Amplitude Analysis of Polarization Modulation Data and 3D-Polarization Demodulation (3D-SPoD)

Abstract

Super-resolution optical fluctuation imaging (SOFI) is a technique that uses the amplitude of fluorescence correlation data for improved resolution of fluorescence images. Here, we explore if also the amplitude of superresolution by polarisation demodulation (SPoD) data can be used to gain additional information about the underlying structures. Highly organized experimental as well a simulated actin filament data demonstrate a principle information gain from this approach. In addition, we explored theoretically the benefits of analyzing the entire 3D-polarization information instead of only 2D-projections thereof. Due to fundamental principles, the probability of finding parallel orientations is approaching zero in 3D-SPoD in contrast to 2D-approaches. Using the modulation-amplitude based analysis we explored systematically simulated 3D-single molecules data (for which the true structures are known) under different conditions that are typically observed in experiments. We found that this approach can significantly improve the distinction, reconstruction and localization. In addition, these approaches are less sensitive to uncertainties in the knowledge about the true experimental point-spread-function (PSF) used for reconstruction compared to approaches using non-modulated data. Finally, they can effectively remove higher levels of nonmodulated back-ground intensity.

Split-HaloTag®Imaging Assay for Sophisticated Microscopy of Protein-Protein Interactionsin planta

“…Depending on the orientation of the illuminated fluorophores (or more specifically the orientation of their transition dipole moments), the observed fluorescence intensity will be phase-shifted, and differently oriented fluorophores will emit periodic signals peaking at different points in time. Therefore, the analyses via deconvolution algorithms allow a high-resolution imaging of cell structures (for details see Albrecht et al ., 2020). During each measurement, 2,000 frames were recorded, which in itself is not problematic when using stable dyes.…”

“…Principles of the experimental set-up are described by Hafi and colleagues (2014) and modifications enabling the analysis of the dye’s 3D-orientations were described by Albrecht et al . (2020).…”

“…Principles of the experimental set-up are described by Hafi and colleagues (2014) and modifications enabling the analysis of the dye´s 3D-orientations were described by Albrecht et al (2020). The cover slip was fixed to the microscope slide with nail polish and directly used for microscopy.…”

Fluorescence polarization immunoassay for the determination and screening of medicines.

“…Одним из распространенных методов тестирования флуоресцентно меченных соединений является поляризация флуоресценции (FP). Благодаря универсальности FP и наличию высококачественных поляризаторов, стабильных детекторов и источников возбуждения, этот метод нашел широкое применение в различных биологических приложениях: исследование клеточного микроокружения [2] и мониторинг клеточных сигнальных путей [3], до 2D и 3D-визуализация, [4], оценка систем доставки лекарств [5], обнаружение органических веществ и лекарственных средств [6][7][8].…”

Combining single-molecule super-resolved localization microscopy with fluorescence polarization imaging to study cellular processes

“…Techniques in which the excitation polarisation is modulated [36]- [40] have promising signal-tobackground characteristics and potential for true 3D reconstruction of molecular orientation [40]. Polarization-based orientation information is also weakly encoded in an emitter's point-spread function [41], although this information is difficult to extract without unequivocal single-molecule emission.…”