scholarly journals An Angstrom Scale Interaction between Plasma Membrane ATP-Gated P2X2 and  4 2 Nicotinic Channels Measured with Fluorescence Resonance Energy Transfer and Total Internal Reflection Fluorescence Microscopy

2005 ◽  
Vol 25 (29) ◽  
pp. 6911-6920 ◽  
Author(s):  
B. S. Khakh
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Fluorescence Microscopy ◽  
Fluorescence Resonance Energy Transfer ◽  
Total Internal Reflection ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Scale Interaction

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.

Förster resonance energy transfer-based total internal reflection fluorescence reader for apoptosis

2009 ◽  
Vol 14 (2) ◽  
pp. 021003 ◽  
Author(s):  
Thomas Bruns ◽  
Brigitte Angres ◽  
Heiko Steuer ◽  
Petra Weber ◽  
Michael Wagner ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Total Internal Reflection ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence

scholarly journals Quantitative Fluorescence Resonance Energy Transfer Measurements Using Fluorescence Microscopy

1998 ◽  
Vol 74 (5) ◽  
pp. 2702-2713 ◽  
Author(s):  
Gerald W. Gordon ◽  
Gail Berry ◽  
Xiao Huan Liang ◽  
Beth Levine ◽  
Brian Herman
Keyword(s):  
Energy Transfer ◽  
Fluorescence Microscopy ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Resonance ◽  
Quantitative Fluorescence

scholarly journals Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET

2016 ◽  
Vol 147 (2) ◽  
pp. 201-216 ◽  
Author(s):  
William N. Zagotta ◽  
Moshe T. Gordon ◽  
Eric N. Senning ◽  
Mika A. Munari ◽  
Sharona E. Gordon
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Transition Metal ◽  
Fluorescence Resonance Energy Transfer ◽  
Metal Ion ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Noncanonical Amino Acid

Despite recent advances, the structure and dynamics of membrane proteins in cell membranes remain elusive. We implemented transition metal ion fluorescence resonance energy transfer (tmFRET) to measure distances between sites on the N-terminal ankyrin repeat domains (ARDs) of the pain-transducing ion channel TRPV1 and the intracellular surface of the plasma membrane. To preserve the native context, we used unroofed cells, and to specifically label sites in TRPV1, we incorporated a fluorescent, noncanonical amino acid, L-ANAP. A metal chelating lipid was used to decorate the plasma membrane with high-density/high-affinity metal-binding sites. The fluorescence resonance energy transfer (FRET) efficiencies between L-ANAP in TRPV1 and Co2+ bound to the plasma membrane were consistent with the arrangement of the ARDs in recent cryoelectron microscopy structures of TRPV1. No change in tmFRET was observed with the TRPV1 agonist capsaicin. These results demonstrate the power of tmFRET for measuring structure and rearrangements of membrane proteins relative to the cell membrane.

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