scholarly journals Tuneable wide-field illumination and single-molecule photoswitching with a single MEMS mirror

2021 ◽  
Author(s):  
Lucas Herdly ◽  
Paul Janin ◽  
Ralf Bauer ◽  
Sebastian van de Linde
Keyword(s):  
Single Molecule ◽  
Microelectromechanical Systems ◽  
Super Resolution ◽  
Cost Effective ◽  
Field Of View ◽  
Wide Field ◽  
Field Mode ◽  
Flat Field ◽  
Kinetics Of ◽  
Single Molecule Localization Microscopy

Homogeneous illumination in single-molecule localization microscopy (SMLM) is key for the quantitative analysis of super-resolution images. Therefore, different approaches for flat-field illumination have been introduced as alternative to the conventional Gaussian illumination. Here, we introduce a single microelectromechanical systems (MEMS) mirror as a tuneable and cost-effective device for adapting wide-field illumination in SMLM. In flat-field mode the MEMS allowed for consistent SMLM metrics across the entire field of view. Employing single-molecule photoswitching, we developed a simple yet powerful routine to benchmark different illumination schemes on the basis of local emitter brightness and ON-state lifetime. Moreover, we propose that tuning the MEMS beyond optimal flat-field conditions enables to study the kinetics of photoswitchable fluorophores within a single acquisition.

scholarly journals Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

2021 ◽  
Vol 22 (4) ◽  
pp. 1903
Author(s):  
Ivona Kubalová ◽  
Alžběta Němečková ◽  
Klaus Weisshart ◽  
Eva Hřibová ◽  
Veit Schubert
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Chromatin Condensation ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Wide Field ◽  
Structured Illumination Microscopy ◽  
Metaphase Chromosomes ◽  
Localization Microscopy ◽  
Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

scholarly journals Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software

2019 ◽  
Vol 16 (5) ◽  
pp. 387-395 ◽  
Author(s):  
Daniel Sage ◽  
Thanh-An Pham ◽  
Hazen Babcock ◽  
Tomas Lukes ◽  
Thomas Pengo ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Fight Club ◽  
2D And 3D ◽  
Single Molecule Localization Microscopy

scholarly journals Maximizing the field of view and accuracy in 3D Single Molecule Localization Microscopy

2018 ◽  
Vol 26 (4) ◽  
pp. 4631 ◽  
Author(s):  
Sohaib Abdul Rehman ◽  
Alexander R. Carr ◽  
Martin O. Lenz ◽  
Steven F. Lee ◽  
Kevin O’Holleran
Keyword(s):  
Single Molecule ◽  
Field Of View ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

scholarly journals Quantitative comparison of camera technologies for cost-effective Super-resolution Optical Fluctuation Imaging (SOFI)

2018 ◽  
Author(s):  
Robin Van den Eynde ◽  
Alice Sandmeyer ◽  
Wim Vandenberg ◽  
Sam Duwé ◽  
Wolfgang Hübner ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Imaging System ◽  
Fluorescent Proteins ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Cost Effective ◽  
Wide Field ◽  
Cmos Camera ◽  
Resolution Imaging ◽  
Cost Efficient

AbstractSuper-Resolution (SR) fluorescence microscopy is typically carried out on high-end research microscopes. Super-resolution Optical Fluctuation Imaging (SOFI) is a fast SR technique capable of live-cell imaging, that is compatible with many wide-field microscope systems. However, especially when employing fluorescent proteins, a key part of the imaging system is a very sensitive and well calibrated camera sensor. The substantial costs of such systems preclude many research groups from employing super-resolution imaging techniques.Here, we examine to what extent SOFI can be performed using a range of imaging hardware comprising different technologies and costs. In particular, we quantitatively compare the performance of an industry-grade CMOS camera to both state-of-the-art emCCD and sCMOS detectors, with SOFI-specific metrics. We show that SOFI data can be obtained using a cost-efficient industry-grade sensor, both on commercial and home-built microscope systems, though our analysis also readily exposes the merits of the per-pixel corrections performed in scientific cameras.

scholarly journals Super-resolution fight club: A broad assessment of 2D & 3D single-molecule localization microscopy software

2018 ◽  
Author(s):  
Daniel Sage ◽  
Thanh-An Pham ◽  
Hazen Babcock ◽  
Tomas Lukes ◽  
Thomas Pengo ◽  
...  
Keyword(s):  
Single Molecule ◽  
Double Helix ◽  
Super Resolution ◽  
Large Set ◽  
Holistic View ◽  
Localization Microscopy ◽  
Fight Club ◽  
Community Effort ◽  
2D And 3D ◽  
Single Molecule Localization Microscopy

ABSTRACTWith the widespread uptake of 2D and 3D single molecule localization microscopy, a large set of different data analysis packages have been developed to generate super-resolution images. To guide researchers on the optimal analytical software for their experiments, we have designed, in a large community effort, a competition to extensively characterise and rank these options. We generated realistic simulated datasets for popular imaging modalities – 2D, astigmatic 3D, biplane 3D, and double helix 3D – and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D single molecule localization microscopy software, provides a holistic view of how the latest 2D and 3D single molecule localization software perform in realistic conditions, and ultimately provides insight into the current limits of the field.

scholarly journals Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

2020 ◽  
Author(s):  
Fabian U. Zwettler ◽  
Sebastian Reinhard ◽  
Davide Gambarotto ◽  
Toby D. M. Bell ◽  
Virginie Hamel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Molecule ◽  
Super Resolution ◽  
Reference Structure ◽  
Positional Error ◽  
Localization Microscopy ◽  
Resolution Imaging ◽  
Endogenous Proteins ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractExpansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining expansion microscopy (ExM) with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

scholarly journals Systematic Tuning of Rhodamine Spirocyclization for Super-Resolution Microscopy

2021 ◽  
Author(s):  
Nicolas Lardon ◽  
Lu Wang ◽  
Aline Tschanz ◽  
Philipp Hoess ◽  
Mai Tran ◽  
...  
Keyword(s):  
Single Molecule ◽  
Large Range ◽  
Dynamic Equilibrium ◽  
Super Resolution ◽  
Live Cell ◽  
Stimulated Emission ◽  
Important Class ◽  
Localization Microscopy ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a non-fluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, which poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell stimulated emission depletion (STED) microscopy, or into a spontaneously blinking dye for single molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.

scholarly journals Multiplane and Spectrally-Resolved Single Molecule Localization Microscopy with Industrial Grade CMOS Cameras

2017 ◽  
Author(s):  
Hazen P. Babcock
Keyword(s):  
Open Source ◽  
Single Molecule ◽  
Open Source Software ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Industrial Grade ◽  
Spectrally Resolved ◽  
Imaging Plane ◽  
Single Molecule Localization Microscopy

ABSTRACTIn this work we explore the use of industrial grade CMOS cameras for single molecule localization microscopy (SMLM). We show that the performance of these cameras in single imaging plane SMLM applications is comparable to much more expensive scientific CMOS (sCMOS) cameras. We show that these cameras can be used in more demanding biplane, multiplane and spectrally resolved SMLM applications. The 10-40× reduction in camera cost makes it practical to build SMLM setups with 4 or more cameras. In addition we provide open-source software for simultaneously controlling multiple CMOS cameras and for the reduction of the movies that are acquired to super-resolution images.

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