Direct-current electric field assisted laser microdissection cell tissue sample collection device and method

Abstract

The invention discloses a direct-current electric field assisted laser microdissection cell tissue sample collection device and method. The device comprises an objective table, a sample collector and a direct-current electric field assembly, a glass slide, a transparent film and a cell tissue sample are fixed on the objective table; a certain distance exists between the cell adhesive on the sample collector and the cell tissue sample; the direct-current electric field assembly comprises a first metal electrode, a second metal electrode and a direct-current voltage source; the first metal electrode is arranged on the objective table; the second metal electrode is arranged in the sample collector; the positive electrode and the negative electrode of the direct-current voltage source are respectively connected with the first metal electrode or the second metal electrode. According to the method disclosed by the invention, the cut cell tissue sample flies to the cell adhesive by utilizing the electrostatic field, so that the cell tissue sample is quickly collected, the defects that an existing electrostatic adsorption scheme has high requirements on transparent films and cell adhesive materials, carried charges are easy to lose, the operation is time-consuming and tedious and the like are overcome, and the method is stable in performance and has no adverse effect on cells.

Description

Technical Field

[0001] The invention belongs to the technical field of laser microdissection, and in particular relates to a device and method for collecting cell tissue samples for direct current electric field-assisted laser microdissection. Background Art

[0002] Laser micro-capture cutting technology can directly separate, capture and preserve specific cell subpopulations or single cells from complex cell tissues, avoiding contamination by other cells, bacteria or other impurities during the collection of experimental samples. The core technical difference between existing laser micro-capture cutting systems lies in the device or method for collecting cells without contamination after the cutting process.

[0003] A Chinese patent (publication number: CN100526453C) discloses a method for collecting cells after cutting using a pre-negatively charged polyimide film and a collector with polar materials. This method relies on a polyimide film that carries static electricity. Its disadvantages are: first, it is only applicable to polyimide films, and the polyimide films are pre-negatively charged. The charge carried by the film is limited, and it cannot meet the collection of thicker or larger samples. The charge loss caused by long-term storage will reduce the collection effect; second, because it is difficult for the industry to produce polyimide films with a thickness of less than 3 microns, thicker films are difficult to cut with lasers or are prone to leaving burn marks on the edges of the cutting line after cutting.

[0004] Chinese patents (Announcement No.: CN102628758B and Publication No.: CN102634455A) and others disclose the technology of charging cell tissue samples using the photoelectric effect, which overcomes the problem of charge loss in polyimide films during long-term storage and further promotes the development of electrostatic capture technology. However, it has the following disadvantages: First, before using the device to cut cells each time, the cell tissue samples need to be charged. The negative charge generated by the photosensitive material per unit time under the irradiation of the light source is limited. It takes a certain amount of time to complete the charging of the cell tissue samples, and it takes a long time to cut multiple cell tissue samples; second, in order for the system to complete the charging, cutting and collection of cell tissue samples, multiple switches need to be operated in a specific order multiple times, and the steps are cumbersome.

[0005] Therefore, although among various collection devices or methods, electrostatic capture technology has the advantages of less damage to biological samples, low risk of contamination, and strong compatibility, it still cannot avoid problems such as high requirements for thin film materials, complex equipment or operations, which limits the application of this technology in laser microdissection technology. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and to provide a DC electric field-assisted laser microdissection cell tissue sample collection device and method, which has low requirements and few restrictions on thin film materials, and the cell tissue sample collection process is simple to operate, and there is no need to use photosensitive materials to charge the cell tissue samples. After cutting the cell tissue samples, electrostatic capture can be completed instantly with a single operation, and the cell tissue samples to be collected will not be adversely affected during the collection process, so that the cell tissue samples can be collected in a pure, pollution-free and damage-free manner, thereby solving the problems in the above-mentioned background technology.

[0007] One of the technical solutions adopted by the present invention to solve the technical problem is: providing a DC electric field assisted laser microdissection cell tissue sample collection device, including a stage, a sample collector and a DC electric field component;

[0008] The stage is fixed with a glass slide, the surface of the glass slide is covered with a transparent film, and the cell tissue sample is attached to the transparent film;

[0009] The sample collector is provided with a cell adhesive on the surface of the side facing the stage to which the cell tissue sample is attached, and there is a certain distance between the cell adhesive and the cell tissue sample;

[0010] The DC electric field component includes a first metal electrode, a second metal electrode and a DC voltage source; the first metal electrode is arranged between the stage and the glass slide and is tightly attached to the glass slide; the second metal electrode is arranged in the sample collector; the positive and negative poles of the DC voltage source are respectively connected to the first metal electrode, the second metal electrode or the second metal electrode and the first metal electrode.

