Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules

Abstract

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

Plasmonics for advance single-molecule fluorescence spectroscopy and imaging in biology

“…Popularization of these fluorescence super-resolution techniques has begun to facilitate discoveries in different field of biology, by resolving protein organization with previously unattainable spatial detail. Whilst these techniques offer lateral resolution in the range of 10-40 nm, axial resolution is typically 2 to 4 times worse (Huang et al, 2008) and therefore different approaches over the years have been developed to achieve isotropic resolution in 3D (Bourg et al, 2015;Szalai et al, 2021;Velas et al, 2021). By levering the distance-dependent fluorescence lifetime modulation as response to the near-field coupling between fluorescent molecules and plasmon surface resonances in a thin metallic film, MIET currently offers a straightforward solution for isotropic 3D super-resolution imaging (Thiele et al, 2021).…”

Through the Eyes of Creators: Observing Artificial Molecular Motors

“…This can drastically improve the signal-to-noise ratio and enable a high vertical spatial resolution at an unchanged lateral resolution; however, an exponential decrease in the fluorescence excitation with vertical distance needs to be taken into account in intensity measurements. This z -dependence of TIRF has been utilized for three-dimensional (3D) microscopy reaching nanometer resolution along the z -axis . An additional capability of TIRF microscopy is that the incident light can be polarized parallel or perpendicular to the glass coverslip.…”

“…This zdependence of TIRF has been utilized for three-dimensional (3D) microscopy reaching nanometer resolution along the zaxis. 90 An additional capability of TIRF microscopy is that the incident light can be polarized parallel or perpendicular to the glass coverslip. Because the probability of photon absorption is a scalar product of the orientation of a fluorophore relative to the polarization of evanescent field, one can use polarization to achieve a selective excitation of fluorophores of a specific orientation.…”

Expansion microscopy with ninefold swelling (NIFS) hydrogel permits cellular ultrastructure imaging on conventional microscope

“…In recent years, novel superresolution techniques provide breakthroughs to the resolution limit. One approach is through innovations in instrumentations, such as STED (stimulated emission depletion) microscopy, SOFI (superresolution optical fluctuation imaging), and STORM (stochastic optical reconstruction microscopy) (9)(10)(11)(12)(13)(14)(15), which could reach resolutions of 30 to 50 nm. However, their widespread applications are still limited by expensive machinery, specific hardware, laborious operation, and sophisticated data processing (7,(16)(17)(18).…”