CN113670799A - Single-cell two-photon Raman recognition and accurate sorting instrument and sorting method thereof - Google Patents
Single-cell two-photon Raman recognition and accurate sorting instrument and sorting method thereof Download PDFInfo
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Abstract
The invention provides a single-cell two-photon Raman recognition and accurate sorting instrument and a sorting method thereof, wherein the sorting method comprises the following steps: the device comprises a pulse laser, a laser control unit, a sample stage, a Raman spectrum unit, a cell sorting unit, a sample cell and a cell receiving unit; the Raman spectrum excitation and detection of the single cells in the complex environment and the accurate sorting of the single cells with low thermal damage are realized; exciting Raman signals by using pulse laser, and sorting single cells; the excitation range of the Raman spectrum can be effectively controlled to be in a single cell range, and the accuracy of Raman specific identification is improved; meanwhile, the heat effect generated in the accurate single cell sorting process is reduced, and the activity of the sorted cells is improved. The system realizes the specific identification and the accurate sorting of the single cells, serves the research of the single cells, and has the advantages of scientific structural design, safe and reliable work, simple and convenient operation and easy popularization.
Description
Technical Field
The invention belongs to the technical field of Raman recognition and sorting of cells, and particularly relates to a single-cell two-photon Raman recognition and accurate sorting instrument and a sorting method thereof.
Background
Cells are the basic unit of life, and there are no two cells in the world, even if there are differences between cells in the same cell population of the same organism; only when biological research is carried out from the single cell level, the essence of life can be dialyzed, and the premise of the single cell research is the specific identification and accurate sorting of the single cells.
As a specific optical identification mode, Raman spectroscopy is widely applied to cell type analysis in the fields of tumor tissue, soil microorganism, pathogenic bacterium identification and the like, and is a single-cell specific identification technology with great potential. Specifically, under laser excitation, biomolecules vibrate and scatter the excitation laser, and a raman spectrum signal is emitted.
Under the influence of optical diffraction limit, the area excited by laser is usually larger than the size space of one cell, so that other cells and substances around the target cell can be excited to generate Raman signals, and the specific recognition of the target single cell is interfered.
On the other hand, laser-induced forward transfer techniques have been used extensively for precise sorting of individual cells. Specifically, the laser launches the metal thin layer through photothermal effect, fixed point location, realizes the accurate sorting of specific position unicell. In this process, the cells are subjected to intense heat, the activity of sorting the cells is severely affected, and biological significance is greatly compromised.
Disclosure of Invention
In order to overcome the technical defects, the invention provides a single-cell two-photon Raman recognition and accurate sorting instrument and a sorting method thereof, which realize Raman spectrum excitation and detection of single cells in complex environment and accurate sorting of single cells with low thermal damage; simultaneously, a pulse laser (such as a picosecond laser, a femtosecond laser and the like which can realize two-photon laser) is used for exciting Raman signals and sorting single cells; the excitation range of the Raman spectrum can be effectively controlled to be in a single cell range, and the accuracy of Raman specific identification is improved; meanwhile, the heat effect generated in the accurate single cell sorting process is reduced, and the activity of the sorted cells is improved. Realizes the specific recognition and the accurate sorting of the single cells, and serves the single cell research.
A single-cell two-photon Raman recognition and accurate sorting instrument and a sorting method thereof, wherein:
single cell two-photon Raman recognition and accurate sorter, comprising:
the device comprises a pulse laser, a laser control unit, a sample stage, a Raman spectrum unit, a cell sorting unit, a sample cell and a cell receiving unit;
further, the pulse laser is used for outputting pulse laser;
further, the laser control unit is configured to receive the pulse laser output by the pulse laser, and control the pulse laser to couple into the raman spectroscopy unit and the cell sorting unit;
as an illustration, the laser control unit may control the frequency, power, wavelength, pulse width of the pulsed laser; the spot shape, size, and beam number of the pulsed laser can also be controlled.
