Abstract

Reflection interference contrast microscopy (RICM) is a technique for measuring the shape and position of microscopic objects in solution; it has many biological and biophysical applications. Use of RICM for long-time acquisitions requires minimizing defocusing effects that are due to thermal and mechanical drift. We present a simple stabilizing method that accomplishes this using an image-analysis-based linear focus function to establish feedback control of the focal position. While implementing this routine, we used RICM for independent measurement of the apparent fluctuation in the vertical position of an immobilized bead: the measured height had a standard deviation of 0.12nm during a 45 min acquisition while under feedback control, demonstrating the high stability achievable with our approach.

© 2008 Optical Society of America

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  1. A. S. G. Curtis, "The mechanism of adhesion of cells to glass: a study by interference reflection microscopy," J. Cell Biol. 20, 199-215 (1964).
    [CrossRef] [PubMed]
  2. J. Rädler and E. Sackmann, "Imaging optical thicknesses and separation distances of phospholipid vesicles at solid surfaces," J. Phys. (Paris) II 3, 727-748 (1993).
  3. Z. Guttenberg, A. R. Bausch, B. Hu, R. Bruinsma, L. Moroder, and E. Sackmann, "Measuring ligand-receptor unbinding forces with magnetic beads: molecular leverage," Langmuir 16, 8984-8993 (2000).
    [CrossRef]
  4. V. Heinrich, K. Ritchie, N. Mohandas, and E. Evans, "Elastic thickness compressibility of the red cell membrane," Biophys. J. 81, 1452-1463 (2001).
    [CrossRef] [PubMed]
  5. A. Zidovska and E. Sackmann, "Brownian motion of nucleated cell envelopes impedes adhesion," Phys. Rev. Lett. 96, 048103 (2006).
    [CrossRef] [PubMed]
  6. D. Cuvelier, M. Théry, Y.-S. Chu, S. Dufour, J.-P. Thiéry, M. Bornens, P. Nassoy, and L. Mahadevan, "The universal dynamics of cell spreading," Curr. Biol. 17, 694-699 (2007).
    [CrossRef] [PubMed]
  7. J. Rädler and E. Sackmann, "On the measurement of weak repulsive and frictional colloidal forces by reflection interference contrast microscopy," Langmuir 8, 848-853 (1992).
    [CrossRef]
  8. G. Wiegand, K. R. Neumaier, and E. Sackmann, "Microinterferometry: three-dimensional reconstruction of surface microtopography for thin-film and wetting studies by reflection interference contrast microscopy (RICM)," Appl. Opt. 37, 6892-6905 (1998).
    [CrossRef]
  9. N. G. Clack and J. T. Groves, "Many-particle tracking with nanometer resolution in three dimensions by reflection interference contrast microscopy," Langmuir 21, 6430-6435 (2005).
    [CrossRef] [PubMed]
  10. S. J. Gokhale, J. L. Plawsky, and P. C. Wayner, "Spreading, evaporation, and contact line dynamics of surfactant-laden microdrops," Langmuir 21, 8188-8197 (2005).
    [CrossRef] [PubMed]
  11. M. Kühner and E. Sackmann, "Ultrathin hydrated dextran films grafted on glass: preparation and characterization of structural, viscous, and elastic properties by quantitative microinterferometry," Langmuir 12, 4866-4876 (1996).
    [CrossRef]
  12. J.-M. Geusebroek, F. Cornelissen, A. W. M. Smeulders, and H. Geerts, "Robust autofocusing in microscopy," Cytometry 39, 1-9 (2000).
    [CrossRef] [PubMed]
  13. Y. Sun, S. Duthaler, and B. J. Nelson, "Autofocusing in computer microscopy: selecting the optimal focus algorithm," Microsc. Res. Tech. 65, 139-149 (2004).
    [CrossRef] [PubMed]
  14. M. Capitanio, R. Cicchi, and F. S. Pavone, "Position control and optical manipulation for nanotechnology applications," Eur. Phys. J. B 46, 1-8 (2005).
    [CrossRef]
  15. H. Oku, M. Ishikawa, Theodorus, and K. Hashimoto, "High-speed autofocusing of a cell using diffraction pattern," Opt. Express 14, 3952-3960 (2006).
    [CrossRef]
  16. A. R. Carter, G. M. King, T. A. Ulrich, W. Halsey, D. Alchenberger, and T. T. Perkins, "Stabilization of an optical microscope to 0.1 nm in three dimensions," Appl. Opt. 46, 421-427(2007).
    [CrossRef] [PubMed]
  17. W. J. Greenleaf, M. T. Woodside, and S. M. Block, "High-resolution, single-molecule measurements of biomolecular motion," Annu. Rev. Biophys. Biomol. Struct. 36, 171-190(2007).
    [CrossRef] [PubMed]
  18. T. Strick, J.-F. Allemand, V. Croquette, and D. Bensimon, "Twisting and stretching single DNA molecules," Prog. Biophys. Mol. Biol. 74, 115-140 (2000).
    [CrossRef] [PubMed]
  19. C. Bustamante, Z. Bryant, and S. B. Smith, "Ten years of tension: single-molecule DNA mechanics," Nature 421, 423-427(2003).
    [CrossRef] [PubMed]
  20. T. Lionnet, A. Dawid, S. Bigot, F.-X. Barre, O. A. Saleh, F. Heslot, J.-F. Allemand, D. Bensimon, and V. Croquette, "DNA mechanics as a tool to probe helicase and translocase activity," Nucleic Acids Res. 34, 4232-4244 (2006).
    [CrossRef] [PubMed]
  21. E. Krotkov, "Focusing," Int. J. Comput. Vision 1, 223-237(1988).
    [CrossRef]
  22. E. Sackmann and R. F. Bruinsma, "Cell adhesion as wetting transition?" ChemPhysChem. 3, 262-269 (2002).
    [CrossRef] [PubMed]
  23. P. C. Nelson, C. Zurla, D. Brogioli, J. F. Beausang, L. Finzi, and D. Dunlap, "Tethered particle motion as a diagnostic of DNA tether length," J. Phys. Chem. B 110, 17260-17267(2006).
    [CrossRef] [PubMed]

