? Eye Organ Specific Project. Comprehensive 3-dimensional biomolecular analysis within tissues requires the coordination of data from many analytical technologies. We will develop a multimodal strategy to obtain spatially specific ?omics? information directly from human whole eye tissue. This work will leverage the unique instrumentation capabilities and expertise of the Mass Spectrometry Research Center (MSRC) in which Professors Spraggins, Caprioli, Schey, Gutierrez and Van de Plas are key personnel. Building from previous work developing atlases of human kidney tissue, we will integrate ultra-high speed, high spatial resolution (<10?m) MALDI timsTOF IMS with high mass resolution (>40,000 Resolving Power, <2 ppm mass accuracy) for molecular imaging of human eyes with an unprecedented combination of spatial fidelity and molecular specificity. The high-throughput capabilities of MALDI timsTOF IMS will be critical for generating high- spatial resolution 3-D molecular images from statistically relevant numbers of normal pancreas samples. MALDI IMS will provide the specificity and mass accuracy necessary to link ion images to orthogonal LC-based fragmentation experiments for molecular identification. Fragmentation will be performed using spatially specific surface sampling approaches to produce LC-MS/MS data from tissues in a manner that is compatible with IMS and capable of being integrated into HuBMAP tissue atlases. To maximize information gleaned from imaging experiments and lay the groundwork for data-driven image fusion and 3-D reconstruction outlined in the Data Analysis Core Research Strategy, we will also develop methods for collecting various microscopy-based image modalities (e.g. autofluorescence and stained microscopy) and pancreas specific, highly multiplexed immunofluorescence using CODEX. Whole eye atlases will be constructed by combining our multimodal IMS/Microscopy pipelines with ex vivo 3-D optical coherence tomography (OCT) imaging. Through the use of our multimodal pipeline, we propose to map spatial relationships of eye cell types from all regions of the eye within healthy tissue across a multitude of demographics.
Aim 1 : To create an eye- specific biospecimen collection and management plan.
Aim 2 : To establish a pre-analytical pipeline for standardizing sample preparation, determining tissue normalcy, and providing quality control metrics.
Aim 3 : To develop a multimodal characterization pipeline for eye-specific 3-D molecular imaging.
Aim 4 : To scale and standardize eye-specific 3-D molecular imaging. To accomplish these aims, we have assembled a highly interactive and established team of investigators consisting of complementary expertise in ophthalmology, analytical chemistry, and data science.
