An all-in-one sorting device for single-cell Raman measurement and laser microdissection

Abstract

The invention provides a sorting device integrating single-cell Raman measurement and laser microdissection, including cell spotting chip, Raman measurement and laser microdissection film and cell recovery chip; Raman measurement and laser microdissection The cutting film is used to fix the cell sample, and perform Raman measurement and laser cutting and sorting of single cells on it; the cell spotting chip is attached and fixed on the front of the Raman measurement and laser microdissection film, and through The micro-holes are used to spot the cell samples; the cell recovery chip is located directly under the back of the Raman measurement and laser micro-dissection film, and the cut and separated single cells are recovered through the adhesion of the micro-pillar structure on the chip. The invention can realize the Raman measurement and laser cutting and sorting of cells; the target cells are directional recovered by adopting the micro-column adhesion recovery structure, and the cell lysis and genome amplification operations are directly realized in the micro-well, realizing single-cell Raman measurement and Integral coupling of laser microdissection sorting.

Description

technical field

[0001] The invention relates to the technical field of single cell analysis and sorting, in particular to an integrated sorting device for realizing the coupling of single cell Raman measurement and laser microdissection. Background technique

[0002] From heterogeneous cell populations or heterogeneous sample mixtures, such as tissue samples, blood, semen samples, environmental sampling microbial samples, it is of great research significance to characterize and isolate a specific small number of cells or even single cells. The laser capture microdissection technology developed for this purpose is just such a sample preparation technology. The principle is to use laser energy to finely cut samples and supporting substrates under microscopic conditions, thereby separating pure samples or even single cells from mixed samples or heterogeneous samples, so as to be used in downstream microgenomics applications (such as next-generation sequencing). , Sanger sequen...

Examples

Embodiment 1

[0039] Embodiment 1: Material selection and fabrication of Raman measurement and laser microdissection thin film

[0040] Raman measurement and laser microdissection of thin films including cut layer and Raman coating. Among them, the cutting layer ( figure 1 a) Using laser-sensitive polymer materials, including but not limited to PC (polycarbonate), EVA (ethylene-vinyl acetate copolymer), PET (polyethylene terephthalate), PEN (polyethylene naphthalate) glycol esters); Raman coatings are made of opaque metallic materials, including but not limited to aluminum, silver, gold. The Raman coating is formed on the surface of the cutting layer by deposition methods such as evaporation and sputtering ( figure 1 b), the thickness of the Raman coating is made to be 10-100nm. The Raman coating itself has no Raman background, and can cover the Raman background of the cutting layer (such as figure 1 shown in c).

Embodiment 2

[0041] Example 2: Fabrication of cell spotting chip, cell recovery chip and integrated device assembly

[0042] The cell spotting chip 2 is made of transparent soft polymer materials, including but not limited to PDMS (polydimethylsiloxane), silicone rubber, PVC (polyvinyl chloride), and the thickness is 0.2-2mm. For the through hole 2-1, the diameter of the micro through hole is made to be 0.5-5 mm.

[0043] The cell recovery chip 3 is made of transparent materials, including but not limited to silicate glass, PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate); the chip contains a microwell 3-1 structure with a certain depth In the center of the micro-well 3-1, a micro-column 3-2 with the same height as the micro-well 3-1 is made, and the surface of the micro-column has a certain degree of adhesion. The positions of the microwells 3-1 and the microcolumns 3-1 on the cell recovery chip 3 are in one-to-one correspondence with the positions of the microthrough holes 2-...

Embodiment 3

[0045] Example 3: Using an integrated device for single-cell Raman measurement and cell sorting

[0046] In the above-mentioned integrated device, a 532nm high-energy pulsed laser can be used for laser microdissection of the Raman measurement and laser microdissection film, and then the cells are separated separately to recover the chip. Due to the adhesion of the microcolumn, the cut Raman measurement And the laser microdissection film 1 remains on the surface of the microcolumn during separation, thereby realizing the separation and recovery of the cut area, wherein, image 3 a is the Raman measurement and laser microdissection film 1 area map after laser cutting, image 3 b is the separated Raman measurement and laser microdissection film 1 area map, image 3 c is the cut Raman measurement and laser microdissection film 1 area diagram of the adhered micropillar 3-2, and the circle area shows the surface of the micropillar. When separating cells, the cell suspension is dri...

technical field

[0001] The invention relates to the technical field of single cell analysis and sorting, in particular to an integrated sorting device for realizing the coupling of single cell Raman measurement and laser microdissection. Background technique

[0002] From heterogeneous cell populations or heterogeneous sample mixtures, such as tissue samples, blood, semen samples, environmental sampling microbial samples, it is of great research significance to characterize and isolate a specific small number of cells or even single cells. The laser capture microdissection technology developed for this purpose is just such a sample preparation technology. The principle is to use laser energy to finely cut samples and supporting substrates under microscopic conditions, thereby separating pure samples or even single cells from mixed samples or heterogeneous samples, so as to be used in downstream microgenomics applications (such as next-generation sequencing). , Sanger sequen...

Examples

Embodiment 1

[0039] Embodiment 1: Material selection and fabrication of Raman measurement and laser microdissection thin film

[0040] Raman measurement and laser microdissection of thin films including cut layer and Raman coating. Among them, the cutting layer ( figure 1 a) Using laser-sensitive polymer materials, including but not limited to PC (polycarbonate), EVA (ethylene-vinyl acetate copolymer), PET (polyethylene terephthalate), PEN (polyethylene naphthalate) glycol esters); Raman coatings are made of opaque metallic materials, including but not limited to aluminum, silver, gold. The Raman coating is formed on the surface of the cutting layer by deposition methods such as evaporation and sputtering ( figure 1 b), the thickness of the Raman coating is made to be 10-100nm. The Raman coating itself has no Raman background, and can cover the Raman background of the cutting layer (such as figure 1 shown in c).

Embodiment 2

[0041] Example 2: Fabrication of cell spotting chip, cell recovery chip and integrated device assembly

[0042] The cell spotting chip 2 is made of transparent soft polymer materials, including but not limited to PDMS (polydimethylsiloxane), silicone rubber, PVC (polyvinyl chloride), and the thickness is 0.2-2mm. For the through hole 2-1, the diameter of the micro through hole is made to be 0.5-5 mm.

[0043] The cell recovery chip 3 is made of transparent materials, including but not limited to silicate glass, PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate); the chip contains a microwell 3-1 structure with a certain depth In the center of the micro-well 3-1, a micro-column 3-2 with the same height as the micro-well 3-1 is made, and the surface of the micro-column has a certain degree of adhesion. The positions of the microwells 3-1 and the microcolumns 3-1 on the cell recovery chip 3 are in one-to-one correspondence with the positions of the microthrough holes 2-...

Embodiment 3

[0045] Example 3: Using an integrated device for single-cell Raman measurement and cell sorting

[0046] In the above-mentioned integrated device, a 532nm high-energy pulsed laser can be used for laser microdissection of the Raman measurement and laser microdissection film, and then the cells are separated separately to recover the chip. Due to the adhesion of the microcolumn, the cut Raman measurement And the laser microdissection film 1 remains on the surface of the microcolumn during separation, thereby realizing the separation and recovery of the cut area, wherein, image 3 a is the Raman measurement and laser microdissection film 1 area map after laser cutting, image 3 b is the separated Raman measurement and laser microdissection film 1 area map, image 3 c is the cut Raman measurement and laser microdissection film 1 area diagram of the adhered micropillar 3-2, and the circle area shows the surface of the micropillar. When separating cells, the cell suspension is dri...

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