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.