Abstract

We report on a simple yet powerful implementation of objective-type total internal reflection fluorescence (TIRF) and highly inclined and laminated optical sheet (HILO, a type of dark-field) illumination. Instead of focusing the illuminating laser beam to a single spot close to the edge of the microscope objective, we are scanning during the acquisition of a fluorescence image the focused spot in a circular orbit, thereby illuminating the sample from various directions. We measure parameters relevant for quantitative image analysis during fluorescence image acquisition by capturing an image of the excitation light distribution in an equivalent objective back-focal plane (BFP). Operating at scan rates above 1 MHz, our programmable light engine allows directional averaging by circular spinning the spot even for sub-millisecond exposure times. We show that restoring the symmetry of TIRF/HILO illumination reduces scattering and produces an evenly lit field-of-view that affords on-line analysis of evanescent-field excited fluorescence without pre-processing. Utilizing crossed acousto-optical deflectors, our device generates arbitrary intensity profiles in BFP, permitting variable-angle, multi-color illumination, or objective lenses to be rapidly exchanged.

© 2008 Optical Society of America

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    [CrossRef] [PubMed]
  2. M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa. "Highly inclined thin illumination enables clear single-molecule imaging in cells," Nat.Methods 5, 159-161 (2008).
    [CrossRef] [PubMed]
  3. H. Chew, D. S. Wang, and M. Kerker, "Elastic scattering of evanescent electromagnetic waves," Appl. Opt. 18, 2679-87 (1979).
    [CrossRef] [PubMed]
  4. A. Rohrbach, "Observing secretory granules with a multiangle evanescent-wave microscope," Biophys. J. 78, 2641-54 (2000).
    [CrossRef] [PubMed]
  5. F. Schapper, J. T. Gonçalves, and M. Oheim, "Fluorescence imaging with two-photon evanescent-wave excitation," Eur. Biophys. J. 32, 635-643 (2003).
    [CrossRef] [PubMed]
  6. M. Oheim and F. Schapper, "Non-linear evanescent-field imaging," J. Phys. D: Appl. Phys. 38, R185-R197 (2005).
    [CrossRef]
  7. A. L. Mattheyses and D. Axelrod, "Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence," J Biomed Opt. 11,014006A (2006).
    [CrossRef]
  8. A. L. Mattheyses and D. Axelrod, "Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle," Microsc. Res. Tech. 69, 642-647 (2006).
    [CrossRef] [PubMed]
  9. R. Fiolka, Y. Belyaev, H. Ewers, and A. Stemmer, "Even illumination in total internal reflection fluorescence microscopy using laser light," Microsc. Res. Tech. 71, 45-50 (2007).
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    [CrossRef] [PubMed]
  17. J. D. Humphries, P. Wang, C. Streuli, B. Geiger, M. J. Humphries, and C. Ballestrem, "Vinculin controls focal adhesion formation by direct interactions with talin and actin," J. Cell Biol. 179, 1043-1057 (2007).
    [CrossRef] [PubMed]
  18. H. Schneckenburger, "Total internal reflection fluorescence microscopy: technical innovations and novel applications," Curr Opin Biotechnol. 16, 13-18 (2005).
    [CrossRef] [PubMed]
  19. W. P. Ambrose, P. M. Goodwin, and J. P. Nolan "Single-molecule detection with total internal reflection 20. excitation: comparing signal-to-background and total signals in different geometries," Cytometry 36224-31 (1999).
  20. P. B. Conibear and C. R. Bagshaw, "A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy," J. Microsc. 200, 218-29 (2000).
  21. B. P. Ölveczky, N. Periasamy, and A. S. Verkman, "Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy," Biophys J. 73,2836-47 (1997).
    [CrossRef] [PubMed]
  22. Y. Kawano, C. Abe, T. Kaneda, Y. Aono, K. Abe, and K. Tamura, "High-numerical aperture objective lenses and optical system improved objective-type total internal reflection fluorescence microscopy," Proc. SPIE 4098, 142-53 (2000).
    [CrossRef] [PubMed]
  23. O. Gilko, G. D. Reddy, B. Anvari, W. E. Brownell, and P. Saggau, "Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells," J. Biomed. Opt. 11, 064013 (2006).
    [CrossRef]
  24. E. Chung, D. K. Kim, and P. T. C. So, "Extended resolution wide-field optical imaging: objective-launched standing wave total internal reflection fluorescence microscopy," Opt. Lett. 31, 945-947 (2006).
    [CrossRef]
  25. M. R. Beversluis, G. W. Bryant, and S. J. Stranick, "Effects of inhomogeneous fields in superresolving structured illumination microscopy," 25, 1371-1377 (2008).
    [CrossRef] [PubMed]

