a three-camera imaging setup and novel cell · pdf filekinetics (figure 1). the novel dyes...
TRANSCRIPT
A THREE-CAMERA IMAGING SETUP AND NOVEL CELL-PREMEABLE DYES FOR MULTIPLEXED SINGLE-MOLECULE LIVE CELL
EXPERIMENTS
Brian P English, Luke D Lavis, Timothée Lionnet, Robert H Singer Janelia Research Campus, Howard Hughes Medical Institute
19700 Helix Drive Ashburn, Virginia 20147, USA
E-mail: [email protected]
KEY WORDS: Living cells, fluorophores, optical reconstruction microscopy (STORM), intracellular labeling, single particle tracking, super-resolution imaging Our aim is to develop quantitative single cell and single molecule assays to study when and where molecules are interacting inside living cells and where enzymes are active. To this end we present a simultaneous three-camera imaging setup for fast tracking of multiple interacting molecules simultaneously. We further present novel bright and cell-
permeable dyes that have enabled live-cell multi-color tracking experiments [1]. This new color palette of bright and photostable fluorophores is a key component for multi-color experiments within the nucleus of a living cell. We also present algorithms that accurately track and co-localize interacting biomolecules. The new setup combined with our co-movement algorithms have made it possible to simultaneously image and track both nascent mRNAs and RNA polymerases, to image where and when mRNA may be translated by ribosomes, and how the chromosome environment affects diffusion kinetics (Figure 1). The novel dyes have facilitated multiplexed single-molecule measurements at a high spatiotemporal resolution inside
living cells. Such experiments will constitute a major tool in testing models relating molecular architecture and biological dynamics. [1] J.B. Grimm; B.P. English; J. Chen; J.P. Slaughter; Z. Zhang; A. Revyakin; R. Patel; J.J. Macklin; D. Normanno; R.H. Singer; T. Lionnet and L.D. Lavis, “A general method to improve fluorophores for live-cell and single-molecule microscopy,”Nature Methods, 10.1038/nmeth.3256 (2015).
Figure 1: Rendering of single-molecule trajectories of SnapTag–TetR–JF549 conjugate overlaid on a dSTORM image of HaloTag–H2B labeled with JF646 in the nucleus of a live U2OS cell [1].