whole monitoring process is compared by analyzing the change of the whole size and the outline of the periphery of the cell, wherein the larger the ellipsoid degree is, the lower the cell activity is.

In a preferred embodiment of the present invention, the vital sign information of the three-dimensional restored image of the target cell is analyzed in the whole period, and the change of the surface roughness of the cell in the whole monitoring process is compared by comparing the change of the small pitch and the tiny peak-valley of the cell surface, so that the higher the cell surface roughness is, the lower the cell activity is.

In a preferred embodiment of the present invention, vital sign information of a three-dimensional restored image of a target cell is analyzed throughout the cycle, and by analyzing cell color and texture information, changes in the color matrix of the cell are compared throughout the monitoring process, with closer color matrix, lower cell viability.

As a preferred embodiment of the invention, the vital sign information of the three-dimensional recovery image of the target cell in the whole period is analyzed, and the change of the pigment overflow rate of the cell in the whole monitoring process is compared by analyzing the pigment overflow degree in the cell, wherein the higher the pigment overflow rate is, the lower the cell activity is.

In conclusion, the cell activity state characterization monitoring device and method based on the three-dimensional image features are intuitively compared to judge the change of cells in the whole monitoring process, so that the activity of the cells is effectively monitored.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monitoring method of a cell activity state characterization monitoring device based on three-dimensional image features is characterized by comprising the following steps:

step S6: observing the rotating cell in the rotating observation chamber through a microscope, and continuously taking images of the cell at a plurality of angles in a time period T; the three-dimensional image of the cells in the time period T is restored in real time through three-dimensional restoration to obtain the image of the target cells in the observation period, so that the vital sign information carried in the image is analyzed; monitoring the activity state of the cells in real time;

the vital sign information comprises an ellipsoid representing the overall size and contour of the periphery of the cell, surface roughness representing the small distance of the cell surface and the unevenness of tiny peaks and valleys, and a color symbiotic matrix representing the color and texture information of the cell, and the image of part of the cell also carries a pigment overflow rate representing the pigment overflow degree in the cell;

the cell activity state characterization monitoring device based on the three-dimensional image characteristics comprises: a PDMS substrate layer and an electrode layer; the electrode layer is arranged on the PDMS substrate layer and is bonded with the clamping groove formed in the PDMS substrate layer.

2. The method of monitoring of claim 1, wherein the card slot comprises:

3. The monitoring method according to claim 1 or 2, wherein the first copper sheet electrode, the second copper sheet electrode, the third copper sheet electrode and the fourth copper sheet electrode in the electrode layer are all cuboid sheets with the same size, and are respectively connected with a multichannel phase generator.

4. The monitoring method according to claim 2, wherein the relative distance between the first copper sheet electrode and the third copper sheet electrode is D1, the relative distance between the second copper sheet electrode and the fourth copper sheet electrode is D2, and D1 is equal to D2; the square groove is a rotary observation chamber, the depth is D3, the length is D4, and the width is D5; the first clamping groove, the second clamping groove, the third clamping groove and the fourth clamping groove are consistent in specification, the length is D6, the width is D7, and the depth is D8.

5. The method of claim 1, further comprising the step of analyzing vital sign information of the three-dimensional restored image of the target cell throughout the cycle, comparing changes in ellipticity of the cell throughout the monitoring process by analyzing changes in overall dimensions and contours of the cell periphery to monitor cell viability.

6. The method of claim 1, further comprising the step of analyzing vital sign information of the three-dimensional restored image of the target cell throughout the cycle, comparing changes in surface roughness of the cell throughout the monitoring process by comparing changes in small pitch and minute peak-valley of the cell surface to monitor cell viability.

7. The method of claim 1, further comprising the step of analyzing vital sign information of the three-dimensional restored image of the target cell throughout the cycle, and comparing changes in the color matrix of the cell throughout the monitoring process by analyzing cell color and texture information to monitor cell viability.

8. The method for monitoring claimed in claim 1, further comprising the step of analyzing vital sign information of the three-dimensional restored image of the target cell throughout the period, and comparing the change of the pigment overflow rate of the cell throughout the monitoring process by analyzing the degree of pigment overflow in the cell to monitor the activity of the cell.