Single-Molecule Light-Sheet Microscopy with Local Nanopipette Delivery

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.