CN114486687A - Multi-scale continuous observation feedback method and device for femtosecond laser processing cells - Google Patents
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Abstract
The invention discloses a multiscale continuous observation feedback method and a multiscale continuous observation feedback device for femtosecond laser processed cells, and belongs to the field of cell laser processing detection. The ultrafast continuous imaging system based on the ultrafast pulse sequence, disclosed by the invention, has the time resolution reaching the femtosecond level, can continuously observe a cell regulation and processing process with biological specificity in real time, simultaneously collects the cell size and shape, extracts the cell size numerical value by utilizing cell image processing, obtains the detection image information in the nanosecond-millisecond level time scale in the cell processing process by coupling the cooperative controller with a time domain broadening method, obtains a multi-scale observation system, establishes a feedback mechanism of cell regulation and processing-detection, realizes the multi-scale continuous observation, and can realize the quick detection and the accurate regulation and control of the processing process. The invention can also guide the technical optimization of femtosecond laser processing cells by utilizing specific images of individual cells, and realizes the application of ultrafast laser regulation and processing cells with high precision, high efficiency, low damage and controllable biological influence.
Description
Technical Field
The invention relates to a multi-scale continuous observation feedback method and device for femtosecond laser processed cells, in particular to a real-time feedback method and device capable of realizing multi-scale continuous observation of a cell processing process, and belongs to the field of cell processing observation.
Background
The cell processing process of high-precision cell stimulation, perforation and other nondestructive and minimally invasive cell processing can be realized by processing the cell by using ultrafast laser, and the method can be widely applied to the biomedical fields of stem cell cultivation, gene therapy research and the like. The femtosecond laser processing cell process is an unbalanced nonlinear dynamic process, and the processing action process and mechanism are yet to be further and deeply researched and discussed. Under different time scales, the electronic dynamic evolution process of the interaction process of the femtosecond laser and the cells is different, and in addition, parameters such as laser flux, frequency and the like play a key role in the dynamic regulation process of cell processing and the influence on the damage, subsequent survival, growth or variation of the cells. Observing the action process of the laser and the cells in different time scales is beneficial to explaining the processing mechanism and improving the processing efficiency. And the biological cell specificity determines the non-repeatability of the processing process, so that the key information in the processing process can be accurately captured by continuous observation in an ultrafast time. However, the current cell processing process by laser can only be observed after processing by using methods such as laser confocal imaging or ultrasonic detection, and cannot be observed continuously in real time in the cell processing process by femtosecond laser, so that the accurate regulation and control of the cell processing process are severely restricted.
Disclosure of Invention
The invention aims to solve the problem that the femtosecond laser processing cell is difficult to accurately regulate and quickly detect, and provides a multi-scale continuous observation feedback method and a device for the femtosecond laser processing cell, an ultrafast continuous imaging system based on an ultrafast pulse sequence has time resolution reaching the femtosecond level, can continuously observe the cell regulation processing process with biological specificity in real time, simultaneously collects the cell size and shape, extracts the cell size numerical value by using cell image processing, is coupled with a time domain broadening method through a cooperative controller, acquires the detection image information in the time scale from the nanosecond level to the millisecond level in the cell processing process, obtains a multi-scale observation system, establishes a feedback mechanism of cell regulation processing-detection, realizes the quick detection and accurate regulation of the processing process, and guides the technical optimization of the femtosecond laser processing cell by using the specific image of an individual cell, the cell processing method realizes the application of ultrafast laser regulation and control processing cells with high precision, high efficiency, low damage and good controllability.
