Abstract

We compare the performance of a total-internal-reflection fluorescence microscope under varying illumination and substrate conditions. The samples are deposited on a standard homogeneous glass slide or on a grating and illuminated by one or two interfering beams at various incident angles. A conjugate gradient with positivity a priori information is used to reconstruct the fluorophore density from the images. Numerical studies demonstrate that when the sample lies on an optimized grating, the lateral resolution of the microscope is greatly improved, up to fourfold, the best result being obtained when the grating is illuminated by two interfering beams.

© 2009 Optical Society of America

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References

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    [CrossRef]
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    [CrossRef]
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    [CrossRef] [PubMed]
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2008 (4)

2007 (3)

P. C. Chaumet, K. Belkebir, and A. Sentenac, “Numerical study of grating-assisted optical diffraction tomography,” Phys. Rev. A 76, 013814-7 (2007).
[CrossRef]

R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating,” Proc. SPIE 3568, 185-196 (2007).
[CrossRef]

S. Hell, “Far-field optical nanoscopy,” Science 25, 1153-1158 (2007).
[CrossRef]

2006 (2)

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

A. Sentenac, P. C. Chaumet, and K. Belkebir, “Beyond the Rayleigh criterion: grating assisted far-field optical diffraction tomography,” Phys. Rev. Lett. 97, 243901-4 (2006).
[CrossRef]

2005 (2)

A. Dubois, K. Belkebir, and M. Saillard, “Retrieval of inhomogeneous targets from experimental frequency diversity data,” Inverse Probl. 21, S65-S79 (2005).
[CrossRef]

M. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102, 13081-13086 (2005).
[CrossRef] [PubMed]

2004 (2)

G. Cox and C. J. R. Sheppard, “Practical limits of resolution in confocal and non-linear microscopy,” Microsc. Res. Tech. 63, 18-22 (2004).
[CrossRef]

P. C. Chaumet, K. Belkebir, and A. Sentenac, “Three-dimensional sub-wavelength optical imaging using the coupled dipole method,” Phys. Rev. B 69, 245405-7 (2004).
[CrossRef]

2003 (1)

2002 (2)

2001 (3)

D. Toomre and D. J. Manstein, “Lighting up the cell surface with evanescent wave microscope,” Trends Cell Biol. 11, 298-303 (2001).
[CrossRef] [PubMed]

K. Belkebir and A. G. Tijhuis, “Modified2 gradient method and modified Born method for solving a two-dimensional inverse scattering problem,” Inverse Probl. 17, 1671-1688 (2001).
[CrossRef]

A. L. Fehrembach, S. Enoch, and A. Sentenac, “Highly directive source devices using slab photonic crystal,” Appl. Phys. Lett. 79, 4280-4282 (2001).
[CrossRef]

2000 (2)

J. Frohn, H. Knapp, and A. Stemmer, “True optical resolution, beyond the Rayleigh limit, achieved by standing wave illumination,” Proc. Natl. Acad. Sci. U.S.A. 97, 7232-7236 (2000).
[CrossRef] [PubMed]

G. Cragg and P. So, “Standing wave total-internal reflection microscopy,” Opt. Lett. 25, 46-48 (2000).
[CrossRef]

1997 (1)

1988 (1)

Ayers, G. R.

Beck, M.

Belkebir, K.

A. Sentenac, K. Belkebir, H. Giovannini, and P. C. Chaumet, “Subdiffraction resolution in total internal reflection fluorescence microscopy with a grating substrate,” Opt. Lett. 33, 255-257 (2008).
[CrossRef] [PubMed]

P. C. Chaumet, K. Belkebir, and A. Sentenac, “Numerical study of grating-assisted optical diffraction tomography,” Phys. Rev. A 76, 013814-7 (2007).
[CrossRef]

A. Sentenac, P. C. Chaumet, and K. Belkebir, “Beyond the Rayleigh criterion: grating assisted far-field optical diffraction tomography,” Phys. Rev. Lett. 97, 243901-4 (2006).
[CrossRef]

A. Dubois, K. Belkebir, and M. Saillard, “Retrieval of inhomogeneous targets from experimental frequency diversity data,” Inverse Probl. 21, S65-S79 (2005).
[CrossRef]

P. C. Chaumet, K. Belkebir, and A. Sentenac, “Three-dimensional sub-wavelength optical imaging using the coupled dipole method,” Phys. Rev. B 69, 245405-7 (2004).
[CrossRef]

K. Belkebir and A. Sentenac, “High-resolution optical diffraction microscopy,” J. Opt. Soc. Am. A 20, 1223-1229 (2003).
[CrossRef]

K. Belkebir and A. G. Tijhuis, “Modified2 gradient method and modified Born method for solving a two-dimensional inverse scattering problem,” Inverse Probl. 17, 1671-1688 (2001).
[CrossRef]

Chaumet, P. C.

