Single-Molecule Imaging in Commercial Stationary Phase Particles Using Highly Inclined and Laminated Optical Sheet Microscopy

We resolve the three-dimensional, nanoscale locations
of single-molecule
analytes within commercial stationary phase materials using highly
inclined and laminated optical sheet (HILO) microscopy. Single-molecule
fluorescence microscopy of chromatography can reveal the molecular
heterogeneities that lead to peak broadening, but past work has focused
on surfaces designed to mimic stationary phases, which have different
physical and chemical properties than the three-dimensional materials
used in real columns and membranes. To extend single-molecule measurements
to commercial stationary phases, we immobilize individual stationary
phase particles and modify our microscope for imaging at further depths
with HILO, a method which was originally developed to resolve single
molecules in cells of comparable size to column packing materials
(∼5–10 μm). We describe and characterize how to
change the angle of incidence to achieve HILO so that other researchers
can easily incorporate this method onto their existing epi- or total
internal reflection fluorescence microscopes. We show improvements
up to a 32% in signal-to-background ratio and 118% in the number of
single molecules detected within stationary phase particles when using
HILO compared to epifluorescence. By controlling the objective position
relative to the sample, we produce three-dimensional maps of molecule
locations throughout entire stationary phase particles at nanoscale
lateral and axial resolutions. The number of localized molecules remains
constant axially throughout isolated stationary phase particles and
between different particles, indicating that heterogeneity in a separation
would not be caused by such affinity differences at microscales but
instead kinetic differences at nanoscales on identifiable and distinct
adsorption sites.