Optimal precision and accuracy in 4Pi-STORM using dynamic spline PSF models.
Mark BatesJan KellerAdrian PrzybylskiAndreas HüperTill StephanPeter IlgenAngel Rafael Cereceda DelgadoElisa D'EsteAlexander EgnerStefan JakobsSteffen J SahlStefan W HellPublished in: Nature methods (2022)
Coherent fluorescence imaging with two objective lenses (4Pi detection) enables single-molecule localization microscopy with sub-10 nm spatial resolution in three dimensions. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation and the challenging nature of the data analysis. Here we report the development of a 4Pi-STORM microscope, which obtains optimal resolution and accuracy by modeling the 4Pi point spread function (PSF) dynamically while also using a simpler optical design. Dynamic spline PSF models incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal evolution of the optical system. Our method reaches the theoretical limits for precision and minimizes phase-wrapping artifacts by making full use of the information content of the data. 4Pi-STORM achieves a near-isotropic three-dimensional localization precision of 2-3 nm, and we demonstrate its capabilities by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.
Keyphrases
- single molecule
- data analysis
- fluorescence imaging
- photodynamic therapy
- nucleic acid
- high resolution
- atomic force microscopy
- high speed
- living cells
- cell death
- high throughput
- optical coherence tomography
- electronic health record
- spinal cord injury
- small molecule
- protein protein
- deep learning
- social media
- quantum dots