A. Sentenac and P. C. Chaumet, “Sub-diffraction light focusing on a grating substrate,” Phys. Rev. Lett. 101, 013901-4 (2008).
[CrossRef] [PubMed]

A. Sentenac, K. Belkebir, H. Giovannini, and P. C. Chaumet, “Subdiffraction resolution in total internal reflection fluorescence microscopy with a grating substrate,” Opt. Lett. 33, 255-257 (2008).
[CrossRef] [PubMed]

P. C. Chaumet, K. Belkebir, and A. Sentenac, “Numerical study of grating-assisted optical diffraction tomography,” Phys. Rev. A 76, 013814-7 (2007).
[CrossRef]

A. Sentenac, P. C. Chaumet, and K. Belkebir, “Beyond the Rayleigh criterion: grating assisted far-field optical diffraction tomography,” Phys. Rev. Lett. 97, 243901-4 (2006).
[CrossRef]

P. C. Chaumet, K. Belkebir, and A. Sentenac, “Three-dimensional sub-wavelength optical imaging using the coupled dipole method,” Phys. Rev. B 69, 245405-7 (2004).
[CrossRef]

Chung, E.

Cox, G.

G. Cox and C. J. R. Sheppard, “Practical limits of resolution in confocal and non-linear microscopy,” Microsc. Res. Tech. 63, 18-22 (2004).
[CrossRef]

Cragg, G.

Cremer, C.

R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating,” Proc. SPIE 3568, 185-196 (2007).
[CrossRef]

R. Heintzmann, T. M. Jovin, and C. Cremer, “Saturated patterned excitation microscopy: a concept for optical resolution improvement,” J. Opt. Soc. Am. A 19, 1599-1609 (2002).
[CrossRef]

Dainty, J. C.

Dubois, A.

A. Dubois, K. Belkebir, and M. Saillard, “Retrieval of inhomogeneous targets from experimental frequency diversity data,” Inverse Probl. 21, S65-S79 (2005).
[CrossRef]

Enoch, S.

A. L. Fehrembach, S. Enoch, and A. Sentenac, “Highly directive source devices using slab photonic crystal,” Appl. Phys. Lett. 79, 4280-4282 (2001).
[CrossRef]

Fehrembach, A. L.

A. L. Fehrembach, D. Maystre, and A. Sentenac, “Phenomenological theory of filtering by resonant dielectric gratings,” J. Opt. Soc. Am. A 19, 1136-1142 (2002).
[CrossRef]

A. L. Fehrembach, S. Enoch, and A. Sentenac, “Highly directive source devices using slab photonic crystal,” Appl. Phys. Lett. 79, 4280-4282 (2001).
[CrossRef]

Fiokla, R.

A. Stemmer, M. Beck, and R. Fiokla, “Widefield fluorescence microsocpy with extended resolution,” Histochem. Cell Biol. 130, 807-617 (2008).
[CrossRef] [PubMed]

Fiolka, R.

Frohn, J.

J. Frohn, H. Knapp, and A. Stemmer, “True optical resolution, beyond the Rayleigh limit, achieved by standing wave illumination,” Proc. Natl. Acad. Sci. U.S.A. 97, 7232-7236 (2000).
[CrossRef] [PubMed]

Giovannini, H.

Gustafsson, M.

M. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102, 13081-13086 (2005).
[CrossRef] [PubMed]

Heintzmann, R.

R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating,” Proc. SPIE 3568, 185-196 (2007).
[CrossRef]

R. Heintzmann, T. M. Jovin, and C. Cremer, “Saturated patterned excitation microscopy: a concept for optical resolution improvement,” J. Opt. Soc. Am. A 19, 1599-1609 (2002).
[CrossRef]

Hell, S.

S. Hell, “Far-field optical nanoscopy,” Science 25, 1153-1158 (2007).
[CrossRef]

Jovin, T. M.

Kim, D.

Knapp, H.