2008 (2)

D. Li, N. Ropert, A. Koulakoff, C. Giaume, and M. Oheim, "Lysosomes are the major vesicular compartment undergoing Ca2+-regulated exocytosis from cortical astrocytes," J. Neurosci. 28, 7648-7658 (2008).
[CrossRef] [PubMed]

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa. "Highly inclined thin illumination enables clear single-molecule imaging in cells," Nat.Methods 5, 159-161 (2008).
[CrossRef] [PubMed]

2007 (3)

R. Fiolka, Y. Belyaev, H. Ewers, and A. Stemmer, "Even illumination in total internal reflection fluorescence microscopy using laser light," Microsc. Res. Tech. 71, 45-50 (2007).
[CrossRef] [PubMed]

N. K. Lee, A. N. Kapanidis, H. R. Koh,  et al, "Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances," Biophys. J. 92, 303-312 (2007).
[CrossRef]

J. D. Humphries, P. Wang, C. Streuli, B. Geiger, M. J. Humphries, and C. Ballestrem, "Vinculin controls focal adhesion formation by direct interactions with talin and actin," J. Cell Biol. 179, 1043-1057 (2007).
[CrossRef] [PubMed]

2006 (4)

A. L. Mattheyses and D. Axelrod, "Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence," J Biomed Opt. 11,014006A (2006).
[CrossRef]

A. L. Mattheyses and D. Axelrod, "Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle," Microsc. Res. Tech. 69, 642-647 (2006).
[CrossRef] [PubMed]

O. Gilko, G. D. Reddy, B. Anvari, W. E. Brownell, and P. Saggau, "Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells," J. Biomed. Opt. 11, 064013 (2006).
[CrossRef]

E. Chung, D. K. Kim, and P. T. C. So, "Extended resolution wide-field optical imaging: objective-launched standing wave total internal reflection fluorescence microscopy," Opt. Lett. 31, 945-947 (2006).
[CrossRef]

2005 (2)

H. Schneckenburger, "Total internal reflection fluorescence microscopy: technical innovations and novel applications," Curr Opin Biotechnol. 16, 13-18 (2005).
[CrossRef] [PubMed]

M. Oheim and F. Schapper, "Non-linear evanescent-field imaging," J. Phys. D: Appl. Phys. 38, R185-R197 (2005).
[CrossRef]

2003 (1)

F. Schapper, J. T. Gonçalves, and M. Oheim, "Fluorescence imaging with two-photon evanescent-wave excitation," Eur. Biophys. J. 32, 635-643 (2003).
[CrossRef] [PubMed]

2000 (3)

A. Rohrbach, "Observing secretory granules with a multiangle evanescent-wave microscope," Biophys. J. 78, 2641-54 (2000).
[CrossRef] [PubMed]

Y. Kawano, C. Abe, T. Kaneda, Y. Aono, K. Abe, and K. Tamura, "High-numerical aperture objective lenses and optical system improved objective-type total internal reflection fluorescence microscopy," Proc. SPIE 4098, 142-53 (2000).
[CrossRef] [PubMed]

P. B. Conibear and C. R. Bagshaw, "A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy," J. Microsc. 200, 218-29 (2000).

1999 (1)

W. P. Ambrose, P. M. Goodwin, and J. P. Nolan "Single-molecule detection with total internal reflection 20. excitation: comparing signal-to-background and total signals in different geometries," Cytometry 36224-31 (1999).

