A technology that enables biomolecules, such as nucleic acids and proteins, to be imaged at nanoscale precision throughout preserved 3-D specimens would enable a greater understanding of life processes and disease detection. This would be beneficial in fields such as neuroscience and oncology where many complex questions remain unanswered. The ability to map the locations of specific types of biomolecules within subcellular compartments would give insight into cellular organization and any altered state in disease. With the advent of expanding microscopy (ExM), it is now possible to obtain nanoscale images using only a diffraction-limited light microscope. This simple yet novel approach uses water-swellable polymers to physically expand biological specimens to be imaged at approximately 70 nm resolution. While the protocol for expansion and the retention of intracellular antigen have progressed rapidly since ExM was first developed, currently available methods are limited by linear expansion of 4- 5 times of their original size. A higher expansion factor is needed to reveal the subtle changes in the size, shape, and texture ratio of subcellular organelles in health status or disease. More importantly, almost all the current ExM methods require a specific anchoring step to ensure targeted biomolecules are covalently linked to the newly synthesized hydrogel. A universal biomolecule anchor that works for thick tissues remains elusive. Additionally, few existing ExM approaches can expand diverse tissue types other than the brain without losing most of the epitopes. Thus there is tremendous pent-up demand for a method of nanoscale imaging for extended 3-D specimens and/or with highly versatile molecular contrast. Given its potential impact, we now propose a new framework called Magnify to bring the power and versatility of ExM to next generation. We will focus on the following aspects: (1) Develop a robust one-shot 11× expansion microscopy with universal biomolecule retention; (2) Further develop Magnify for expanding mm-scale thick tissues and whole small animals; (3) Extend Magnify for super-resolution vibrational imaging, including label-free, metabolic and multicolor super-resolution imaging. We will demonstrate the potential of Magnify as a powerful tool for mapping subcellular proteomic changes in diverse tissues, cells, and organelles by visualizing molecular spatial patterns at unprecedented high spatial resolution throughout preserved specimens.

Public Health Relevance Statement The research seeks to develop super-resolution imaging technologies that allow for versatile nanoscale imaging of biological specimens such as cells and tissues. This would enable profiling of how biomolecules are spatially organized in healthy and disease states, and discovery of new therapeutic targets.