CN109612388B - Optical measurement system and method for cell perforation - Google Patents

Abstract

An optical measurement system and method for cell perforation. A cell culture dish and a reflector are arranged on a three-dimensional motion platform, and a 642nm laser is installed on the horizontal right side of the reflector; an objective lens and an A dichroic mirror are arranged in sequence directly below the three-dimensional motion platform. , B dichroic mirror, C dichroic mirror and photoelectric detection device; A dichroic mirror is installed with two focusing lenses in the horizontal right direction, and laser is installed on the side of focusing lens and dichroic mirror; the laser is connected with the delay device ; Install a high-speed camera on the right side of the dichroic mirror, and the high-speed camera and photodetection device are connected to the DSP processing system; the method is: culture the cells in a cell culture dish, and add nano-gold and voltage-sensitive dyes; The laser continuously irradiates the cells and The voltage-sensitive dye is excited, the image is collected by a high-speed camera, and the change trend of the cell transmembrane potential is calculated and drawn by the DSP processing system. The invention can realize the recoverable perforation of the cell at low cost.

Description

Optical measurement system and method for cell perforation

technical field

The invention relates to the technical field of cell photoperforation and biological tissue potential measurement, in particular to an optical measurement system and method for cell perforation.

Background technique

Gene therapy is a promising and effective approach in the treatment of diseases that are difficult to cure by drugs (such as cancer, AIDS, genetic diseases, etc.). Gene therapy is to load exogenous drugs or genes into cells to fundamentally treat diseases. In this process, reversible perforation of cells is a key step in realizing gene therapy.

Traditional cell perforation methods include biological methods, chemical methods, mechanical methods and physical methods. However, biological methods are potentially toxic and easily infect healthy cells around the targeted cells; chemical methods have relatively low transfection efficiency; and mechanical methods require a particularly high level of proficiency for operating physicians. In recent years, scholars have focused their research on physical methods. The physical method is to form tiny perforations on the cell membrane with the help of physical force, and then load foreign genes and drugs. The main physical methods include electroporation, acoustic perforation, magnetic perforation and optical perforation. Among them, electroporation is easy to damage tissue; sonoporation has low accuracy and poor repeatability; magnetic perforation efficiency is relatively low, and transfection reagents are easy to aggregate. While overcoming the above shortcomings, photoperforation has the advantages of non-invasive and non-contact, and can also directly kill the mutant cells. Existing photoporation methods mainly use femtosecond lasers to excite cells to generate perforations, and the cost of femtosecond lasers is very expensive, which is the main disadvantage that limits the application of photoporation in the field of gene transfection.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned defects of the prior art, the purpose of the present invention is to provide an optical measurement system and method for cell perforation, which records the change trend of transmembrane potential in the process of cell perforation in combination with optical measurement technology, and obtains cell perforation by detecting scattered light signals. It has the characteristics of non-invasive, non-contact and low cost.

In order to achieve the above object, the technical scheme adopted in the present invention is:

An optical measurement system for cell perforation, comprising a three-dimensional motion platform 4, a cell culture dish 3 is arranged on the three-dimensional motion platform 4, a circular hole with a diameter of 40 mm is arranged in the center of the three-dimensional motion platform 4, and a reflector 2 is installed in a direction perpendicular to the three-dimensional motion. Just above the circular hole of the platform 4, the angle between the mirror 2 and the vertical direction perpendicular to the cell culture dish 3 is 45°; a 642nm laser 1 is installed in the horizontal right direction of the mirror 2; the three-dimensional motion platform 4 The objective lens 5, the A dichroic mirror 6, the B dichroic mirror 12, the C dichroic mirror 13 and the photodetector 16 are arranged vertically in turn directly below the circular hole; A focusing lens 7 and B focusing lens 8 are installed in the right direction, and a 440nm A laser 9 is installed in the horizontal rightward direction of the B focusing lens 8; 532nm B laser 11 is installed in the horizontal rightward direction of the B dichroic mirror 12; The A laser 9 and the B laser 11 are respectively connected with the delay device 10; a high-speed camera 14 is installed in the horizontal rightward direction of the C dichroic mirror 13, and the high-speed camera 14 and the photoelectric detection device 16 are respectively connected with the DSP processing system 15.

The distance between the objective lens 5 and the three-dimensional motion platform 4 is 10.6 mm.

The distance between the A focusing lens 7 and the B focusing lens 8 is the sum of the focal lengths of the two lenses.