1997 (1)

B. P. Ölveczky, N. Periasamy, and A. S. Verkman, "Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy," Biophys J. 73,2836-47 (1997).
[CrossRef] [PubMed]

1989 (1)

1979 (1)

Abe, C.

Y. Kawano, C. Abe, T. Kaneda, Y. Aono, K. Abe, and K. Tamura, "High-numerical aperture objective lenses and optical system improved objective-type total internal reflection fluorescence microscopy," Proc. SPIE 4098, 142-53 (2000).
[CrossRef] [PubMed]

Abe, K.

Y. Kawano, C. Abe, T. Kaneda, Y. Aono, K. Abe, and K. Tamura, "High-numerical aperture objective lenses and optical system improved objective-type total internal reflection fluorescence microscopy," Proc. SPIE 4098, 142-53 (2000).
[CrossRef] [PubMed]

Ambrose, W. P.

W. P. Ambrose, P. M. Goodwin, and J. P. Nolan "Single-molecule detection with total internal reflection 20. excitation: comparing signal-to-background and total signals in different geometries," Cytometry 36224-31 (1999).

Anvari, B.

O. Gilko, G. D. Reddy, B. Anvari, W. E. Brownell, and P. Saggau, "Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells," J. Biomed. Opt. 11, 064013 (2006).
[CrossRef]

Aono, Y.

Y. Kawano, C. Abe, T. Kaneda, Y. Aono, K. Abe, and K. Tamura, "High-numerical aperture objective lenses and optical system improved objective-type total internal reflection fluorescence microscopy," Proc. SPIE 4098, 142-53 (2000).
[CrossRef] [PubMed]

Axelrod, D.

A. L. Mattheyses and D. Axelrod, "Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence," J Biomed Opt. 11,014006A (2006).
[CrossRef]

A. L. Mattheyses and D. Axelrod, "Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle," Microsc. Res. Tech. 69, 642-647 (2006).
[CrossRef] [PubMed]

A. L. Stout and D. Axelrod, "Evanescent field excitation of fluorescence by epi-illumination microscopy," Appl. Opt. 28, 5237-5242 (1989).
[CrossRef] [PubMed]

Bagshaw, C. R.

P. B. Conibear and C. R. Bagshaw, "A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy," J. Microsc. 200, 218-29 (2000).

Ballestrem, C.

J. D. Humphries, P. Wang, C. Streuli, B. Geiger, M. J. Humphries, and C. Ballestrem, "Vinculin controls focal adhesion formation by direct interactions with talin and actin," J. Cell Biol. 179, 1043-1057 (2007).
[CrossRef] [PubMed]

Belyaev, Y.

R. Fiolka, Y. Belyaev, H. Ewers, and A. Stemmer, "Even illumination in total internal reflection fluorescence microscopy using laser light," Microsc. Res. Tech. 71, 45-50 (2007).
[CrossRef] [PubMed]

Beversluis, M. R.

M. R. Beversluis, G. W. Bryant, and S. J. Stranick, "Effects of inhomogeneous fields in superresolving structured illumination microscopy," 25, 1371-1377 (2008).
[CrossRef] [PubMed]

Brownell, W. E.

O. Gilko, G. D. Reddy, B. Anvari, W. E. Brownell, and P. Saggau, "Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells," J. Biomed. Opt. 11, 064013 (2006).
[CrossRef]

Bryant, G. W.

M. R. Beversluis, G. W. Bryant, and S. J. Stranick, "Effects of inhomogeneous fields in superresolving structured illumination microscopy," 25, 1371-1377 (2008).
[CrossRef] [PubMed]

Chew, H.

Chung, E.

Conibear, P. B.

P. B. Conibear and C. R. Bagshaw, "A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy," J. Microsc. 200, 218-29 (2000).

Ewers, H.

R. Fiolka, Y. Belyaev, H. Ewers, and A. Stemmer, "Even illumination in total internal reflection fluorescence microscopy using laser light," Microsc. Res. Tech. 71, 45-50 (2007).
[CrossRef] [PubMed]

Fiolka, R.