J. Frohn, H. Knapp, and A. Stemmer, “True optical resolution, beyond the Rayleigh limit, achieved by standing wave illumination,” Proc. Natl. Acad. Sci. U.S.A. 97, 7232-7236 (2000).
[CrossRef] [PubMed]

Li, L.

Mandula, O.

O. Mandula, “Patterned excitation microscopy,” Master's thesis (Institute of Physics, King's College, 2008).

Manstein, D. J.

D. Toomre and D. J. Manstein, “Lighting up the cell surface with evanescent wave microscope,” Trends Cell Biol. 11, 298-303 (2001).
[CrossRef] [PubMed]

Maystre, D.

Rost, F. W. D.

F. W. D. Rost, Fluorescence Microscopy, Vol. I (Cambridge Univ. Press, 1992).

F. W. D. Rost, Fluorescence Microscopy, Vol. II (Cambridge Univ. Press, 1995).

Saillard, M.

A. Dubois, K. Belkebir, and M. Saillard, “Retrieval of inhomogeneous targets from experimental frequency diversity data,” Inverse Probl. 21, S65-S79 (2005).
[CrossRef]

Sentenac, A.

A. Sentenac, K. Belkebir, H. Giovannini, and P. C. Chaumet, “Subdiffraction resolution in total internal reflection fluorescence microscopy with a grating substrate,” Opt. Lett. 33, 255-257 (2008).
[CrossRef] [PubMed]

A. Sentenac and P. C. Chaumet, “Sub-diffraction light focusing on a grating substrate,” Phys. Rev. Lett. 101, 013901-4 (2008).
[CrossRef] [PubMed]

P. C. Chaumet, K. Belkebir, and A. Sentenac, “Numerical study of grating-assisted optical diffraction tomography,” Phys. Rev. A 76, 013814-7 (2007).
[CrossRef]

A. Sentenac, P. C. Chaumet, and K. Belkebir, “Beyond the Rayleigh criterion: grating assisted far-field optical diffraction tomography,” Phys. Rev. Lett. 97, 243901-4 (2006).
[CrossRef]

P. C. Chaumet, K. Belkebir, and A. Sentenac, “Three-dimensional sub-wavelength optical imaging using the coupled dipole method,” Phys. Rev. B 69, 245405-7 (2004).
[CrossRef]

K. Belkebir and A. Sentenac, “High-resolution optical diffraction microscopy,” J. Opt. Soc. Am. A 20, 1223-1229 (2003).
[CrossRef]

A. L. Fehrembach, D. Maystre, and A. Sentenac, “Phenomenological theory of filtering by resonant dielectric gratings,” J. Opt. Soc. Am. A 19, 1136-1142 (2002).
[CrossRef]

A. L. Fehrembach, S. Enoch, and A. Sentenac, “Highly directive source devices using slab photonic crystal,” Appl. Phys. Lett. 79, 4280-4282 (2001).
[CrossRef]

Sheppard, C. J. R.

G. Cox and C. J. R. Sheppard, “Practical limits of resolution in confocal and non-linear microscopy,” Microsc. Res. Tech. 63, 18-22 (2004).
[CrossRef]

So, P.

Stemmer, A.

R. Fiolka, M. Beck, and A. Stemmer, “Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator,” Opt. Lett. 33, 1629-1631 (2008).
[CrossRef] [PubMed]

A. Stemmer, M. Beck, and R. Fiokla, “Widefield fluorescence microsocpy with extended resolution,” Histochem. Cell Biol. 130, 807-617 (2008).
[CrossRef] [PubMed]

J. Frohn, H. Knapp, and A. Stemmer, “True optical resolution, beyond the Rayleigh limit, achieved by standing wave illumination,” Proc. Natl. Acad. Sci. U.S.A. 97, 7232-7236 (2000).
[CrossRef] [PubMed]

Tijhuis, A. G.

K. Belkebir and A. G. Tijhuis, “Modified2 gradient method and modified Born method for solving a two-dimensional inverse scattering problem,” Inverse Probl. 17, 1671-1688 (2001).
[CrossRef]

Toomre, D.

