Flat-Field Super-Resolution Localization Microscopy with a Low-Cost Refractive Beam-Shaping Element

Rowlands, Christopher J.; Ströhl, Florian; Ramirez, Pedro P. Vallejo; Scherer, Katharina M.; Kaminski, Clemens F.

doi:10.1038/s41598-018-24052-4

Cited by 28 publications

(25 citation statements)

References 14 publications

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“…If super-resolution microscopy is to be used for population based imaging, the number of cells imaged, and their diversity must be increased. To achieve this, researchers have been making efforts toward increasing the throughput of an average super-resolution experiment by automating the process [47,48], and by using flat field illumination to increase the number of cells collectable in each image [49,50].…”

Section: High Throughput Trans-scale Microscopymentioning

confidence: 99%

Bridging the Nanoscopy-Immunology Gap

Shannon

Owen

2019

Front. Phys.

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Bridging the gap between traditional immunology and nanoscale biophysics has proved more difficult than originally thought. For cell biology applications however, super-resolution microscopy has already facilitated considerable advances. From neuronal segmentation to nuclear pores and 3D focal adhesion structure-nanoscopy has begun to illuminate links between nanoscale organization and function. With immunology, the explanation must go further, relating nanoscale biophysical phenomena to the manifestation of specific diseases, or the altered activity of specific immune cell types in a bodily compartment. What follows is a summary of how nanoscopy has elucidated single cell immunological function, and what might be achieved in the future to link quantifiable, nanoscale, biophysical phenomena with cell and whole tissue functionality. We explore where the gaps in our understanding occur, and how they might be addressed.

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Section: High Throughput Trans-scale Microscopymentioning

confidence: 99%

Bridging the Nanoscopy-Immunology Gap

Shannon

Owen

2019

Front. Phys.

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“…Akin to the SIM reconstruction plugin FairSIM, single molecule localisation microscopy (SMLM) software has been made freely available by a vibrant community (for a comprehensive list see [11][12][13]). Similar to SIM, SMLM techniques can be regarded as hybrid nanoscopy methods due to the requirements of multiple high-power lasers and additional optical elements for field flattening [14] or when 3D information is desired [15]. A notable exception are 3D SMLM techniques with purely computational 3D information extraction based on aberrations in the microscope's point spread function (PSF) -these are also available in Fiji [16].…”

Section: Introductionmentioning

confidence: 99%

MusiJ: an ImageJ plugin for video nanoscopy

Acuña¹,

Ströhl²,

Opstad³

et al. 2020

Biomed. Opt. Express

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We present an open-source implementation of the fluctuation-based nanoscopy method MUSICAL for ImageJ. This implementation improves the algorithm's computational efficiency and takes advantage of multi-threading to provide orders of magnitude faster reconstructions than the original MATLAB implementation. In addition, the plugin is capable of generating super-resolution videos from large stacks of time-lapse images via an interleaved reconstruction, thus enabling easy-to-use multi-color super-resolution imaging of dynamic systems.

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“…Thus, several recent studies have aimed at obtaining a uniform excitation over a large FOV. Classical solutions revolve around beam-reshapers [20][21][22][23] , multimode fibers [24][25][26] and Spatial light modulators (piSMLM 27 ). These methods illuminate the whole field at once, so provide larger FOVs under the premise of using higher laser power.…”

Section: Introductionmentioning

confidence: 99%

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

Mau

Friedl

Leterrier

et al. 2020

Preprint

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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.

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Flat-Field Super-Resolution Localization Microscopy with a Low-Cost Refractive Beam-Shaping Element

Cited by 28 publications

References 14 publications

Bridging the Nanoscopy-Immunology Gap

Bridging the Nanoscopy-Immunology Gap

MusiJ: an ImageJ plugin for video nanoscopy

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

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