Nanometric axial localization of single fluorescent molecules with modulated excitation

Abstract

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Global fitting for high-accuracy multi-channel single-molecule localization

“…Compared to the single-channel SMLM, data analysis for all these methods is complicated by the fact that measures from two or more channels have to be combined to result in the additional information (color, z-position, polarization state, interference phase, etc.). Typically, this is achieved by first fitting the fluorophores individually in each channel to extract corresponding parameters, and then combining the returned parameters from different channels to obtain the extra information [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] . Separate fitting of an individual fluorophore present in two channels is not optimal, as we neglect the information that the fitting parameters (e.g., 3D positions and photons) are highly correlated.…”

“…different phases of the pattern on multiple parts of a camera, was used to increase the resolution of SMLM by a factor of two [13][14][15][16] .…”

Fluorescence nanoscopy at the sub-10 nm scale

“…Particularly, methods using graphene-covered substrates (graphene energy transfer, GET) reach sub-10 nm localization precision (Ghosh et al 2019;Kaminska et al 2019;Kamińska et al 2021). Finally, the SML-SSI methods described before can also be applied to localize molecules in the axial direction with sub-10 nm resolution, as demonstrated by 3D-MINFLUX (Gwosch et al 2020), ModLoc (Jouchet et al 2021), and ROSE-Z (Gu et al 2021).…”

“…They all inject the information of the spatial distribution of the excitation light in the localization process. Some of these techniques, namely, modulated localization (ModLoc) (Jouchet, et al 2021), Repetitive Optical Selective Exposure (ROSE) (Gu et al 2019), SIMFLUX (Cnossen et al 2020), and Structured Illumination based Point Localization Estimator with enhanced precision (SIMPLE) (Reymond et al 2019) have been recently reviewed (Reymond et al 2020) and a common framework to describe SML-SSI methods has been developed (Masullo et al 2021). While they should be considered single-molecule localization techniques, they differ from SMLM methods in the illumination used, which is not homogenous but carefully structured and characterized to enhance the localization precision.…”

Imaging therapeutic peptide transport across intestinal barriers

“…Notably, MINFLUX 109,110 [111][112][113] . Very recently, similar methods have been developed for improved axial localization using illumination structured along that dimension 114,115 . These wide-field methods are yet to be applied to imaging dynamics, however.…”