The invention is realized by the following technical scheme:
the invention discloses a multi-scale continuous observation feedback method for femtosecond laser processing cells, which comprises the following steps:
firstly, ultrafast laser generated by an ultrashort pulse laser passes through a multi-frequency pulse sequence generator and then is changed into a pulse sequence with femtosecond time delay, the pulse sequence is divided into two beams by a beam splitting method, wherein one beam is focused through an objective lens and acts on a cell sample with cell flow velocity controlled by a microfluidic device, and multi-frequency pulse femtosecond laser nondestructive or minimally invasive regulation and processing of cells is carried out; another laser pulse sequence carries ultrafast information after passing through the sample, and is received by the CCD to generate ultrafast continuous images and stored in the computer;
and step two, extracting cell images which are captured by the CCD camera in the step one and are restricted in the microfluidic chip, wherein each image comprises one or more cells, calculating a cell equivalent diameter value d by a cell image processing method, and outputting the cell equivalent diameter value d to the cooperative controller. The temporal resolution t of the temporal broadening probe image is calculated as the inverse of the laser repetition frequency f, i.e., f is 1/t. According to the time span T of the required detection image and the matching relation of the cell equivalent diameter d and the cell flow velocity v:
calculating the cell flow velocity v, and setting parameters of the microfluidic device to realize feedback regulation of the detection process;
triggering a time domain broadening pulse generator by the cooperative controller, generating pulse laser with the repetition frequency f obtained by calculation in the step two, generating laser pulse with any time delay in a range from nanosecond to millisecond magnitude through time domain broadening, collecting a time domain broadening detection image of cells under the time resolution by an image detector and a signal generator after acting on a sample, storing the time domain broadening detection image in a computer, obtaining multi-scale cell continuous observation information by combining an ultrafast continuous image received by a CCD camera, observing a cell biological reaction process, and guiding optimization of femtosecond laser cell processing. The cellular biological reaction process comprises cell morphology, cell flow, cell membrane perforation and healing.
The concrete implementation method of the cell image processing method in the second step is as follows:
(1) converting the ultrafast continuous image containing the cell morphology in the step one into a gray level picture, and drawing a gray level histogram of the image;
(2) selecting a threshold value from the wave trough of the histogram for binary segmentation, and processing the picture into a binary image;
(3) filling holes in the binary image, and deleting connecting lines among cells to achieve the purpose of cell adhesion segmentation;
(4) performing morphological opening operation to improve the visual smooth effect of the image;
(5) calculating the size of the coverage area of each cell, and excluding block outlines with too small or too large area;
(6) then, calculating the center of gravity of the connected domain and drawing numbers at the coordinate point of the center of gravity, and finally generating a cell counting mark image;
(7) the cell equivalent diameter d is output.
The invention discloses a multi-scale continuous observation feedback device for femtosecond laser processing cells, which is used for realizing the multi-scale continuous observation feedback method for the femtosecond laser processing cells, and comprises an ultrashort pulse laser, a multi-frequency pulse sequence generator, a processing objective lens, a microfluidic device for controlling the flow speed of a cell sample, a cooperative controller, a time domain broadening pulse generator, a space optical splitter, a space optical beam combiner, an image detector, a plurality of beam splitters, a CCD camera and a computer. Ultrafast pulse laser by ultrashort pulse laser production passes through multifrequency pulse sequence generator and converts the pulse sequence of being become by a plurality of multifrequency pulse that have time delay, is divided into two bundles through the beam splitter, wherein a bundle of is used for carrying out laser process to the cell through processing objective, another bundle is behind the sample, gather the cell signal in the femto second time scale of cell process, the different frequency pulse laser that carry imaging information passes through a plurality of beam splitters and separates, get into CCD camera collection signal, it saves in the computer to generate ultrafast continuous image. The above-mentioned image is passed through the cell image processing method to identify cell equivalent diameter, and transferred into the synergistic controller, calculating the laser pulse repetition frequency of the time domain broadening pulse generator according to the repetition frequency calculation method in the step two, the method comprises the steps of regulating parameters of a microfluidic device to regulate the flow rate of cells according to the required flow rate of the cells, triggering a time-domain broadening pulse generator by using a cooperative controller to generate pulse lasers with different frequencies with time delay of nanosecond to millisecond magnitude, separating the pulses with different frequencies by using a spatial light splitter and acting on a sample, collecting signals of any time scale from nanosecond to millisecond in the cell processing process, converging imaging information carried by the pulse lasers at different moments at the same spatial position by using a spatial light beam combiner, receiving the signals by using an image detector in sequence, forming a time-domain broadening detection image and storing the time-domain broadening detection image in a computer. After being processed by a computer, the multi-scale cell processing imaging information is obtained, the light stimulation and ablation processes in the cell processing process are observed, and the cell processing process and results are fed back. The multi-scale includes femtoseconds, nanoseconds, microseconds, or milliseconds.
The ultra-short pulse laser is an ultra-fast laser capable of generating laser with single wavelength and femtosecond pulse width.
The multi-frequency pulse sequence generator comprises a dispersion device, a delay device, a beam splitter, a reflector and an objective lens; the color dispersion device is a nonlinear crystal or an optical fiber; the time delay device is a quartz slice or a grating.
