CN114519716A - Online monitoring method and system for NK cell culture form - Google Patents
Online monitoring method and system for NK cell culture form Download PDFInfo
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Abstract
The invention relates to the technical field of cell culture monitoring methods, and discloses an NK cell culture form on-line monitoring method and system, which comprise a signal acquisition sensor, a signal conversion circuit, a data analysis system, a communication circuit and a human-computer interaction interface; the signal acquisition sensor comprises a temperature sensor, a microwave sensor and a microscopic monitoring sensor; the signal conversion circuit comprises an amplifying circuit, a filter circuit and an AD conversion circuit; the data analysis system analyzes the real-time culture form of the NK cells through a software algorithm according to the information acquired by the signal acquisition sensor, and uploads the analysis result to a human-computer interaction interface through a communication circuit; the human-computer interaction interface comprises real-time image display of NK cell culture form, display of cell temperature information, display of medium information in a culture dish, calling of historical data, comparative analysis of data, statistics of data change trend and waveform drawing. The invention realizes intelligent and high-precision monitoring of the culture form of the NK cells.
Description
Technical Field
The invention relates to the technical field of monitoring methods of cell culture, in particular to an online monitoring method and system of NK cell culture form.
Background
Natural killer cells (NK) are important immune cells of the body, not only involved in anti-tumor, anti-viral infection and immune regulation, but also involved in the development of hypersensitivity reactions and autoimmune diseases in some cases, capable of recognizing target cells, killing mediators. As a common experimental technique, NK cell culture has a great influence on research and application of various subjects such as cytology, virology and immunology. When the cell culture is performed, it is usually observed every day in real time to know the growth state, the consumption and evaporation of the culture solution in the culture dish, and the like. To date, there have been a variety of techniques available for analyzing cells, mainly conventional optical techniques and dielectric spectroscopy, which, while enabling cell monitoring, have advantages and disadvantages. In the existing NK cell culture form online detection technology, the intelligent degree is low, and the accuracy of detected data is low.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides an online monitoring method and system for NK cell culture form. The technical scheme adopted by the invention comprises the following steps:
an NK cell culture form on-line monitoring system comprises a signal acquisition sensor, a signal conversion circuit, a data analysis system, a communication circuit and a human-computer interaction interface; wherein the content of the first and second substances,
the signal acquisition sensor comprises a temperature sensor, a microwave sensor and a microscopic monitoring sensor, and is used for respectively acquiring temperature information of NK cells in the culture process, medium information in a culture dish and morphological evolution information of the NK cells in the culture process, converting the temperature information and the microwave information into voltage signals and then outputting the voltage signals to the signal conversion circuit, and directly connecting signals of the microscopic monitoring sensor with the data analysis system;
the signal conversion circuit comprises an amplifying circuit, a filtering circuit and an AD conversion circuit, and is used for amplifying and filtering voltage signals output by the temperature sensor and the microwave sensor, inputting the amplified and filtered voltage signals into the AD conversion circuit, converting analog quantity into digital quantity and then inputting the digital quantity into the data analysis system;
the data analysis system analyzes the real-time culture form of the NK cells according to the NK cell temperature information, the medium information in the culture dish and the NK cell form evolution information acquired by the signal acquisition sensor through a built-in processing strategy and a microscopic image recognition tracking algorithm, and uploads the analysis result to a human-computer interaction interface through a communication circuit; meanwhile, the data analysis system also has a real-time data storage function and continuously stores the NK cell temperature information, the medium information in the culture dish and the NK cell form evolution information;
the human-computer interaction interface comprises real-time image display of NK cell culture form, display of NK cell temperature information, display of medium information in a culture dish, calling of historical data, comparative analysis of data, statistics of data change trend and waveform drawing; the real-time image display of the NK cell culture form is a core module of a human-computer interaction interface, the display of the NK cell temperature information and the display of medium information in a culture dish are integrated in the real-time image of the NK cell culture form, and the real-time image display is amplified and displayed when a user needs; calling historical data, clicking an operation button in a human-computer interaction interface by a user, sending a control instruction to a data analysis system by the human-computer interaction interface, and feeding back the stored historical data to the human-computer interaction interface for display; the data comparison and analysis method comprises the steps that a user clicks an operation button in a human-computer interaction interface, the human-computer interaction interface sends a control instruction to a data analysis system, the data analysis system calls related historical data and performs comparison and analysis according to the user requirements, and then the result is uploaded to the human-computer interaction interface to be displayed; and (3) counting the data change trend and drawing a waveform, clicking an operation button in a human-computer interaction interface by a user, sending a control instruction to a data analysis system by the human-computer interaction interface, calling related historical data by the data analysis system, counting according to the user requirement, drawing a result into a waveform diagram, transmitting the result back to the human-computer interaction interface for display, adding an analysis line or an analysis point in the waveform diagram of the human-computer interaction interface by the user according to the requirement, and printing and outputting the analysis result.
