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.