The B laser 11 and the B dichroic mirror 12 form a cell excitation device, and the cell excitation device is used to excite cells and generate perforations; the B laser 11 is a pulsed laser with an output wavelength of 532 nm; B dichroic mirror 12 reflects, transmits through A dichroic mirror 6, and focuses on the cell surface through the objective lens; B dichroic mirror 12 and A dichroic mirror 6 are both long-pass dichroic mirrors, and the cut-off wavelengths are respectively 505nm and 550nm.

A laser 9, B focusing lens 8, A focusing lens 7 and A dichroic mirror 6 form a voltage-sensitive dye excitation device, and the voltage-sensitive dye excitation device is used to excite the voltage-sensitive dye to generate fluorescence; A laser 9 has an output wavelength of 440 nm. Pulse laser, A laser 9 outputs a laser beam, after beam expansion through B focusing lens 8 and A focusing lens 7, it is reflected by A dichroic mirror 6, and focused on the cell surface through objective lens 5; the emission wavelength of the voltage-sensitive dye is 610nm .

Laser 1 and mirror 2 form a scattered light excitation device. The scattered light excitation device is used to continuously irradiate cells to generate scattered light, so as to obtain the perforation size information after cell perforation; Laser 1 is a continuous laser with an output wavelength of 642 nm; Laser 1 A continuous laser beam is output, which is reflected by the mirror 2 and then irradiated to the cell surface.

The sequence control device 10 is composed of one FPGA to form a processing system, which is used to control the working sequence and working sequence of the cell excitation device and the voltage-sensitive dye excitation device.

The photodetection device 16 is composed of a photodiode; the scattered light excitation device irradiates the scattered light generated by the cells to detect the scattered light signal; The dichroic mirror 13 is then captured by the photodetector 16; the cut-off wavelength of the long-pass C dichroic mirror 13 is 625 nm.

The high-speed camera 14 and the C dichroic mirror 13 form an image acquisition device, which is used to acquire the fluorescence image of the cell membrane transmembrane potential change; after the cells are perforated, the 610nm fluorescence generated by the voltage-sensitive dye excitation device excited by the voltage-sensitive dye passes through the objective lens 5, A two The dichroic mirror 6 and the B dichroic mirror 12 are reflected by the C dichroic mirror 13 and enter the high-speed camera to obtain the fluorescence image of the cells after perforation; the high-speed camera adopts the Andor_iXon3_897 single-photon EMCCD.

The DSP processing system 15 is composed of a DSP processing system, which is used for real-time processing of the acquired fluorescent images after cell perforation to obtain the cell transmembrane potential, and processing the scattered light signal obtained by the photoelectric detection device to obtain the cell photoperforation. Size Information.

A measurement method based on the above-mentioned optical measurement system for cell perforation, comprising the following steps:

S1. Cultivate 1×10 ⁵ cells in cell culture dish 3, add 1.8 microliters of gold nanoparticles with a concentration of 5.6×10 ⁹ cells/ML, and incubate for three hours;

S2. Add 2ML of voltage-sensitive dye at a concentration of 10 μL/ML to the cell culture dish 3;

S3. Laser 1 continuously irradiates the cells with a 642nm laser for 3s;

S4 and B laser 11 hit the cells with a 532nm pulsed laser, and after 5ns, A laser 9 excites the voltage-sensitive dye with a 440nm pulsed laser for 10ms;

S5. After exciting the voltage-sensitive dye, the image acquisition device composed of the high-speed camera 14 and the C dichroic mirror 13 collects images; step S4 is performed before each image acquisition, and the time delay of each image acquisition is the excitation of the voltage-sensitive dye In the last 5t ns, t is a natural number, and as the number of acquisitions increases, t increases by 1 each time;

S6. After acquiring the cell fluorescence image, the DSP processing system 15 is used to calculate and draw the change trend of the cell transmembrane potential.

The specific calculation steps of the S6 include:

(1) In the acquired cell fluorescence image, select the pixel at the edge of the cell as the fluorescence signal. The selection principle is to select the gray value of three pixels at the edge of the cell, select a circle along the edge of the cell, and perform The average value of the fluorescence signal is obtained by averaging;

(2), select ten pixels as the noise signal at the position where the distance from the cell is greater than two cell diameters, and average the selected pixels to obtain the average value of the noise signal;

(3), subtract the average value of the noise signal from the average value of the fluorescence signal to obtain the fluorescence signal value corresponding to the membrane potential of an image;

(4) Calculate the transmembrane potential value of each image according to the corresponding relationship between the fluorescence signal intensity and the potential.