In a preferred embodiment, the temperature sensor comprises a thermistor substrate, a Wheatstone bridge measuring circuit and a voltage amplifying circuit; the thermistor substrate is made of a platinum material, two connecting wires are led out and connected to one arm of the Wheatstone bridge; the Wheatstone bridge matches a bridge arm resistor 1, a bridge arm resistor 2 and a bridge arm resistor 3 according to the resistance value change range of the thermistor substrate; the positive pole of a power supply of the power supply voltage is connected with the input ends of the bridge arm resistor 1 and the bridge arm resistor 2, and the reference ground end is connected with the output ends of the bridge arm resistor 3 and the thermistor; the signal output voltage is led out from the connection point of the bridge arm resistor 1 and the bridge arm resistor 3 and the connection point of the bridge arm resistor 2 and the thermistor, amplified by the voltage amplifying circuit and then output outwards.
In a preferred embodiment, the microscopic monitoring sensor comprises a point light source, a photoelectric coupling element, an objective lens and a data acquisition card; the point light source is located below the NK cell culture dish, the objective lens is located above the NK cell culture dish, the photoelectric coupling element is located above the objective lens, and the data acquisition card is connected to the photoelectric coupling element and sends acquired and converted data to the data analysis system.
In a preferred embodiment, the signal conversion circuit and the data analysis system are integrated in a group of computers, and the hardware part of the computer consists of 4 PCB boards, including 1 motherboard, 2 signal conversion circuit boards and 1 data analysis circuit board; the 2 signal conversion circuit boards are divided into a temperature signal conversion circuit board and a microwave signal conversion circuit board, the 2 signal conversion circuit boards and the data analysis circuit board are plugged on a mother board, and a data acquisition card of the microscopic monitoring sensor is directly plugged and communicated with the data analysis circuit board.
In a preferred embodiment, the data analysis system comprises a temperature analysis module, a microwave analysis module and a microscopic image recognition and tracking module.
In a preferred embodiment, the temperature analysis module adopts a linear curve fitting method, a start point value and an end point value of a linear curve are calibrated when the data analysis system leaves a factory and are input into the storage device, an abscissa of the linear curve fitting is a digital quantity obtained by performing AD conversion on temperature information by the signal conversion circuit, and a ordinate of the linear curve fitting is a temperature value.
In a preferred embodiment, the microwave analysis module receives two paths of voltage signals, which are respectively a digital quantity obtained by converting the microwave signal and a digital quantity obtained by converting the output voltage of the reference circuit, and performs homodromous component cancellation on the two paths of digital quantity signals to obtain a single microwave signal transformation quantity.
In a preferred embodiment, the method for on-line monitoring of NK cell culture morphology comprises the steps of:
s1: and (3) carrying out threshold segmentation on the microscopic image by using a maximum between-class variance algorithm, and separating the NK cell image from the background image, wherein the realization principle is as follows: let the gray scale value range of the microscopic image be 0, L]And the gray value is
Has a number of pixels of
Total number of pixels in the micrograph
The gray value is further obtained as
The probability of occurrence of the pixel of (a) is:
setting the gray threshold value of the microscopic image divided by the maximum inter-class variance method as TThreshold(s)The gray value of the microscopic image is divided into two sections U according to the gray value smaller than the gray threshold value and larger than the gray threshold value1And U2Wherein the interval U1Has K gray values in the interval U2If there are L-K gray values, the average gray value calculation method in two intervals is as follows:
by traversing all gray values, when a certain gray value causes a variance value
When the maximum value is reached, the optimal threshold value is set;
s2: filtering noise signals which are distributed in a disorder manner in a background image by adopting a median filtering algorithm, carrying out binarization processing on an NK cell outline by combining an OTSU algorithm, and carrying out non-operation on the segmented NK cell image and the background image to obtain a complete NK cell image;
s3: NK cell contours were extracted using Sobel operators, which included two sets of 3 × 3 computational matrices:
after the NK cell contour is obtained, in order to improve the definition of the NK cell contour, the OTSU algorithm is used again for binarization processing, and thus a complete NK cell image is obtained.
