Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first expansion microscopy method was unable to retain native proteins in the gel and used custom made reagents not widely available. Here, we describe protein retention ExM (proExM), a variant of ExM that anchors proteins to the swellable gel allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validate and demonstrate utility of proExM for multi-color super-resolution (~70 nm) imaging of cells and mammalian tissues on conventional microscopes.

Mutations in alpha-tropomyosin are a rare cause of familial hypertrophic cardiomyopathy, accounting for approximately 3 percent of cases. Mutations in cardiac troponin T account for approximately 15 percent of cases of familial hypertrophic cardiomyopathy in this referral-center population. These mutations are characterized by relatively mild and sometimes subclinical hypertrophy but a high incidence of sudden death. Genetic testing may therefore be especially important in this group.

We here present a new way to engineer complex proteins toward multidimensional specifications, through a simple yet scalable directed evolution strategy. By robotically picking mammalian cells that are identified, under a microscope, to express proteins that simultaneously exhibit several specific properties, we can screen through hundreds of thousands of proteins in a library in a matter of a few hours, evaluating each along multiple performance axes. We demonstrate the power of this approach by identifying a novel genetically encoded fluorescent voltage indicator, simultaneously optimizing brightness and membrane localization of the protein using our microscopy-guided cell picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1, and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices as well as in larval zebrafish in vivo. We also demonstrate measurement of postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.