Abstract

We combine single molecule fluorescence orientation imaging with single-pair fluorescence resonance energy transfer microscopy, using a total internal reflection microscope. We show how angles and FRET efficiencies can be determined for membrane proteins at the single molecule level and provide data from the epidermal growth factor receptor system in cells.

© 2008 Optical Society of America

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  1. J. N. Forkey, M. E. Quinlan, and Y. E. Goldman, "Protein structural dynamics by single-molecule fluorescence polarization," Prog. Biophys. Mol. Biol. 74, 1-35 (2000).
    [CrossRef] [PubMed]
  2. J. N. Forkey, M. E. Quinlan, M. A. Shaw, J. E. T. Corrie, and Y. E. Goldman, "Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization," Nature 422, 399-404 (2003).
    [CrossRef] [PubMed]
  3. E. A. Jares-Erijman and T. M. Jovin, "FRET imaging," Nat. Biotechnol. 21, 1387-1395 (2003).
    [CrossRef] [PubMed]
  4. T. Ha, "Single-molecule fluorescence resonance energy transfer," Methods 25, 78-86 (2001).
    [CrossRef] [PubMed]
  5. M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, "Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy," Biochem. Biophys. Res. Comm. 235, 47-53 (1997).
    [CrossRef] [PubMed]
  6. S. E. D. Webb, S. R. Needham, S. K. Roberts, and M. L. Martin-Fernandez, "Multidimensional single-molecule imaging in live cells using total-internal-reflection fluorescence microscopy," Opt. Lett. 31, 2157-2159 (2006).
    [CrossRef] [PubMed]
  7. S. Hohng, C. Joo, and T. Ha, "Single-molecule three-color FRET," Biophys. J. 87, 1328-1337 (2004).
    [CrossRef] [PubMed]
  8. J. Schlessinger, "Cell signaling by receptor tyrosine kinases," Cell 103, 211-225 (2000).
    [CrossRef] [PubMed]
  9. S. E. D. Webb, S. K. Roberts, S. R. Needham, C. J. Tynan, D. J. Rolfe, M. D. Winn, D. T. Clarke, R. Barraclough, and M. L. Martin-Fernandez, "Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high- and low-affinity epidermal growth factor receptors in A431 cells," Biophys. J. 94, 808-819 (2008).
    [CrossRef]
  10. R. S. Chandran, S. E. John, and M. Amit, "Using fluorescence resonance energy transfer to measure distances along individual DNA molecules: Corrections due to nonideal transfer," J. Chem. Phys. 122, 061103 (2005).
    [CrossRef]

2008 (1)

S. E. D. Webb, S. K. Roberts, S. R. Needham, C. J. Tynan, D. J. Rolfe, M. D. Winn, D. T. Clarke, R. Barraclough, and M. L. Martin-Fernandez, "Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high- and low-affinity epidermal growth factor receptors in A431 cells," Biophys. J. 94, 808-819 (2008).
[CrossRef]

2006 (1)

2005 (1)

R. S. Chandran, S. E. John, and M. Amit, "Using fluorescence resonance energy transfer to measure distances along individual DNA molecules: Corrections due to nonideal transfer," J. Chem. Phys. 122, 061103 (2005).
[CrossRef]

2004 (1)

S. Hohng, C. Joo, and T. Ha, "Single-molecule three-color FRET," Biophys. J. 87, 1328-1337 (2004).
[CrossRef] [PubMed]

2003 (2)

J. N. Forkey, M. E. Quinlan, M. A. Shaw, J. E. T. Corrie, and Y. E. Goldman, "Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization," Nature 422, 399-404 (2003).
[CrossRef] [PubMed]

E. A. Jares-Erijman and T. M. Jovin, "FRET imaging," Nat. Biotechnol. 21, 1387-1395 (2003).
[CrossRef] [PubMed]

2001 (1)

T. Ha, "Single-molecule fluorescence resonance energy transfer," Methods 25, 78-86 (2001).
[CrossRef] [PubMed]

2000 (2)

J. Schlessinger, "Cell signaling by receptor tyrosine kinases," Cell 103, 211-225 (2000).
[CrossRef] [PubMed]

J. N. Forkey, M. E. Quinlan, and Y. E. Goldman, "Protein structural dynamics by single-molecule fluorescence polarization," Prog. Biophys. Mol. Biol. 74, 1-35 (2000).
[CrossRef] [PubMed]

1997 (1)

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, "Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy," Biochem. Biophys. Res. Comm. 235, 47-53 (1997).
[CrossRef] [PubMed]

Amit, M.

