Toward Single-Molecule Localization Microscopy (SMLM) Acquisitions Assisted by Real-Time Quality Control

AbstractFluorescence imaging techniques such as single molecule localization microscopy, high-content screening and light-sheet microscopy are producing ever-larger datasets, which poses increasing challenges in data handling and data sharing. Here, we introduce a real-time compression library that allows for very fast (beyond 1 GB/s) compression and de-compression of microscopy datasets during acquisition. In addition to an efficient lossless mode, our algorithm also includes a lossy option, which limits pixel deviations to the intrinsic noise level of the image and yields compression ratio of up to 100-fold. We present a detailed performance analysis of the different compression modes for various biological samples and imaging modalities.

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Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

10.26434/chemrxiv.7992980.v1 ◽

2019 ◽

Author(s):

Zacharias Thiel ◽

Pablo Rivera-Fuentes

Keyword(s):

Subcellular Localization ◽

Fluorescent Probe ◽

Single Molecule ◽

Mammalian Cells ◽

Fluorescent Sensor ◽

Biological Functions ◽

Localization Microscopy ◽

Drug Activation ◽

Nitroreductase Activity ◽

Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

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Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

10.26434/chemrxiv.7992980 ◽

2019 ◽

Author(s):

Zacharias Thiel ◽

Pablo Rivera-Fuentes

Keyword(s):

Subcellular Localization ◽

Fluorescent Probe ◽

Single Molecule ◽

Mammalian Cells ◽

Fluorescent Sensor ◽

Biological Functions ◽

Localization Microscopy ◽

Drug Activation ◽

Nitroreductase Activity ◽

Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

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Automated Analysis of Fluorescence Kinetics in Single-Molecule Localization Microscopy Data Reveals Protein Stoichiometry

The Journal of Physical Chemistry B ◽

10.1021/acs.jpcb.1c01130 ◽

2021 ◽

Author(s):

Alon Saguy ◽

Tim N. Baldering ◽

Lucien E. Weiss ◽

Elias Nehme ◽

Christos Karathanasis ◽

...

Keyword(s):

Single Molecule ◽

Automated Analysis ◽

Fluorescence Kinetics ◽

Localization Microscopy ◽

Microscopy Data ◽

Single Molecule Localization Microscopy

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Dual-Labeled Graphene Quantum Dot-Based Förster Resonance Energy Transfer Nanoprobes for Single-Molecule Localization Microscopy

ACS Omega ◽

10.1021/acsomega.0c05417 ◽

2021 ◽

Author(s):

Ju Lu ◽

Shenfei Zong ◽

Zhuyuan Wang ◽

Chen Chen ◽

Yizhi Zhang ◽

...

Keyword(s):

Energy Transfer ◽

Quantum Dot ◽

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Förster Resonance Energy Transfer ◽

Localization Microscopy ◽

Graphene Quantum Dot ◽

Single Molecule Localization Microscopy ◽

Förster Resonance

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Single-molecule localization microscopy

Nature Reviews Methods Primers ◽

10.1038/s43586-021-00038-x ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Mickaël Lelek ◽

Melina T. Gyparaki ◽

Gerti Beliu ◽

Florian Schueder ◽

Juliette Griffié ◽

...

Keyword(s):

Single Molecule ◽

Localization Microscopy ◽

Single Molecule Localization Microscopy

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Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules

Nature Communications ◽

10.1038/s41467-020-20863-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Alan M. Szalai ◽

Bruno Siarry ◽

Jerónimo Lukin ◽

David J. Williamson ◽

Nicolás Unsain ◽

...

Keyword(s):

Single Molecule ◽

Total Internal Reflection ◽

Single Molecules ◽

Fluorescence Microscope ◽

Internal Reflection ◽

Total Internal Reflection Fluorescence ◽

Axial Position ◽

Localization Microscopy ◽

Localization Precision ◽

Single Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

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