WO2019015674A1 - 一种高通量微生物单细胞自动化分选及接收系统 - Google Patents

一种高通量微生物单细胞自动化分选及接收系统 Download PDF

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WO2019015674A1
WO2019015674A1 PCT/CN2018/096483 CN2018096483W WO2019015674A1 WO 2019015674 A1 WO2019015674 A1 WO 2019015674A1 CN 2018096483 W CN2018096483 W CN 2018096483W WO 2019015674 A1 WO2019015674 A1 WO 2019015674A1
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cell
module
receiving
laser
automated
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PCT/CN2018/096483
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French (fr)
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勾洪磊
籍月彤
徐健
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中国科学院青岛生物能源与过程研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G01N21/85—Investigating moving fluids or granular solids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00584—Control arrangements for automatic analysers

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  • the invention relates to the field of microbial single cell sorting instrument equipment, and more particularly to an instrument system capable of coupling multi-modal cell measurement while realizing high-throughput, rapid, automated sorting and receiving of microorganism single cells.
  • Microorganisms are important resources for Earth's species, and their biomass accounts for about half of the world's total biomass. Humans have made tremendous value in the fields of industry, agriculture, medicine, and environmental protection through the transformation of microbial cell engineering and genetic engineering. However, human understanding and utilization of the Earth's microbes is still very limited. More than 90% of the microbial species still belong to the “dark matter” of the biological world because they cannot be cultured. Currently, there are two main methods for obtaining microbial cells. One is the theoretical dilution method; the other is the flow cytometry method.
  • flow cytometry can analyze individual cells at high speed and high throughput, it also results in less cell information that can be obtained in a very short detection time, and often needs to be richer by fluorescent labeling. Accurate information, and it is not possible to visually observe long-term observations of individual cells and other modes such as Raman spectroscopy.
  • the present invention provides an instrument system capable of coupling multi-modal cell measurements while achieving high-throughput, rapid, automated sorting and receiving of microbial cells.
  • the invention provides a high-throughput single-cell automated sorting and receiving system, comprising a microscopic imaging module, a pulsed laser module, a cell separation module, a cell receiving module and a control terminal, wherein the microscopic imaging module is used for observation or imaging,
  • the pulsed laser module is coupled to the microscopic imaging module via an optical fiber or optical path for ejection sorting of single cells, the cell separation module being disposed in a microscopic imaging module for single cell localization and separation, a cell receiving module disposed under the cell separation module for receiving a single cell, the cell separation module comprising a laser ejection substrate and a first automated displacement carrier platform, the laser ejection substrate being placed in a first automated displacement
  • the cell receiving module comprises a multiwell plate and a second automated displacement carrier platform, the perforated plate being placed on a second automated displacement carrier platform.
  • the microscopic imaging module, the pulsed laser module, the cell separation module, and the cell receiving module are independently controlled by the control terminal.
  • the microscopic imaging module includes an upright microscope system.
  • the upright microscope system includes a camera or CCD that is imaged in real time, and a focusing objective lens of different magnifications.
  • the focusing objective magnification includes one or more of 10X, 20X, 40X, 50X, 60X, 100X, preferably: 50X, 60X, 100X.
  • the microscopic imaging module is capable of coupling the laser source, spectrometer, and optical path necessary for observation or imaging.
  • the imaging includes one or more of optical imaging, Raman spectroscopy imaging, and fluorescence imaging.
  • the pulsed laser module is coupled to the microscopic imaging module via an optical fiber or optical path.
  • the pulsed laser module emits a pulsed laser having a wavelength of from 266 nm to 1064 nm, preferably 266 nm, 532 nm or 1064 nm, more preferably 532 nm.
  • the pulse laser has a pulse width of one of 1 fs to 1 ⁇ s, preferably 1 ns.
  • the pulsed laser has an energy in the range of 1 ⁇ J to 100 ⁇ J, preferably 10 ⁇ J.
  • the first automated displacement carrier platform is capable of free movement in three dimensions, driven by a motor, preferably driven by a piezoelectric ceramic.
  • the first automated displacement carrier platform has a displacement accuracy of less than 1 ⁇ m, preferably less than 500 nm, and more preferably 100 nm.