In a preferred embodiment, the method for on-line monitoring of NK cell culture morphology further comprises:
r1: the microwave emitter is used as an excitation source and generates a high-frequency microwave signal into the circuit, the frequency of the high-frequency microwave signal is between 100MHz and 1GHz, and the amplitude of the high-frequency microwave signal is between 5dB and 50 dB;
r2: the high-frequency microwave signal enters a power amplifier, the amplification factor is between 30 times and 100 times, and the amplified signal is input into a power distributor;
r3: the power distributor equally divides the high-frequency microwave signal into two parts which are respectively input into the microstrip inductor and the reference circuit;
r4: the microstrip inductor is arranged below the NK cell culture dish, the reference circuit is arranged adjacent to the microstrip inductor, an electromagnetic field is enhanced when a high-frequency microwave signal passes through the microstrip inductor, the high-frequency microwave signal is attenuated after the electromagnetic field passes through the NK cell culture dish, and the attenuation coefficient is related to a solution and a medium in the NK cell culture dish;
r5: and shaping the attenuated high-frequency microwave signal and the output signal of the reference circuit, converting the shaped signal into a voltage signal, and inputting the voltage signal into a data analysis system after passing through a signal conversion circuit for judging the real-time state of the NK cell culture dish.
In a preferred embodiment, the data analysis circuit board comprises a DSP processor, an SRAM static memory and a NorFlash memory; the DSP processor is used for data analysis, the SRAM static memory is used for storing a temperature fitting curve and a microscopic image recognition tracking algorithm, and the NorFlash memory is used for storing historical data.
Compared with the prior art, the on-line monitoring method and the system for the culture form of the NK cells have the advantages that:
the invention provides an NK cell culture form on-line monitoring method and system based on the image recognition technology and the intelligent monitoring technology which are rapidly developed at present, so that the temperature information of NK cells, the medium information in a culture dish and the form evolution information of the NK cells in the culture process are monitored on line, historical data are stored in real time, a user is supported to call the historical data when needed and analyze and process the historical data, and the intelligent and high-precision monitoring of the NK cell culture form is realized.
Drawings
FIG. 1 is a schematic diagram of an on-line monitoring system for NK cell culture morphology;
FIG. 2 is a schematic diagram of a microscopic image recognition and tracking algorithm based on threshold segmentation and image enhancement techniques;
FIG. 3 is a schematic diagram of the microwave sensor assembly;
FIG. 4 is a schematic diagram of a data analysis circuit board assembly;
FIG. 5 is a schematic diagram of the installation of a microstrip inductor;
FIG. 6 is a schematic of a linear curve fit of a temperature analysis module;
FIG. 7 is a schematic view of the installation of the microscopic monitoring sensor;
FIG. 8 is an assembly view of PCB boards;
FIG. 9 is a schematic diagram of the measurement principle of a Wheatstone bridge;
FIG. 10 is a schematic diagram of the components of the human-computer interface.
Detailed Description
In the following description, for purposes of explanation and not limitation, specific details are set forth, such as particular system structures, techniques, etc. in order to provide a thorough understanding of the embodiments of the invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced in other embodiments that depart from these specific details. In other instances, detailed descriptions of well-known systems, devices, circuits, and methods are omitted so as not to obscure the description of the present disclosure with unnecessary detail.
