System and method for laser lysis

Patent metadata

Field	Value
Publication number	US-2016237476-A1
Application number	US-201615135150-A
Country	US
Kind code	A1
Filing date	Apr 21, 2016
Priority date	Sep 27, 2013
Publication date	Aug 18, 2016
Grant date	—

Abstract

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.

First claim

We claim:

1 . A method for three-dimensional spatial mapping of a target molecule expression in a cell in a multicellular environment, comprising:
lysing a cell of interest with a laser; collecting cellular contents of the lysed cell; performing RT-qPCR on said contents; and sequentially reconstructing a 3D spatial map of said target molecule expression in said cell by performing a 3D image segmentation.

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 a SQUIRT-PCR system.

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 otherwise preserved.

6 . The method of claim 1 , wherein said cell is part of a tissue sample and said 3D spatial map is generated for more than one cell in the tissue.

7 . A method of analyzing an intracellular analyte of interest using one or more analytical platforms at the single-cell level in situ, comprising.
lysing a cell of interest with a laser; collecting cellular contents of the lysed cell; performing RT-qPCR on said contents; and analyzing said analyte of interest using said one or more analytical platforms of interest.

8 . The method of claim 7 , wherein said one or more analytical platforms of interest are selected from the group consisting of enzymatic activity assay apparatus, sequencing apparatus, and mass spectroscopy apparatus.

9 . The method of claim 7 , wherein said analyte of interest is selected from the group consisting of DNA, RNA, and protein.

10 . The method of claim 7 , wherein said performing RT-qPCR utilizes a SQUIRT-PCR system.

11 . The method of claim 7 , wherein said lysing utilizes a two-photon (2P) process.

12 . The method of claim 7 , wherein the cell is live, fixed, frozen or otherwise preserved.

Assignees

• Univ Arizona State

Inventors

• Meldrum Deirdre
• Chao Shih-Hui (Joseph)
• Tran Thai
• Kelbauskas Laimonas
• Houkal Jeff
• Hatch Andrew
• Gao Weimin
• Richardson David

Classifications

• C12Q1/6841

  In situ hybridisation · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12M47/06

  Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material (lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003) · CPC title

• B01L3/502761

  specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules · CPC title

• B01L2300/0816

  Cards, e.g. flat sample carriers usually with flow in two horizontal directions · CPC title