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

Abstract

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.

Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells

“…Early light sheet methods such as selective plane illumination microscopy (SPIM) [ 38 , 40 ] were designed for imaging of large samples at low magnification. Other early methods producing a thin tilted beam are highly inclined and laminated optical sheet (HILO/pseudo-TIR) [ 41 , 42 ] and variable-angle epi-fluorescence microscopy (VAEM) [ 43 ]. However, in these techniques, the thickness, intensity, position, and depth of the excitation light pattern are highly coupled.…”

Single-Molecule Fluorescence Investigations of Solute Transport Dynamics in Nanostructured Membrane Separation Materials

“…The ensemble averaging of solute diffusion measurements can be circumvented by direct observation of the motions of the individual molecules. Advanced fluorescence microscopic techniques provide means for in situ observation of the dynamics of individual fluorescent molecules not only in nanoporous materials, ,,− but also at various interfaces relevant to chemical separations. − These techniques can be classified to two approaches, confocal single-point measurements and wide-field microscopic imaging: , The confocal single-point measurements record fluorescence intensity fluctuations from single (or a small number of) molecules in a femtoliter-scale detection volume at submicrosecond time resolution. If the fluctuations are associated with solute passage across the detection volume, an autocorrelation function from the fluctuations provides the characteristic time scales of molecular transport processes and the average number of solute molecules involved in the fluctuations (fluorescence correlation spectroscopy, FCS). ,, The wide-field microscopic imaging is based on direct video recording of the motions of individual fluorescent molecules (single molecule tracking, SMT). , SMT can offer both the trajectories of single molecules with positional precision beyond the diffraction limit and the speed of their motions at millisecond time resolution, ,, permitting the investigation of relationship between the structure and function of nanostructured materials.…”

Mechano-Electron Spin Coupling Modulates the Reactivity of Individual Coronazymes

“…While the force modulation is achieved by manipulating the corona phase, the electron spin can be controlled by a combined effect of the chiral plasmonic phase of the Au nanocore and the chiral sense of DNA in the peripheral corona phase. To characterize individual coronazyme catalysis, we have innovated a 3D magnetic tweezers (MT) with a highly inclined and laminated optical sheet (HILO) excitation to reduce the fluorescence background from an MT trapped super-paramagnetic bead, which is used to levitate the coronazyme 19,20 . The super-paramagnetic bead not only serves as a handle to apply mechanical forces on the coronazyme, but they also help to polarize electron spins in the coronazyme.…”