Multidimensional single-molecule imaging in live cells using total-internal-reflection fluorescence microscopy.
We have developed a wide-field total-internal-reflection fluorescence microscope capable of imaging single molecules in live cells, resolved in both wavelength and polarization. We show fluorescence resonance energy transfer between single pairs of fluorescent molecules bound to signaling receptors in the plasma membrane of live cells and demonstrate the importance of polarization discrimination in addition to wavelength separation.
Webb
S E D
SE
CCLRC Daresbury Laboratory, Warrington, UK. stephen.webb@dl.ac.uk
Needham
S R
SR
Roberts
S K
SK
Martin-Fernandez
M L
ML
D000818:Animals; D002478:Cells, Cultured; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods; D005456:Fluorescent Dyes; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D008565:Membrane Proteins