[0011] In a preferred embodiment of the present invention, an insulating layer is provided between the first metal electrode and the stage.

[0012] In a preferred embodiment of the present invention, a through hole is provided on the stage, and the size of the through hole meets the field of view requirement of laser microdissection; the insulating layer and the first metal electrode are in a ring shape arranged around the through hole.

[0013] In a preferred embodiment of the present invention, the second metal electrode is circular, and is disposed on a side of the sample collector away from the stage, and the size of the second metal electrode is not larger than the sample collector.

[0014] In a preferred embodiment of the present invention, the glass slide is connected to the transparent film via an adhesive, and the adhesive includes UV-curable glue or other light-curable glue, and the thickness of the adhesive is less than 2 mm.

[0015] In a preferred embodiment of the present invention, the first metal electrode and the second metal electrode form a DC electrostatic field when the DC voltage source switch is closed, and the electric field direction of the DC electrostatic field is consistent with the connection direction between the cell tissue sample and the cell adhesive.

[0016] In a preferred embodiment of the present invention, the stage is horizontally disposed, and the sample collector is located directly above the stage.

[0017] In a preferred embodiment of the present invention, the transparent film is made of thermoplastic polymer material and has a thickness of 1 micron to 6 microns.

[0018] In a preferred embodiment of the present invention, the sample collector is made of insulating material, including a PCR tube cover; the cell adhesive includes agar; and the first metal electrode and the second metal electrode include copper foil.

[0019] The second technical solution adopted by the present invention to solve the technical problem is: providing a method for collecting cell tissue samples by direct current electric field assisted laser microdissection, using the above-mentioned direct current electric field assisted laser microdissection cell tissue sample collection device to collect direct current electric field assisted laser microdissection cell tissue samples, comprising the following steps:

[0020] (1) Adhere the cell tissue sample to the surface of the transparent film and fix it on the stage;

[0021] (2) selecting a cutting area of ​​the cell tissue sample, controlling the laser microdissection device to perform cutting, and irradiating the laser beam along the boundary of the selected area for one cycle to achieve cutting of the cell tissue sample;

[0022] (3) Turn on the DC voltage source to form an electrostatic field of a certain intensity between the glass slide and the sample collector. The cut transparent film and the cell tissue sample on its surface fly from the surface of the glass slide to the cell adhesive on the surface of the sample collector under the action of the electrostatic field, thereby collecting the cell tissue sample.

[0023] In a preferred embodiment of the present invention, when a transparent thin film material with strong electron-accepting ability (electrophilic) is used, the positive electrode of the DC voltage source is connected to the second metal electrode, and the negative electrode is connected to the first metal electrode; the transparent thin film material is such as polyimide PI;

[0024] When a transparent film material with weak electron-accepting ability (electron-losing ability) is used, the positive electrode of the DC voltage source is connected to the first metal electrode, and the negative electrode is connected to the second metal electrode; the transparent film material is, for example, polyethylene terephthalate (PET).

[0025] Compared with the background technology, this technical solution has the following advantages:

[0026] 1. The present invention breaks through the limitations of the prior art on transparent film materials and solves the problems of high requirements on the charging capacity of film materials and easy loss of charge during long-term storage;

[0027] 2. The present invention adopts a stable direct current electric field to assist the electrostatic adsorption of cell tissue samples, which solves the problem that the prior art method needs to introduce photosensitive materials to charge the cell tissue samples in advance using a charging light source, and the cutting of multiple tissue samples takes a long time;

[0028] 3. The collection device of the present invention can be introduced into an upright or inverted laser microscopic cell cutting system, and has strong compatibility. The system has stable functions and will not cause adverse effects on cell tissue samples during the collection process. It can achieve pure, pollution-free, and damage-free collection of cell tissue samples, which is beneficial to subsequent analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings described herein are used to provide further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0030] Figure 1 This is a schematic diagram of the collection device and method of Example 1;

[0031] Figure 2 This is a schematic diagram of the collection device and method of Example 2.

