Clinical research is incorporating single cell analysis methods into various evaluations. During 75N91019C00029 (Phase I) of this proposed program, we intended to validate the performance of a novel single cell transcriptomic (i.e. sc-RNA-Seq) sample preparation platform which integrates imaging into the overall workflow. SCOPE-Seq on the AIR FLOW System will link imaging data to sc-RNA-Seq data on a cell by-cell basis allowing clinical outcomes such as resection margins and therapeutic success to be linked to a cell’s transcriptomic profile. Here, we will acquire primary biopsy samples from glioblastoma patients undergoing surgical resection during a clinical evaluation of dyes used in fluorescent-guided surgery. One of the dyes, 5-ALA, has been reported to preferentially label tumor cells over non-disease subpopulations. Patient-derived cells will be processed on Cell Microsystems' AIR FLOW System using the novel Single Cell Optical Phenotyping and Expression Sequencing (SCOPESeq) method. Using SCOPE-Seq’s optical barcoding methods on the automated AIR FLOW System, we will link the imaging data (e.g. 5-ALA positive vs. negative) with sc-RNA-Seq data on a cell-by-cell basis. While not part of the research endpoints, the resulting data set is intended to determine tumor-cell staining specificity of dyes while using sc-RNA-Seq analysis to reveal cellular subpopulations with high resolution. The goal of the program is to examine clinically derived glioma cells for presence of the fluorescent dye 5-ALA which is used as a visual guide during tumor resection. The AIR FLOW System will be used to determine which cells are 5-ALA positive and which exhibit transcriptomic properties of glioma cells as a means of testing the sensitivity and specificity of 5-ALA as a visual guide during surgery. The proposal also seeks to validate the use of the CITE-Seq/TotalSeq workflow, a means of using oligo-tagged antibodies for protein quantification using next generation sequencing, in the overall glioma phenotyping assay.