Total internal reflection energy transfer (TIRET) microscopy for analysis of focal adhesions in living cells

2006 ◽  
Author(s):  
Brigitte Angres ◽  
Heiko Steuer ◽  
Michael Wagner ◽  
Petra Weber ◽  
Herbert Schneckenburger
Keyword(s):  
Energy Transfer ◽  
Total Internal Reflection ◽  
Focal Adhesions ◽  
Living Cells ◽  
Internal Reflection

Uniform Total Internal Reflection Fluorescence Illumination Enables Live Cell Fluorescence Resonance Energy Transfer Microscopy

2013 ◽  
Vol 19 (2) ◽  
pp. 350-359 ◽  
Author(s):  
Jia Lin ◽  
Adam D. Hoppe
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Focal Plane ◽  
Total Internal Reflection ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Fluorescence Resonance

AbstractFluorescence resonance energy transfer (FRET) microscopy is a powerful technique to quantify dynamic protein-protein interactions in live cells. Total internal reflection fluorescence (TIRF) microscopy can selectively excite molecules within about 150 nm of the glass-cell interface. Recently, these two approaches were combined to enable high-resolution FRET imaging on the adherent surface of living cells. Here, we show that interference fringing of the coherent laser excitation used in TIRF creates lateral heterogeneities that impair quantitative TIRF-FRET measurements. We overcome this limitation by using a two-dimensional scan head to rotate laser beams for donor and acceptor excitation around the back focal plane of a high numerical aperture objective. By setting different radii for the circles traced out by each laser in the back focal plane, the penetration depth was corrected for different wavelengths. These modifications quell spatial variations in illumination and permit calibration for quantitative TIRF-FRET microscopy. The capability of TIRF-FRET was demonstrated by imaging assembled cyan and yellow fluorescent protein–tagged HIV-Gag molecules in single virions on the surfaces of living cells. These interactions are shown to be distinct from crowding of HIV-Gag in lipid rafts.

Visualization of Membrane Sorting and Fusion in Living Cells using Total Internal Reflection (TIR) and Multicolor Video Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 34-35
Author(s):  
Derek Toomre ◽  
Patrick Keller ◽  
Elena Diaz ◽  
Jamie White ◽  
Kai Simons
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Video Microscopy ◽  
Internal Reflection ◽  
Fast Time ◽  
Cellular Polarity ◽  
Microscopy Technique

Post-Golgi sorting of different classes of newly synthesized proteins and lipids is central to the generation and maintenance of cellular polarity. to directly visualize the dynamics and location of apical/basolateral sorting and trafficking we used fast time-lapse multicolor video microscopy in living cells. Specifically, green fluorescent protein color variants (cyan, CFP; yellow, YFP) of apical cargo (GPI-anchored) and basolateral cargo (vesicular stomatitis virus glycoprotein, VSVG) were generated; see FIG 1. Fast dual color fluorescence video microscopy allowed visualization with high temporal and spatial resolution. Our studies revealed that apical and basolateral cargo progressively segregated into large domains in Golgi/TGN structures, excluded resident proteins, and exited in separate transport containers. These carries remained distinct and did not merge with endocytic structures en route to the plasma membrane. Interestingly, our data suggest that the primary sorting occurs by lateral segregation in the Golgi, prior to budding (FIG 2). Further characterization of morphological differences of apical versus basolateral transport carriers was achieved using a specialized microscopy technique called total internal reflection (TIR) microscopy. with this approach only the bottom of the cell (<100 nm) was illuminated by an exponentially decaying evanescent “wave” of light. A series of images, taken at ∼1 second intervals, shows a bright “flash” of fluorescence when the vesicle fuse with the plasma membrane and the fluorophore diffuses into the plasma membrane (FIG 3).

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.

Fluorescence intensity, lifetime, and anisotropy screening of living cells based on total internal reflection techniques

2009 ◽  
Author(s):  
Thomas Bruns ◽  
Brigitte Angres ◽  
Heiko Steuer ◽  
Wolfgang S. L. Strauss ◽  
Herbert Schneckenburger
Keyword(s):  
Fluorescence Intensity ◽  
Total Internal Reflection ◽  
Living Cells ◽  
Internal Reflection
Sign in / Sign up

Export Citation Format

Share Document