  • the laser ejection substrate comprises a base layer and a plating film, and the material of the base layer comprises silicate glass, quartz glass, calcium fluoride glass and a hard polymer material.
  • the coated film is vaporized or melted by a pulsed laser.
  • the plating film material includes a metal, a metal oxide, a metal semiconductor oxide or a high molecular polymer, preferably aluminum, ITO or PEN.
  • the plating film has a thickness of 10 to 100 nm.
  • the pulsed laser light emitted by the pulsed laser module is concentrated by a focusing objective lens to a laser ejection substrate to eject a single cell.
  • the cell receiving module is located directly below the cell separation module and includes a second automated displacement carrier platform and a multiwell plate.
  • the second automated displacement carrier platform is capable of free movement in a two-dimensional direction, driven by a motor, preferably a stepper motor.
  • the displacement accuracy of the second automated displacement carrier platform is less than 1 mm, preferably less than 100 ⁇ m, more preferably 5 ⁇ m.
  • the porous plate is a transparent material including one or more of silicate glass, PDMS (polydimethylsiloxane), and PMMA (polymethyl methacrylate).
  • More than one receiving microhole is provided on the perforated plate.
  • the single cells include microbial single cells having a diameter or length of less than 5 ⁇ m or spherical microbial cells having a diameter of less than 30 ⁇ m.
  • the microbial cells include bacteria, fungi, and microalgae cells.
  • the control terminal controls the cell separation module and the cell receiving module to move autonomously, so that the single cells on the laser ejection substrate and the receiving micropores on the corresponding porous plate are located directly below the ejection laser spot, and the control terminal performs single cell Recognition of optical images, fluorescent images or Raman spectra, generating and transmitting a status signal triggers a pulsed laser module to emit a pulsed laser, ejecting a single cell from a laser ejection substrate into a corresponding receiving microwell in a multiwell plate of a cell receiving module.
  • the present invention also provides a method of automated single cell rapid sorting comprising sorting single cells using the high throughput single cell automated sorting and receiving system.
  • the specific method comprises: placing a laser-ejection substrate coated with a single cell on a first automated displacement carrier platform, the microscopic imaging module performing optical image observation on a single cell or collecting a fluorescence image or a Raman spectrum,
  • the control terminal controls the cell separation module and the cell receiving module to move autonomously, so that the single cells on the laser ejection substrate and the receiving micropores on the corresponding porous plate are located directly below the ejection laser spot, and the control terminal performs optical image on the single cell.
  • Recognition of fluorescence images or Raman spectra after identifying a single cell, generating and transmitting a status signal triggers the pulsed laser module to turn on the pulsed laser to separate and eject a single cell from the laser ejection substrate into the corresponding receiving microwell.
  • the invention has the beneficial effects that the sorting instrument for microbial single cells of the invention can automatically identify and sort microbial single cells with diameter or length less than 5 ⁇ m and spherical microbial cells with diameter less than 30 ⁇ m, and can couple optical images and fluorescence
  • a variety of observation modes, such as images and Raman spectroscopy, enable fast, automated sorting and reception of microbial cells with low hardware cost and high processing throughput, helping to compensate for some of the flow cytometry techniques. Disadvantages.
  • Figure 1 is a schematic diagram of the system configuration.
  • Figure 2 is an example of single cell sorting based on fluorescence image recognition.
  • 2a is the fluorescence picture of E. coli before ejection sorting
  • 2b is the fluorescence map of the same position after ejection sorting.
  • Figure 3 is an example of single cell sorting based on Raman spectroscopy identification.
  • 3a is a microscopic image of carbon-fixing microorganisms collected in seawater
  • 3b is a microscopic image after bombardment sorting
  • 3c is a single-cell Raman spectrum of carbon-fixing microorganisms.
  • Figure 4 shows the results of single cell genomic MDA amplification and PCR verification based on single cell ejection sorting.
  • Example 1 Fluorescence imaging-based microbial single cell sorting
  • Figure 1 shows a schematic diagram of a high-throughput single-cell automated sorting and receiving system.
  • the present invention provides a high-throughput single-cell automated sorting and receiving system, including a microscopic imaging module 1, a pulsed laser module 2, and a cell.