R. S. Chandran, S. E. John, and M. Amit, "Using fluorescence resonance energy transfer to measure distances along individual DNA molecules: Corrections due to nonideal transfer," J. Chem. Phys. 122, 061103 (2005).
[CrossRef]

Barraclough, R.

S. E. D. Webb, S. K. Roberts, S. R. Needham, C. J. Tynan, D. J. Rolfe, M. D. Winn, D. T. Clarke, R. Barraclough, and M. L. Martin-Fernandez, "Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high- and low-affinity epidermal growth factor receptors in A431 cells," Biophys. J. 94, 808-819 (2008).
[CrossRef]

Chandran, R. S.

R. S. Chandran, S. E. John, and M. Amit, "Using fluorescence resonance energy transfer to measure distances along individual DNA molecules: Corrections due to nonideal transfer," J. Chem. Phys. 122, 061103 (2005).
[CrossRef]

Clarke, D. T.

S. E. D. Webb, S. K. Roberts, S. R. Needham, C. J. Tynan, D. J. Rolfe, M. D. Winn, D. T. Clarke, R. Barraclough, and M. L. Martin-Fernandez, "Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high- and low-affinity epidermal growth factor receptors in A431 cells," Biophys. J. 94, 808-819 (2008).
[CrossRef]

Corrie, J. E. T.

J. N. Forkey, M. E. Quinlan, M. A. Shaw, J. E. T. Corrie, and Y. E. Goldman, "Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization," Nature 422, 399-404 (2003).
[CrossRef] [PubMed]

Forkey, J. N.

J. N. Forkey, M. E. Quinlan, M. A. Shaw, J. E. T. Corrie, and Y. E. Goldman, "Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization," Nature 422, 399-404 (2003).
[CrossRef] [PubMed]

J. N. Forkey, M. E. Quinlan, and Y. E. Goldman, "Protein structural dynamics by single-molecule fluorescence polarization," Prog. Biophys. Mol. Biol. 74, 1-35 (2000).
[CrossRef] [PubMed]

Goldman, Y. E.

J. N. Forkey, M. E. Quinlan, M. A. Shaw, J. E. T. Corrie, and Y. E. Goldman, "Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization," Nature 422, 399-404 (2003).
[CrossRef] [PubMed]

J. N. Forkey, M. E. Quinlan, and Y. E. Goldman, "Protein structural dynamics by single-molecule fluorescence polarization," Prog. Biophys. Mol. Biol. 74, 1-35 (2000).
[CrossRef] [PubMed]

Ha, T.

S. Hohng, C. Joo, and T. Ha, "Single-molecule three-color FRET," Biophys. J. 87, 1328-1337 (2004).
[CrossRef] [PubMed]

T. Ha, "Single-molecule fluorescence resonance energy transfer," Methods 25, 78-86 (2001).
[CrossRef] [PubMed]

Hohng, S.

S. Hohng, C. Joo, and T. Ha, "Single-molecule three-color FRET," Biophys. J. 87, 1328-1337 (2004).
[CrossRef] [PubMed]

Iwane, A. H.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, "Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy," Biochem. Biophys. Res. Comm. 235, 47-53 (1997).
[CrossRef] [PubMed]

Jares-Erijman, E. A.

E. A. Jares-Erijman and T. M. Jovin, "FRET imaging," Nat. Biotechnol. 21, 1387-1395 (2003).
[CrossRef] [PubMed]

John, S. E.

R. S. Chandran, S. E. John, and M. Amit, "Using fluorescence resonance energy transfer to measure distances along individual DNA molecules: Corrections due to nonideal transfer," J. Chem. Phys. 122, 061103 (2005).
[CrossRef]

Joo, C.

S. Hohng, C. Joo, and T. Ha, "Single-molecule three-color FRET," Biophys. J. 87, 1328-1337 (2004).
[CrossRef] [PubMed]

Jovin, T. M.

E. A. Jares-Erijman and T. M. Jovin, "FRET imaging," Nat. Biotechnol. 21, 1387-1395 (2003).
[CrossRef] [PubMed]

Kitamura, K.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, "Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy," Biochem. Biophys. Res. Comm. 235, 47-53 (1997).
[CrossRef] [PubMed]

Martin-Fernandez, M. L.

