CN110736639A - Single cell autosampler - Google Patents

Abstract

Translated fromChinese

本发明公开了一种单细胞自动取样仪，包括正置成像系统、显微切割系统、样品台系统和样品提取系统，正置成像系统包括设置在CCD调节支架上的高清成像CCD和显示器，显微切割系统包括空心切割针头、切割探头调节架，与空心切割针头连接的高速马达，空心切割针头和高速马达设置在切割探头调节架上，样品台系统包括样品台和设置在样品台上的样品加热台，样品加热台上设置样品片，空心切割针头通过切割探头调节架对准样品片待切割区域正上方，通过CCD调节支架调节高清成像CCD将待切割的区域成像于显示器上，品提取系统包括与空心切割针头连接的吸管，以及与吸管连接的真空泵。样品加热台上设置有刻度标尺。本发明产品具有微小区域精密切割、不影响切割区域内细胞及组织活性、操作简单方便、切割效率高、使用成本低等优点。

The invention discloses a single-cell automatic sampler, comprising an upright imaging system, a microdissection system, a sample stage system and a sample extraction system. The upright imaging system includes a high-definition imaging CCD and a display arranged on a CCD adjusting bracket, and the display The micro-cutting system includes a hollow cutting needle, a cutting probe adjusting frame, and a high-speed motor connected with the hollow cutting needle. The hollow cutting needle and the high-speed motor are arranged on the cutting probe adjusting frame. The sample stage system includes a sample stage and a sample set on the sample stage. The heating table, the sample piece is set on the sample heating table, the hollow cutting needle is aligned directly above the area to be cut on the sample piece through the cutting probe adjustment frame, and the high-definition imaging CCD is adjusted through the CCD adjustment bracket to image the area to be cut on the monitor. Includes a suction tube attached to the hollow cut needle, and a vacuum pump attached to the suction tube. A scale ruler is provided on the sample heating stage. The product of the invention has the advantages of precise cutting of a small area, no influence on the activity of cells and tissues in the cutting area, simple and convenient operation, high cutting efficiency, low use cost, and the like.

Description

Translated fromChinese

单细胞自动取样仪Single cell autosampler

技术领域technical field

本发明涉及一种单细胞自动取样仪，属于精密自动化与图像识别技术在生物检测技术领域的应用。The invention relates to a single-cell automatic sampling instrument, which belongs to the application of precision automation and image recognition technology in the field of biological detection technology.

背景技术Background technique

单细胞分离属于单细胞研究的样品前处理重要技术，而在组织或活体上直接获取单细胞或微区样本更加接近于细胞实际生存环境，对于临床、生物学、药剂学领域具有积极意义。在组织或活体水平进行微区取样，由于其样品的特殊性，对取样设备的精度具有极高要求。目前组织水平的单细胞取样设备主要依赖于高功率激光切割技术，但该设备成本极高，尤其是高功率激光束会灼伤样品的边缘，同时由于高温操作，被切割样品区域的生物活性也会受到极大影响，对于后期的研究十分不利。因此，开发一种既不影响样本生物活性，价格适中，高效快速的单细胞取样仪显得尤为必要。Single-cell isolation is an important sample pretreatment technology for single-cell research, and the direct acquisition of single-cell or micro-area samples from tissues or living bodies is closer to the actual living environment of cells, which is of positive significance for the fields of clinical, biology and pharmacy. Micro-sampling at the tissue or living body level has extremely high requirements on the precision of the sampling equipment due to the particularity of the sample. The current tissue-level single-cell sampling equipment mainly relies on high-power laser cutting technology, but the cost of this equipment is extremely high, especially the high-power laser beam will burn the edge of the sample, and due to the high temperature operation, the biological activity of the cut sample area will also be It has been greatly affected and is very unfavorable for later research. Therefore, it is particularly necessary to develop an affordable, efficient and fast single-cell sampler that does not affect the biological activity of the sample.