And the cooperative controller sets the laser repetition frequency of the time-domain broadening pulse generator by calculating the equivalent diameter of the cell, the laser repetition frequency of the time-domain broadening pulse generator and the cell flow velocity relation by using the repetition frequency calculation method in the second step, and controls the frequency of the time-domain broadening pulses acting on the cell so that the time resolution of the time-domain broadening detection image reaches nanosecond or millisecond magnitude.
The time-domain stretched pulse generator is a combination of a high repetition frequency pulse laser capable of generating a predetermined center wavelength and a prism to generate stretched laser pulses.
The image detector is a high-speed camera according to different time resolutions or a combination of a photoelectric detector and an oscilloscope.
Has the advantages that:
1. the invention discloses a multi-scale continuous observation feedback method for femtosecond laser processing cells, which couples a femtosecond laser ultrafast continuous imaging and time domain broadening image detection method to the femtosecond laser cell processing process, and acquires information of the femtosecond laser cell processing process in a nanosecond, microsecond or even millisecond time scale range by cooperating with cooperative control of cell images and laser frequency, so as to realize real-time multi-scale continuous observation of the femtosecond laser processing cell process.
2. The multi-scale continuous observation feedback method for processing the cells by the femtosecond laser disclosed by the invention fills up the blank of performing femtosecond time scale continuous shooting on unrepeatable cell processing process by observing the ultrafast dynamic action process of the femtosecond laser and the cells at different moments.
3. The invention discloses a multi-scale continuous observation feedback method for femtosecond laser processing cells, which obtains an intensity map and a high-resolution state image of each cell in real time by accurately controlling the resolution of imaging information, establishes a feedback mechanism real-time feedback processing effect of cell regulation processing-detection by combining the obtained cell data information with an image processing method, can deeply research a laser regulation cell processing method, timely adjusts processing parameters, and finally realizes the application of ultrafast laser regulation processing cells with high precision, high efficiency, low damage and controllable biological influence.
Drawings
FIG. 1 is a flow chart of the multi-scale continuous observation feedback method for femtosecond laser processing cells.
Wherein: the system comprises a 1-ultrashort pulse laser, a 2-multifrequency pulse sequence generator, a 3-beam splitter, a 4-objective lens, a 5-cell sample constrained by a microfluidic device, a 6-9-reflector, a 10-CCD camera, a 11-computer, a 12-cooperative controller, a 13-time domain broadening pulse generator, a 14-space beam splitter, a 15-space beam combiner and a 16-image detector.
Detailed Description
For a better understanding of the objects and advantages of the present invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings and examples.
Example 1
In order to realize the observation of the cell states and the ultrafast dynamic characteristics at different times in the femtosecond laser cell processing process, the multi-scale continuous observation feedback method for the femtosecond laser cell processing disclosed by the embodiment comprises the following steps:
firstly, ultrafast laser generated by an ultrashort pulse laser passes through a multi-frequency pulse sequence generator and then is changed into a pulse sequence with femtosecond time delay, the pulse sequence is divided into two beams by a beam splitting method, wherein one beam is focused through an objective lens and acts on a cell sample with cell flow velocity controlled by a microfluidic device, and multi-frequency pulse femtosecond laser nondestructive or minimally invasive regulation and processing of cells is carried out; after another beam of laser pulse sequence passes through the sample, the light carrying ultrafast information is received by the CCD to generate ultrafast continuous images and stored in the computer;
and step two, extracting the cell images which are captured by the CCD camera in the step one and are restricted in the microfluidic chip, wherein each image comprises one or more cells. The method comprises the steps of firstly converting an image containing cells into a gray picture by using a cell image processing algorithm, drawing a gray histogram of the image, then selecting a threshold value from a trough of the histogram for binary segmentation, processing the picture into a binary image, calling a hole filling function to fill holes in the binary image, and deleting connecting lines among the cells, thereby effectively segmenting cell adhesion, then performing morphological open operation, improving the visual smoothness effect of the image, calculating the size of the coverage area of each cell, excluding block outlines with too small or too large areas, then solving the gravity center of a connected domain and drawing numbers at gravity center coordinate points, finally generating a cell counting label image, completing the whole image processing process and outputting the equivalent diameter of the cells. Calculating the cell equivalent by the cell image processing methodOutputting the diameter value d to a cooperative controller; and calculating the time resolution t of the time domain broadening detection image as the reciprocal of the laser repetition frequency f. According to the time span T of the required detection image and the matching relation of the cell equivalent diameter d and the cell flow velocity v
Calculating the cell flow velocity v, and setting parameters of the microfluidic device to realize feedback regulation of the detection process;
and step three, triggering a time domain broadening pulse generator by the cooperative controller, generating pulse laser with the repetition frequency f obtained by calculation in the step two, generating laser pulses with nanosecond time delay through time domain broadening, acting on a flowing sample after space and time separation, wherein each laser pulse carries part of sample imaging information, collecting a time domain broadening detection image of the cell under the time resolution by an image detector and a signal generator after space combination, and storing the time domain broadening detection image in a computer. The method combines the ultrafast continuous images received by the CCD camera to obtain multi-scale cell continuous detection information, can directly observe biological reaction processes such as cell morphology, cell flow, cell membrane perforation, healing and the like, visually reveals morphological changes generated after the cells are irradiated by the femtosecond laser in an image mode, and can analyze a continuous evolution process of the femtosecond laser acting with cell membranes and organelles within an ultrashort time.