S. E. D. Webb, S. K. Roberts, S. R. Needham, C. J. Tynan, D. J. Rolfe, M. D. Winn, D. T. Clarke, R. Barraclough, and M. L. Martin-Fernandez, "Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high- and low-affinity epidermal growth factor receptors in A431 cells," Biophys. J. 94, 808-819 (2008).
[CrossRef]

S. E. D. Webb, S. R. Needham, S. K. Roberts, and M. L. Martin-Fernandez, "Multidimensional single-molecule imaging in live cells using total-internal-reflection fluorescence microscopy," Opt. Lett. 31, 2157-2159 (2006).
[CrossRef] [PubMed]

Needham, S. R.

S. E. D. Webb, S. K. Roberts, S. R. Needham, C. J. Tynan, D. J. Rolfe, M. D. Winn, D. T. Clarke, R. Barraclough, and M. L. Martin-Fernandez, "Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high- and low-affinity epidermal growth factor receptors in A431 cells," Biophys. J. 94, 808-819 (2008).
[CrossRef]

S. E. D. Webb, S. R. Needham, S. K. Roberts, and M. L. Martin-Fernandez, "Multidimensional single-molecule imaging in live cells using total-internal-reflection fluorescence microscopy," Opt. Lett. 31, 2157-2159 (2006).
[CrossRef] [PubMed]

Quinlan, M. E.

J. N. Forkey, M. E. Quinlan, M. A. Shaw, J. E. T. Corrie, and Y. E. Goldman, "Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization," Nature 422, 399-404 (2003).
[CrossRef] [PubMed]

J. N. Forkey, M. E. Quinlan, and Y. E. Goldman, "Protein structural dynamics by single-molecule fluorescence polarization," Prog. Biophys. Mol. Biol. 74, 1-35 (2000).
[CrossRef] [PubMed]

Roberts, S. K.

S. E. D. Webb, S. K. Roberts, S. R. Needham, C. J. Tynan, D. J. Rolfe, M. D. Winn, D. T. Clarke, R. Barraclough, and M. L. Martin-Fernandez, "Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high- and low-affinity epidermal growth factor receptors in A431 cells," Biophys. J. 94, 808-819 (2008).
[CrossRef]

S. E. D. Webb, S. R. Needham, S. K. Roberts, and M. L. Martin-Fernandez, "Multidimensional single-molecule imaging in live cells using total-internal-reflection fluorescence microscopy," Opt. Lett. 31, 2157-2159 (2006).
[CrossRef] [PubMed]

Rolfe, D. J.

S. E. D. Webb, S. K. Roberts, S. R. Needham, C. J. Tynan, D. J. Rolfe, M. D. Winn, D. T. Clarke, R. Barraclough, and M. L. Martin-Fernandez, "Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high- and low-affinity epidermal growth factor receptors in A431 cells," Biophys. J. 94, 808-819 (2008).
[CrossRef]

Saito, K.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, "Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy," Biochem. Biophys. Res. Comm. 235, 47-53 (1997).
[CrossRef] [PubMed]

Schlessinger, J.

J. Schlessinger, "Cell signaling by receptor tyrosine kinases," Cell 103, 211-225 (2000).
[CrossRef] [PubMed]

Shaw, M. A.

J. N. Forkey, M. E. Quinlan, M. A. Shaw, J. E. T. Corrie, and Y. E. Goldman, "Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization," Nature 422, 399-404 (2003).
[CrossRef] [PubMed]

Tokunaga, M.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, "Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy," Biochem. Biophys. Res. Comm. 235, 47-53 (1997).
[CrossRef] [PubMed]

Tynan, C. J.

S. E. D. Webb, S. K. Roberts, S. R. Needham, C. J. Tynan, D. J. Rolfe, M. D. Winn, D. T. Clarke, R. Barraclough, and M. L. Martin-Fernandez, "Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high- and low-affinity epidermal growth factor receptors in A431 cells," Biophys. J. 94, 808-819 (2008).
[CrossRef]

Webb, S. E. D.

S. E. D. Webb, S. K. Roberts, S. R. Needham, C. J. Tynan, D. J. Rolfe, M. D. Winn, D. T. Clarke, R. Barraclough, and M. L. Martin-Fernandez, "Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high- and low-affinity epidermal growth factor receptors in A431 cells," Biophys. J. 94, 808-819 (2008).
[CrossRef]

S. E. D. Webb, S. R. Needham, S. K. Roberts, and M. L. Martin-Fernandez, "Multidimensional single-molecule imaging in live cells using total-internal-reflection fluorescence microscopy," Opt. Lett. 31, 2157-2159 (2006).
[CrossRef] [PubMed]

Winn, M. D.