Referring to fig. 1, an online monitoring method and system for NK cell culture morphology is provided, including a signal acquisition sensor, a signal conversion circuit, a data analysis system, a communication circuit, and a human-computer interaction interface; wherein the content of the first and second substances,
the signal acquisition sensor comprises a temperature sensor, a microwave sensor and a microscopic monitoring sensor, and is used for respectively acquiring temperature information of NK cells in the culture process, medium information in a culture dish and morphological evolution information of the NK cells in the culture process, converting the temperature information and the microwave information into voltage signals and then outputting the voltage signals to the signal conversion circuit, and directly connecting signals of the microscopic monitoring sensor with the data analysis system;
the temperature sensor comprises a thermistor substrate, a Wheatstone bridge measuring circuit and a voltage amplifying circuit; specifically, referring to fig. 9, the thermistor substrate is made of a platinum material, two connection wires are led out, and are connected to one arm of the wheatstone bridge; the Wheatstone bridge matches a bridge arm resistor 1, a bridge arm resistor 2 and a bridge arm resistor 3 according to the resistance value change range of the thermistor substrate; the positive pole of a power supply of the power supply voltage is connected with the input ends of the bridge arm resistor 1 and the bridge arm resistor 2, and the reference ground end is connected with the output ends of the bridge arm resistor 3 and the thermistor; the signal output voltage is led out from the connection point of the bridge arm resistor 1 and the bridge arm resistor 3 and the connection point of the bridge arm resistor 2 and the thermistor, amplified by the voltage amplifying circuit and then output outwards.
Referring to fig. 3, the microwave sensor is based on the microwave principle, and includes a microwave transmitter, a power amplifier, a power distributor, and a microstrip inductor, and the operation steps are as follows:
r1: the microwave emitter is used as an excitation source to generate a high-frequency microwave signal into the circuit, the frequency of the high-frequency microwave signal is between 100MHz and 1GHz, and the amplitude is between 5dB and 50 dB;
r2: the high-frequency microwave signal enters a power amplifier, the amplification factor is between 30 times and 100 times, and the amplified signal is input into a power distributor;
r3: the power distributor equally divides the high-frequency microwave signal into two parts which are respectively input into the microstrip inductor and the reference circuit;
r4: the microstrip inductor is arranged below the NK cell culture dish, the reference circuit is arranged adjacent to the microstrip inductor, an electromagnetic field is enhanced when a high-frequency microwave signal passes through the microstrip inductor, the high-frequency microwave signal is attenuated after the electromagnetic field passes through the NK cell culture dish, and the attenuation coefficient is related to a solution and a medium in the NK cell culture dish;
r5: and shaping the attenuated microwave signal and the output signal of the reference circuit, converting the shaped microwave signal into a voltage signal, and inputting the voltage signal into a data analysis system after passing through a signal conversion circuit for judging the real-time state of the NK cell culture dish.
The microscopic monitoring sensor comprises a point light source, a photoelectric coupling element, an objective lens and a data acquisition card; further, referring to fig. 5 and 7, the point light source preferably uses an LED lamp, and the LED lamp is disposed below the NK cell culture dish, the objective lens is disposed above the NK cell culture dish, the photoelectric coupling element is disposed above the objective lens, and the data acquisition card is connected to the photoelectric coupling element and transmits the acquired and converted data to the data analysis system.
The signal conversion circuit comprises an amplifying circuit, a filtering circuit and an AD conversion circuit, and is used for amplifying and filtering voltage signals output by the temperature sensor and the microwave sensor, inputting the amplified and filtered voltage signals into the AD conversion circuit, converting analog quantity into digital quantity and then inputting the digital quantity into the data analysis system;
referring to fig. 8, the signal conversion circuit and the data analysis system are integrated in a set of computer, and the hardware part of the computer is composed of 4 PCB boards, including 1 motherboard, 2 signal conversion circuit boards, and 1 data analysis circuit board; the 2 signal conversion circuit boards are divided into a temperature signal conversion circuit board and a microwave signal conversion circuit board, the 2 signal conversion circuit boards and the data analysis circuit board are plugged on a mother board, and a data acquisition card of the microscopic monitoring sensor is directly plugged and communicated with the data analysis circuit board.
Further, referring to fig. 4, the data analysis circuit board includes a DSP processor, an SRAM static memory, and a NorFlash memory, where the DSP processor is used for data analysis, the SRAM static memory is used for storing a temperature fitting curve and a microscopic image recognition tracking algorithm, and the NorFlash memory is used for storing historical data.