  • the separation module 3, the cell receiving module 4 and the control terminal 5, the connection relationship of the above components is as follows:
  • the microscopic imaging module 1 is mainly composed of an upright microscope, and includes a CCD 1-1 for imaging and a plurality of focusing objective lenses 1-2.
  • the pulsed laser module 2 is coupled to the microscopic imaging module 1 through the optical path 2-1, and the cells are coupled.
  • the separation module 3 is placed in the microscopic imaging module 1.
  • the cell separation module 3 includes a first automated displacement carrier platform and a laser ejection substrate. The pulsed laser light emitted by the pulsed laser module 2 is focused by the focusing objective lens 1-2 onto the laser ejection substrate.
  • a cell receiving module 4 comprising a second automated displacement carrier platform and a multiwell plate, the microscopic imaging module 1, the pulsed laser module 2, the cell separation module 3 and the cell receiving module 4 It is connected to the control terminal 5 separately, and automatic operation can be realized.
  • the micro imaging module 1 includes a commercial upright fluorescence microscope system (model BX-41, available from Olympus) with a focusing objective magnification of 50X;
  • the pulsed laser module 2 is a pulsed laser with a wavelength of 532 nm (model PULSELAS-P-532-300, available from ALPHALAS), with a laser pulse width of 1 nm, a maximum single pulse energy of 10 ⁇ J, and a pulse frequency of 5-16.6 kHz;
  • the cell separation module 3 comprises a first automated displacement carrier platform and a laser ejection substrate, the base layer material of the laser ejection substrate is silicate glass; the plating film material is ITO;
  • the cell receiving module 4 includes a second automated displacement carrier platform and a 384 well plate.
  • the E. coli water droplet with green fluorescent protein label was sprayed onto the surface of the laser ejecting substrate coating film and air-dried at room temperature, inverted on the first automated displacement platform, and the E. coli fluorescence image was imaged by CCD. 5
  • the individual cells are automatically identified by detecting the fluorescent image of the E. coli shape, the 384-well plate in the cell receiving module 4 is placed under the laser ejection substrate, the receiving micro-hole is located directly below the ejection laser spot, and the control terminal 5 recognizes an E. coli cell.
  • the trigger pulse laser module turns on the pulse laser to eject the cells into the lower receiving micro-hole, and then the control terminal 5 controls the movement of the first automatic displacement platform according to the cell identification image, so that the ejection laser center position is moved to the second single cell, and at the same time
  • the cell receiving module 4 is driven by the stepping motor and moved to the second receiving micro hole.
  • the pulse laser is turned on again to eject the second single cell into the receiving micro hole of the 384-well plate; the above steps are performed until the setting The program terminates or is manually stopped.
  • Fig. 2 is a photomicrograph of (2a) and (2b) before and after laser ejection sorting by E. coli with green fluorescent protein labeling by the control terminal 5. It can be seen that after the completion of the ejection sorting, the position of the original cells is already empty, indicating that the cells have been successfully separated.
  • Example 2 Microbial single cell sorting based on Raman spectroscopy
  • the micro imaging module 1 includes a commercial upright Raman microscope system (model BX-41, available from Olympus);
  • the pulsed laser module 2 is a pulsed laser with a wavelength of 532 nm (model PULSELAS-P-532-300, available from ALPHALAS), with a laser pulse width of 1 nm, a maximum single pulse energy of 10 ⁇ J, and a pulse frequency of 5-16.6 kHz;
  • the cell separation module 3 comprises a first automated displacement carrier platform and a laser ejection substrate, the base layer material of the laser ejection substrate is silicate glass; the plating film material is ITO;
  • the cell receiving module 4 includes a second automated displacement carrier platform and a 384 well plate.
  • the seawater sample was added to 13 C-labeled NaHCO 3 for photosynthetic culture, and then all the microbial cells in the sample were centrifuged, and the pure water resuspension was applied to the surface of the laser ejecting substrate coating film and air-dried at room temperature, and inverted in the first automatic displacement carrier.
  • the control terminal 5 acquires a single cell Raman spectrum and recognizes it. Since the cells with photosynthetic carbon fixation absorb 13 C elements, the peak position of the pigment in the single-cell Raman spectrum will produce a fixed drift, so the carbon-fixing microorganisms can be screened according to whether the Raman peak shifts.