S. E. D. Webb, S. K. Roberts, S. R. Needham, C. J. Tynan, D. J. Rolfe, M. D. Winn, D. T. Clarke, R. Barraclough, and M. L. Martin-Fernandez, "Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high- and low-affinity epidermal growth factor receptors in A431 cells," Biophys. J. 94, 808-819 (2008).
[CrossRef]

Yanagida, T.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, "Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy," Biochem. Biophys. Res. Comm. 235, 47-53 (1997).
[CrossRef] [PubMed]

Biochem. Biophys. Res. Comm. (1)

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, "Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy," Biochem. Biophys. Res. Comm. 235, 47-53 (1997).
[CrossRef] [PubMed]

Biophys. J. (2)

S. Hohng, C. Joo, and T. Ha, "Single-molecule three-color FRET," Biophys. J. 87, 1328-1337 (2004).
[CrossRef] [PubMed]

S. E. D. Webb, S. K. Roberts, S. R. Needham, C. J. Tynan, D. J. Rolfe, M. D. Winn, D. T. Clarke, R. Barraclough, and M. L. Martin-Fernandez, "Single-molecule imaging and fluorescence lifetime imaging microscopy show different structures for high- and low-affinity epidermal growth factor receptors in A431 cells," Biophys. J. 94, 808-819 (2008).
[CrossRef]

Cell (1)

J. Schlessinger, "Cell signaling by receptor tyrosine kinases," Cell 103, 211-225 (2000).
[CrossRef] [PubMed]

J. Chem. Phys. (1)

R. S. Chandran, S. E. John, and M. Amit, "Using fluorescence resonance energy transfer to measure distances along individual DNA molecules: Corrections due to nonideal transfer," J. Chem. Phys. 122, 061103 (2005).
[CrossRef]

Methods (1)

T. Ha, "Single-molecule fluorescence resonance energy transfer," Methods 25, 78-86 (2001).
[CrossRef] [PubMed]

Nat. Biotechnol. (1)

E. A. Jares-Erijman and T. M. Jovin, "FRET imaging," Nat. Biotechnol. 21, 1387-1395 (2003).
[CrossRef] [PubMed]

Nature (1)

J. N. Forkey, M. E. Quinlan, M. A. Shaw, J. E. T. Corrie, and Y. E. Goldman, "Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization," Nature 422, 399-404 (2003).
[CrossRef] [PubMed]

Opt. Lett. (1)

Prog. Biophys. Mol. Biol. (1)

J. N. Forkey, M. E. Quinlan, and Y. E. Goldman, "Protein structural dynamics by single-molecule fluorescence polarization," Prog. Biophys. Mol. Biol. 74, 1-35 (2000).
[CrossRef] [PubMed]

Supplementary Material (2)

» Media 1: MPG (4834 KB)     
» Media 2: MPG (3354 KB)     

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Figures (4)

Fig. 1.
Fig. 1.

Reference frame of molecules in the microscope.

Fig. 2.
Fig. 2.

Schematic of single molecule TIRF microscope for simultaneous orientation and FRET microscopy.

Fig. 3.
Fig. 3.

Single molecule orientation and FRET data for a spot showing low SNR. (a) (Media 1) showing a small region of interest extracted from the images acquired, with all 8 channels shown. The square superimposed on the image highlights a fluorescent spot detected and the intensity v time traces for this spot are shown below. Images with the two excitation polarisations cannot be acquired simultaneously; the square is green for the ‘current’ image (Media 1). (b) Intensity v time traces following correction for bleedthrough (top panel), FRET efficiency and axial and azimuthal angles for Cy3 and Atto 647N - these latter are only calculated for the part of the trace where a fluorescent spot has been detected.

Fig. 4.
Fig. 4.

Single molecule orientation and FRET data for a fluorescent spot with high SNR (Media 2). Details as for Fig. 3.

Equations (2)

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P a y , Donor = sin 2 θ a sin 2 φ a P a z , Donor = ω sin 2 θ a cos 2 φ a + ( 1 ω ) cos 2 θ a
P e x = C 1 sin 2 θ e cos 2 φ e + C 2 sin 2 θ e sin 2 φ e + C 3 cos 2 θ e P e y = C 1 sin 2 θ e sin 2 φ e + C 2 sin 2 θ e cos 2 φ e + C 3 cos 2 θ e

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