Abstract

We present a simple modification of a standard total internal reflection fluorescence microscope to achieve nanometric axial resolution, typically 10nm. The technique is based on a normalization of total internal reflection images by conventional epi-illumination images. We demonstrate the potential of our method to study the adhesion of phopholipid giant unilamellar vesicles.

© 2014 Optical Society of America

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References

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  1. K. Hu, L. Ji, K. T. Applegate, G. Danuser, and C. M. Waterman-Storer, Science 315, 111 (2007).
    [CrossRef]
  2. A. Hassanzadeh, S. Armstrong, S. F. Dixon, and S. Mittler, Appl. Phys. Lett. 94, 033503 (2009).
    [CrossRef]
  3. D. Axelrod, E. H. Hellen, and R. M. Fulbright, in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed., (Plenum, 1992), Vol. 3, pp. 289–343.
  4. W. Lukosz and R. E. Kunz, J. Opt. Soc. Am. 67, 1607 (1977).
    [CrossRef]
  5. L. Limozin and K. Sengupta, Biophys. J. 93, 3300 (2007).
    [CrossRef]
  6. C. Vézy, G. Massiera, and A. Viallat, Soft Matter 3, 844 (2007).
  7. A. L. Stout and D. Axelrod, Appl. Opt. 28, 5237 (1989).
    [CrossRef]
  8. J. Mertz, Introduction to Optical Microscopy (Roberts & Company, 2010).
  9. J. S. Burmeister, G. A. Truskey, and W. M. Reichert, J. Microsc. 173, 39 (1994).
    [CrossRef]
  10. M. Abkarian and A. Viallat, Biophys. J. 89, 1055 (2005).
    [CrossRef]
  11. C. Boutin, R. Jaffiol, J. Plain, and P. Royer, J. Fluoresc. 18, 1115 (2008).
    [CrossRef]
  12. L. Limozin and K. Sengupta, Chem. Phys. Chem. 10, 2752 (2009).
    [CrossRef]

2009 (2)

A. Hassanzadeh, S. Armstrong, S. F. Dixon, and S. Mittler, Appl. Phys. Lett. 94, 033503 (2009).
[CrossRef]

L. Limozin and K. Sengupta, Chem. Phys. Chem. 10, 2752 (2009).
[CrossRef]

2008 (1)

C. Boutin, R. Jaffiol, J. Plain, and P. Royer, J. Fluoresc. 18, 1115 (2008).
[CrossRef]

2007 (3)

K. Hu, L. Ji, K. T. Applegate, G. Danuser, and C. M. Waterman-Storer, Science 315, 111 (2007).
[CrossRef]

L. Limozin and K. Sengupta, Biophys. J. 93, 3300 (2007).
[CrossRef]

C. Vézy, G. Massiera, and A. Viallat, Soft Matter 3, 844 (2007).

2005 (1)

M. Abkarian and A. Viallat, Biophys. J. 89, 1055 (2005).
[CrossRef]

1994 (1)

J. S. Burmeister, G. A. Truskey, and W. M. Reichert, J. Microsc. 173, 39 (1994).
[CrossRef]

1989 (1)

1977 (1)

Abkarian, M.

M. Abkarian and A. Viallat, Biophys. J. 89, 1055 (2005).
[CrossRef]

Applegate, K. T.

K. Hu, L. Ji, K. T. Applegate, G. Danuser, and C. M. Waterman-Storer, Science 315, 111 (2007).
[CrossRef]

Armstrong, S.

A. Hassanzadeh, S. Armstrong, S. F. Dixon, and S. Mittler, Appl. Phys. Lett. 94, 033503 (2009).
[CrossRef]

Axelrod, D.

A. L. Stout and D. Axelrod, Appl. Opt. 28, 5237 (1989).
[CrossRef]

D. Axelrod, E. H. Hellen, and R. M. Fulbright, in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed., (Plenum, 1992), Vol. 3, pp. 289–343.

Boutin, C.

C. Boutin, R. Jaffiol, J. Plain, and P. Royer, J. Fluoresc. 18, 1115 (2008).
[CrossRef]

Burmeister, J. S.

J. S. Burmeister, G. A. Truskey, and W. M. Reichert, J. Microsc. 173, 39 (1994).
[CrossRef]

Danuser, G.