  • the 384-well plate of the cell receiving module 4 is placed on the second automated displacement carrier platform, and the receiving microhole is located directly below the ejection laser spot.
  • the control terminal 5 controls the Raman microscope system to acquire a single cell Raman spectrum, and the control terminal 5 recognizes whether the single cell Raman spectrum has a Raman peak shift of the carbon fixation characteristic, and if the single cell is controlled by the terminal 5, automatic Raman spectroscopy Recognizing the carbon sequestration feature, the pulsed laser module 2 turns on the pulsed laser to eject a single cell into the lower receiving micropore; then the control terminal 5 controls the movement of the first automated displacement platform according to the cell identification image, so that the pulsed laser center position is moved to The second single cell collects its Raman spectrum, while the cell receiving module 4 is driven by the stepping motor to move to the second receiving microwell; the control terminal 5 controls the Raman microscope system to acquire single cell Raman spectroscopy, and the control terminal 5 Automatically recognize whether single-cell Raman spectroscopy has carbon-fixing characteristics
  • Figure 3 shows the single-cell Raman spectroscopy and microscopic images before and after sorting for Raman spectroscopy automatic identification and ejection sorting of carbon-fixing microorganisms in the ocean.
  • Figure 3a is a micrograph of the three carbon-fixing microbial cells in the ocean before laser ejection sorting.
  • Figure 3b shows the microscopic images of the single-cell carbon-fixing micro-cells in the above five oceans after laser ejection. It can be seen from Fig. 3b that the position of the original cells becomes a blank point, indicating that the above five single cells have been separated from the laser ejection substrate, and Fig. 3c is a Raman spectrum of the above five single cells collected by the microscopic imaging module.