The multi-scale continuous observation feedback device for femtosecond laser processing cells disclosed by the embodiment is used for realizing the multi-scale continuous observation feedback method for femtosecond laser processing cells disclosed by the embodiment, and the working method of the multi-scale continuous observation feedback device and the device comprises the following steps: ultrafast pulse laser that is produced by ultrashort pulse laser 1 passes through multifrequency pulse sequence generator 2 and converts the pulse sequence that comprises a plurality of multifrequency pulses that have certain time delay into two bundles through beam splitter 3, and one of them is used for carrying out laser process to the cell through processing objective 4, and another bundle is behind sample 5, gathers the cell signal in the femto second time scale of cell process, and the different frequency pulse laser that carries imaging information passes through a plurality of beam splitters 6-9 and separates, gets into CCD camera 10 and gathers the signal to save ultrafast continuous image in computer 11. The cell image processing method is utilized to convert the ultrafast continuous image containing the cell morphology into a gray level picture, a gray level histogram of the image is drawn, the picture is processed into a binary image, cell adhesion is further segmented, the visual smoothness effect of the image is improved, the size of the coverage area of each cell is calculated, a cell counting mark image is generated, and finally the equivalent diameter of the cell is output. According to the calculation method in the second step, the laser pulse frequency and the cell flow velocity of the time-domain broadening pulse generator 13 are calculated and regulated, the cooperative controller 12 triggers the time-domain broadening pulse generator 13 to generate pulse lasers with different frequencies and nanosecond-level time delay, the pulses with different frequencies are separated and act on the sample 5 through the spatial light splitter 14, signals in the nanosecond time scale range in the cell processing process are collected, the imaging information carried by the pulse lasers at different moments is converged at the same spatial position through the spatial light combiner 15, the signals are received through the image detector 16 in sequence, and a time-domain broadening detection image is formed and stored in the computer 11. After the cell processing by a computer, the femtosecond-nanosecond multiscale cell processing imaging information is obtained, the light stimulation and ablation processes in the cell processing process are observed, and the cell processing imaging information can also be used for observing biological reaction processes such as cell morphology, cell flow, cell membrane perforation, healing and the like, and guiding the optimization of the cell processing technology.
The specific implementation process of the embodiment is as follows:
the pulse laser 1 generates ultrashort pulse laser with the wavelength of 800nm and the pulse width of 35fs, the ultrashort pulse passes through the multi-pulse sequence generator 2, specifically, the ultrashort pulse with the central wavelength of 800nm is broadened into a supercontinuum laser pulse covering a 400nm-1100nm wave band through a photonic crystal fiber, a pulse sequence consisting of 4 laser pulses with the wavelengths of 400nm, 600nm, 800nm and 1000nm is generated through a dispersion delay medium quartz slice, and the time delay between adjacent pulses is 200 fs. The pulse sequence is divided into two paths after passing through a first beam splitter 3, one path is focused on a cell sample 5 through an objective lens 4, and the sample 5 is restrained by a microfluidic device and controls the flow rate; the other path of laser pulse sequence acts on a sample 5 according to a time delay sequence, four beams of pulse laser are separated from the space after carrying cell imaging information at different processing moments through 4 beam splitters 6-9, and are imaged through a CCD camera 10 to obtain ultrafast continuous images of cells when the laser processing is carried out at 0fs, 200fs, 400fs and 600fs, and the ultrafast continuous images are stored in a computer 11.