R. Fiolka, Y. Belyaev, H. Ewers, and A. Stemmer, "Even illumination in total internal reflection fluorescence microscopy using laser light," Microsc. Res. Tech. 71, 45-50 (2007).
[CrossRef] [PubMed]

Geiger, B.

J. D. Humphries, P. Wang, C. Streuli, B. Geiger, M. J. Humphries, and C. Ballestrem, "Vinculin controls focal adhesion formation by direct interactions with talin and actin," J. Cell Biol. 179, 1043-1057 (2007).
[CrossRef] [PubMed]

Giaume, C.

D. Li, N. Ropert, A. Koulakoff, C. Giaume, and M. Oheim, "Lysosomes are the major vesicular compartment undergoing Ca2+-regulated exocytosis from cortical astrocytes," J. Neurosci. 28, 7648-7658 (2008).
[CrossRef] [PubMed]

Gilko, O.

O. Gilko, G. D. Reddy, B. Anvari, W. E. Brownell, and P. Saggau, "Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells," J. Biomed. Opt. 11, 064013 (2006).
[CrossRef]

Gonçalves, J. T.

F. Schapper, J. T. Gonçalves, and M. Oheim, "Fluorescence imaging with two-photon evanescent-wave excitation," Eur. Biophys. J. 32, 635-643 (2003).
[CrossRef] [PubMed]

Goodwin, P. M.

W. P. Ambrose, P. M. Goodwin, and J. P. Nolan "Single-molecule detection with total internal reflection 20. excitation: comparing signal-to-background and total signals in different geometries," Cytometry 36224-31 (1999).

Humphries, J. D.

J. D. Humphries, P. Wang, C. Streuli, B. Geiger, M. J. Humphries, and C. Ballestrem, "Vinculin controls focal adhesion formation by direct interactions with talin and actin," J. Cell Biol. 179, 1043-1057 (2007).
[CrossRef] [PubMed]

Humphries, M. J.

J. D. Humphries, P. Wang, C. Streuli, B. Geiger, M. J. Humphries, and C. Ballestrem, "Vinculin controls focal adhesion formation by direct interactions with talin and actin," J. Cell Biol. 179, 1043-1057 (2007).
[CrossRef] [PubMed]

Imamoto, N.

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa. "Highly inclined thin illumination enables clear single-molecule imaging in cells," Nat.Methods 5, 159-161 (2008).
[CrossRef] [PubMed]

Kaneda, T.

Y. Kawano, C. Abe, T. Kaneda, Y. Aono, K. Abe, and K. Tamura, "High-numerical aperture objective lenses and optical system improved objective-type total internal reflection fluorescence microscopy," Proc. SPIE 4098, 142-53 (2000).
[CrossRef] [PubMed]

Kapanidis, A. N.

N. K. Lee, A. N. Kapanidis, H. R. Koh,  et al, "Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances," Biophys. J. 92, 303-312 (2007).
[CrossRef]

Kawano, Y.

Y. Kawano, C. Abe, T. Kaneda, Y. Aono, K. Abe, and K. Tamura, "High-numerical aperture objective lenses and optical system improved objective-type total internal reflection fluorescence microscopy," Proc. SPIE 4098, 142-53 (2000).
[CrossRef] [PubMed]

Kerker, M.

Kim, D. K.

Koh, H. R.

N. K. Lee, A. N. Kapanidis, H. R. Koh,  et al, "Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances," Biophys. J. 92, 303-312 (2007).
[CrossRef]

Koulakoff, A.

D. Li, N. Ropert, A. Koulakoff, C. Giaume, and M. Oheim, "Lysosomes are the major vesicular compartment undergoing Ca2+-regulated exocytosis from cortical astrocytes," J. Neurosci. 28, 7648-7658 (2008).
[CrossRef] [PubMed]

Lee, N. K.

N. K. Lee, A. N. Kapanidis, H. R. Koh,  et al, "Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances," Biophys. J. 92, 303-312 (2007).
[CrossRef]

Li, D.

