pixOL: pixel-wise dipole-spread function engineering for simultaneously measuring the 3D orientation and 3D localization of dipole-like emitters

Abstract

Interactions between biomolecules are characterized by both where they occur and how they are organized, e.g., the alignment of lipid molecules to form a membrane. However, spatial and angular information are mixed within the image of a fluorescent molecule-the microscopy's dipole spread function (DSF). We demonstrate the pixOL algorithm for simultaneously optimizing all pixels within a phase mask to produce an engineered Green's tensor-the dipole extension of point-spread function engineering. The pixOL DSF achieves optimal precision for measuring simultaneously the 3D orientation and 3D location of a single molecule, i.e., 1.14 degree orientation, 0.24 sr wobble angle, 8.17 nm lateral localization, and 12.21 nm axial localization precisions over an 800-nm depth range using 2500 detected photons. The pixOL microscope accurately and precisely resolves the 3D positions and 3D orientations of Nile red within a spherical supported lipid bilayer, resolving both membrane defects and differences in cholesterol concentration, in 6 dimensions.

Monitoring Non-Specific Adsorption at Solid-Liquid Interfaces by Supercritical Angle Fluorescence Microscopy

“…Finally, the facile nature of the new SAF module suggests the potential for straightforward improvements in the future. For example, integration of filters or a monochromator should allow for multi-colour SAF measurements, allowing one to characterize exchange dynamics and competitive adsorption phenomena while polarized SAF measurements may provide novel insights into emission dipole orientation 10,32 .…”

6D single-fluorogen orientation-localization microscopy for elucidating the architecture of beta-sheet assemblies and biomolecular condensates

“…We present an overview of six-dimensional single-molecule orientation-localization microscopy (SMOLM) 1 and its applications to imaging β-sheet peptide assemblies 2 and biomolecular condensates. 3 This paper focuses on recent work performed at Washington University and is not intended to be a comprehensive review [4][5][6][7] of the field. SMOLM is a form of super-resolution microscopy that combines single-molecule localization microscopy (SMLM) 8 with vectorial imaging models 9,10 and multi-dimensional estimation algorithms 11 to measure the orientations of single fluorophores.…”

Six-Dimensional Single-Molecule Imaging with Isotropic Resolution using a Multi-View Reflector Microscope

“…Silica spheres coated with supported lipid bilayers (SLBs) composed of DPPC (1,2-Dipalmitoyl-sn-glycero-3-phosphocholine) and 40% cholesterol (Figure 3a) have been previously shown 22 to be excellent calibration targets for SMOLM since we are able to systematically control both the morphology and ordering of the lipids within the SLB. By imaging Nile red (NR, Figure S15) molecules transiently bound within the SLBs (using the PAINT blinking mechanism 36 ), we resolve spherical structures with radii as small as 150 nm and as large as 1 µm without scanning the sample (Figures 3b,c).…”

“…We follow previously reported procedures 16,17,22 for preparing lipid-coated silica spheres. First, we prepare large unilamellar vesicles (LUVs) composed of DPPC and 40% cholesterol.…”

“…Limited by challenging signal-to-background ratios (SBRs) in typical single molecule (SM) experiments, microscopists have developed various SMOLM techniques to improve measurement performance, 18 approaching the theoretical limits under ideal conditions. [19][20][21] To use precious fluorescence photons more efficiently and maximize signal-to-noise ratios (SNRs), most recently developed techniques create dipolespread functions (DSFs), or images of dipole emitters, with compact footprints 11,12,15,17,22 and thusly achieve higher precision and accuracy. One key challenge with these designs is that orientation estimation requires fitting subtle features within noisy images.…”