As an illustration, the pulsed laser is coupled into the laser of the raman spectroscopy unit and output through the focusing structure of the raman spectroscopy unit as an excitation laser;
as an illustration, the pulsed laser is coupled into the cell sorting unit as a sorting laser for a sorting operation of target cells;
further, the sample stage is in electrical signal connection with the raman spectroscopy unit and is used for receiving the identification result output by the raman spectroscopy unit, and the sample stage adjusts the position of the focus of the excitation laser output by the raman spectroscopy unit in the sample pool through self displacement; moving the sample stage again according to the recognition result, and adjusting the sorting laser output by the cell sorting unit to focus on the position of the sample pool corresponding to the target cell;
as an illustration, the sample stage has one or more dimensions of movement; the moving precision interval of the sample stage is as follows: between 1 nanometer and 1 micrometer;
as a preferred example, the moving precision interval of the sample stage is: between 1 nanometer and 100 nanometers;
further, the raman spectrum unit is used for outputting excitation laser to excite a raman signal of the sample, and then collecting the raman spectrum signal of the sample; judging whether the sample is a target cell or not by analyzing a Raman spectrum signal of the sample; when the Raman spectrum unit analyzes the target cells, the identification result can be obtained, and the identification result is output to the sample stage; the recognition result comprises: size, shape, and location of the target cell;
as an illustration, the raman spectroscopy unit includes: an optical microscopic imaging module;
further, the cell sorting unit is positioned at the suspension above the sample table, and can sort target cells based on a laser-induced forward transfer sorting method;
further, the sample cell is arranged below the sample table and used for loading a sample to be sorted;
further, the cell receiving unit is arranged below the sample pool and used for receiving the target cells;
the sorting method of the single-cell two-photon Raman recognition and accurate sorter comprises the following steps:
loading a sample into a sample cell, and fixing the sample cell on a sample table;
step two, starting a pulse laser and outputting pulse laser;
thirdly, the laser control unit controls the pulse laser to be coupled into two paths of laser, wherein one path of laser is input into the Raman spectrum unit to be used as excitation laser, and the other path of laser is input into the cell sorting unit to be used as sorting laser;
fourthly, controlling the sample stage to move so that the focus of the excitation laser falls on the position of the sample in the sample cell;
exciting, collecting and identifying the Raman spectrum signal of the sample in the sample pool by the Raman spectrum unit with the resolution exceeding the diffraction limit, analyzing the sample to obtain the identification result of the target cell, and outputting the identification result to the sample platform;
as an illustration, the recognition result further includes: the size, shape, location, and type of target cell;
sixthly, the sample stage moves again according to the recognition result, the sorting laser output by the cell sorting unit is adjusted to focus on the position of the sample pool corresponding to the target cell, and the target cell is sorted based on a laser-induced forward transfer sorting method;
as an example, compared with other types of sorting lasers, the sorting laser generated after coupling of the pulse laser has a lower thermal effect, so that damage to target cells caused by the thermal effect is reduced while a stable sorting effect is ensured;
seventhly, the cell receiving unit is matched with the cell sorting unit and the sample pool to receive the target cells;
the invention has the beneficial effects that:
the invention is based on the nonlinear effect, the pulse laser can excite the Raman signal with the resolution exceeding the diffraction limit, the action range can be controlled in the size range of single cells, and the invention can be completely used for exciting the Raman signal of the single cells in a cell group.
The pulse width of the pulse laser is extremely short, the duration time is far shorter than the time of the thermal effect of the metal thin layer, and the thermal effect can be inhibited. The pulsed laser is used as a laser source for laser induction forward transfer sorting of single cells, so that heating of the cells can be obviously reduced, and the activity of the sorted single cells is improved.
The invention has scientific structural design, safe and reliable work, simple and convenient operation and easy popularization.
Drawings
FIG. 1 is a schematic diagram of the overall structure of the single-cell two-photon Raman recognition and precision sorter of the present invention
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Detailed description referring to fig. 1, a single-cell two-photon raman identification and precision sorter and a sorting method thereof, wherein:
single cell two-photon Raman recognition and accurate sorter, comprising: a pulse laser 101, a laser control unit 102, a sample stage 103, a raman spectroscopy unit 104, a cell sorting unit 105, a sample cell 106, and a cell receiving unit 107;
further, the pulse laser 101 is configured to output pulse laser light;
further, the laser control unit 102 is configured to receive the pulsed laser output by the pulsed laser 101, and control the pulsed laser to be coupled into the raman spectroscopy unit 104 and the cell sorting unit 105;
as an illustration, the laser control unit 102 may control the frequency, power, wavelength, pulse width of the pulsed laser; the spot shape, size, and beam number of the pulsed laser can also be controlled.