The data analysis system analyzes the real-time culture form of the NK cells according to the NK cell temperature information, the medium information in the culture dish and the NK cell form evolution information acquired by the signal acquisition sensor through a built-in processing strategy and a microscopic image recognition tracking algorithm, and uploads the analysis result to a human-computer interaction interface through a communication circuit; the data analysis system also has a real-time data storage function and continuously stores the NK cell temperature information, the medium information in the culture dish and the NK cell form evolution information;
the data analysis system comprises a temperature analysis module, a microwave analysis module and a microscopic image recognition tracking module.
The temperature analysis module adopts a linear curve fitting method, referring to fig. 6, the start point value and the end point value of the linear curve are calibrated when the data analysis system leaves the factory and are input into the storage device, the abscissa of the linear curve fitting is a digital quantity obtained by performing AD conversion on temperature information by the signal conversion circuit, and the ordinate is a temperature value.
The microwave analysis module receives the two paths of voltage signals, respectively converts the digital quantity of the microwave signals and the digital quantity of the reference circuit output voltage, and cancels the homodromous components of the two paths of digital quantity signals to obtain a single microwave signal conversion quantity.
The microscopic image recognition and tracking module specifically relates to a microscopic image recognition and tracking algorithm based on threshold segmentation and image enhancement technology, and further, with reference to fig. 2, the implementation steps are as follows:
s1: and (3) carrying out threshold segmentation on the microscopic image by using a maximum between-class variance algorithm, and separating the NK cell image from the background image, wherein the realization principle is as follows: assume that the range of gray values of the microscopic image is [0, L]And the gray value is
Has a number of pixels of
Total number of pixels in the microscopic image is
The gray value is further obtained as
The probability of occurrence of the pixel of (a) is:
setting the gray threshold value of the microscopic image divided by the maximum inter-class variance method as TThreshold(s)The gray value of the microscopic image is divided into two sections U according to the gray value smaller than the gray threshold value and larger than the gray threshold value1And U2Wherein the interval U1Has K gray values in the interval U2If there are L-K gray values, the average gray value calculation method in two intervals is as follows:
by traversing all gray values, when a certain gray value causes a variance value
When the maximum value is reached, the optimal threshold value is set;
s2: filtering noise signals which are distributed in a disorder manner in a background image by adopting a median filtering algorithm, carrying out binarization processing on an NK cell contour by combining an OTSU algorithm, and carrying out non-operation on the segmented NK cell image and the background image to obtain a complete NK cell image;
s3: NK cell contours were extracted using Sobel operators, which included two sets of 3 × 3 computational matrices, as follows:
after the NK cell contour is obtained, in order to improve the definition of the NK cell contour, the OTSU algorithm is used again for binarization processing, and thus a complete NK cell image is obtained.
Referring to fig. 10, the human-computer interface includes real-time image display of NK cell culture morphology, display of NK cell temperature information, display of medium information in a culture dish, retrieval of historical data, comparative analysis of data, statistics of data variation trend, and waveform drawing; the real-time image display of the NK cell culture form is a core module of a human-computer interaction interface, the display of the NK cell temperature information and the display of medium information in a culture dish are integrated in the real-time image of the NK cell culture form, and the real-time image display is amplified and displayed when a user needs; calling historical data, clicking an operation button in a human-computer interaction interface by a user, sending a control instruction to a data analysis system by the human-computer interaction interface, and feeding back the stored historical data to the human-computer interaction interface for display; the data comparison and analysis method comprises the steps that a user clicks an operation button in a human-computer interaction interface, the human-computer interaction interface sends a control instruction to a data analysis system, the data analysis system calls related historical data and performs comparison and analysis according to the user requirements, and then the result is uploaded to the human-computer interaction interface to be displayed; and the user clicks an operation button in the human-computer interaction interface to send a control instruction to the data analysis system, the data analysis system calls related historical data and performs statistics according to the user requirements, then draws the result into a waveform diagram and transmits the waveform diagram back to the human-computer interaction interface to be displayed, and the user adds analysis lines or analysis points in the waveform diagram of the human-computer interaction interface as required and prints and outputs the analysis result.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various equivalent changes, modifications, substitutions and alterations can be made herein without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims (10)