The invention overcomes the shortcomings of the existing photo-perforation methods, and can realize the recoverable perforation of cells at low cost. In addition, the size information of cell perforations can be obtained by detecting scattered light signals. The method realizes the acquisition of cell photoporation information, and provides experimental information for gene therapy. Specifically, it has the following advantages:

(1) The voltage value of the cell transmembrane potential and the change trend of the transmembrane voltage during the cell perforation and recovery process can be obtained;

(2) The size information of cell perforation can be obtained;

(3) It has high spatial and temporal resolution;

(4) The requirements for the operator's operating proficiency are relatively low.

Description of drawings

FIG. 1 is a structural block diagram of an optical measurement system for cell perforation according to an embodiment of the present invention.

FIG. 2 is a flowchart of an optical measurement method for cell perforation according to an embodiment of the present invention.

FIG. 3 is a flowchart of a method for calculating cell transmembrane potential according to an embodiment of the present invention.

Detailed ways

The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

As shown in FIG. 1, an optical measurement system for cell perforation includes a three-dimensional motion platform 4, a cell culture dish 3 is arranged on the three-dimensional motion platform 4, a circular hole with a diameter of 40 mm is arranged in the center of the three-dimensional motion platform 4, and a mirror 2 Installed right above the circular hole perpendicular to the three-dimensional motion platform 4, the angle between the mirror 2 and the vertical direction perpendicular to the cell culture dish 3 is 45°; a 642nm laser is installed in the horizontal right direction of the mirror 2 1; The object lens 5, the A dichroic mirror 6, the B dichroic mirror 12, the C dichroic mirror 13 and the photoelectric detection device 16 are vertically arranged successively below the circular hole of the three-dimensional motion platform 4; A focusing lens 7 and B focusing lens 8 are installed in the horizontal rightward direction of the color mirror 6, and a 440nm A laser 9 is installed in the horizontal rightward direction of the B focusing lens 8; in the horizontal rightward direction of the B dichroic mirror 12 Install 532nm B laser 11; A laser 9 and B laser 11 are connected with delay device 10 respectively; Install high-speed camera 14 in the horizontal rightward direction of C dichroic mirror 13, high-speed camera 14 and photoelectric detection device 16 are respectively connected with DSP The processing system 15 is connected.

The distance between the objective lens 5 and the three-dimensional motion platform 4 is 10.6 mm. The three-dimensional motion platform 4 is a 100mm*100mm*10mm flat plate with a circular hole with a diameter of 40mm in the middle of the flat plate, and the cell culture dish 3 is fixed on the sample table. The laser that excites the cells and the laser that excites the voltage-sensitive dye irradiate the sample from a small hole below the sample stage.

The distance between the A focusing lens 7 and the B focusing lens 8 is the sum of the focal lengths of the two lenses.

The B laser 11 and the B dichroic mirror 12 form a cell excitation device, and the cell excitation device is used to excite cells and generate perforations; the B laser 11 is a pulsed laser with an output wavelength of 532 nm; B dichroic mirror 12 reflects, transmits through A dichroic mirror 6, and focuses on the cell surface through the objective lens; B dichroic mirror 12 and A dichroic mirror 6 are both long-pass dichroic mirrors, and the cut-off wavelengths are respectively 505nm and 550nm.

A laser 9, B focusing lens 8, A focusing lens 7 and A dichroic mirror 6 form a voltage-sensitive dye excitation device, and the voltage-sensitive dye excitation device is used to excite the voltage-sensitive dye to generate fluorescence; A laser 9 has an output wavelength of 440 nm. pulsed laser. The A laser 9 outputs a beam of laser light, which is expanded by the B focusing lens 8 and the A focusing lens 7, reflected by the A dichroic mirror 6, and focused on the cell surface through the objective lens. The emission wavelength of the voltage-sensitive dye is 610 nm.

Laser 1 and mirror 2 form a scattered light excitation device. The scattered light excitation device is used to continuously irradiate cells to generate scattered light, so as to obtain the perforation size information after cell perforation; Laser 1 is a continuous laser with an output wavelength of 642 nm; Laser 1 A continuous laser beam is output, which is reflected by the mirror 2 and then irradiated to the cell surface.

The sequence control device 10 is composed of one FPGA to form a processing system, which is used to control the working sequence and working sequence of the cell excitation device and the voltage-sensitive dye excitation device.

The photodetection device 16 is composed of a photodiode; the scattered light excitation device irradiates the scattered light generated by the cells to detect the scattered light signal; After the dichroic mirror 13 is captured by the photodetection device 16 . The cutoff wavelength of the long-pass C dichroic mirror 13 is 625 nm.