The imaging is processed by a cell image processing method, an ultrafast continuous image containing cells is converted into a gray picture by a cell image processing algorithm, a gray histogram of the image is drawn, then a threshold value is selected from a trough of the histogram for binary segmentation, the picture is processed into a binary image, a hole filling function is called to fill holes in the binary image, connecting lines among the cells are deleted, cell adhesion is effectively segmented, morphological opening operation is carried out, the visual effect of the image is improved, the size of the coverage area of each cell is calculated smoothly, block outlines with too small or too large areas are excluded, then the gravity center of a connected domain and drawing numbers at the gravity center coordinate points are solved, and the equivalent diameter of a single cell is about 10 mu m. In order to obtain cell processing information with 1ns time resolution and 10ns time span of detected image, the repetition frequency of the time domain broadening pulse generator is calculated to be 109Hz, cell flow rate of the microfluidic device should be 103m/s, the cell flow rate of the microfluidic device is adjusted to the above value. The time domain stretched pulse generator 13 is a combination of a high repetition frequency pulsed laser and a prism, controlled by a co-controller 12, producing a repetition frequency of 109Hz, the laser with the center wavelength of 800nm is dispersed by a prism, the pulse laser with the wavelength range of 790nm-810nm is separated in time, the broadened pulse laser is separated in the space position by a space beam splitter 14, the space beam splitter is selected as a grating and acts on a flowing sample 5, the 810nm laser firstly reaches the sample position, the 790nm laser finally reaches the sample position, the pulse laser carries the sample imaging information and enters a space beam combiner 15 through a necessary reflector, the pulse laser with different space positions at different times is converged to the same position of an image detector 16, the image detector 16 is a photoelectric detector and a matched oscilloscope, and each time, the pulse laser with the center wavelength of 800nm, the spatial light beam combiner 15 and the photodetector 16 are connected with each other through the prism, and the prism is used for realizing the functions of the photoelectric detector and the matched oscilloscopeA laser pulse carries part of information of a cell sample, and a time domain broadening detection image is formed by splicing and is finally stored in the computer 11. The morphology change of cells in the femtosecond scale ultrafast continuous image, which is formed by stimulation of laser under different time delays, is combined, the phenomenon that cell membranes are sunken and modified under the action of laser is observed, the cell membranes generate regular perforation after the femtosecond laser processes the cells for 1 nanosecond according to the time domain broadening detection image, the femtosecond laser processing process is fed back to be effective for the membrane perforation of the cells, and the method can be used as a pretreatment method of biological processes such as cell transfection and the like to realize the application of biological detection and the like.
Example 2
In order to detect the influence of different laser fluxes on the cell activity after the cells are processed by laser, the device is set up and implemented as follows: the pulse laser 1 generates ultrashort pulse laser with the wavelength of 800nm and the pulse width of 50fs, the ultrashort pulse passes through the multi-pulse sequence generator 2, specifically, a pulse sequence consisting of 3 laser pulses with the wavelengths of 650nm, 800nm and 950nm is generated through broadening of a nonlinear crystal and dispersion of an optical fiber, and the time delay between adjacent pulses is 300 fs. The pulse sequence is divided into two paths after passing through the first beam splitter 3, one path is focused on a cell sample 5 through the objective lens 4, the sample 5 is restrained by the microfluidic device and the flow speed is controlled, and the laser flux irradiating on the cell is 0.5mJ/cm at the moment2(ii) a The other path of laser pulse sequence acts on a sample 5 according to a time delay sequence, carries cell imaging information at different processing moments, separates four beams of pulse laser from space after passing through 4 beam splitters 6-9, images through a CCD camera 10 to obtain ultrafast continuous images of cells when the laser processes 0fs, 300fs and 600fs, and records the ultrafast continuous images into a computer 11 to represent the ultrafast dynamic process of the cells. Next, the cell image processing algorithm is used for processing the cell ultrafast continuous image, gray processing, cell segmentation, cell filling and area identification calculation are carried out, finally, the equivalent diameter of a single cell is output to be about 20 μm, the cell flow velocity of the microfluidic device is 10m/s, in order to obtain a detection