System and method for laser lysis
Systems and methods for in situ laser lysis for analysis of biological tissue (live, fixed, frozen or otherwise preserved) at single cell resolution in 3D. For example, a system and method for lysing individual cells in situ, including the steps of capturing a tissue sample comprising a cellular content, subjecting the tissue sample to a stream of continuous fluid flow, lysing a selected area of the tissue sample with a laser, thereby releasing at least a portion of the cellular content from the tissue sample, recovering at least one target molecule from the cellular content in the stream, and processing at least one target molecule is provided. The system collects cellular contents, performs highly multiplexed (RT-qPCR or RNA-seq), and sequentially (cell-by-cell) reconstructs a 3D spatial map of mRNA expression of the tissue with a large number of genes. A 3D spatial map of the DNA, RNA, and/or proteins can be generated for each cell in the tissue.
1. A method for three-dimensional spatial mapping of a target molecule expression in a cell in a multicellular environment without disaggregation of said cell from said multicellular environment, comprising:
(1) lysing a first cell of interest from an imaged cluster of cells with a laser, wherein said cluster of cells is located in a containment device configured for flow through of lysate;
(2) collecting cellular contents of the lysed cell from said containment device by flowing said lysate to an outlet;
(3) performing RT-qPCR on said cellular contents of the lysed cell;
(4) repeating steps (1)-(3) with a second cell of interest from said cluster of cells; and
(5) constructing a 3D spatial map of said target molecule expression in said first and second cells based on a RT-qPCR result on a respective content of each lysed cell and known coordinates of each cell determined from said imaged cluster of cells.
2. The method of claim 1 , wherein said target molecule of interest is selected from the group consisting of DNA, RNA, and protein.
3. The method of claim 1 , wherein said performing RT-qPCR utilizes SQUIRT-PCR.
4. The method of claim 1 , wherein said lysing utilizes a two-photon (2P) process.
5. The method of claim 1 , wherein the cell is live, fixed, frozen or preserved.