The high-speed camera 14 and the C dichroic mirror 13 form an image acquisition device, which is used to acquire the fluorescence image of the cell membrane transmembrane potential change; after the cells are perforated, the 610nm fluorescence generated by the voltage-sensitive dye excitation device excited by the voltage-sensitive dye passes through the objective lens 5, A two The dichroic mirror 6 and the B dichroic mirror 12 are reflected by the C dichroic mirror 13 and enter the high-speed camera to obtain the fluorescence image of the cells after perforation. The high-speed camera uses an Andor_iXon3_897 single-photon EMCCD.

DSP processing system 15 is composed of a DSP processing system, which is used for real-time processing of the acquired fluorescent images after perforation of cells to obtain cell transmembrane potential, automatic real-time drawing of the changing trend of cell membrane transmembrane potential, and detection of photoelectric detection devices. The obtained scattered light signal is processed to obtain the size information of cell photoporation.

As shown in FIG. 2 , an embodiment of the present invention provides an optical mapping method for cell perforation, characterized in that the method includes the following steps:

S1. Incubate 1×10 ⁵ cells in cell culture dish 3, add 1.8 microliters of gold nanoparticles with a concentration of 5.6×10 ⁹ cells/ML, and incubate for three hours;

S2. Add 2ML of voltage-sensitive dye at a concentration of 10 μL/ML to the cell culture dish 3;

S3. Laser 1 continuously irradiates the cells with a 642nm laser for 3s;

S4 and B laser 11 hit the cells with a 532nm pulsed laser, and after 5ns, A laser 9 excites the voltage-sensitive dye with a 440nm pulsed laser for 10ms;

S5. After exciting the voltage-sensitive dye, the image acquisition device composed of the high-speed camera 14 and the C dichroic mirror 13 collects images; step S4 is performed before each image acquisition, and the time delay of each image acquisition is the excitation of the voltage-sensitive dye In the last 5t ns, t is a natural number, and as the number of acquisitions increases, t increases by 1 each time;

S6. After acquiring the cell fluorescence image, the DSP processing system 15 is used to calculate and draw the change trend of the cell transmembrane potential.

The specific calculation steps of the S6 include:

(1) In the acquired cell fluorescence image, select the pixel at the edge of the cell as the fluorescence signal. The selection principle is to select the gray value of three pixels at the edge of the cell, select a circle along the edge of the cell, and perform The average value of the fluorescence signal is obtained by averaging;

(2), select ten pixels as the noise signal at the position where the distance from the cell is greater than two cell diameters, and average the selected pixels to obtain the average value of the noise signal;

(3), subtract the average value of the noise signal from the average value of the fluorescence signal to obtain the fluorescence signal value corresponding to the membrane potential of an image;

(4) Calculate the transmembrane potential value of each image according to the corresponding relationship between the fluorescence signal intensity and the potential.

The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that the foregoing embodiments can still be used for The recorded technical solutions are modified, or some technical features thereof are equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (5)

1. An optical measurement system for cell perforation is characterized by comprising a three-dimensional motion platform (4), wherein a cell culture dish (3) is arranged on the three-dimensional motion platform (4), a circular hole with the diameter of 40mm is formed in the center of the three-dimensional motion platform (4), a reflector (2) is arranged right above the circular hole vertical to the three-dimensional motion platform (4), and the included angle between the reflector (2) and the vertical direction vertical to the cell culture dish (3) is 45 degrees; a 642nm laser (1) is arranged on the horizontal right direction of the reflector (2); an objective lens (5), an A dichroic mirror (6), a B dichroic mirror (12), a C dichroic mirror (13) and a photoelectric detection device (16) are vertically arranged right below a circular hole of the three-dimensional motion platform (4) in sequence; an A focusing lens (7) and a B focusing lens (8) are installed on the horizontal direction of the A dichroic mirror (6), and a 440nm A laser (9) is installed on the horizontal direction of the B focusing lens (8); a 532nm B laser (11) is installed in the horizontal direction of the B dichroic mirror (12); the A laser (9) and the B laser (11) are respectively connected with a time delay device (10); a high-speed camera (14) is installed on the C dichroic mirror (13) in the horizontal right direction, and the high-speed camera (14) and the photoelectric detection device (16) are respectively connected with a DSP processing system (15);

the B laser (11) and the B dichroic mirror (12) form a cell excitation device, and the cell excitation device is used for exciting cells and generating perforations; the B laser (11) is a pulse laser with the output wavelength of 532nm, and the B laser (11) outputs a laser beam, and the laser beam is reflected by the B dichroic mirror 12, transmitted by the A dichroic mirror (6) and focused on the surface of a cell by the objective lens (5); the dichroic mirror B (12) and the dichroic mirror A (6) are both long-wave-pass dichroic mirrors, and the cut-off wavelengths are 505nm and 550nm respectively;