K. Hu, L. Ji, K. T. Applegate, G. Danuser, and C. M. Waterman-Storer, Science 315, 111 (2007).
[CrossRef]

Dixon, S. F.

A. Hassanzadeh, S. Armstrong, S. F. Dixon, and S. Mittler, Appl. Phys. Lett. 94, 033503 (2009).
[CrossRef]

Fulbright, R. M.

D. Axelrod, E. H. Hellen, and R. M. Fulbright, in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed., (Plenum, 1992), Vol. 3, pp. 289–343.

Hassanzadeh, A.

A. Hassanzadeh, S. Armstrong, S. F. Dixon, and S. Mittler, Appl. Phys. Lett. 94, 033503 (2009).
[CrossRef]

Hellen, E. H.

D. Axelrod, E. H. Hellen, and R. M. Fulbright, in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed., (Plenum, 1992), Vol. 3, pp. 289–343.

Hu, K.

K. Hu, L. Ji, K. T. Applegate, G. Danuser, and C. M. Waterman-Storer, Science 315, 111 (2007).
[CrossRef]

Jaffiol, R.

C. Boutin, R. Jaffiol, J. Plain, and P. Royer, J. Fluoresc. 18, 1115 (2008).
[CrossRef]

Ji, L.

K. Hu, L. Ji, K. T. Applegate, G. Danuser, and C. M. Waterman-Storer, Science 315, 111 (2007).
[CrossRef]

Kunz, R. E.

Limozin, L.

L. Limozin and K. Sengupta, Chem. Phys. Chem. 10, 2752 (2009).
[CrossRef]

L. Limozin and K. Sengupta, Biophys. J. 93, 3300 (2007).
[CrossRef]

Lukosz, W.

Massiera, G.

C. Vézy, G. Massiera, and A. Viallat, Soft Matter 3, 844 (2007).

Mertz, J.

J. Mertz, Introduction to Optical Microscopy (Roberts & Company, 2010).

Mittler, S.

A. Hassanzadeh, S. Armstrong, S. F. Dixon, and S. Mittler, Appl. Phys. Lett. 94, 033503 (2009).
[CrossRef]

Plain, J.

C. Boutin, R. Jaffiol, J. Plain, and P. Royer, J. Fluoresc. 18, 1115 (2008).
[CrossRef]

Reichert, W. M.

J. S. Burmeister, G. A. Truskey, and W. M. Reichert, J. Microsc. 173, 39 (1994).
[CrossRef]

Royer, P.

C. Boutin, R. Jaffiol, J. Plain, and P. Royer, J. Fluoresc. 18, 1115 (2008).
[CrossRef]

Sengupta, K.

L. Limozin and K. Sengupta, Chem. Phys. Chem. 10, 2752 (2009).
[CrossRef]

L. Limozin and K. Sengupta, Biophys. J. 93, 3300 (2007).
[CrossRef]

Stout, A. L.

Truskey, G. A.

J. S. Burmeister, G. A. Truskey, and W. M. Reichert, J. Microsc. 173, 39 (1994).
[CrossRef]

Vézy, C.

C. Vézy, G. Massiera, and A. Viallat, Soft Matter 3, 844 (2007).

Viallat, A.

C. Vézy, G. Massiera, and A. Viallat, Soft Matter 3, 844 (2007).

M. Abkarian and A. Viallat, Biophys. J. 89, 1055 (2005).
[CrossRef]

Waterman-Storer, C. M.

K. Hu, L. Ji, K. T. Applegate, G. Danuser, and C. M. Waterman-Storer, Science 315, 111 (2007).
[CrossRef]

Appl. Opt. (1)

Appl. Phys. Lett. (1)

A. Hassanzadeh, S. Armstrong, S. F. Dixon, and S. Mittler, Appl. Phys. Lett. 94, 033503 (2009).
[CrossRef]

Biophys. J. (2)

L. Limozin and K. Sengupta, Biophys. J. 93, 3300 (2007).
[CrossRef]

M. Abkarian and A. Viallat, Biophys. J. 89, 1055 (2005).
[CrossRef]

Chem. Phys. Chem. (1)