image with 1 μ s time resolution and 2 μ s time span, the frequency of the time domain broadening pulse laser is 10 μ s8Hz, microfluidizationThe control device speed was set to 10 m/s. The time domain stretched pulse generator 13 is a combination of a high repetition frequency pulse laser and a prism, and is controlled by the cooperative controller 12 to generate a repetition frequency of 10 according to the above calculation result8Hz, laser with the center wavelength of 800nm is dispersed by a prism, pulse laser with the wavelength range of 790nm-810nm is separated in time, the broadened pulse laser is separated in the space position through a space beam splitter 14, the space beam splitter is selected as a grating and acts on a flowing sample 5, the 810nm laser firstly reaches the sample, the 790nm laser finally reaches the sample, the pulse laser carries sample imaging information, the pulse laser enters a space beam combiner 15 through a necessary reflector, the pulse laser at different space positions at different times is converged to the same position of an image detector 16, the image detector 16 is a high-speed camera, and finally the shot cell information is output and stored in a computer 11. Combining femtosecond scale ultrafast continuous imaging information to obtain excited morphological change of cells in femtosecond time scale during femtosecond laser cell processing, and real-time processing result with multiple time scales capable of indicating cell activity after 2 μ s, determining cell damage under the processing parameter according to the result, adjusting parameter in time, reducing power of pulse laser 1 to 50% of original power, and making laser flux focused on sample 5 be 0.25mJ/cm2And repeating the processing and observation process again, analyzing information from the ultrafast continuous image and the time domain broadening detection image, realizing real-time feedback of cell processing results and optimization of processing parameters, obtaining the influence rule of laser flux on the effect of femtosecond laser processing cells, supporting the technical development of high-precision lossless processing cells, and solving the technical problems of related engineering in the field.
The above detailed description is intended to illustrate the objects, aspects and advantages of the present invention, and it should be understood that the above detailed description is only exemplary of the present invention and is not intended to limit the scope of the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (8)
1. The multi-scale continuous observation feedback method for femtosecond laser processing cells is characterized in that: comprises the following steps of (a) carrying out,
firstly, ultrafast laser generated by an ultrashort pulse laser passes through a multi-frequency pulse sequence generator and then is changed into a pulse sequence with femtosecond time delay, the pulse sequence is divided into two beams by a beam splitting method, wherein one beam is focused through an objective lens and acts on a cell sample with cell flow velocity controlled by a microfluidic device, and multi-frequency pulse femtosecond laser nondestructive or minimally invasive regulation and processing of cells is carried out; another laser pulse sequence carries ultrafast information after passing through the sample, and is received by the CCD to generate ultrafast continuous images and stored in the computer;
step two, extracting cell images which are captured by the CCD camera in the step one and are restricted in the microfluidic chip, wherein each image comprises one or more cells, calculating a cell equivalent diameter value d by a cell image processing method, and outputting the cell equivalent diameter value d to a cooperative controller; calculating the time resolution t of the time domain broadening detection image, wherein t is the reciprocal of the laser repetition frequency f, namely f is 1/t; according to the time span T of the required detection image and the matching relation of the cell equivalent diameter d and the cell flow velocity v:
calculating the cell flow velocity v, and setting parameters of the microfluidic device to realize feedback regulation of the detection process;
triggering a time domain broadening pulse generator by the cooperative controller, generating pulse laser with the repetition frequency f obtained by calculation in the step two, generating laser pulse with any time delay in a range from nanosecond to millisecond magnitude through time domain broadening, collecting a time domain broadening detection image of cells under the time resolution by an image detector and a signal generator after acting on a sample, storing the time domain broadening detection image in a computer, obtaining multi-scale cell continuous observation information by combining an ultrafast continuous image received by a CCD (charge coupled device) camera, observing a cell biological reaction process, and guiding optimization of femtosecond laser cell processing; the cellular biological reaction process comprises cell morphology, cell flow, cell membrane perforation and healing.
