NIH Research Festival
FARE Award Winner
Although the plasma membrane and associated protein complexes are involved in critical cellular functions, including cell-cell interactions and information transduction, current imaging techniques are unable to conduct multiple imaging rounds on the same sample, limiting the ability to multiplex protein labels. One approach resolving this complication is resin-embedded multicycle imaging (REMI), which stabilizes protein structure and antigenicity in acrylic resin, allowing labels to be removed and replaced, leading to imaging large numbers of proteins of interest on the same specimen. The current implementation of this technology, array tomography, is of great benefit to problems involving protein complexes and organelles up to about a micron in size, but imaging large, distributed organelles such as plasma membranes rapidly encounters increased complexity in imaging and analysis. We have developed novel preparative methods for imaging multiple proteins in larger areas of individual cells. After affixing cells to glass-bottomed dishes, we embed whole cells, or isolate their basal membranes using physical shear forces. We fix and embed these cells or membranes in hydrophilic resin in an oxygen-free environment, forming an ultra-thin resin sheet around the specimen. The result is a stable platform in which epitopes are trapped in-situ, with minimal barrier to immunohistochemical label diffusion. We have identified conditions for removing and relabeling antibodies, enabling multiplexed imaging. By sequential imaging the same specimen we can repeat this process as needed. Here we describe this method and highlight a few potential applications, including fluorophore reuse with precision localization microscopy.
Scientific Focus Area: Cell Biology
This page was last updated on Friday, March 26, 2021