Single Molecule Detection at Surfaces: Dual-Color Fluorescence Fluctuation Spectroscopy with Total Internal Reflection Excitation

We develop a sensitive and selective method for the multiplex detection of histone-modifying enzymes using total internal reflection fluorescence-based single-molecule detection.

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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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Optimal Practices for Surface-Tethered Single Molecule Total Internal Reflection Fluorescence Resonance Energy Transfer Analysis

DNA Nanotechnology - Methods in Molecular Biology ◽

10.1007/978-1-61779-142-0_19 ◽

2011 ◽

pp. 273-289 ◽

Cited By ~ 5

Author(s):

Matt V. Fagerburg ◽

Sanford H. Leuba

Keyword(s):

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

Single Molecule ◽

Total Internal Reflection ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Internal Reflection ◽

Total Internal Reflection Fluorescence ◽

Fluorescence Resonance

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Total Internal Reflection Fluorescence Microscopy (TIRFM) – novel techniques and applications

Medical Research Archives ◽

10.18103/mra.v8i11.2287 ◽

2020 ◽

Vol 8 (11) ◽

Author(s):

Verena Richter ◽

Michael Wagner ◽

Herbert Schneckenburger

Keyword(s):

Fluorescence Microscopy ◽

Single Molecule ◽

Total Internal Reflection ◽

Plasma Membranes ◽

Focal Adhesions ◽

Internal Reflection ◽

Total Internal Reflection Fluorescence ◽

Thin Layers ◽

Structured Illumination ◽

Structured Illumination Microscopy

Total Internal Reflection Fluorescence Microscopy (TIRFM) has been established almost 40 years ago for studies of plasma membranes or membrane proximal sites of living cells. The method is based on light incidence at an angle above the critical angle of total internal reflection and generation of an evanescent electromagnetic field penetrating about 100 nm into a sample and permitting selective excitation of membrane proximal fluorophores. Two techniques are presented here: prism-type TIRFM and objective-type TIRFM with high aperture microscope objective lenses. Furthermore, numerous applications are summarized, e.g. measurement of focal adhesions, cell-substrate topology, endocytosis or exocytosis of vesicles as well as single molecule detection within thin layers. Finally, highly innovative combinations of TIRFM with Förster Resonance Energy Transfer (FRET) measurements as well as with Structured Illumination Microscopy (SIM) and fluorescence reader technologies are presented.

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