the laser (9) A, the focusing lens (8) B, the focusing lens (7) A and the dichroic mirror (6) A form a voltage sensitive dye excitation device which is used for exciting the voltage sensitive dye to generate fluorescence; the A laser (9) is a pulse laser with the output wavelength of 440nm, the A laser (9) outputs a laser beam, the laser beam is expanded by a B focusing lens (8) and an A focusing lens (7), reflected by an A dichroic mirror (6) and focused to the surface of a cell by an objective lens, and the emission wavelength of the voltage sensitive dye is 610 nm;

the laser (1) and the reflector (2) form a scattered light excitation device, and the scattered light excitation device is used for continuously irradiating cells to generate scattered light so as to obtain perforation size information after cell perforation; the laser (1) is a continuous laser with an output wavelength of 642 nm; the laser (1) outputs a beam of continuous laser, and the continuous laser is reflected by the reflector (2) and then irradiates the cell surface;

the time delay device (10) is a processing system consisting of 1 FPGA and is used for controlling the working time sequence and the working sequence of the cell excitation device and the voltage sensitive dye excitation device;

the photoelectric detection device (16) consists of 1 photodiode; scattered light generated after the scattered light excitation device irradiates the cells is used for detecting scattered light signals; the long wave passes through an objective lens (5), an A dichroic mirror (6), a B dichroic mirror (12) and a C dichroic mirror (13) and then is acquired by a photoelectric detection device (16), and the cut-off wavelength of the long wave passing through the C dichroic mirror (13) is 625 nm;

the high-speed camera (14) and the C dichroic mirror (13) form an image acquisition device for acquiring a fluorescence image of transmembrane potential change of a cell membrane; after the cell is perforated, 610nm fluorescence generated by exciting a voltage sensitive dye by a voltage sensitive dye excitation device passes through an objective lens (5), an A dichroic mirror (6) and a B dichroic mirror (12), is reflected by a C dichroic mirror (13), and enters a high-speed camera to obtain a fluorescence image of the perforated cell, wherein the high-speed camera adopts an Andor _ iXon3_897 single-photon EMCCD;

and the DSP processing system (15) consists of 1 DSP and is used for processing the obtained fluorescence image after cell perforation in real time to obtain cell transmembrane potential and processing the scattered light signal obtained by the photoelectric detection device to obtain the size information of the cell photoperforation.

2. An optical measurement system for cell perforation according to claim 1, wherein the distance between the objective lens (5) and the three-dimensional moving platform (4) is 10.6 mm.

3. An optical measurement system for cell perforation according to claim 1, wherein the distance between the A focusing lens (7) and the B focusing lens (8) is the sum of the focal lengths of the two lenses.

4. The method of claim 1, wherein the step of measuring comprises:

s1, culturing 1 × 10 cells in a cell culture dish (3)⁵One cell, and 1.8. mu.l of 5.6X 10. mu.l was added⁹Nanogold per ML, incubated for three hours;

s2, adding a voltage sensitive dye with the concentration of 2ML being 10 muL/ML into the cell culture dish (3);

s3, continuously irradiating cells for 3S by a laser (1) with 642nm laser;

s4, a B laser (11) uses pulse laser with the wavelength of 532nm to strike cells, and after 5ns, an A laser (9) uses pulse laser with the wavelength of 440nm to excite a voltage sensitive dye for 10 ms;

s5, after the voltage sensitive dye is excited, an image acquisition device consisting of a high-speed camera (14) and a C dichroic mirror (13) acquires an image; step S4 is carried out before each image acquisition, the time delay of each image acquisition is 5 tons after the voltage sensitive dye is excited, t is a natural number, and t is increased by 1 time each time along with the increment of the acquisition times;

s6, after the cell fluorescence image is obtained, the change trend of the cell transmembrane potential is calculated and drawn through the DSP processing system (15).

5. The method as claimed in claim 4, wherein the step of S6 comprises:

(1) selecting pixels at the edge of the cell as fluorescent signals in the obtained cell fluorescent image, selecting gray values of three pixels at the edge of the cell according to a selection principle, selecting a circle along the edge of the cell, and averaging the selected pixel points to obtain an average value of the fluorescent signals;

(2) selecting ten pixels at a position which is more than two cell diameters away from the cells as noise signals, and averaging the selected pixels to obtain an average value of the noise signals;

(3) subtracting the average value of the noise signal from the average value of the fluorescence signal to obtain a fluorescence signal value corresponding to the membrane potential of the image;

(4) and calculating the transmembrane potential value of each image according to the corresponding relation between the fluorescence signal intensity and the potential.

Images