  • Example 3 Single cell sorting coupled single cell genome manipulation
  • the single cells obtained by sorting are subjected to genome operation, and the operation process is as follows:
  • Example 1 The E. coli single cell ejection in Example 1 was sorted into 9 receiving microwells, and the alkaline lysate was added to lyse the cells, and then transferred to a centrifuge tube and a nucleic acid amplification mixing reagent and an amplification enzyme were added.
  • the MDA amplification method amplifies the single-cell genome and performs PCR verification on the product to obtain a gel electrophoresis pattern as shown in Fig. 4.
  • #1 to #9 are the results of single-cell ejection by ejection sorting, N1.
  • N2 is a negative control and does not contain any cells. The results show that the system can successfully sort single microbial cells, and can directly couple single-cell genome amplification operations and perform downstream DNA sequencing to obtain single-cell genomic information.

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Abstract

一种高通量微生物单细胞自动化分选及接收系统，包括显微成像模块（1）、细胞分离模块（2）、脉冲激光模块（3）、细胞接收模块（4）及控制终端（5）。用于微生物单细胞的分选仪可自动识别微生物单细胞，同时可耦合光学图像、荧光图像及拉曼光谱等多种观测模式，以较低的硬件成本和较高的处理通量，实现对微生物单细胞的快速自动化分选和接收。

Description

一种高通量微生物单细胞自动化分选及接收系统 技术领域
本发明涉及微生物单细胞分选仪器装备领域，更具体涉及一种能够耦合多模态细胞测量，同时实现微生物单细胞高通量、快速、自动化分选与接收的仪器系统。
背景技术
微生物是地球物种的重要资源，其生物量大约占据了全球总生物量的一半。人类通过对微生物进行细胞工程和基因工程的改造，已在工业、农业、医药、环保等领域产生了巨大的价值。然而，目前人类对地球微生物的了解和利用仍然十分有限，90％以上的微生物物种，由于尚不能人工培养仍然属于生物界的“暗物质(dark matter)”。目前，主要有两种方法用以获得微生物单细胞。其一，是理论稀释法；其二，是流式细胞法。流式细胞仪虽然能够高速、高通量地对单个细胞进行分析，然而这也造成在极短的检测时间内能够获得的细胞信息较少，且往往需要通过荧光标记的方式获得更为丰富和准确的信息，而无法直观地对单个细胞进行长时间的观察和其他模式如拉曼光谱的检测。
发明内容
针对以上技术问题，本发明提供一种能够耦合多模态细胞测量，同时实现微生物单细胞高通量、快速、自动化分选与接收的仪器系统。
本发明提供一种高通量单细胞自动化分选及接收系统，包括显微成像模块、脉冲激光模块、细胞分离模块、细胞接收模块及控制终端，所述显微成像模块用于观测或成像，所述脉冲激光模块通过光纤或光路与显微成像模块耦合，用于对单细胞进行弹射分选，所述细胞分离模块置于显微成像模块中，用于单细胞的定位和分离，所述细胞接收模块置于所述细胞分离模块的下方，用于接收单细胞，所述细胞分离模块包括激光弹射基片和第一自动化位移载物平台，所述激光弹射基片置于第一自动化位移载物平台上，所述细胞接收模块包括多孔板和第二自动化位移载物平台，所述多孔板置于第二自动化位移载物平台上。
所述显微成像模块、脉冲激光模块、细胞分离模块及细胞接收模块由控制 终端进行独立控制。