发明内容SUMMARY OF THE INVENTION

本发明所要解决的技术问题是提供一种快速、对样品生物活性影响极低、价格适中的单细胞或微区生物样本的提取仪。The technical problem to be solved by the present invention is to provide an extractor for single-cell or micro-zone biological samples that is fast, has very low influence on the biological activity of the sample, and is moderately priced.

本发明的技术方案是：The technical scheme of the present invention is:

一种单细胞自动取样仪，包括正置成像系统、显微切割系统、样品台系统和样品提取系统，正置成像系统包括设置在CCD调节支架上的高清成像CCD和显示器，显微切割系统包括空心切割针头、切割探头调节架，与空心切割针头连接的高速马达，空心切割针头和高速马达设置在切割探头调节架上，样品台系统包括样品台和设置在样品台上的样品加热台，样品加热台上设置样品片，空心切割针头通过切割探头调节架对准样品片待切割区域正上方，通过CCD调节支架调节高清成像CCD将待切割的区域成像于显示器上，品提取系统包括与空心切割针头连接的吸管，以及与吸管连接的真空泵。A single-cell automatic sampler, including an upright imaging system, a microdissection system, a sample stage system and a sample extraction system, the upright imaging system includes a high-definition imaging CCD and a display set on a CCD adjustment bracket, and the microdissection system includes The hollow cutting needle, the cutting probe adjusting frame, the high-speed motor connected with the hollow cutting needle, the hollow cutting needle and the high-speed motor are arranged on the cutting probe adjusting frame, the sample stage system includes a sample stage and a sample heating stage arranged on the sample stage, the sample The sample piece is set on the heating table, and the hollow cutting needle is aligned with the cutting probe adjustment frame directly above the area to be cut on the sample piece, and the high-definition imaging CCD is adjusted through the CCD adjustment bracket to image the area to be cut on the display. The sample extraction system includes and hollow cutting. A suction tube to which the needle is attached, and a vacuum pump to which the suction tube is attached.

样品加热台上设置有刻度标尺。A scale scale is provided on the sample heating stage.

本发明的有益效果是：The beneficial effects of the present invention are:

微区图像精准定位：样品前处理时，在待切割区域上做好标记，正置成像系统可进行精确定位，为切割探头提供准确的坐标位置。Precise positioning of micro-area images: When the sample is pre-processed, mark the area to be cut, and the upright imaging system can perform precise positioning to provide accurate coordinate positions for the cutting probe.

切割探头微区精准定位：空心切割探头通过切割探头调节架精准移动到正置成像系统定位的待切割微区。Precise positioning of the cutting probe micro-area: The hollow cutting probe is precisely moved to the micro-area to be cut positioned by the upright imaging system through the cutting probe adjusting frame.

微米级别空心切割探头：空心切割探头采用复合新型材料，通过微纳米半导体加工技术制备而成，具有微米尺寸，任性好，不易破碎等技术优势，本发明通过微针机械切割样品，具有损伤小，切割位置及大小可控，操作简单，成本低等特点。Micron-level hollow cutting probe: The hollow cutting probe is made of composite new materials and prepared by micro-nano semiconductor processing technology. It has the technical advantages of micron size, good willfulness, and not easy to break. The cutting position and size are controllable, the operation is simple, and the cost is low.

图像识别与微区切割联动：当图像识别到待切割区域后，信号反馈到切割系统，马达和切割探头调节架开始自动对位到图像识别区域，通过距离设置后开始进行切割，保证了一致性和准确性。Linkage between image recognition and micro area cutting: When the image recognizes the area to be cut, the signal is fed back to the cutting system, the motor and the cutting probe adjustment frame start to automatically align to the image recognition area, and the cutting starts after setting the distance to ensure consistency. and accuracy.

附图说明Description of drawings

图1是本发明的结构示意图；Fig. 1 is the structural representation of the present invention;

图2是样品切割完毕后整体形貌俯视图；Figure 2 is a top view of the overall morphology after the sample is cut;

图3是附带刻度标识的加热板的俯视图。Fig. 3 is a plan view of a heating plate with scale marks.