D. Toomre and D. J. Manstein, “Lighting up the cell surface with evanescent wave microscope,” Trends Cell Biol. 11, 298-303 (2001).
[CrossRef] [PubMed]

Appl. Phys. Lett. (1)

A. L. Fehrembach, S. Enoch, and A. Sentenac, “Highly directive source devices using slab photonic crystal,” Appl. Phys. Lett. 79, 4280-4282 (2001).
[CrossRef]

Histochem. Cell Biol. (1)

A. Stemmer, M. Beck, and R. Fiokla, “Widefield fluorescence microsocpy with extended resolution,” Histochem. Cell Biol. 130, 807-617 (2008).
[CrossRef] [PubMed]

Inverse Probl. (2)

K. Belkebir and A. G. Tijhuis, “Modified2 gradient method and modified Born method for solving a two-dimensional inverse scattering problem,” Inverse Probl. 17, 1671-1688 (2001).
[CrossRef]

A. Dubois, K. Belkebir, and M. Saillard, “Retrieval of inhomogeneous targets from experimental frequency diversity data,” Inverse Probl. 21, S65-S79 (2005).
[CrossRef]

J. Opt. Soc. Am. A (4)

Microsc. Res. Tech. (1)

G. Cox and C. J. R. Sheppard, “Practical limits of resolution in confocal and non-linear microscopy,” Microsc. Res. Tech. 63, 18-22 (2004).
[CrossRef]

Opt. Lett. (5)

Phys. Rev. A (1)

P. C. Chaumet, K. Belkebir, and A. Sentenac, “Numerical study of grating-assisted optical diffraction tomography,” Phys. Rev. A 76, 013814-7 (2007).
[CrossRef]

Phys. Rev. B (1)

P. C. Chaumet, K. Belkebir, and A. Sentenac, “Three-dimensional sub-wavelength optical imaging using the coupled dipole method,” Phys. Rev. B 69, 245405-7 (2004).
[CrossRef]

Phys. Rev. Lett. (2)

A. Sentenac and P. C. Chaumet, “Sub-diffraction light focusing on a grating substrate,” Phys. Rev. Lett. 101, 013901-4 (2008).
[CrossRef] [PubMed]

A. Sentenac, P. C. Chaumet, and K. Belkebir, “Beyond the Rayleigh criterion: grating assisted far-field optical diffraction tomography,” Phys. Rev. Lett. 97, 243901-4 (2006).
[CrossRef]

Proc. Natl. Acad. Sci. U.S.A. (2)

M. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102, 13081-13086 (2005).
[CrossRef] [PubMed]

J. Frohn, H. Knapp, and A. Stemmer, “True optical resolution, beyond the Rayleigh limit, achieved by standing wave illumination,” Proc. Natl. Acad. Sci. U.S.A. 97, 7232-7236 (2000).
[CrossRef] [PubMed]

Proc. SPIE (1)

R. Heintzmann and C. Cremer, “Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating,” Proc. SPIE 3568, 185-196 (2007).
[CrossRef]

Science (1)

S. Hell, “Far-field optical nanoscopy,” Science 25, 1153-1158 (2007).
[CrossRef]

Trends Cell Biol. (1)

D. Toomre and D. J. Manstein, “Lighting up the cell surface with evanescent wave microscope,” Trends Cell Biol. 11, 298-303 (2001).
[CrossRef] [PubMed]

Other (5)

O. Mandula, “Patterned excitation microscopy,” Master's thesis (Institute of Physics, King's College, 2008).

C.Zander, J.Enderlein, and R.A.Keller, eds., Single Molecule Detection in Solution: Methods and Applications (Wiley-VCH Verlag, 2003).

F. W. D. Rost, Fluorescence Microscopy, Vol. I (Cambridge Univ. Press, 1992).

F. W. D. Rost, Fluorescence Microscopy, Vol. II (Cambridge Univ. Press, 1995).

D. Maystre, Diffraction Gratings, SPIE Milestones Series (SPIE Press, 1993).

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Figures (7)

Fig. 1
Fig. 1

Schematic view of the TIRFM that is simulated in this work. (a) The fluorophores deposited on the substrate (glass slide: CG) are illuminated by a parallel beam that is totally reflected at the glass/air interface. IO is an inversion objective, and the image of each fluorophore is formed on the CCD camera, through the optical system (IO, L). (b) θ inc is the incident polar angle and ϕ is the azimuthal angle. k inc is the tangential component of the incident wave vector.

Fig. 2
Fig. 2

Illumination configurations. (a) Glass slide illuminated homogeneously by a plane wave under total internal reflection. (b) Glass slide illuminated by a standing-wave pattern. The periodic intensity pattern above the substrate is generated by the interference of two plane waves. (c) Grating illuminated by one plane wave. The period of the intensity pattern just above the grating can be much smaller than that obtained in (b). (d) Grating illuminated by two interfering plane waves. The intensity pattern above the grating is not periodic in general.