D. Li, N. Ropert, A. Koulakoff, C. Giaume, and M. Oheim, "Lysosomes are the major vesicular compartment undergoing Ca2+-regulated exocytosis from cortical astrocytes," J. Neurosci. 28, 7648-7658 (2008).
[CrossRef] [PubMed]

Mattheyses, A. L.

A. L. Mattheyses and D. Axelrod, "Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle," Microsc. Res. Tech. 69, 642-647 (2006).
[CrossRef] [PubMed]

A. L. Mattheyses and D. Axelrod, "Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence," J Biomed Opt. 11,014006A (2006).
[CrossRef]

Nolan, J. P.

W. P. Ambrose, P. M. Goodwin, and J. P. Nolan "Single-molecule detection with total internal reflection 20. excitation: comparing signal-to-background and total signals in different geometries," Cytometry 36224-31 (1999).

Oheim, M.

D. Li, N. Ropert, A. Koulakoff, C. Giaume, and M. Oheim, "Lysosomes are the major vesicular compartment undergoing Ca2+-regulated exocytosis from cortical astrocytes," J. Neurosci. 28, 7648-7658 (2008).
[CrossRef] [PubMed]

M. Oheim and F. Schapper, "Non-linear evanescent-field imaging," J. Phys. D: Appl. Phys. 38, R185-R197 (2005).
[CrossRef]

F. Schapper, J. T. Gonçalves, and M. Oheim, "Fluorescence imaging with two-photon evanescent-wave excitation," Eur. Biophys. J. 32, 635-643 (2003).
[CrossRef] [PubMed]

Ölveczky, B. P.

B. P. Ölveczky, N. Periasamy, and A. S. Verkman, "Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy," Biophys J. 73,2836-47 (1997).
[CrossRef] [PubMed]

Periasamy, N.

B. P. Ölveczky, N. Periasamy, and A. S. Verkman, "Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy," Biophys J. 73,2836-47 (1997).
[CrossRef] [PubMed]

Reddy, G. D.

O. Gilko, G. D. Reddy, B. Anvari, W. E. Brownell, and P. Saggau, "Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells," J. Biomed. Opt. 11, 064013 (2006).
[CrossRef]

Rohrbach, A.

A. Rohrbach, "Observing secretory granules with a multiangle evanescent-wave microscope," Biophys. J. 78, 2641-54 (2000).
[CrossRef] [PubMed]

Ropert, N.

D. Li, N. Ropert, A. Koulakoff, C. Giaume, and M. Oheim, "Lysosomes are the major vesicular compartment undergoing Ca2+-regulated exocytosis from cortical astrocytes," J. Neurosci. 28, 7648-7658 (2008).
[CrossRef] [PubMed]

Saggau, P.

O. Gilko, G. D. Reddy, B. Anvari, W. E. Brownell, and P. Saggau, "Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells," J. Biomed. Opt. 11, 064013 (2006).
[CrossRef]

Sakata-Sogawa, K.

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa. "Highly inclined thin illumination enables clear single-molecule imaging in cells," Nat.Methods 5, 159-161 (2008).
[CrossRef] [PubMed]

Schapper, F.

M. Oheim and F. Schapper, "Non-linear evanescent-field imaging," J. Phys. D: Appl. Phys. 38, R185-R197 (2005).
[CrossRef]

F. Schapper, J. T. Gonçalves, and M. Oheim, "Fluorescence imaging with two-photon evanescent-wave excitation," Eur. Biophys. J. 32, 635-643 (2003).
[CrossRef] [PubMed]

Schneckenburger, H.

H. Schneckenburger, "Total internal reflection fluorescence microscopy: technical innovations and novel applications," Curr Opin Biotechnol. 16, 13-18 (2005).
[CrossRef] [PubMed]

So, P. T. C.

Stemmer, A.

R. Fiolka, Y. Belyaev, H. Ewers, and A. Stemmer, "Even illumination in total internal reflection fluorescence microscopy using laser light," Microsc. Res. Tech. 71, 45-50 (2007).
[CrossRef] [PubMed]

Stout, A. L.

