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

2011 ◽  
pp. 273-289 ◽  
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

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.

Single molecule fluorescence resonance energy transfer scanning near-field optical microscopy: potentials and challenges

2015 ◽  
Vol 184 ◽  
pp. 51-69 ◽  
Author(s):  
S. K. Sekatskii ◽  
K. Dukenbayev ◽  
M. Mensi ◽  
A. G. Mikhaylov ◽  
E. Rostova ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Single Molecule ◽  
Near Field ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nitrogen Vacancy ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fret ◽  
Fluorescence Resonance

A few years ago, single molecule Fluorescence Resonance Energy Transfer Scanning Near-Field Optical Microscope (FRET SNOM) images were demonstrated using CdSe semiconductor nanocrystal–dye molecules as donor–acceptor pairs. Corresponding experiments reveal the necessity to exploit much more photostable fluorescent centers for such an imaging technique to become a practically used tool. Here we report the results of our experiments attempting to use nitrogen vacancy (NV) color centers in nanodiamond (ND) crystals, which are claimed to be extremely photostable, for FRET SNOM. All attempts were unsuccessful, and as a plausible explanation we propose the absence (instability) of NV centers lying close enough to the ND border. We also report improvements in SNOM construction that are necessary for single molecule FRET SNOM imaging. In particular, we present the first topographical images of single strand DNA molecules obtained with fiber-based SNOM. The prospects of using rare earth ions in crystals, which are known to be extremely photostable, for single molecule FRET SNOM at room temperature and quantum informatics at liquid helium temperatures, where FRET is a coherent process, are also discussed.

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