Direct supercritical angle localization microscopy for nanometer 3D superresolution

Abstract

Abstract3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. In supercritical angle localization microscopy (SALM), z-positions of single fluorophores are extracted from the intensity of supercritical angle fluorescence, which strongly depends on their distance to the coverslip. Here, we realize the full potential of SALM and improve its z-resolution by more than four-fold compared to the state-of-the-art by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying fitting routines to extract precise intensities of single emitters. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.

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

“…It is usually realized using two spectral channels 1-3 or one spatial channel combined with spectral detection in a second channel 4 . Three-dimensional (3D) SMLM techniques using two or more detection channels, such as biplane 5 or multi-plane 6 detection, self-bending point spread functions 7 (PSFs), supercritical-angle fluorescence detection 8,9 and multi-phase interference 10,11 , are powerful in investigating the intrinsic 3D organization of biological structures. Two or more fluorescence polarization channels are used to probe the orientation of fluorophores 12 , offering insight into the orientation of proteins in a molecular machinery.…”

“…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.…”

Fluorescence nanoscopy at the sub-10 nm scale

“…In Supercritical Angle Localization Microscopy (SALM), the super-critical angular emission is the key property that encodes the axial position (Bourg et al 2015). Performed with high numerical aperture objectives (NA = 1.70) and high-refractive index coverslips and immersion oil, SALM can reach sub-10 nm axial precision (Dasgupta et al 2021). Methods that exploit the distance-dependent energy transfer to a conductive surface, determine the axial position of fluorophores from lifetime measurements (Chizhik et al 2014).…”

Answering some questions about structured illumination microscopy

“…near-field emission that is converted to propagating light by interacting with the boundary) to further enhance their resolution calculation (i.e. the detection NA is limited to the refractive index of the sample), despite other measurements with the same objective showing that significant super-critical light can be present [ 40 ].…”