By using microscopy images of cells to guide multiplexed spatial indexing of sequencing reads, Light-Seq allows combined imaging and spatially resolved next generation sequencing (NGS) measurements to be captured from fixed biological samples. This is achieved through the combination of spatially targeted, rapid photocrosslinking of DNA barcodes onto complementary DNAs in situ with a one-step DNA stitching reaction to create pooled, spatially indexed sequencing libraries. Selection of cells can be done manually by pointing and clicking on the regions of interest (ROIs) to be sequenced, or automatically through the use of computer vision for cell type identification and segmentation. This foundational capability opens up a broad range of applications for multi-omic analysis unifying microscopy and NGS measurements from intact biological samples.