1. An on-line monitoring system of NK cell culture form, characterized in that, the on-line monitoring system of NK cell culture form includes:
the signal acquisition sensor is used for respectively acquiring temperature information of NK cells in the culture process, medium information in a culture dish and morphological evolution information of the NK cells in the culture process through a temperature sensor, a microwave sensor and a microscopic monitoring sensor, converting the temperature information and the microwave information into voltage signals and then outputting the voltage signals to the signal conversion circuit, and the microscopic monitoring sensor signals are directly connected with the data analysis system;
the signal conversion circuit amplifies and filters voltage signals output by the temperature sensor and the microwave sensor through the amplifying circuit, the filtering circuit and the AD conversion circuit, inputs the amplified and filtered voltage signals into the AD conversion circuit, converts analog quantity into digital quantity and then inputs the digital quantity into the data analysis system;
the data analysis system analyzes the real-time culture form of the NK cells through a built-in processing strategy and a microscopic image recognition tracking algorithm according to the NK cell temperature information, the medium information in the culture dish and the NK cell form evolution information acquired by the signal acquisition sensor, and uploads an analysis result to a human-computer interaction interface through a communication circuit; the data analysis system also has a real-time data storage function, and continuously stores the NK cell temperature information, the medium information in the culture dish and the NK cell form evolution information;
the human-computer interaction interface is used for realizing real-time image display of the culture form of the NK cells, display of temperature information of the NK cells, display of medium information in a culture dish, calling of historical data, comparative analysis of data, statistics of data change trend and waveform drawing; the display of the NK cell temperature information and the display of the medium information in the culture dish are integrated in a real-time image of the NK cell culture form; the human-computer interaction interface sends a control instruction to the data analysis system, and the stored historical data is fed back to the human-computer interaction interface for display; the data comparison and analysis is implemented by clicking an operation button in a human-computer interaction interface by a user, the human-computer interaction interface sends a control instruction to a data analysis system, and the data analysis system calls related historical data and performs comparison and analysis according to the user requirements, and then uploads the result to the human-computer interaction interface for display; and the user clicks an operation button in the human-computer interaction interface to send a control instruction to the data analysis system, the data analysis system calls related historical data and performs statistics according to the user requirements, then draws the result into a waveform diagram and transmits the waveform diagram back to the human-computer interaction interface to be displayed, and the user adds analysis lines or analysis points in the waveform diagram of the human-computer interaction interface as required and prints and outputs the analysis result.
2. The system for on-line monitoring of NK cell culture morphology according to claim 1, wherein said temperature sensor comprises: the device comprises a thermistor substrate, a Wheatstone bridge measuring circuit and a voltage amplifying circuit;
the thermistor substrate is made of a platinum material, two connecting wires are led out and connected to one arm of the Wheatstone bridge; the Wheatstone bridge matches a bridge arm resistor 1, a bridge arm resistor 2 and a bridge arm resistor 3 according to the resistance value change range of the thermistor substrate; the positive pole of a power supply of the power supply voltage is connected with the input ends of the bridge arm resistor 1 and the bridge arm resistor 2, and the reference ground end is connected with the output ends of the bridge arm resistor 3 and the thermistor; the signal output voltage is led out from the connection point of the bridge arm resistor 1 and the bridge arm resistor 3 and the connection point of the bridge arm resistor 2 and the thermistor, amplified by the voltage amplifying circuit and then output outwards.
3. The system for on-line monitoring of NK cell culture morphology according to claim 1, wherein said microscopic monitoring sensor comprises: the device comprises a point light source, a photoelectric coupling element, an objective lens and a data acquisition card; the pointolite is located NK cell culture dish below, objective is located NK cell culture dish top, optoelectronic coupling component is located the objective top, data acquisition card is connected to optoelectronic coupling component to data transmission after will gathering the conversion to data analysis system.
4. The system for on-line monitoring of NK cell culture morphology according to claim 1, wherein the signal conversion circuit and the data analysis system are integrated in a set of computer, the hardware part of the computer is composed of 4 PCB boards, including 1 motherboard, 2 signal conversion circuit boards, and 1 data analysis circuit board, the 2 signal conversion circuit boards are respectively a temperature signal conversion circuit board and a microwave signal conversion circuit board, the 2 signal conversion circuit boards and the data analysis circuit board are plugged on the motherboard, and the data acquisition card of the microscopic monitoring sensor is directly plugged in and communicated with the data analysis circuit board.
