We introduce single molecule light field microscopy (SMLFM), a new class of three-dimensional (3D) single molecule localization microscopy. By segmenting the back focal plane of a microscope objective with an array of microlenses to generate multiple 2D perspective views, the same single fluorophore can be imaged from different angles. These views, in combination with a bespoke fitting algorithm, enable the 3D positions of single fluorophores to be determined from parallax. SMLFM achieves up to 20 nm localization precision throughout an extended 6 µ m depth of field. The capabilities of SMLFM are showcased by imaging membranes of fixed eukaryotic cells and DNA nanostructures below the optical diffraction limit.

We introduce single molecule light field microscopy (SMLFM), a novel 3D single molecule localization technique that is capable of up to 20 nm lateral and axial precision across a 6 µm depth of field. SMLFM can be readily implemented by installing a refractive microlens array into the conjugate back focal plane of any widefield single molecule localization system. We demonstrate that 3D localization can be performed by post-processing 2D localization data generated by common, widely-used, algorithms. In this work we benchmark the performance of SMLFM and finally showcase its capabilities by imaging fluorescently labeled membranes of fixed eukaryotic cells below the diffraction limit.

Points for accumulation in nanoscale topography (PAINT) allows practically unlimited measurements in localisation microscopy but is limited by background fluorescence at high probe concentrations, especially in volumetric imaging. We present reservoir-PAINT (resPAINT), which combines PAINT and active control of probe photophysics. In resPAINT, an activatable probe "reservoir" accumulates on target, enabling a 50-fold increase in localisation rate versus conventional PAINT, without compromising contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate super-resolution imaging of entire cell surfaces. We generalise the approach by implementing various switching strategies and 3D imaging techniques. Finally, we use resPAINT with a Fab to image membrane proteins, extending the operating regime of PAINT to include a wider range of biological interactions.

Points for accumulation in nanoscale topography (PAINT) allows practically unlimited measurements in localisation microscopy but is limited by background fluorescence at high probe concentrations, especially in volumetric imaging. We present reservoir-PAINT (resPAINT), which combines PAINT and active control of probe photophysics. In resPAINT, an activatable probe "reservoir" accumulates on target, enabling a 50-fold increase in localisation rate versus conventional PAINT, without compromising contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate super-resolution imaging of entire cell surfaces. We generalise the approach by implementing various switching strategies and 3D imaging techniques. Finally, we use resPAINT with a Fab to image membrane proteins, extending the operating regime of PAINT to include a wider range of biological interactions.

Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting the widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, that can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field-of-view errors, used simulations to optimize experimental design, and developed a fast algorithm based on Stokes parameter estimation which can operate over 1000 fold faster than the state of the art, enabling near-instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software; a napari plugin for visualization of high-dimensional diffraction-limited polarization camera datasets, and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study both alpha-synuclein fibrils and the actin cytoskeleton of mammalian cells. To demonstrate that POLCAM also allows diffraction-limited imaging, we demonstrate POLCAM imaging actin in fibroblast-like cells and the plasma membrane of live human T cells.