As an illustration, the pulsed laser is coupled into the laser of the raman spectrum unit 104 and output through the focusing structure of the raman spectrum unit 104 as an excitation laser;
as an illustration, the pulsed laser is coupled into the cell sorting unit 105 as a sorting laser for a sorting operation of the target cells 108;
further, the sample stage 103 is electrically connected to the raman spectroscopy unit 104, and is configured to receive the identification result output by the raman spectroscopy unit 104, and the sample stage 103 adjusts, through self-displacement, a position where a focal point of the excitation laser output by the raman spectroscopy unit 104 falls in the sample cell 106; moving the sample stage 103 again according to the recognition result, and adjusting the sorting laser output by the cell sorting unit 105 to focus on the position of the sample cell 106 corresponding to the target cell 108;
as an illustration, the sample stage 103 has one or more dimensions of movement; the moving precision interval of the sample stage 103 is as follows: between 1 nanometer and 1 micrometer;
as an illustration, the one dimension is a movement on a rectilinear coordinate system; the plurality of dimensions includes two dimensions and three dimensions; the two dimensions are movement on a plane coordinate system; the three dimensions are movement on a three-dimensional coordinate system;
as a preferred example, the moving precision interval of the sample stage 103 is: between 1 nanometer and 100 nanometers;
further, the raman spectrum unit 104 is configured to output excitation laser to excite a raman signal of the sample, and then collect the raman spectrum signal of the sample; judging whether the sample is a target cell 108 by analyzing a Raman spectrum signal of the sample; when the raman spectrum unit 104 analyzes the target cell 108, it may obtain an identification result and output the identification result to the sample stage 103; the recognition result comprises: size, shape, and location of the target cell;
as an illustration, the raman spectroscopy unit 104 includes: an optical microscopic imaging module;
further, the cell sorting unit 105 is located in the suspension above the sample stage 103, and can sort the target cells 108 based on a laser-induced forward transfer sorting method;
further, the sample cell 106 is arranged below the sample stage 103 and used for loading a sample to be sorted;
further, the cell receiving unit 107 is disposed below the sample cell 106, and is configured to receive the target cell 108;
the sorting method of the single-cell two-photon Raman recognition and accurate sorter comprises the following steps:
loading a sample into a sample cell 106, and fixing the sample cell 106 on a sample table 103;
step two, starting the pulse laser 101 and outputting pulse laser;
thirdly, the laser control unit 102 controls the pulse laser to be coupled into two paths of laser, wherein one path of laser is input into the Raman spectrum unit 104 and is used as the excitation laser of the Raman spectrum unit 104, and the other path of laser is input into the cell sorting unit 105 and is used as the sorting laser;
fourthly, controlling the sample stage 103 to move so that the focal point of the excitation laser falls on the position of the sample in the sample cell 106;
step five, the raman spectrum unit 104 excites, collects and identifies the raman spectrum signal of the sample in the sample cell 106 with the resolution exceeding the diffraction limit, analyzes the sample to obtain the identification result of the target cell 108, and outputs the identification result 108 to the sample stage;
as an illustration, the recognition result further includes: the size, shape, location, and type of target cell;
sixthly, the sample stage 103 moves again according to the recognition result, the position of the sorting laser output by the cell sorting unit 105 is adjusted to be focused on the sample pool 106 corresponding to the target cell 108, and the target cell 108 is sorted based on a laser-induced forward transfer sorting method;
as an example, compared with other types of sorting lasers, the sorting laser generated after coupling of the pulse laser has a lower thermal effect, so that damage to the target cells 108 caused by the thermal effect is reduced while a stable sorting effect is ensured;
seventhly, the cell receiving unit 107 is matched with the cell sorting unit 105 and the sample cell 106 to receive the target cells 108;
as an illustration, the pulsed laser is used to output pulsed laser including: picosecond, femtosecond and nanosecond lasers;
the invention is based on the nonlinear effect, the pulse laser can excite the Raman signal with the resolution exceeding the diffraction limit, the action range can be controlled in the size range of single cells, and the invention can be completely used for exciting the Raman signal of the single cells in a cell group. The pulse width of the pulse laser is extremely short, the duration time is far shorter than the time of the thermal effect of the metal thin layer, and the thermal effect can be inhibited. The pulsed laser is used as a laser source for laser induction forward transfer sorting of single cells, so that heating of the cells can be obviously reduced, and the activity of the sorted single cells is improved. The invention has scientific structural design, safe and reliable work, simple and convenient operation and easy popularization.