[0032] Among them, there are a stage 1, an insulating layer 2, a first metal electrode 3, a glass slide 4, a transparent film 5, a cell tissue sample 6, a cell adhesive 7, a sample collector 8, a second metal electrode 9, and a DC voltage source 10. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments. The terms "upper", "lower", "vertical", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0034] Example 1

[0035] The present embodiment provides a DC electric field assisted laser microdissection cell tissue sample 6 collection device, which can be introduced into an upright or inverted laser microdissection system, and includes a stage 1, a sample collector 8 and a DC electric field component.

[0036] The stage 1 is arranged horizontally, and a glass slide 4 is fixed on the stage 1. The glass slide 4 can be a commercially available film glass slide 4 or a frame glass slide 4, or a commercially available transparent film 5 can be purchased and attached to the surface of the glass slide 4 by using an adhesive. A cell tissue sample 6 is attached to the transparent film 5. When the frame glass slide 4 is selected, the cell tissue sample 6 can be placed on the surface of the transparent film 5 facing the sample collector 8, or on the surface of the transparent film 5 facing the laser light source L.

[0037] The stage 1 of this embodiment is provided with a central through hole. Under the condition of the upright system, the central through hole faces the laser light source L. The size of the central through hole meets the cutting field requirements of the laser microdissection equipment, and the cell tissue sample 6 is located in the central through hole.

[0038] The sample collector 8 is attached with a cell adhesive 7 on the surface of the side facing the stage 1 to which the cell tissue sample 6 is attached, and there is a certain distance between the cell adhesive 7 and the cell tissue sample 6; in this embodiment, the sample collector 8 is arranged directly above the stage 1.

[0039] The DC electric field component includes a first metal electrode 3, a second metal electrode 9 and a DC voltage source 10; the first metal electrode 3 is arranged between the stage 1 and the glass slide 4, and is tightly fitted with the glass slide 4, and an insulating layer 2 is provided between the stage 1 and the first metal electrode 3. In this embodiment, the insulating layer 2 and the first metal electrode 3 are annularly arranged around the central through hole; the second metal electrode 9 is arranged on the upper surface of the sample collector 8. In this embodiment, the second metal electrode 9 and the sample collector 8 are both circular, and the radius of the second metal electrode 9 is not larger than that of the sample collector 8.

[0040] The first metal electrode 3 and the second metal electrode 9 form a DC electrostatic field when the DC voltage source 10 is switched on, and the electric field direction of the DC electrostatic field is consistent with the direction of the connection line between the cell tissue sample 6 and the cell adhesive 7. In this embodiment, there is a certain vertical distance between the cell tissue sample 6 and the cell adhesive 7, and the electric field direction is vertical.

[0041] The transparent film 5 is made of thermoplastic polymer material, including but not limited to polyethylene terephthalate resin (PET), polyethylene naphthalate (PEN) or polyimide (PI) and other materials that have a certain absorption capacity for the ultraviolet laser beam used. The sample collector 8 is made of insulating material, including a PCR tube cover; the cell adhesive 7 includes agar; the first metal electrode 3 and the second metal electrode 9 include copper foil.

[0042] The DC voltage source 10 is provided with a switch, which can provide a stable DC voltage, and its positive and negative poles are respectively connected to two metal electrodes, and a stable electrostatic field is formed between the cell tissue sample 6 and the cell adhesive 7 at the moment of closing the switch. The DC voltage source 10 has an adjustable voltage range of 0 to 10 kV. By adjusting the applied voltage to a suitable value, the electrostatic field strength can be ensured to achieve electrostatic adsorption and collection of the cell tissue sample 6 without damaging the cells.

[0043] This embodiment also provides a method for collecting cell tissue samples 6 by direct current electric field assisted laser microdissection, using a PET transparent film 5 with a thickness of 1-6 μm. When the material is a transparent film 5 material with weak electron-accepting ability, the positive pole of the direct current voltage source 10 is connected to the first metal electrode 3, and the negative pole is connected to the second metal electrode 9.

[0044] The method of this embodiment includes the following steps:

[0045] (1) Adhere the cell tissue sample 6 to the surface of the transparent film 5 and fix it on the stage 1;

[0046] (2) selecting a cutting area of ​​the cell tissue sample 6, controlling the laser microdissection device to perform cutting, and irradiating the laser beam along the boundary of the selected area for one cycle to achieve cutting of the cell tissue sample 6;

[0047] (3) Turn on the DC voltage source 10 to form an electrostatic field of a certain intensity between the glass slide 4 and the sample collector 8. The cut transparent film 5 and the cell tissue sample 6 on its surface fly from the surface of the glass slide 4 to the cell adhesive 7 on the surface of the sample collector 8 under the action of the electrostatic field, thereby collecting the cell tissue sample 6.