L. Limozin and K. Sengupta, Chem. Phys. Chem. 10, 2752 (2009).
[CrossRef]

J. Fluoresc. (1)

C. Boutin, R. Jaffiol, J. Plain, and P. Royer, J. Fluoresc. 18, 1115 (2008).
[CrossRef]

J. Microsc. (1)

J. S. Burmeister, G. A. Truskey, and W. M. Reichert, J. Microsc. 173, 39 (1994).
[CrossRef]

J. Opt. Soc. Am. (1)

Science (1)

K. Hu, L. Ji, K. T. Applegate, G. Danuser, and C. M. Waterman-Storer, Science 315, 111 (2007).
[CrossRef]

Soft Matter (1)

C. Vézy, G. Massiera, and A. Viallat, Soft Matter 3, 844 (2007).

Other (2)

J. Mertz, Introduction to Optical Microscopy (Roberts & Company, 2010).

D. Axelrod, E. H. Hellen, and R. M. Fulbright, in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed., (Plenum, 1992), Vol. 3, pp. 289–343.

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Figures (4)

Fig. 1.
Fig. 1.

(a) Experimental setup. M, rotatable mirror; L1, lens of focal f1; DM, dichroic mirror; Ob, microscope objective; S, sample; L2: tube lens. Inset: the image of the back focal plane, corresponding to the emission of a monolayer of quantum dots on a glass-air interface at θ=0. The photoluminescence of the QD appears in orange; the focused laser beam in blue. (b) Calibration curve of the incident angle θ versus α. (c) Laser irradiance γ versus θ at a glass-water interface, for s-polarized incident light (γ is normalized to unity at θ=0). Black dots are the experimental data; red lines the fitting curve (b) or the theoretical curve (c).

Fig. 2.
Fig. 2.

Schematic view of a GUV in adhesion (in red, the lipid bi-layer membrane). In blue, the PSFdet for an arbitrary pixel position of the CCD.

Fig. 3.
Fig. 3.

Background correction factor β in epi-illumination versus (x,y) apparent diameter D of the GUV. Square and circle dots give, respectively, the increase of β for a spherical or an half-spherical 3D-contour of the GUV. The dotted lines correspond to an oblate profile with a ratio between the long and the small axis equal to 2. These simulations were done according to Eq. (1), taking into account a Gaussian–Lorentzian expression of the PSF: PSFdet(x,y,z)=(ω02/ω(z)2)e(2(x2+y2)/ω(z)2) with ω(z)=ω01+(z2λL2/π2ω04), ω0=200nm, λL=525nm.

Fig. 4.
Fig. 4.

(a) and (b) TIRF images; (c) and (d) epi-illumination images; (e) and (f) corresponding calculated z0 images. The pictures (a), (c), (e) were obtained on a functionalized surface n°1, and (b), (d), (f) on a surface n°2. The acquisition time is 100 ms for (a) and (c), 40 ms for (b) and (d). The laser irradiance is 40W/cm2.

Equations (9)

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fd(xd,yd,θ)=dxsdysdzsfs(xs,ys,zs,θ)×PSFdet(xdxs,ydys,zs),
fs(xs,ys,zs,θ)=ηdσabsϕfIexc(xs,ys,zs,θ)hνLC(xs,ys,zs),
Iexc(xs,ys,zs,θ=0)=I0e2(xs2+ys2)ωL,
Iexc(xs,ys,zs,θ>θc)=γ(θ)I0e2(xs2+ys2)ωLe(zs+z0)κ(θ),
κ(θ)=λL4π1ng2sin2θneff2,
fd(xd,yd,θ=0)=ηdσabsϕfI0hνLc0dxsdyse2(xs2+ys2)ωL×PSFdet(xdxs,ydys,0),
fd(xd,yd,θ>θc)=ηdσabsϕfI0hνLc0γ(θ)ez0κ(θ)dxsdys×e2(xs2+ys2)ωLPSFdet(xdxs,ydys,0).
z0(xd,yd)=κ(θ)lnfd(xd,yd,θ>θc)γ(θ)fd(xd,yd,θ=0).
z0(xd,yd)=κ(θ)lnfd(xd,yd,θ>θc)γ(θ)β(D)fd(xd,yd,θ=0).

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