2007

D. Cuvelier, M. Théry, Y.-S. Chu, S. Dufour, J.-P. Thiéry, M. Bornens, P. Nassoy, and L. Mahadevan, "The universal dynamics of cell spreading," Curr. Biol. 17, 694-699 (2007).
[CrossRef] [PubMed]

A. R. Carter, G. M. King, T. A. Ulrich, W. Halsey, D. Alchenberger, and T. T. Perkins, "Stabilization of an optical microscope to 0.1 nm in three dimensions," Appl. Opt. 46, 421-427(2007).
[CrossRef] [PubMed]

W. J. Greenleaf, M. T. Woodside, and S. M. Block, "High-resolution, single-molecule measurements of biomolecular motion," Annu. Rev. Biophys. Biomol. Struct. 36, 171-190(2007).
[CrossRef] [PubMed]

2006

H. Oku, M. Ishikawa, Theodorus, and K. Hashimoto, "High-speed autofocusing of a cell using diffraction pattern," Opt. Express 14, 3952-3960 (2006).
[CrossRef]

A. Zidovska and E. Sackmann, "Brownian motion of nucleated cell envelopes impedes adhesion," Phys. Rev. Lett. 96, 048103 (2006).
[CrossRef] [PubMed]

T. Lionnet, A. Dawid, S. Bigot, F.-X. Barre, O. A. Saleh, F. Heslot, J.-F. Allemand, D. Bensimon, and V. Croquette, "DNA mechanics as a tool to probe helicase and translocase activity," Nucleic Acids Res. 34, 4232-4244 (2006).
[CrossRef] [PubMed]

P. C. Nelson, C. Zurla, D. Brogioli, J. F. Beausang, L. Finzi, and D. Dunlap, "Tethered particle motion as a diagnostic of DNA tether length," J. Phys. Chem. B 110, 17260-17267(2006).
[CrossRef] [PubMed]

2005

M. Capitanio, R. Cicchi, and F. S. Pavone, "Position control and optical manipulation for nanotechnology applications," Eur. Phys. J. B 46, 1-8 (2005).
[CrossRef]

N. G. Clack and J. T. Groves, "Many-particle tracking with nanometer resolution in three dimensions by reflection interference contrast microscopy," Langmuir 21, 6430-6435 (2005).
[CrossRef] [PubMed]

S. J. Gokhale, J. L. Plawsky, and P. C. Wayner, "Spreading, evaporation, and contact line dynamics of surfactant-laden microdrops," Langmuir 21, 8188-8197 (2005).
[CrossRef] [PubMed]

2004

Y. Sun, S. Duthaler, and B. J. Nelson, "Autofocusing in computer microscopy: selecting the optimal focus algorithm," Microsc. Res. Tech. 65, 139-149 (2004).
[CrossRef] [PubMed]

2003

C. Bustamante, Z. Bryant, and S. B. Smith, "Ten years of tension: single-molecule DNA mechanics," Nature 421, 423-427(2003).
[CrossRef] [PubMed]

2002

E. Sackmann and R. F. Bruinsma, "Cell adhesion as wetting transition?" ChemPhysChem. 3, 262-269 (2002).
[CrossRef] [PubMed]

2001

V. Heinrich, K. Ritchie, N. Mohandas, and E. Evans, "Elastic thickness compressibility of the red cell membrane," Biophys. J. 81, 1452-1463 (2001).
[CrossRef] [PubMed]

2000

Z. Guttenberg, A. R. Bausch, B. Hu, R. Bruinsma, L. Moroder, and E. Sackmann, "Measuring ligand-receptor unbinding forces with magnetic beads: molecular leverage," Langmuir 16, 8984-8993 (2000).
[CrossRef]

J.-M. Geusebroek, F. Cornelissen, A. W. M. Smeulders, and H. Geerts, "Robust autofocusing in microscopy," Cytometry 39, 1-9 (2000).
[CrossRef] [PubMed]

T. Strick, J.-F. Allemand, V. Croquette, and D. Bensimon, "Twisting and stretching single DNA molecules," Prog. Biophys. Mol. Biol. 74, 115-140 (2000).
[CrossRef] [PubMed]

1998

1996

M. Kühner and E. Sackmann, "Ultrathin hydrated dextran films grafted on glass: preparation and characterization of structural, viscous, and elastic properties by quantitative microinterferometry," Langmuir 12, 4866-4876 (1996).
[CrossRef]

1993

J. Rädler and E. Sackmann, "Imaging optical thicknesses and separation distances of phospholipid vesicles at solid surfaces," J. Phys. (Paris) II 3, 727-748 (1993).