图中：1-高清成像CCD、2-显示器、3-CCD调节支架、4-空心切割探头、5-马达、6-切割探头调节架、7-样品台、8-样品加热台、9-吸管、10-真空泵、11-微区切割后的样品、12-样品片、13-样品待切割的微小区域。In the picture: 1-HD imaging CCD, 2-display, 3-CCD adjustment bracket, 4-hollow cutting probe, 5-motor, 6-cutting probe adjustment frame, 7-sample stage, 8-sample heating stage, 9-pipette , 10-vacuum pump, 11-sample after micro-section cutting, 12-sample piece, 13-micro area of sample to be cut.

具体实施方式Detailed ways

下面结合具体附图和实施例对本发明作进一步说明。The present invention will be further described below with reference to the specific drawings and embodiments.

一种单细胞自动取样仪，包括正置成像系统、显微切割系统、样品台系统和样品提取系统，正置成像系统包括设置在CCD调节支架3上的高清成像CCD1和显示器2，显微切割系统包括空心切割针头4、切割探头调节架6，与空心切割针头4连接的高速马达5，空心切割针头4和高速马达5设置在切割探头调节架6上，样品台系统包括样品台7和设置在样品台7上的样品加热台8，样品加热台8上设置样品片12，空心切割针头4通过切割探头调节架6对准样品片12待切割区域正上方，通过CCD调节支架3调节高清成像CCD 1将待切割的区域成像于显示器2上，品提取系统包括与空心切割针头4连接的吸管9，以及与吸管9连接的真空泵10。A single-cell automatic sampling instrument, including an upright imaging system, a microdissection system, a sample stage system and a sample extraction system, the upright imaging system includes a high-definition imaging CCD1 and adisplay 2 arranged on a CCD adjustment bracket 3, and the microdissection The system includes a hollow cutting needle 4, a cuttingprobe adjusting frame 6, a high-speed motor 5 connected with the hollow cutting needle 4, the hollow cutting needle 4 and the high-speed motor 5 are arranged on the cuttingprobe adjusting frame 6, and the sample stage system includes a sample stage 7 and a set On the sample heating table 8 on the sample table 7, asample piece 12 is set on the sample heating table 8, the hollow cutting needle 4 is aligned with thesample piece 12 directly above the area to be cut through the cuttingprobe adjustment frame 6, and the high-definition imaging is adjusted through the CCD adjustment bracket 3 TheCCD 1 images the area to be cut on thedisplay 2 , and the product extraction system includes a suction pipe 9 connected with the hollow cutting needle 4 , and avacuum pump 10 connected with the suction pipe 9 .

样品加热台8上设置有刻度标尺。A scale scale is provided on thesample heating stage 8 .