Stranick, S. J.

M. R. Beversluis, G. W. Bryant, and S. J. Stranick, "Effects of inhomogeneous fields in superresolving structured illumination microscopy," 25, 1371-1377 (2008).
[CrossRef] [PubMed]

Streuli, C.

J. D. Humphries, P. Wang, C. Streuli, B. Geiger, M. J. Humphries, and C. Ballestrem, "Vinculin controls focal adhesion formation by direct interactions with talin and actin," J. Cell Biol. 179, 1043-1057 (2007).
[CrossRef] [PubMed]

Tamura, K.

Y. Kawano, C. Abe, T. Kaneda, Y. Aono, K. Abe, and K. Tamura, "High-numerical aperture objective lenses and optical system improved objective-type total internal reflection fluorescence microscopy," Proc. SPIE 4098, 142-53 (2000).
[CrossRef] [PubMed]

Tokunaga, M.

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa. "Highly inclined thin illumination enables clear single-molecule imaging in cells," Nat.Methods 5, 159-161 (2008).
[CrossRef] [PubMed]

Verkman, A. S.

B. P. Ölveczky, N. Periasamy, and A. S. Verkman, "Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy," Biophys J. 73,2836-47 (1997).
[CrossRef] [PubMed]

Wang, D. S.

Wang, P.

J. D. Humphries, P. Wang, C. Streuli, B. Geiger, M. J. Humphries, and C. Ballestrem, "Vinculin controls focal adhesion formation by direct interactions with talin and actin," J. Cell Biol. 179, 1043-1057 (2007).
[CrossRef] [PubMed]

Appl. Opt. (2)

Biophys J. (1)

B. P. Ölveczky, N. Periasamy, and A. S. Verkman, "Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy," Biophys J. 73,2836-47 (1997).
[CrossRef] [PubMed]

Biophys. J. (2)

N. K. Lee, A. N. Kapanidis, H. R. Koh,  et al, "Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances," Biophys. J. 92, 303-312 (2007).
[CrossRef]

A. Rohrbach, "Observing secretory granules with a multiangle evanescent-wave microscope," Biophys. J. 78, 2641-54 (2000).
[CrossRef] [PubMed]

Curr Opin Biotechnol. (1)

H. Schneckenburger, "Total internal reflection fluorescence microscopy: technical innovations and novel applications," Curr Opin Biotechnol. 16, 13-18 (2005).
[CrossRef] [PubMed]

Cytometry (1)

W. P. Ambrose, P. M. Goodwin, and J. P. Nolan "Single-molecule detection with total internal reflection 20. excitation: comparing signal-to-background and total signals in different geometries," Cytometry 36224-31 (1999).

Eur. Biophys. J. (1)

F. Schapper, J. T. Gonçalves, and M. Oheim, "Fluorescence imaging with two-photon evanescent-wave excitation," Eur. Biophys. J. 32, 635-643 (2003).
[CrossRef] [PubMed]

J Biomed Opt. (1)

A. L. Mattheyses and D. Axelrod, "Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence," J Biomed Opt. 11,014006A (2006).
[CrossRef]

J. Biomed. Opt. (1)

O. Gilko, G. D. Reddy, B. Anvari, W. E. Brownell, and P. Saggau, "Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells," J. Biomed. Opt. 11, 064013 (2006).
[CrossRef]

J. Cell Biol. (1)

J. D. Humphries, P. Wang, C. Streuli, B. Geiger, M. J. Humphries, and C. Ballestrem, "Vinculin controls focal adhesion formation by direct interactions with talin and actin," J. Cell Biol. 179, 1043-1057 (2007).
[CrossRef] [PubMed]

J. Microsc. (1)

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Figures (6)

Fig. 1.
Fig. 1.

Dielectric interface (a) and beam parameters (b) for objective-type total internal reflection. (c) Typical light distributions in the objective back focal plane (BFP) for eccentric-spot (left), crescent-(middle) and ring-shaped illumination (bottom). See text for details.

