Unraveling cellular complexity with transient adapters in highly multiplexed super-resolution imaging

• Published in: Cell Vol. 187; no. 7; pp. 1769 - 1784.e18
• Main Authors: Schueder, Florian, Rivera-Molina, Felix, Su, Maohan, Marin, Zach, Kidd, Phylicia, Rothman, James E., Toomre, Derek, Bewersdorf, Joerg
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.03.2024
• Subjects: Animals; DNA; DNA-PAINT; FLASH-PAINT; fluorescence microscopy; Golgi Apparatus; Mammals; Microscopy, Fluorescence - methods; multiplexing; nanoscopy; Oligonucleotides; Proteins; spatial omics; super-resolution; nanoscopy; FLASH-PAINT; multiplexing; spatial omics; DNA-PAINT; super-resolution
• ISSN: 0092-8674; 1097-4172; 1097-4172
• DOI: 10.1016/j.cell.2024.02.033

Mapping the intricate spatial relationships between the many different molecules inside a cell is essential to understanding cellular functions in all their complexity. Super-resolution fluorescence microscopy offers the required spatial resolution but struggles to reveal more than four different targets simultaneously. Exchanging labels in subsequent imaging rounds for multiplexed imaging extends this number but is limited by its low throughput. Here, we present a method for rapid multiplexed super-resolution microscopy that can, in principle, be applied to a nearly unlimited number of molecular targets by leveraging fluorogenic labeling in conjunction with transient adapter-mediated switching for high-throughput DNA-PAINT (FLASH-PAINT). We demonstrate the versatility of FLASH-PAINT with four applications: mapping nine proteins in a single mammalian cell, elucidating the functional organization of primary cilia by nine-target imaging, revealing the changes in proximity of thirteen different targets in unperturbed and dissociated Golgi stacks, and investigating and quantifying inter-organelle contacts at 3D super-resolution. [Display omitted] •FLASH-PAINT enables highly multiplexed super-resolution imaging•Transient adapters and erasers allow for fast, efficient, and gentle label exchange•Multiplexed super-resolution imaging reveals complex cilia and Golgi organization•3D FLASH-PAINT allows for quantification of organelle contact site numbers and areas Development of FLASH-PAINT enables rapid and efficient visualization of cellular organelles, using a potentially unlimited number of labels.