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Single-Molecule Light-Sheet Microscopy with Local Nanopipette Delivery.

Accepted version
Peer-reviewed

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Type

Article

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Authors

Ponjavic, Aleks 
Chen, Wei-Hsin 
Hopkins, Lee 
Hughes, Craig 

Abstract

The detection of single molecules in biological systems has rapidly increased in resolution over the past decade. However, the delivery of single molecules remains to be a challenge. Currently, there is no effective method that can both introduce a precise amount of molecules onto or into a single cell at a defined position and then image the cellular response. Here, we have combined light-sheet microscopy with local delivery, using a nanopipette, to accurately deliver individual proteins to a defined position. We call this method local-delivery selective-plane illumination microscopy (ldSPIM). ldSPIM uses a nanopipette and ionic feedback current at the nanopipette tip to control the position from which the molecules are delivered. The number of proteins delivered can be controlled by varying the voltage applied. For single-molecule detection, we implemented single-objective SPIM using a reflective atomic force microscopy cantilever to create a 2 μm thin sheet. Using this setup, we demonstrate that ldSPIM can deliver single fluorescently labeled proteins onto the plasma membrane of HK293 cells or into the cytoplasm. Next, we deposited the aggregates of amyloid-β, which causes proteotoxicity relevant to Alzheimer's disease, onto a single macrophage stably expressing a MyDD88-eGFP fusion construct. Whole-cell imaging in the three-dimensional (3D) mode enables the live detection of MyDD88 accumulation and the formation of myddosome signaling complexes, as a result of the aggregate-induced triggering of toll-like receptor 4. Overall, we demonstrate a novel multifunctional imaging system capable of precise delivery of single proteins to a specific location on the cell surface or inside the cytoplasm and high-speed 3D detection at single-molecule resolution within live cells.

Description

Keywords

Cell Membrane, Microscopy, Atomic Force, Nanotechnology, Single Molecule Imaging

Journal Title

Anal Chem

Conference Name

Journal ISSN

0003-2700
1520-6882

Volume Title

93

Publisher

American Chemical Society (ACS)

Rights

All rights reserved
Sponsorship
Royal Society (RP150066)
Engineering and Physical Sciences Research Council (EP/L027631/1)
Biotechnology and Biological Sciences Research Council (BB/L006227/1)
Single-molecule light-sheet microscopy with local nanopipette de-livery.