所述显微成像模块包括正置显微镜系统。
所述正置显微镜系统包括实时成像的照相机或者CCD，以及不同倍率的聚焦物镜。
聚焦物镜倍率包括10X，20X，40X，50X，60X，100X中的一种或多种，优选地：为50X，60X，100X。
所述显微成像模块能够耦合观测或成像所必须的激光光源、光谱仪及光路。
所述成像包括光学成像、拉曼光谱成像、荧光成像中的一种或多种。
所述脉冲激光模块通过光纤或光路与显微成像模块耦合。
所述脉冲激光模块发射的脉冲激光波长为266nm-1064nm，优选地，为266nm、532nm或1064nm，更优选地，为532nm。
所述脉冲激光的脉宽为1fs-1μs中的一种，优选地，为1ns。
所述脉冲激光的能量范围为1μJ-100μJ，优选地，为10μJ。
所述第一自动化位移载物平台能够进行三维方向的自由移动，由电机驱动，优选地，由压电陶瓷驱动。
所述第一自动化位移载物平台位移精度小于1μm，优选地，小于500nm，更优选地，为100nm。
所述激光弹射基片包括基底层和镀层膜，所述基底层的材料包括硅酸盐玻璃、石英玻璃、氟化钙玻璃及硬质高分子材料。
所述镀层膜在脉冲激光作用下气化或融化。
所述镀层膜材料包括金属、金属氧化物、金属半导体氧化物或高分子聚合物，优选地，为铝、ITO或PEN。
所述镀层膜的厚度为10-100nm。
所述脉冲激光模块发射的脉冲激光通过聚焦物镜汇聚至激光弹射基片弹射分离单细胞。
所述细胞接收模块位于细胞分离模块正下方，包括第二自动化位移载物平台及多孔板。
所述第二自动化位移载物平台能够进行二维方向的自由移动，由电机驱动，优选地，为步进马达驱动。
所述第二自动化位移载物平台的位移精度小于1mm，优选地，小于100μm， 更优选地，为5μm。
所述多孔板为透明材料，包括硅酸盐玻璃、PDMS(聚二甲基硅氧烷)、PMMA(聚甲基丙烯酸甲酯)中的一种或多种。
所述多孔板上设有一个以上接收微孔。
所述单细胞包括直径或长度小于5μm的微生物单细胞或者直径小于30μm的球形微生物细胞。
所述微生物单细胞包括细菌、真菌和微藻细胞。
所述控制终端控制细胞分离模块和细胞接收模块分别自主移动，使激光弹射基片上的单细胞与对应的多孔板上的接收微孔都位于弹射激光点正下方，所述控制终端对单细胞进行光学图像、荧光图像或拉曼光谱的识别，生成并发送状态信号触发脉冲激光模块发射脉冲激光，将单细胞从激光弹射基片分离弹射至细胞接收模块的多孔板中对应的接收微孔中。
本发明还提供了一种自动化单细胞快速分选的方法，包括用所述的高通量单细胞自动化分选及接收系统分选单细胞。具体方法包括：将滴涂有单细胞的激光弹射基片放置在第一自动化位移载物平台上，所述显微成像模块对单细胞进行光学图像观察或者采集荧光图像或拉曼光谱，所述控制终端控制细胞分离模块和细胞接收模块分别自主移动，使激光弹射基片上的单细胞与对应的多孔板上的接收微孔都位于弹射激光点正下方，所述控制终端对单细胞进行光学图像、荧光图像或拉曼光谱的识别，识别单细胞后，生成并发送状态信号触发脉冲激光模块开启脉冲激光，将单细胞从激光弹射基片分离弹射至对应的接收微孔中。
本发明的有益效果是：本发明用于微生物单细胞的分选仪可自动识别和分选直径或长度小于5μm的微生物单细胞和直径小于30μm的球形微生物单细胞，同时可耦合光学图像、荧光图像及拉曼光谱等多种观测模式，以较低的硬件成本和较高的处理通量，实现对微生物单细胞的快速、自动化的分选和接收，有助于弥补流式细胞技术的一些缺点。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图做简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为系统构造示意图。
图2为基于荧光图像识别的单细胞分选示例。其中，2a为大肠杆菌经弹射分选前细胞荧光图，2b为经弹射分选后同一位置荧光图。
图3为基于拉曼光谱识别的单细胞分选示例。其中，3a为海水中采获的固碳微生物显微图像，3b为经弹射分选后的显微图像，3c为固碳微生物单细胞拉曼图谱。
图4为基于单细胞弹射分选后的单细胞基因组MDA扩增及PCR验证结果。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外，附图为示意图，因此本发明装置和设备的并不受所述示意图的尺寸或比例限制。
需要说明的是，在本专利的权利要求和说明书中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下，由语句“包括一个”限定的要素，并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
实施例1：基于荧光成像的微生物单细胞分选
