Light Sheet Microscopy for Tracking Single Molecules
on the Apical Surface of Living Cells
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Abstract
Single particle tracking is a powerful
tool to study single molecule
dynamics in living biological samples. However, current tracking techniques,
which are based mainly on epifluorescence, confocal, or TIRF microscopy,
have difficulties in tracking single molecules on the apical surface
of a cell. We present here a three-dimensional (3D) single particle
tracking technique that is based on prism coupled light-sheet microscopy
(PCLSM). This novel design provides a signal-to-noise ratio comparable
to confocal microscopy while it has the capability of illuminating
at arbitrary depth. We demonstrate tracking of single EGF molcules
on the apical surface of live cell membranes from their binding to
EGF receptors until they are internalized or photobleached. We found
that EGF exhibits multiple diffusion behaviors on live A549 cell membranes.
At room temperature, the average diffusion coefficient of EGF on A549
cells was measured to be 0.13 μm<sup>2</sup>/s. Depletion of
cellular cholesterol with methyl-β-cyclodextrin leads to a broader
distribution of diffusion coefficients and an increase of the average
diffusion coefficient at room temperature. This light-sheet based
3D single particle tracking technique solves the technique difficulty
of tracking single particles on apical membranes and is able to document
the whole “lifetime” of a particle from binding till
photobleaching or internalization