2. The multi-scale continuous observation feedback method of femtosecond laser processed cells as claimed in claim 1, wherein: the method for processing the cell image in the second step is realized by the following steps,
(1) converting the ultrafast continuous image containing the cell morphology in the step one into a gray level picture, and drawing a gray level histogram of the image;
(2) selecting a threshold value from the wave trough of the histogram for binary segmentation, and processing the picture into a binary image;
(3) filling holes in the binary image, and deleting connecting lines among cells to achieve the purpose of cell adhesion segmentation;
(4) performing morphological opening operation to improve the visual smooth effect of the image;
(5) calculating the size of the coverage area of each cell, and excluding block outlines with too small or too large area;
(6) then, calculating the center of gravity of the connected domain and drawing numbers at the coordinate point of the center of gravity, and finally generating a cell counting mark image;
(7) the cell equivalent diameter d is output.
3. A multi-scale continuous observation feedback device of femtosecond laser processed cells, which is used for realizing the multi-scale continuous observation feedback method of the femtosecond laser processed cells as set forth in claim 1 or 2, and is characterized in that: the system comprises an ultrashort pulse laser, a multi-frequency pulse sequence generator, a processing objective, a microfluidic device for controlling the flow speed of a cell sample, a cooperative controller, a time domain broadening pulse generator, a spatial beam splitter, a spatial light beam combiner, an image detector, a plurality of beam splitters, a CCD camera and a computer; ultrafast pulse laser generated by an ultrashort pulse laser is converted into a pulse sequence consisting of a plurality of multi-frequency pulses with time delay through a multi-frequency pulse sequence generator, the pulse sequence is divided into two beams through a beam splitter, one beam is used for carrying out laser processing on cells through a processing objective, the other beam is used for collecting cell signals in a femtosecond time scale in the cell processing process after passing through a sample, pulse laser with different frequencies carrying imaging information is separated through the beam splitters and enters a CCD camera to collect signals, and ultrafast continuous images are generated and stored in a computer; the above-mentioned image is passed through the cell image processing method to identify cell equivalent diameter, and transferred into the synergistic controller, calculating the laser pulse repetition frequency of the time domain broadening pulse generator according to the repetition frequency calculation method in the step two, adjusting parameters of a microfluidic device to adjust the flow velocity of cells according to the required flow velocity of the cells, triggering a time-domain broadening pulse generator by using a cooperative controller to generate pulse lasers with different frequencies with time delay of nanosecond to millisecond magnitude, separating the pulses with different frequencies by using a spatial light splitter and acting on a sample, collecting signals of any time scale from nanosecond to millisecond in the cell processing process, converging imaging information carried by the pulse lasers at different moments at the same spatial position by using a spatial light beam combiner, receiving the signals by using an image detector in sequence, forming a time-domain broadening detection image and storing the time-domain broadening detection image in a computer; after being processed by a computer, multi-scale cell processing imaging information is obtained, the light stimulation and ablation processes in the cell processing process are observed, and the cell processing process and results are fed back; the multi-scale includes femtoseconds, nanoseconds, microseconds, or milliseconds.
4. The multi-scale continuous observation feedback device of femtosecond laser processed cells as claimed in claim 3, wherein: the ultra-short pulse laser is an ultra-fast laser capable of generating laser with single wavelength and femtosecond pulse width.
5. The multi-scale continuous observation feedback device of femtosecond laser processed cells as claimed in claim 3, wherein: the multi-frequency pulse sequence generator comprises a dispersion device, a delay device, a beam splitter, a reflector and an objective lens; the color dispersion device is a nonlinear crystal or an optical fiber; the time delay device is a quartz slice or a grating.
6. The multi-scale continuous observation feedback device of femtosecond laser processed cells as claimed in claim 3, wherein: and the cooperative controller sets the laser repetition frequency of the time-domain broadening pulse generator by calculating the equivalent diameter of the cell, the laser repetition frequency of the time-domain broadening pulse generator and the cell flow velocity relation by using the repetition frequency calculation method in the second step, and controls the frequency of the time-domain broadening pulses acting on the cell so that the time resolution of the time-domain broadening detection image reaches nanosecond or millisecond magnitude.
7. The multi-scale continuous observation feedback device of femtosecond laser processed cells as claimed in claim 3, wherein: the time-domain stretched pulse generator is a combination of a high repetition frequency pulse laser capable of generating a predetermined center wavelength and a prism to generate stretched laser pulses.
8. The multi-scale continuous observation feedback device of femtosecond laser processed cells as claimed in claim 3, wherein: the image detector is a high-speed camera according to different time resolutions or a combination of a photoelectric detector and an oscilloscope.
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