图1所示为高通量单细胞自动化分选及接收系统示意图，由图可见，本发明提供高通量单细胞自动化分选及接收系统，包括显微成像模块1，脉冲激光模块2，细胞分离模块3，细胞接收模块4和控制终端5，上述部件的连接关系如下：
所述显微成像模块1以正置显微镜为主体，包括了用于成像的CCD1-1和多个聚焦物镜1-2，脉冲激光模块2通过光路2-1与显微成像模块1耦合，细胞分离模块3置于显微成像模块1中，细胞分离模块3包括第一自动化位移载物平台和激光弹射基片，脉冲激光模块2发射的脉冲激光由聚焦物镜1-2聚焦于激光弹射基片，细胞分离模块3下方为细胞接收模块4，细胞分离模块3包括第二自动化位移载物平台和多孔板，所述显微成像模块1，脉冲激光模块2，细胞分离模块3和细胞接收模块4分别与控制终端5连接，可实现自动化操作。
下面是一个基于荧光成像的微生物单细胞分选的实施例：其中：
显微成像模块1包括一台商用正置荧光显微镜系统(型号BX-41，购自Olympus公司)，聚焦物镜倍率为50X；
脉冲激光模块2为波长532nm的脉冲激光器(型号PULSELAS-P-532-300，购自ALPHALAS公司)，激光脉宽为1nm，最大单脉冲能量为10μJ，脉冲频率为：5-16.6kHz；
细胞分离模块3包括第一自动化位移载物平台和激光弹射基片，激光弹射基片的基底层材料为硅酸盐玻璃；镀层膜材料为ITO；
细胞接收模块4包括第二自动化位移载物平台和384孔板。
利用上述高通量单细胞自动化分选及接收系统进行自动化单细胞快速分选的方法，包括下列过程：
将带有绿色荧光蛋白标记的大肠杆菌水悬液滴涂至激光弹射基片镀层膜表面并室温风干，倒置在第一自动化位移载物平台上，采用CCD对大肠杆菌荧光图像进行成像，控制终端5通过检测大肠杆菌形状的荧光图像自动识别单个细胞，细胞接收模块4中的384孔板放置于激光弹射基片下方，接收微孔位于弹射激光点正下方，控制终端5识别一个大肠杆菌细胞后触发脉冲激光模块开启脉冲激光，将细胞弹射至下方接收微孔中，随即控制终端5根据细胞识别图像控制第一自动化位移载物平台移动，使弹射激光中心位置移至第二个单细胞，同时细胞接收模块4被步进马达驱动，移至第二个接收微孔，此时脉冲激光再次开启将第二个单细胞弹射至384孔板的接收微孔中；上述步骤依此进行直至 设定程序终止或人工停止。图2为带有绿色荧光蛋白标记的大肠杆菌经控制终端5识别荧光图像进行激光弹射分选前(2a)、后(2b)的显微图片。可以看到完成弹射分选后，在原有细胞的位置已经为空，说明细胞已被成功分离。
实施例2：基于拉曼光谱的微生物单细胞分选
下面是一个基于拉曼光谱的微生物单细胞分选的实施例：其中：
显微成像模块1包括一台商用正置拉曼显微镜系统(型号BX-41，购自Olympus公司)；
脉冲激光模块2为波长532nm的脉冲激光器(型号PULSELAS-P-532-300，购自ALPHALAS公司)，激光脉宽为1nm，最大单脉冲能量为10μJ，脉冲频率为：5-16.6kHz；
细胞分离模块3包括第一自动化位移载物平台和激光弹射基片，激光弹射基片的基底层材料为硅酸盐玻璃；镀层膜材料为ITO；
细胞接收模块4包括第二自动化位移载物平台和384孔板。
利用上述高通量单细胞自动化分选及接收系统进行自动化单细胞快速分选的方法，包括下列过程：
海水样品加入 13C标记的NaHCO 3进行光合培养后离心分离出样本中所有微生物细胞，将其纯水重悬液涂至激光弹射基片镀层膜表面并室温风干，倒置在第一自动化位移载物平台上，控制终端5采集单细胞拉曼光谱并识别。由于具有光合固碳功能的细胞吸收利用了 13C元素，其单细胞拉曼光谱中色素峰位将产生固定漂移，因此可根据拉曼峰位是否发生漂移从中筛选出固碳功能微生物。细胞接收模块4的384孔板放置于第二自动化位移载物平台上，接收微孔位于弹射激光点正下方。控制终端5控制拉曼显微镜系统采集单细胞拉曼光谱，并由控制终端5识别单细胞拉曼光谱中是否具有固碳特征的拉曼峰位漂移，若单细胞经控制终端5自动拉曼光谱识别具有固碳特征，脉冲激光模块2开启脉冲激光，将单细胞弹射至下方接收微孔中；随即控制终端5根据细胞识别图像控制第一自动化位移载物平台移动，使脉冲激光中心位置移至第二个单细胞并采集其拉曼光谱，同时细胞接收模块4被步进马达驱动，移至第二个接收微孔；控制终端5控制拉曼显微镜系统采集单细胞拉曼光谱，控制终端5自动识别单细胞拉曼光谱是否具有固碳特征，若单细胞经控制终端5自动拉曼光谱识别具有 固碳特征，则脉冲激光模块2再次开启脉冲激光将第二个单细胞弹射至下方对应接收微孔中；上述步骤依次进行直至设定程序终止或人工停止。图3所示为对海洋中固碳功能微生物进行拉曼光谱自动识别与弹射分选的单细胞拉曼光谱及分选前后的显微图像。图3a圆圈标注的为5个海洋中固碳功能微生物单细胞进行激光弹射分选前的显微图像，图3b为上述5个海洋中固碳功能微生物单细胞经激光弹射后的显微图像，从图3b中可以看到原有细胞的位置成为空白点，说明上述5个单细胞已从激光弹射基片中分离，图3c为显微成像模块采集的上述5个单细胞的拉曼图谱。
