Although cellular heterogeneity has been characterized with single-cell sequencing, it needs to assessed in the tissue context defined by spatially resolved molecular profiles to better understand the role of spatial heterogeneity in biological, physiological and pathological processes. In this talk, I will be discussing a new technology platform called DBiT-seq – microfluidic Deterministic Barcoding in Tissue for spatial multi-omics sequencing (DBiT-seq). Parallel microfluidic channels (10μm, 25μm, or 50μm in width) are used to deliver molecular barcodes to the surface of a formaldehyde fixed tissue slide in a spatially confined manner. Crossflow of two sets of barcodes A1-A100 and B1-B100 followed by ligation in situ yields a 2D array of tixels (tissue pixels), each containing a unique combination of full barcode AiBj (i=1-100, j=1-100), which can be resolved by paired-end NGS sequencing to reconstruct a spatial map of biomolecules in tissue. First, I will show the application to spatial transcriptome and protein mapping of whole mouse embryo tissues that revealed all major tissue types in early organogenesis, brain microvascular networks, and a single-cell-layer of melanocytes lining an optical vesicle. Second, I will discuss spatial transcriptome mapping of FFPE tissue slides including archival human tumor specimens. Third, I will show the power of integration with single-cell RNA-seq for cell type annotation in relation to spatial location in tissue. Fourth, I will discuss DBiT as a platform technology to enable spatial epigenome sequencing (spatial-ATAC-seq, spatial-CUT&Tag, etc) at single cell level. Finally, emerging opportunities and future perspectives in spatial omics will be discussed with regard to the impact on biomarker discovery and therapeutic development.