Single-molecule interaction between circularly permuted green fluorescent protein and trypsin by total internal reflection fluorescence microscopy

2006 ◽  
Vol 284-285 ◽  
pp. 395-400 ◽  
Author(s):  
Tong Wang ◽  
Takashi Isoshima ◽  
Atsushi Miyawaki ◽  
Masahiko Hara
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Green Fluorescent ◽  
Molecule Interaction

scholarly journals Imaging Constitutive Exocytosis with Total Internal Reflection Fluorescence Microscopy

2000 ◽  
Vol 149 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Jan Schmoranzer ◽  
Mark Goulian ◽  
Dan Axelrod ◽  
Sanford M. Simon
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Fluorescence Microscopy ◽  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Green Fluorescent ◽  
Kinetics Of ◽  
Flattening Process

Total internal reflection fluorescence microscopy has been applied to image the final stage of constitutive exocytosis, which is the fusion of single post-Golgi carriers with the plasma membrane. The use of a membrane protein tagged with green fluorescent protein allowed the kinetics of fusion to be followed with a time resolution of 30 frames/s. Quantitative analysis allowed carriers undergoing fusion to be easily distinguished from carriers moving perpendicularly to the plasma membrane. The flattening of the carriers into the plasma membrane is seen as a simultaneous rise in the total, peak, and width of the fluorescence intensity. The duration of this flattening process depends on the size of the carriers, distinguishing small spherical from large tubular carriers. The spread of the membrane protein into the plasma membrane upon fusion is diffusive. Mapping many fusion sites of a single cell reveals that there are no preferred sites for constitutive exocytosis in this system.

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

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.

scholarly journals Single-Molecule Total Internal Reflection Fluorescence Microscopy

2016 ◽  
Vol 2016 (5) ◽  
pp. pdb.top077800 ◽  
Author(s):  
Emily M. Kudalkar ◽  
Trisha N. Davis ◽  
Charles L. Asbury
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Total Internal Reflection ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence

scholarly journals Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation

Science ◽  
2018 ◽  
Vol 361 (6404) ◽  
pp. eaau1504 ◽  
Author(s):  
Annapurna Vemu ◽  
Ewa Szczesna ◽  
Elena A. Zehr ◽  
Jeffrey O. Spector ◽  
Nikolaus Grigorieff ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Total Internal Reflection ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
High Density ◽  
Guanosine Triphosphate ◽  
Microtubule Severing

Spastin and katanin sever and destabilize microtubules. Paradoxically, despite their destructive activity they increase microtubule mass in vivo. We combined single-molecule total internal reflection fluorescence microscopy and electron microscopy to show that the elemental step in microtubule severing is the generation of nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers. These damage sites are repaired spontaneously by guanosine triphosphate (GTP)–tubulin incorporation, which rejuvenates and stabilizes the microtubule shaft. Consequently, spastin and katanin increase microtubule rescue rates. Furthermore, newly severed ends emerge with a high density of GTP-tubulin that protects them against depolymerization. The stabilization of the newly severed plus ends and the higher rescue frequency synergize to amplify microtubule number and mass. Thus, severing enzymes regulate microtubule architecture and dynamics by promoting GTP-tubulin incorporation within the microtubule shaft.

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