The above embodiments are only preferred embodiments of the present invention, and it should be understood that the above embodiments are only for assisting understanding of the method and the core idea of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (8)
1. Unicellular two-photon Raman discernment and accurate sorter, its characterized in that includes: the device comprises a pulse laser, a laser control unit, a sample stage, a Raman spectrum unit, a cell sorting unit, a sample cell and a cell receiving unit;
the pulse laser is used for outputting pulse laser;
the laser control unit is used for receiving the pulse laser output by the pulse laser and controlling the pulse laser to be coupled into the Raman spectrum unit and the cell sorting unit;
the pulse laser is coupled into the laser of the Raman spectrum unit and is output through the focusing structure of the Raman spectrum unit to serve as excitation laser; the pulse laser is coupled into the cell sorting unit and is used as sorting laser for sorting operation of target cells;
the sample stage is in electrical signal connection with the Raman spectrum unit and is used for receiving the identification result output by the Raman spectrum unit, and the position of the focus of the excitation laser output by the Raman spectrum unit in the sample pool is adjusted through self displacement of the sample stage; moving the sample stage again according to the recognition result, and adjusting the sorting laser output by the cell sorting unit to focus on the position of the sample pool corresponding to the target cell;
the Raman spectrum unit is used for outputting excitation laser to excite a Raman signal of the sample and then collecting the Raman spectrum signal of the sample; judging whether the sample is a target cell or not by analyzing a Raman spectrum signal of the sample; when the Raman spectrum unit analyzes the target cells, the identification result can be obtained, and the identification result is output to the sample stage;
the cell sorting unit is positioned at the suspension above the sample table and can sort target cells based on a laser-induced forward transfer sorting method;
the sample cell is arranged below the sample table and used for loading a sample to be sorted;
the cell receiving unit is arranged below the sample pool and used for receiving target cells.
2. The single-cell two-photon raman identification and precision sorter of claim 1, wherein the laser control unit can control the frequency, power, wavelength, pulse width of the pulsed laser; the spot shape, size, and beam number of the pulsed laser can also be controlled.
3. The single-cell two-photon raman identification and precision sorter of claim 1, wherein the sample stage has one or more moving dimensions; the moving precision interval of the sample stage is as follows: between 1 nanometer and 1 micrometer.
4. The single-cell two-photon raman identification and precision sorter of claim 1, wherein the moving precision interval of the sample stage is: between 1 nm and 100 nm.
5. The single-cell two-photon raman identification and precision sorter of claim 1, wherein the raman spectroscopy unit comprises: an optical microscopic imaging module.
6. The single-cell two-photon raman identification and precision sorter of claim 1, wherein the identification results comprise: size, shape, and location of the target cell.
7. The sorting method of the single-cell two-photon Raman recognition and accurate sorter is characterized by comprising the following steps of:
loading a sample into a sample cell, and fixing the sample cell on a sample table;
step two, starting a pulse laser and outputting pulse laser;
thirdly, the laser control unit controls the pulse laser to be coupled into two paths of laser, wherein one path of laser is input into the Raman spectrum unit to be used as excitation laser, and the other path of laser is input into the cell sorting unit to be used as sorting laser;
fourthly, controlling the sample stage to move so that the focus of the excitation laser falls on the position of the sample in the sample cell;
exciting, collecting and identifying the Raman spectrum signal of the sample in the sample pool by the Raman spectrum unit with the resolution exceeding the diffraction limit, analyzing the sample to obtain the identification result of the target cell, and outputting the identification result to the sample platform;
sixthly, the sample stage moves again according to the recognition result, the sorting laser output by the cell sorting unit is adjusted to focus on the position of the sample pool corresponding to the target cell, and the target cell is sorted based on a laser-induced forward transfer sorting method;
and seventhly, the cell receiving unit is matched with the cell sorting unit and the sample pool to receive the target cells.
8. The sorting method of the single-cell two-photon Raman recognition and precision sorter of claim 7, wherein the sorting laser generated after coupling the pulsed laser has a lower thermal effect than other types of sorting lasers, thereby ensuring a stable sorting effect and reducing damage to target cells due to the thermal effect.
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