5. The system for on-line monitoring of NK cell culture morphology according to claim 1, characterized in that the data analysis system comprises a temperature analysis module, a microwave analysis module, and a microscopic image recognition tracking module.
6. The system for on-line monitoring of the culture morphology of NK cells according to claim 5, wherein the temperature analysis module adopts a linear curve fitting method, and the starting point value and the end point value of a linear curve are calibrated and input into a storage device when the data analysis system leaves a factory; the abscissa of the linear curve fitting is a digital quantity obtained by performing AD conversion on the temperature information by the signal conversion circuit, and the ordinate is a temperature value.
7. The system for on-line monitoring of NK cell culture form according to claim 5, wherein the microwave analysis module receives two voltage signals, which are respectively a digital quantity after microwave signal conversion and a digital quantity after reference circuit output voltage conversion, and performs homodromous component cancellation on the two digital quantity signals to obtain a single microwave signal transformation quantity.
8. The system for on-line monitoring of NK cell culture morphology according to claim 4, characterized in that the data analysis circuit board comprises a DSP processor, an SRAM static memory and a NorFlash memory, wherein the DSP processor is used for data analysis, the SRAM static memory is used for storing a temperature fitting curve and a microscopic image recognition tracking algorithm, and the NorFlash memory is used for storing historical data.
9. The on-line monitoring method of the NK cell culture morphology on-line monitoring system according to any of claims 1 to 8, characterized in that it comprises the following steps:
s1: and (3) carrying out threshold segmentation on the microscopic image by using a maximum between-class variance algorithm, and separating the NK cell image from the background image, wherein the realization principle is as follows: assuming a gray value range of a microscopic imageIs enclosed as [0, L]And the gray value is
Has a number of pixels of
Total number of pixels in the micrograph
The gray value is further obtained as
The probability of occurrence of the pixel of (a) is:
dividing the microscopic image by the maximum inter-class variance method to obtain a gray threshold value TThreshold(s)Dividing the gray value of the microscopic image into two intervals U according to the gray value of the microscopic image being smaller than the gray threshold value and larger than the gray threshold value1And U2Wherein the interval U1Has K gray values in the interval U2If there are L-K gray values, the average gray value calculation method in two intervals is as follows:
by traversing all gray values, when a certain gray value causes a variance value
When the maximum value is reached, the value is set as the optimal threshold value;
s2: filtering noise signals which are distributed in a background image in a disorder manner by adopting a median filtering algorithm, carrying out binarization processing on the NK cell outline by combining an OTSU algorithm, and carrying out non-operation on the segmented NK cell image and the background image to obtain a complete NK cell image;
s3: NK cell contours were extracted using Sobel operators, which included two sets of 3 × 3 computational matrices:
after the NK cell contour is obtained, in order to improve the definition of the NK cell contour, the OTSU algorithm is used again for binarization processing, so that a complete NK cell image is obtained.
10. The method of on-line monitoring of NK cell culture morphology according to claim 9, further comprising:
r1: the microwave emitter is used as an excitation source and generates a high-frequency microwave signal into the circuit, the frequency of the high-frequency microwave signal is between 100MHz and 1GHz, and the amplitude is between 5dB and 50 dB;
r2: the high-frequency microwave signal enters a power amplifier, the amplification factor is between 30 times and 100 times, and the amplified signal is input into a power distributor;
r3: the power distributor equally divides the high-frequency microwave signal into two parts which are respectively input into the micro-strip inductor and the reference circuit;
r4: the microstrip inductor is arranged below the NK cell culture dish, the reference circuit is arranged adjacent to the microstrip inductor, an electromagnetic field is enhanced when a high-frequency microwave signal passes through the microstrip inductor, the high-frequency microwave signal is attenuated after the electromagnetic field passes through the NK cell culture dish, and the attenuation coefficient is related to a solution and a medium in the NK cell culture dish;
r5: and shaping the attenuated high-frequency microwave signal and the output signal of the reference circuit, converting the shaped signal into a voltage signal, and inputting the voltage signal into a data analysis system after passing through a signal conversion circuit for judging the real-time state of the NK cell culture dish.
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