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

Cite this: Anal. Chem. 2023, 95, 4, 2245–2252
Publication Date (Web):January 18, 2023
https://doi.org/10.1021/acs.analchem.2c03753
Copyright © 2023 American Chemical Society

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    Abstract

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    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.

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    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.2c03753.

    • Further details on calculations, sources of error, axial and lateral resolutions, additional 3D maps of particles, comparison to DH-PSFs, visual aids for 3D map elongation, and HILO illumination (PDF)

    • (Video S1) A 3D CSP scan (AVI)

    • (Video S2) Isolated CSPs imaged in epi (AVI)

    • (Video S3) Isolated CSPs imaged HILO (AVI)

    • (Video S4) Grouped CSPs imaged in epi (AVI)

    • (Video S5) Grouped CSPs imaged in HILO (AVI)

    • (Video S6) Applying DH-PSFs to CSP (AVI)

    • TIF Image Stacks S1–S6 (ZIP)

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    Cited By

    This article is cited by 2 publications.

    1. Takashi Ito. Single-Molecule Fluorescence Investigations of Solute Transport Dynamics in Nanostructured Membrane Separation Materials. The Journal of Physical Chemistry B 2023, 127 (26) , 5733-5741. https://doi.org/10.1021/acs.jpcb.3c02807
    2. Khanh-Hoa Tran-Ba, Kathryn Foreman. Single-molecule tracking of dye diffusion in synthetic polymers: A tutorial review. Journal of Applied Physics 2023, 133 (10) , 101101. https://doi.org/10.1063/5.0139719

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