样品片12放置于样品台7上，如需加热可启动加热装置8，加热温度20-80度，上表面带有刻度标识，方便放置样品；通过调节在CCD调节支架33的位置来调节高清成像CCD1的焦距，高清成像CCD1可前后移动，也可360度旋转，样品待切割的微小区域13（常用范围：100um-2500um区域，可根据实际需求，提前标注好其他带切割小区域）即可清晰成像，图像可通过显示器2直接观察；调节空心切割探头4、和切割探头调节架6，即调节切割探头调节架6可前后及左右移动，使空心切割探头4垂直于样品片12表面，通过切割探头调节架6，将空心切割探头4调节至待切割区域正上方，当空心切割探头4接触到样品片12上表面时，空心切割探头4高度坐标设置为0，同时设置切割深度、切割转速及空心切割探头4下降速度（一般在样品的厚度上增加2-5um），设置完毕后，启动切割系统的马达5及切割探头调节架6，转动空心切割探头4开始切割；切割完毕后，切割出来的微区切割后的样品11会存留在空心切割探头4里面，通过样品提取系统的吸管9由真空泵10提供正压将样品吹出来，放置到样品储存位置。Thesample sheet 12 is placed on the sample stage 7. If heating is required, theheating device 8 can be activated. The heating temperature is 20-80 degrees. The focal length of CCD1, high-definition imaging CCD1 can move back and forth, and can also rotate 360 degrees, thesmall area 13 of the sample to be cut (commonly used range: 100um-2500um area, other small areas with cutting can be marked in advance according to actual needs) to be clear Imaging, the image can be directly observed through thedisplay 2; adjust the hollow cutting probe 4 and the cuttingprobe adjusting frame 6, that is, adjust the cuttingprobe adjusting frame 6 to move back and forth and left and right, so that the hollow cutting probe 4 is perpendicular to the surface of thesample piece 12, through cutting Theprobe adjustment frame 6 adjusts the hollow cutting probe 4 to be just above the area to be cut. When the hollow cutting probe 4 contacts the upper surface of thesample piece 12, the height coordinate of the hollow cutting probe 4 is set to 0, and the cutting depth, cutting speed and The descending speed of the hollow cutting probe 4 (generally increases by 2-5um on the thickness of the sample), after the setting is completed, start the motor 5 of the cutting system and the cuttingprobe adjusting frame 6, and turn the hollow cutting probe 4 to start cutting; after cutting, cut out Themicro-cut sample 11 will remain in the hollow cutting probe 4, and the positive pressure is provided by thevacuum pump 10 through the suction pipe 9 of the sample extraction system to blow the sample out and place it in the sample storage position.

实施例：Example:

实验材料：样品为小鼠新鲜脑切片（厚度约为45um,大小15´12mm）；Experimental materials: The samples are fresh mouse brain slices (thickness is about 45um, size is 15´12mm);

CCD1放大倍数：40倍；CCD1 magnification: 40 times;

马达5：轻噪音，震动小，最小转速120rpm；Motor 5: light noise, small vibration, minimum speed 120rpm;

移动CCD调节支架3、切割探头调节架6：最小精度10um；Moving CCD adjustment bracket 3, cutting probe adjustment bracket 6: minimum accuracy 10um;

空心切割探头4：内径1000um，外径1500um；Hollow cutting probe 4: inner diameter 1000um, outer diameter 1500um;

高清成像CCD 1：可实现在线成像功能；High-definition imaging CCD 1: can realize online imaging function;

样品加热台8：温度25摄氏度，样品放置于中间位置。Sample heating stage 8: The temperature is 25 degrees Celsius, and the sample is placed in the middle position.

实验过程：experiment procedure:

组织样品切片平铺置于样品加热台8中央，在高清成像CCD 1引导下，对待切割的位置进行标记；The tissue sample slices are laid flat in the center of thesample heating stage 8, and under the guidance of the high-definition imaging CCD 1, the position to be cut is marked;

位置调节：通过移动CCD调节支架3、切割探头调节架6精确调节空心切割探头4至标记的区域，然后调节切割探头调节架6中的Z轴将空心切割探头4接触到小鼠脑切片的上表面；Position adjustment: precisely adjust the hollow cutting probe 4 to the marked area by moving the CCD adjustment bracket 3 and the cuttingprobe adjustment frame 6, and then adjust the Z axis in the cuttingprobe adjustment frame 6 to contact the hollow cutting probe 4 to the top of the mouse brain slice. surface;

参数设置：探头高度坐标设置为0，同时设置切割深度:47um、切割转速:125rpm，探头下降速度:50um/min；Parameter setting: The height coordinate of the probe is set to 0, and the cutting depth: 47um, the cutting speed: 125rpm, and the descending speed of the probe: 50um/min;

切割：设置完毕后，启动马达5及切割探头调节架6，转动空心切割探头4开始切割；Cutting: After setting, start the motor 5 and the cuttingprobe adjusting frame 6, and rotate the hollow cutting probe 4 to start cutting;

样品处理： 1分钟后，切割完毕，切割出来的微区切割后的样品11会存留在空心切割探头4里面，通过样品提取系统的吸管9由真空泵10提供正压将样品吹出来，放置到样品储存位置。Sample processing: After 1 minute, the cutting is completed, and themicro-cut sample 11 will remain in the hollow cutting probe 4. Thevacuum pump 10 provides positive pressure through the suction pipe 9 of the sample extraction system to blow the sample out and place it in the sample. storage location.

