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Three-Dimensional Single Molecule Localization Microscopy Reveals the Topography of the Immunological Synapse at Isotropic Precision below 15 nm.

ABSTRACT: T-cells engage with antigen-presenting cells in search for antigenic peptides and form transient interfaces termed immunological synapses. Synapse topography affects receptor binding rates and the mutual segregation of proteins due to size exclusion effects. It is hence important to determine the 3D topography of the immunological synapse at high precision. Current methods provide only rather coarse images of the protein distribution within the synapse. Here, we applied supercritical angle fluorescence microscopy combined with defocused imaging, which allows three-dimensional single molecule localization microscopy (3D-SMLM) at an isotropic localization precision below 15 nm. Experiments were performed on hybrid synapses between primary T-cells and functionalized glass-supported lipid bilayers. We used 3D-SMLM to quantify the cleft size within the synapse by mapping the position of the T-cell receptor (TCR) with respect to the supported lipid bilayer, yielding average distances of 18 nm up to 31 nm for activating and nonactivating bilayers, respectively.

SUBMITTER: Velas L

PROVIDER: S-EPMC8587899 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Publications

Three-Dimensional Single Molecule Localization Microscopy Reveals the Topography of the Immunological Synapse at Isotropic Precision below 15 nm.

Velas Lukas L Brameshuber Mario M Huppa Johannes B JB Kurz Elke E Dustin Michael L ML Zelger Philipp P Jesacher Alexander A Schütz Gerhard J GJ

Nano letters 20211028 21

T-cells engage with antigen-presenting cells in search for antigenic peptides and form transient interfaces termed immunological synapses. Synapse topography affects receptor binding rates and the mutual segregation of proteins due to size exclusion effects. It is hence important to determine the 3D topography of the immunological synapse at high precision. Current methods provide only rather coarse images of the protein distribution within the synapse. Here, we applied supercritical angle fluor ...[more]

PMID: 34709845

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Project description:Modulation enhanced single-molecule localization microscopy (meSMLM) methods improve the localization precision by using patterned illumination to encode additional position information. Iterative meSMLM (imeSMLM) methods iteratively generate prior information on emitter positions, used to locally improve the localization precision during subsequent iterations. The Cramér-Rao lower bound cannot incorporate prior information to bound the best achievable localization precision because it requires estimators to be unbiased. By treating estimands as random variables with a known prior distribution, the Van Trees inequality (VTI) can be used to bound the best possible localization precision of imeSMLM methods. An imeSMLM method is considered, where the positions of in-plane standing-wave illumination patterns are controlled over the course of multiple iterations. Using the VTI, we analytically approximate a lower bound on the maximum localization precision of imeSMLM methods that make use of standing-wave illumination patterns. In addition, we evaluate the maximally achievable localization precision for different illumination pattern placement strategies using Monte Carlo simulations. We show that in the absence of background and under perfect modulation, the information content of signal photons increases exponentially as a function of the iteration count. However, the information increase is no longer exponential as a function of the iteration count under non-zero background, imperfect modulation, or limited mechanical resolution of the illumination positioning system. As a result, imeSMLM with two iterations reaches at most a fivefold improvement over SMLM at 8 expected background photons per pixel and 95% modulation contrast. Moreover, the information increase from imeSMLM is balanced by a reduced signal photon rate. Therefore, SMLM outperforms imeSMLM when considering an equal measurement time and illumination power per iteration. Finally, the VTI is an excellent tool for the assessment of the performance of illumination control and is therefore the method of choice for optimal design and control of imeSMLM methods.

Project description:Single-molecule localization microscopy (SMLM) can decipher fine details that are otherwise impossible using diffraction-limited microscopy. Often, the reconstructed super-resolved images suffer from noise, strong background and are prone to false detections that may impact quantitative imaging. To overcome these limitations, we propose a technique (corrSMLM) that recognizes and detects fortunate molecules (molecules with long blinking cycles) from the recorded data. The method uses correlation between two or more consecutive frames to identify and isolate fortunate molecules that blink longer than the standard blinking period of a molecule. The corrSMLM is based on the fact that random fluctuations (noise) do not last longer (usually limited to a single frame). In contrast, fortunate molecules consistently fluoresce for extended periods and hence appear on more than one frame. Accordingly, strongly correlated spots (representing fortunate molecules) are compared in the consecutive frames, followed by data integration to determine their position and localization precision. The technique addresses two significant problems that plague existing SMLM : (1) false detection due to random noise that contributes to a strong background and (2) poor localization leading to overall low resolution. To demonstrate, corrSMLM is used for imaging fixed NIH3T3 cells (transfected with Dendra2-Actin, Dendra2-Tubulin, and mEos-Tom20 plasmid DNA). The super-resolved images show a significant reduction in background noise ( > 1.5 fold boost in SBR) and > 2-fold improvement in localization precision as compared to standard SMLM. Intensity analysis based on the number of molecules suggests that corrSMLM better corroborates the raw data and preserves finer features (e.g., edges), which are wiped out in standard SMLM. Overall, an improvement is noted in the localization precision and spatial resolution. The proposed technique is anticipated to advance SMLM and is expected to contribute to a better understanding of single-molecule dynamics in a cellular environment.

Dataset Information

Three-Dimensional Single Molecule Localization Microscopy Reveals the Topography of the Immunological Synapse at Isotropic Precision below 15 nm.

Publications

Three-Dimensional Single Molecule Localization Microscopy Reveals the Topography of the Immunological Synapse at Isotropic Precision below 15 nm.

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