Fast scanned widefield scheme provides tunable and uniform illumination for optimized SMLM on large fields of view

Abstract

Quantitative analyses in classical fluorescence microscopy and Single Molecule Localization Microscopy (SMLM) require uniform illumination over the field of view; ideally coupled with optical sectioning techniques such as Total Internal Reflection Fluorescence (TIRF) to remove out of focus background. In SMLM, high irradiances (several kW/cm²) are crucial to drive the densely labeled sample into the single molecule regime, and conventional gaussianshaped lasers will typically restrain the usable field of view to around 40 µm x 40 µm. Here we present Adaptable Scanning for Tunable Excitation Regions (ASTER), a novel and versatile illumination technique that generates uniform illumination over adaptable fields of view and is compatible with illumination schemes from epifluorescence to speckle-free TIRF. For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power, providing constant precision and higher throughput over fields of view 25 times larger than typical (up to 200 µm x 200 µm). This allows improved clustering analysis and uniform size measurements on sub-100 nm objects, as we demonstrate by imaging nanorulers, microtubules and clathrin-coated pits in COS cells, as well as periodic β2spectrin along the axons of neurons. ASTER's sharp, quantitative TIRF and SMLM images up to 200 µm x 200 µm in size pave the way for high-throughput quantification of cellular structures and processes.

Enabling single-molecule localization microscopy in turbid food emulsions

“…Examples include the use of a pair of micro lenses array consisting of identical spherical lenslets [24,25], the use of multimode optical fibres for illumination (MMF) [26] in combination with speckle reducers [27] or rotating diffusers [28] with the latter being less suitable for total internal reflection fluorescence (TIRF) microscopy due to the degradation of spatial coherence that prevents diffraction limited focusing. Recent work further demonstrated flat field illumination over variable field sizes using two galvanometer scanning mirrors placed in a plane conjugated to the back focal plane of the microscope objective in epifluorescence or TIRF mode [29]. In our implementation, we added a top-hat beam shaper in the excitation path that converts the Gaussian shaped intensity distribution of the excitation beam into a homogeneous flat field profile (top hat) enabling quantitative microscopy [30,31].…”

Enabling single-molecule localization microscopy in turbid food emulsions

“…Examples include the use of a pair of micro lenses array consisting of identical spherical lenslets (Douglass et al, 2016; Scholtens et al, 2011), the use of multimode optical fibers for illumination (MMF) (Deschamps et al, 2016) in combination with speckle reducers (Kwakwa et al, 2016) or rotating diffusers (Ma et al, 2017) with the latter being less suitable for total internal reflection fluorescence (TIRF) microscopy due to the degradation of spatial coherence prevention diffraction limited focusing. Recent work further demonstrated flat field illumination over variable field sizes using two galvanometer scanning mirrors placed in a plane conjugated to the back focal plane of the microscope objective in epifluorescence or TIRF mode (Mau et al, 2020). In our implementation, we added a top-hat beam shaper in the excitation path that converts the Gaussian shaped intensity distribution of the excitation beam into a homogeneous flat field profile (top hat) enabling quantitative microscopy (Khaw et al, 2018; Stehr et al, 2019).…”

Large Field-of-View Super-Resolution Optical Microscopy Based on Planar Polymer Waveguides

“…Techniques such as light-sheet and total-internal-reflection fluorescence (TIRF) microscopy utilize excitation profiles with a width at the sample plane below 1 μm and below 100 nm, respectively, but suffer from distortions and illumination heterogeneity. Illumination homogeneity can be increased using beam-shaping optics. − Objective-type TIRF microscopy uses shared excitation and detection pathways, which limits the use to only a few high-numerical-aperture (NA) oil immersion objective lenses. This results in high spatial resolution imaging, but also in an unavoidable rather small field of view.…”