7 research outputs found
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
(ā¼5ā10 Ī¼m). 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
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
(ā¼5ā10 Ī¼m). 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
Direct Imaging of Protein Stability and Folding Kinetics in Hydrogels
We
apply fast relaxation imaging (FReI) as a novel technique for investigating
the folding stability and dynamics of proteins within polyacrylamide
hydrogels, which have diverse and widespread uses in biotechnology.
FReI detects protein unfolding in situ by imaging changes in fluorescence
resonance energy transfer (FRET) after temperature jump perturbations.
Unlike bulk measurements, diffraction-limited epifluorescence imaging
combined with fast temperature perturbations reveals the impact of
local environment effects on proteinābiomaterial compatibility.
Our experiments investigated a crowding sensor protein (CrH2) and
phosphoglycerate kinase (PGK), which undergoes cooperative unfolding.
The crowding sensor quantifies the confinement effect of the cross-linked
hydrogel: the 4% polyacrylamide hydrogel is similar to aqueous solution
(no confinement), while the 10% hydrogel is strongly confining. FRAP
measurements and protein concentration gradients in the 4% and 10%
hydrogels further support this observation. PGK reveals that noncovalent
interactions of the protein with the polymer surface are more important
than confinement for determining protein properties in the gel: the
mere presence of hydrogel increases protein stability, speeds up folding
relaxation, and promotes irreversible binding to the polymer even at the solutionāgel
interface, whereas the difference between the 4% and the 10% hydrogels
is negligible despite their large difference in confinement. The imaging
capabilities of FReI, demonstrated to be diffraction limited, further
revealed spatially homogeneous protein unfolding across the hydrogels
at 500 nm length scales and revealed differences in protein properties at
the gelāsolution boundary
Improved Analysis for Determining Diffusion Coefficients from Short, Single-Molecule Trajectories with Photoblinking
Two maximum likelihood estimation (MLE) methods were
developed
for optimizing the analysis of single-molecule trajectories that include
phenomena such as experimental noise, photoblinking, photobleaching,
and translation or rotation out of the collection plane. In particular,
short, single-molecule trajectories with photoblinking were studied,
and our method was compared to existing analytical techniques applied
to simulated data. The optimal method for various experimental cases
was established, and the optimized MLE method was applied to a real
experimental system: single-molecule diffusion of fluorescent molecular
machines known as nanocars
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Characterization of Porous Materials by Fluorescence Correlation Spectroscopy Super-resolution Optical Fluctuation Imaging
Porous materials such as cellular cytosol, hydrogels, and block copolymers have nanoscale features that determine macroscale properties. Characterizing the structure of nanopores is difficult with current techniques due to imaging, sample preparation, and computational challenges. We produce a super-resolution optical image that simultaneously characterizes the nanometer dimensions of and diffusion dynamics within porous structures by correlating stochastic fluctuations from diffusing fluorescent probes in the pores of the sample, dubbed here as āfluorescence correlation spectroscopy super-resolution optical fluctuation imagingā or āfcsSOFIā. Simulations demonstrate that structural features and diffusion properties can be accurately obtained at sub-diffraction-limited resolution. We apply our technique to image agarose hydrogels and aqueous lyotropic liquid crystal gels. The heterogeneous pore resolution is improved by up to a factor of 2, and diffusion coefficients are accurately obtained through our method compared to diffraction-limited fluorescence imaging and single-particle tracking. Moreover, fcsSOFI allows for rapid and high-throughput characterization of porous materials. fcsSOFI could be applied to soft porous environments such hydrogels, polymers, and membranes in addition to hard materials such as zeolites and mesoporous silica
Charge-Dependent Transport Switching of Single Molecular Ions in a Weak Polyelectrolyte Multilayer
The
tunable nature of weak polyelectrolyte multilayers makes them
ideal candidates for drug loading and delivery, water filtration,
and separations, yet the lateral transport of charged molecules in
these systems remains largely unexplored at the single molecule level.
We report the direct measurement of the charge-dependent, pH-tunable,
multimodal interaction of single charged molecules with a weak polyelectrolyte
multilayer thin film, a 10 bilayer film of polyĀ(acrylic acid) and
polyĀ(allylamine hydrochloride) PAA/PAH. Using fluorescence microscopy
and single-molecule tracking, two modes of interaction were detected:
(1) adsorption, characterized by the molecule remaining immobilized
in a subresolution region and (2) diffusion trajectories characteristic
of hopping (<i>D</i> ā¼ 10<sup>ā9</sup> cm<sup>2</sup>/s). Radius of gyration evolution analysis and comparison
with simulated trajectories confirmed the coexistence of the two transport
modes in the same single molecule trajectories. A mechanistic explanation
for the probe and condition mediated dynamics is proposed based on
a combination of electrostatics and a reversible, pH-induced alteration
of the nanoscopic structure of the film. Our results are in good agreement
with ensemble studies conducted on similar films, confirm a previously-unobserved
hopping mechanism for charged molecules in polyelectrolyte multilayers,
and demonstrate that single molecule spectroscopy can offer mechanistic
insight into the role of electrostatics and nanoscale tunability of
transport in weak polyelectrolyte multilayers
Optimization of Spectral and Spatial Conditions to Improve Super-Resolution Imaging of Plasmonic Nanoparticles
Interactions
between fluorophores and plasmonic nanoparticles modify
the fluorescence intensity, shape, and position of the observed emission
pattern, thus inhibiting efforts to optically super-resolve plasmonic
nanoparticles. Herein, we investigate the accuracy of localizing dye
fluorescence as a function of the spectral and spatial separations
between fluorophores (Alexa 647) and gold nanorods (NRs). The distance
at which Alexa 647 interacts with NRs is varied by layer-by-layer
polyelectrolyte deposition while the spectral separation is tuned
by using NRs with varying localized surface plasmon resonance (LSPR)
maxima. For resonantly coupled Alexa 647 and NRs, emission to the
far field through the NR plasmon is highly prominent, resulting in
underestimation of NR sizes. However, we demonstrate that it is possible
to improve the accuracy of the emission localization when both the
spectral and spatial separations between Alexa 647 and the LSPR are
optimized