Fig. 3
Fig. 3

Excitation light patterns generated by the illumination configurations depicted in Fig. 2, (a), (b) Simulation of the field intensity just above the glass slide when the illumination is made of two interfering plane waves with θ inc = ± 65 deg and relative phase 0 and 2 π 3 respectively. (c), (d) Simulation of the field intensity just above the grating when the illumination is an s-polarized plane wave with θ inc = 65 deg and azimuthal angle ϕ = π 2 and ϕ = 3 π 2 , respectively. The intensity is calculated with the rigorous Fourier modal method [17]. (e), (f) Same as (c), (d) but the grating is illuminated by two s-polarized plane waves with θ inc = ± 65 deg , ϕ = π 2 , same amplitude and relative phase 0 and π, respectively;

Fig. 4
Fig. 4

Simulation of a TIRFM experiment. (a) Theoretical sample: (beads with a diameter of 20 nm , randomly scattered on a glass slide). (b) Simulation of the image recorded on the CCD camera using a homogeneous illumination. (c) Deconvolution of (b) with the conjugate-gradient algorithm depicted in Section 4 without assuming the positivity of the density of fluorescence. (d) Same as (c) but the algorithm accounts for the positivity.

Fig. 5
Fig. 5

Illustration of the resolution improvement brought by the grating. (a) Theoretical sample: beads with a diameter of 20 nm that are separated by λ 2 , λ 4 , λ 6 , λ 8 from bottom to top on the left side and λ 10 , λ 14 from top to bottom on the right side. (b) Simulation of an image recorded on the CCD camera using homogeneous illumination. (c) Normalized density of fluorescence obtained with the iterative inversion procedure accounting for the positivity (Section 4) in the homogeneous configuration; see Fig. 2a. (d) Same as (b) but in the standing-wave configuration; see Fig. 2b. (e) Same as (b) but in the grating configuration; see Fig. 2c. (f) Same as (b) but in the standing-wave grating configuration; see Fig. 2d.

Fig. 6
Fig. 6

Reconstructed fluorescence density of the random sample depicted in Fig. 4. (a) Standing-wave configuration; see Fig. 2b. (b) Grating configuration; see Fig. 2c. (c) Standing-wave grating configuration; see Fig. 2d. (d), (e), and (f) zoom of (a), (b), and (c) respectively.

Fig. 7
Fig. 7

Schematic representation of the grating. (a) Top view. (b) Side view; d = 157 nm , e = 50 nm , h = 88 nm , l = 19 nm , p = 50 nm , incident wavelength λ = 630 nm .

Equations (14)

Equations on this page are rendered with MathJax. Learn more.

I l mes ( r ) = [ ( O × E l ) P ] ( r ) + B ( r ) , for l = 1 , , N ,
P ( r ) = ( J 1 ( k 0 NA r ) r ) 2 ,
E l ( r ) = A | exp ( i k inc r ) + exp ( i k inc r + i ψ ) | 2 ,
E l ( r ) = | exp ( i k inc r ) n , m Z 2 A n , m exp ( i K n , m r ) | 2 ,
E l ( r ) = | exp ( i k inc r ) n , m Z 2 A n , m exp ( i K n , m r ) + exp ( i k inc r + i ψ ) n , m Z 2 A n , m exp ( i K n , m r ) | 2 .
F ( O ̂ ) = l = 1 L I l mes A ( O ̂ E l ) Ω 2 l = 1 L I l mes Ω 2 = W l = 1 L I l mes A ( O ̂ E l ) Ω 2 ,
I l = ( O ̂ × E l ) P = A ( O ̂ E l ) .
O ̂ n = O ̂ n 1 + α n d n ,
d n = g n + γ n d n 1 , with γ n = g n | g n g n 1 Ω g n 1 Ω 2 ,
g n = W l = 1 L E l A h l , n 1 ,
g n ; ξ ̂ = 2 W l = 1 L E l ξ ̂ A h l , n 1 .
E l ( r ) | A 0 , 0 + A 1 , 0 exp ( i K 1 , 0 r ) | 2 .
E l ( r ) | A 0 , 0 cos ( k inc r + ψ 2 ) + A 1 , 0 cos [ ( k inc + K 1 , 0 ) r + ψ 2 ] | 2 .
| k inc + K 1 , 0 | k mode ,

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