Fig. 2.
Fig. 2.

Schematic representation of the optical set-up. Solid lines – laser light; dotted – fluorescence; dashed – bright-field/epi-illumination. See text for details

Fig. 3.
Fig. 3.

BFP imaging allows quantitative TIRF microscopy. (a) Left, schematic view of the objective BFP. (x 0,y 0) defines the optical axis, r=|r|=[(x-x 0)2+(y-y 0)2]1/2 the off-axis displacement and hence beam angle α. TIR occurs for r>r c (dashed). r=r·e- iϕ (t) corresponds to spinning the spot at a constant radius r, ϕ=arccos(x/r). Middle, fluorescence generated in a dilute fluorescein (FITC) solution, for eccentric single-spot illumination Inset shows coreesponding BFP image. Red curve graphs the resulting asymmetric intensity line profile along the dashed line across the field-of-view. Right, overlay of 16 images acquired at different ϕ values. Scale bars are 5 µm. (b) Left, measurement of beam angles with ~1° precision. Objective 60× PlanApochromat TIRFM. Measured values of α (circles) compared favorably with theory (solid line) and generated an 8-bit look-up table α (r). Right, fluorescence generated in FITC, as a function of α. Dashed part differs from the usual transmitted intensity given by Fresnel coefficient for refraction because of the finite thickness of the FITC layer. (c) Left, sum projection of six BFP images acquired at different r, corresponding α=14.5, 28.5, 45, and 72°. Middle, plot of the average intensity (red) and SD (dashed). The angle-dependent diffraction efficiency of the AODs causes a ~20% intensity modulation) along the circular ROI (red). Right, cross-sectional intensity profile along the blue ROI. Fit of a Gaussian function with the 2nd ring yields r=2.09 µm, a width Δr=136 µm and angular dispersion of Δα≈±0.8°. α is obtained from the multi-pixel fit with a precision of ~1 µm, corresponding to ≈0.01°.

Fig. 4.
Fig. 4.

(a). HILO (α=59.5°) fluorescence images of a mouse cortical astrocyte labeled with FM1-43, as well as the simultaneously captured BFP images (insets), for different propagation directions of the wave vector. Note the absence of flare and the enlarged and homogenously lit field-of-view on fluorescence images excited with spinning-spot illumination (centre image) compared to the four cardinal images taken with conventional eccentric-spot illumination. North, east, south and west images display pronounced directionality (arrows). (b). Fine morphological detail is resolved on this enlarged view of a circular-spin TIRF image (α=63°). Individual lysosomes (bright spots) and mitochondria (dimmer tubules) are easily recognized and tracked without further image processing. Bottom curves display intensity profiles along the dashed lines shown. Scale is 5 µm. Exposure times were 1 ms and 100 ms for the BFP and fluorescence images, respectively. 60× PlanApochromat TIRFM objective.

Fig. 5.
Fig. 5.

Spinning TIRFs image mouse cortical astrocyte transiently expressing vinculin-EGFP. Fluorescence images of the same cell at different magnification. All objectives had NA1.45, see table 1. Scale is 5 µm. Insets show corresponding BFP images.

Fig. 6.
Fig. 6.

Quantum dots (QD565-ITK) immobilized on a biotinylated cover slip showed rapid blinking photoluminescent emission upon 405-nm excitation. Note the evenly lit field-of-view giving comparable intensities across the field-of-view. Exposure time 2 ms, scale 5 µm.

Tables (1)

Tables Icon

Table 1: TIRF objectives used in this study (Olympus Europe, Hamburg, Germany) had NA-1.45 and 0.1-mm working distance. We calculated pupil diameters 2NA f TL /M. h is the position of the BFP above the objective shoulder. See Fig. 5 for an example.

Equations (1)

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P = { P x cos 2 ( ω t x n 1 ω c sin α + θ ) P y = 0 P z sin ( ω t x n 1 ω c sin α + θ ) cos ( ω t x n 1 ω c sin α + θ )

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