[0048] Example 2

[0049] The difference between Example 2 and Example 1 is: a DC electric field assisted laser microdissection cell tissue sample 6 collection method, using a PI transparent film 5 with a thickness of 1-6μm, which is a transparent film 5 material with strong electron-accepting ability, the positive pole of the DC voltage source 10 is connected to the second metal electrode 9, and the negative pole is connected to the first metal electrode 3.

[0050] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A DC electric field assisted laser microdissection cell tissue sample collection device, characterized in that: It includes a stage, a sample collector and a DC electric field assembly; The stage is fixed with a glass slide, the surface of the glass slide is covered with a transparent film, and the cell tissue sample is attached to the transparent film; The sample collector is provided with a cell adhesive on the surface of the side facing the stage to which the cell tissue sample is attached, and there is a certain distance between the cell adhesive and the cell tissue sample; The DC electric field component includes a first metal electrode, a second metal electrode and a DC voltage source; the first metal electrode is arranged between the stage and the glass slide and is tightly attached to the glass slide; the second metal electrode is arranged in the sample collector; the positive and negative poles of the DC voltage source are respectively connected to the first metal electrode, the second metal electrode or the second metal electrode and the first metal electrode.

2. The DC electric field assisted laser microdissection cell tissue sample collection device according to claim 1, characterized in that: An insulating layer is provided between the first metal electrode and the stage.

3. The DC electric field assisted laser microdissection cell tissue sample collection device according to claim 2, characterized in that: The stage is provided with a through hole, the size of which meets the field of view requirement of laser microdissection; the insulating layer and the first metal electrode are in a ring shape arranged around the through hole.

4. The DC electric field assisted laser microdissection cell tissue sample collection device according to claim 1, characterized in that: The second metal electrode is circular and is disposed on a side of the sample collector away from the stage, and the size of the second metal electrode is not larger than the sample collector.

5. The DC electric field assisted laser microdissection cell tissue sample collection device according to claim 1, characterized in that: The glass slide is connected to the transparent film through an adhesive, and the adhesive includes light-curing glue, and the thickness of the glue coating is less than 2 mm.

6. The DC electric field assisted laser microdissection cell tissue sample collection device according to claim 1, characterized in that: The first metal electrode and the second metal electrode form a DC electrostatic field when the DC voltage source switch is closed, and the electric field direction of the DC electrostatic field is consistent with the direction of the connection line between the cell tissue sample and the cell adhesive.

7. The DC electric field assisted laser microdissection cell tissue sample collection device according to claim 1, characterized in that: The transparent film is made of thermoplastic polymer material and has a thickness of 1 micron to 6 microns.

8. The DC electric field assisted laser microdissection cell tissue sample collection device according to claim 1, characterized in that: The sample collector is made of insulating material and includes a PCR tube cover; the cell adhesive includes agar; and the first metal electrode and the second metal electrode include copper foil.

9. A method for collecting cell tissue samples by direct current electric field assisted laser microdissection, characterized in that: The DC electric field assisted laser microdissection cell tissue sample collection device as claimed in any one of claims 1 to 8 is used to collect DC electric field assisted laser microdissection cell tissue samples, comprising the following steps: (1) Adhere the cell tissue sample to the surface of the transparent film and fix it on the stage; (2) selecting a cutting area of ​​the cell tissue sample, controlling the laser microdissection device to perform cutting, and irradiating the laser beam along the boundary of the selected area for one cycle to achieve cutting of the cell tissue sample; (3) Turn on the DC voltage source to form an electrostatic field of a certain intensity between the glass slide and the sample collector. The cut transparent film and the cell tissue sample on its surface fly from the surface of the glass slide to the cell adhesive on the surface of the sample collector under the action of the electrostatic field, thereby collecting the cell tissue sample.

10. The method for collecting cell tissue samples by direct current electric field assisted laser microdissection according to claim 9, characterized in that: When polyimide is used as the transparent film material, the positive pole of the DC voltage source is connected to the second metal electrode, and the negative pole is connected to the first metal electrode; when polyethylene terephthalate is used as the transparent film material, the positive pole of the DC voltage source is connected to the first metal electrode, and the negative pole is connected to the second metal electrode.

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