1-高清成像CCD、2-显示器、3-CCD调节支架、4-空心切割探头、5-马达、6-切割探头调节架、7-样品台、8-样品加热台、9-吸管、10-真空泵、11-微区切割后的样品、12-样品片、13-样品待切割的微小区域。1-HD imaging CCD, 2-monitor, 3-CCD adjustment bracket, 4-hollow cutting probe, 5-motor, 6-cutting probe adjustment bracket, 7-sample stage, 8-sample heating stage, 9-pipette, 10- Vacuum pump, 11-sample after micro-section cutting, 12-sample piece, 13-micro area of sample to be cut.

高清成像CCD1由深圳ANDONSTAR公司生产，型号：A1；显示器2由深圳盈利兴公司生产，型号:HDV20；CCD调节支架3由江苏瑞明生物生产，型号：WCJ100；空心切割探头4由江苏瑞明生物生产，型号：MPC300；马达5由上海惠利得机电有限公司生产，型号：775A；切割探头调节架6由江苏瑞明生物生产，型号：MPC500；样品台7由江苏瑞明生物生产，型号：SP100）；样品加热台 8由格丹纳仪器有限公司，型号：HT-200；吸管9由北京东方精华苑科技有限公司生产，型号：SGK-2LB-T；真空泵10由北京东方精华苑科技有限公司生产，型号：SGK-2LB。High-definition imaging CCD1 is produced by Shenzhen ANDONSTAR Company, model: A1;monitor 2 is produced by Shenzhen Yiyingxing Company, model: HDV20; CCD adjusting bracket 3 is produced by Jiangsu Ruiming Bio, model: WCJ100; hollow cutting probe 4 is produced by Jiangsu Ruiming Bio, Model: MPC300; Motor 5 is produced by Shanghai Huili De Electromechanical Co., Ltd., Model: 775A; Cutting ProbeAdjustment Frame 6 is produced by Jiangsu Ruiming Biology, Model: MPC500; Sample Stage 7 is produced by Jiangsu Ruiming Biology, Model: SP100); Sample heating Station 8 is produced by Gedana Instrument Co., Ltd., model: HT-200; suction tube 9 is produced by Beijing Dongfang Jingyuan Technology Co., Ltd., model: SGK-2LB-T;vacuum pump 10 is produced by Beijing Dongfang Jingyuan Technology Co., Ltd., model: SGK-2LB.

Claims (2)

1. The automatic sampling instrument for the single cells comprises a positive imaging system, a microdissection system, a sample stage system and a sample extraction system, and is characterized in that the positive imaging system comprises a high-definition imaging CCD (1) and a display (2) which are arranged on a CCD adjusting bracket (3), the microdissection system comprises a hollow cutting needle head (4), a cutting probe adjusting bracket (6), a high-speed motor (5) connected with the hollow cutting needle head (4), the hollow cutting needle head (4) and the high-speed motor (5) are arranged on the cutting probe adjusting bracket (6), the sample stage system comprises a sample stage (7) and a sample heating stage (8) arranged on the sample stage (7), a sample sheet (12) is arranged on the sample heating stage (8), the hollow cutting needle head (4) is aligned to a position right above a region to be cut of the sample sheet (12) through the cutting probe adjusting bracket (6), the high-definition imaging CCD to be cut region is imaged on the display (2) through the CCD adjusting bracket (3), and the sample extraction system comprises a suction pipe (9) connected with the hollow cutting probe adjusting bracket (4) and a vacuum pump (10).
2. 2. The automatic single cell sampler as claimed in claim 1, wherein a graduated scale is provided on the sample heating stage (8).

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