In Situ Laser Lysis for Single-Cell Spatial Transcriptomics
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Solution Overview
Problem
Current methods for single-cell analysis, such as microfluidic high-throughput systems, require cells to be dissociated from their native environments, which can obscure biological states and are not capable of capturing lysate from individual cells in situ, especially due to the short-lived nature of RNA and fast response times of cellular gene-expression machinery.
Innovation Solution
A system and method for in situ laser lysis using a microfluidic chip with a cage to capture tissue samples, where a two-photon laser is used to lyse selected areas, releasing cellular content into a fluid channel for downstream processing, allowing for rapid and precise release of cellular contents for analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If mechanical probe is used to continuously release lysis buffer through microchannel, then cell lysis can be achieved, but the probe head size creates physical limitation in accessing target cells and lysis time is lengthy (around 1 minute)
Solution Approach 1:
The patent replaces the mechanical probe-based lysis system with a laser-based optical system. The laser focuses energy directly onto target cells through the microchannel without requiring physical contact, eliminating the probe head size limitation and enabling rapid lysis (within seconds) by directly disrupting cell membranes through photothermal or photomechanical effects.
Solution Approach 2:
The patent utilizes fluid flow dynamics to deliver lysis buffer through the microchannel to the target cell location. The continuous fluid flow carries the lysis buffer precisely to the laser-lysed cell, enabling rapid and efficient lysis without the mechanical constraints of a large probe head.
2Quantity of substance
If mechanical probe is used for cell lysis, then lysis can be performed, but harvesting sufficient cells for statistically significant results takes over one hour, triggering cellular stress responses
Solution Approach 1:
The patent implements continuous laser lysis of cells as they flow through the microchannel, followed by immediate collection of lysate in the fluid stream. This continuous process allows rapid harvesting of sufficient cell numbers (within minutes rather than hours) while maintaining physiological conditions, preventing cellular stress responses that would occur with prolonged harvesting times.
3Productivity
If cells are dissociated from native environment for analysis, then single-cell analysis can be performed, but valuable biological states influenced by multicellular complexity are obscured
Solution Approach 1:
The patent performs laser lysis and lysate collection on cells while they remain within the intact tissue structure in the microchannel. The tissue architecture and cellular context are preserved until the moment of lysis, allowing single-cell analysis to be performed without dissociating cells from their native environment, thereby retaining valuable information about cellular context and multicellular interactions.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables single-cell analysis with minimal cellular stress and carryover contamination, allowing for comprehensive genomic, transcriptional, and protein expression level analysis, and provides a better understanding of cell-cell communication in tissues by capturing cellular contents at biologically relevant conditions and timescales.
Implementation Method 1
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
Data Source
AI summary
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.