High-density volumetric super-resolution microscopy Sam Daly 1 , João Ferreira Fernandes 2 , Anoushka Handa 1 , Ezra Bruggeman 1 , Sarah Benaissa 1 , Boya Zhang 1 , Edward W. Sanders 1 , Ruth Sims 3 , David Klenerman 1 , Kevin O’Holleran 1 , Simon Davis 2 , Steven F. Lee 1 1 University of Cambridge, UK, 2 University of Oxford, UK, 3 Institut de la Vision, France Observing systems in 3D is crucial in order to understand chemistry happening in complex environments, such as biological structures. Imaging complex natural morphologies in only 2D can lead to artifacts, such as artificial clustering and the underestimation of diffusion dynamics. Single-molecule localisation microscopy (SMLM) is a super-resolution microscopy technique that enables the observation of a sample with a resolution beyond the diffraction limit of light (~250 nm). 1–3 SMLM has captured sub-diffraction biological architecture in 3D by encoding axial position into the fluorescence image of a single molecule (known as the point spread function, or PSF). However, 3D techniques suffer from lower spatial and temporal resolution compared to 2D, which significantly extends experimental duration and presents limitations in biological sample preparation. As a result, to have broad applicability to the biological community there is a fundamental need for physical scientists to develop new strategies to perform 3D-SMLM at high speed. Single-molecule light field microscopy (SMLFM) 4 is a novel microscopy technique based on the principle of parallax combined with SMLM to enable imaging of sub-diffraction biological structures in 3D. 5 An array of microlenses placed in the emission path of a typical widefield microscope enables observation of the sample from multiple perspective views and reconstruction in 3D. Conceptually, SMLFM facilitates imaging of biological systems with highly dense fluorescent labelling through two attributes: (1) the minimisation of the area of the PSF recorded on the detector, and (2) the separation of overlapping PSFs by imaging the same emitter from multiple angles. This new approach in 3D microscopy leads to a significant improvement in temporal resolution, which opens the door to real-time 3D super-resolution imaging. We report the first hexagonal SMLFM platform with a large 8 µm depth-of-focus, suitable for both whole-cell super-resolution imaging and single particle tracking at high densities. Firstly, we address how different state-of- the-art 3D-SMLM techniques perform at resolving single molecules at increasing molecular density, and show comparatively that SMLFM performs substantially higher in emitter identification and temporal resolution. We then show that SMLFM is insensitive to PSF overlap through the scan-free imaging and tracking of individual B cell receptors (BCRs) and the imaging of tubulin in HeLa cells. In doing so, we demonstrate that existing labelling methods can now be directly transferred to 3D imaging pipelines by obtaining 29 nm isotropic resolution in 3D (Fourier Shell Correlation) over ~8 minutes. References 1. Lelek, et al. , Nat. Rev. Methods Primer, 2021, 1 , 39.
2. Rust, et al. , Nat. Methods, 2006, 3 , 793–796. 3. Betzig, et al. , Science, 2006, 313 , 1642–1645. 4. Sims, et al. , Optica, 2020, 7 , 1065. 5. Guo, et al. , Opt. Express, 2019, 27 , 25573–25594.
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