实施例3：单细胞分选耦合单细胞基因组操作
为了进一步验证经上述高通量单细胞自动化分选及接收系统分选后的单细胞为所需细胞，对分选获得的单细胞进行基因组操作，操作过程为：
将实施例1中的大肠杆菌单细胞弹射分选至9个接收微孔中，加入碱性裂解液对细胞进行裂解，随后，转移至离心管并加入核酸扩增混合试剂及扩增酶，采用MDA扩增方法对单细胞基因组进行扩增，并对产物进行PCR验证，获得如图4所示的凝胶电泳图，图中#1至#9为弹射分选获得单细胞的结果，N1、N2为阴性对照，不包含任何细胞。结果显示，该系统可成功对单个微生物细胞进行快速分选，同时可直接耦合单细胞基因组扩增操作，并进行下游DNA测序，从而获得单细胞基因组信息。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种高通量单细胞自动化分选及接收系统，其特征在于，所述系统包括显微成像模块(1)、脉冲激光模块(2)、细胞分离模块(3)、细胞接收模块(4)以及控制终端(5)；
    所述显微成像模块(1)用于观测或者成像；
    所述脉冲激光模块(2)通过光纤或光路(2-1)与所述显微成像模块(1)耦合，用于对单细胞进行弹射分选；
    所述细胞分离模块(3)置于所述显微成像模块(1)中，用于单细胞的定位和分离；
    所述细胞接收模块(4)置于所述细胞分离模块(3)的下方，用于接收单细胞；
    所述显微成像模块(1)、脉冲激光模块(2)、细胞分离模块(3)以及细胞接收模块(4)均由所述控制终端(5)进行独立控制；
    所述细胞分离模块(3)包括激光弹射基片和第一自动化位移载物平台，所述激光弹射基片置于第一自动化位移载物平台上；
    所述细胞接收模块(4)包括多孔板和第二自动化位移载物平台，所述多孔板置于第二自动化位移载物平台上。
  2. 根据权利要求1所述的高通量单细胞自动化分选及接收系统，其特征在于，所述第一自动化位移载物平台能够进行三维方向的自由移动。
  3. 根据权利要求2所述的高通量单细胞自动化分选及接收系统，其特征在于，所述激光弹射基片包括基底层和镀层膜，所述基底层的材料包括硅酸盐玻璃、石英玻璃、氟化钙玻璃及硬质高分子材料；
    所述镀层膜在脉冲激光作用下气化或融化；以及
    所述镀层膜材料包括金属、金属氧化物、金属半导体氧化物或高分子聚合物。
  4. 根据权利要求3所述的高通量单细胞自动化分选及接收系统，其特征在于，所述镀层膜的厚度为10-100nm。
  5. 根据权利要求1所述的高通量单细胞自动化分选及接收系统，其特征在于，所述第二自动化位移载物平台能够进行二维方向的自由移动。
  6. 根据权利要求5所述的高通量单细胞自动化分选及接收系统，其特征在 于，所述多孔板的透光率大于80％，由硅酸盐玻璃、PDMS(聚二甲基硅氧烷)、PMMA(聚甲基丙烯酸甲酯)中的一种或多种材料制成。
  7. 根据权利要求1-6任一所述的高通量单细胞自动化分选及接收系统，其特征在于，所述显微成像模块(1)包括正置显微镜系统。
  8. 根据权利要求7所述的高通量单细胞自动化分选及接收系统，其特征在于，所述控制终端(5)控制细胞分离模块(3)和细胞接收模块(4)分别自主移动，且移动到所述激光弹射基片上的单细胞与所述多孔板上的接收微孔都位于弹射激光点正下方，控制终端(5)对单细胞进行光学图像、荧光图像或拉曼光谱的识别，生成并发送状态信号触发脉冲激光模块(2)发射脉冲激光。
  9. 根据权利要求7所述的高通量单细胞自动化分选及接收系统，其特征在于，所述脉冲激光模块(2)发射的脉冲激光的波长为266nm-1064nm，脉宽为1fs-1μs。
  10. 利用权利要求1所述的高通量单细胞自动化分选及接收系统进行自动化单细胞快速分选的方法，其特征在于，将滴涂有单细胞的所述激光弹射基片放置在所述第一自动化位移载物平台上，所述显微成像模块(1)对单细胞进行光学图像观察或采集单细胞的荧光图像或拉曼光谱，所述控制终端(5)控制所述细胞分离模块(3)和所述细胞接收模块(4)分别自主移动，且移动到所述激光弹射基片上的单细胞与对应的所述多孔板上的接收微孔都位于弹射激光点正下方，所述控制终端(5)对单细胞进行光学图像、荧光图像或拉曼光谱的识别，识别单细胞后，生成并发送状态信号触发所述脉冲激光模块(2)开启脉冲激光，将单细胞从所述激光弹射基片分离弹射至所述多孔板上对应的接收微孔中。
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