1992

J. Rädler and E. Sackmann, "On the measurement of weak repulsive and frictional colloidal forces by reflection interference contrast microscopy," Langmuir 8, 848-853 (1992).
[CrossRef]

1988

E. Krotkov, "Focusing," Int. J. Comput. Vision 1, 223-237(1988).
[CrossRef]

1964

A. S. G. Curtis, "The mechanism of adhesion of cells to glass: a study by interference reflection microscopy," J. Cell Biol. 20, 199-215 (1964).
[CrossRef] [PubMed]

Annu. Rev. Biophys. Biomol. Struct.

W. J. Greenleaf, M. T. Woodside, and S. M. Block, "High-resolution, single-molecule measurements of biomolecular motion," Annu. Rev. Biophys. Biomol. Struct. 36, 171-190(2007).
[CrossRef] [PubMed]

Appl. Opt.

Biophys. J.

V. Heinrich, K. Ritchie, N. Mohandas, and E. Evans, "Elastic thickness compressibility of the red cell membrane," Biophys. J. 81, 1452-1463 (2001).
[CrossRef] [PubMed]

ChemPhysChem.

E. Sackmann and R. F. Bruinsma, "Cell adhesion as wetting transition?" ChemPhysChem. 3, 262-269 (2002).
[CrossRef] [PubMed]

Curr. Biol.

D. Cuvelier, M. Théry, Y.-S. Chu, S. Dufour, J.-P. Thiéry, M. Bornens, P. Nassoy, and L. Mahadevan, "The universal dynamics of cell spreading," Curr. Biol. 17, 694-699 (2007).
[CrossRef] [PubMed]

Cytometry

J.-M. Geusebroek, F. Cornelissen, A. W. M. Smeulders, and H. Geerts, "Robust autofocusing in microscopy," Cytometry 39, 1-9 (2000).
[CrossRef] [PubMed]

Eur. Phys. J. B

M. Capitanio, R. Cicchi, and F. S. Pavone, "Position control and optical manipulation for nanotechnology applications," Eur. Phys. J. B 46, 1-8 (2005).
[CrossRef]

Int. J. Comput. Vision

E. Krotkov, "Focusing," Int. J. Comput. Vision 1, 223-237(1988).
[CrossRef]

J. Cell Biol.

A. S. G. Curtis, "The mechanism of adhesion of cells to glass: a study by interference reflection microscopy," J. Cell Biol. 20, 199-215 (1964).
[CrossRef] [PubMed]

J. Phys. (Paris) II

J. Rädler and E. Sackmann, "Imaging optical thicknesses and separation distances of phospholipid vesicles at solid surfaces," J. Phys. (Paris) II 3, 727-748 (1993).

J. Phys. Chem. B

P. C. Nelson, C. Zurla, D. Brogioli, J. F. Beausang, L. Finzi, and D. Dunlap, "Tethered particle motion as a diagnostic of DNA tether length," J. Phys. Chem. B 110, 17260-17267(2006).
[CrossRef] [PubMed]

Langmuir

Z. Guttenberg, A. R. Bausch, B. Hu, R. Bruinsma, L. Moroder, and E. Sackmann, "Measuring ligand-receptor unbinding forces with magnetic beads: molecular leverage," Langmuir 16, 8984-8993 (2000).
[CrossRef]

N. G. Clack and J. T. Groves, "Many-particle tracking with nanometer resolution in three dimensions by reflection interference contrast microscopy," Langmuir 21, 6430-6435 (2005).
[CrossRef] [PubMed]

S. J. Gokhale, J. L. Plawsky, and P. C. Wayner, "Spreading, evaporation, and contact line dynamics of surfactant-laden microdrops," Langmuir 21, 8188-8197 (2005).
[CrossRef] [PubMed]

M. Kühner and E. Sackmann, "Ultrathin hydrated dextran films grafted on glass: preparation and characterization of structural, viscous, and elastic properties by quantitative microinterferometry," Langmuir 12, 4866-4876 (1996).
[CrossRef]

J. Rädler and E. Sackmann, "On the measurement of weak repulsive and frictional colloidal forces by reflection interference contrast microscopy," Langmuir 8, 848-853 (1992).
[CrossRef]

Microsc. Res. Tech.

Y. Sun, S. Duthaler, and B. J. Nelson, "Autofocusing in computer microscopy: selecting the optimal focus algorithm," Microsc. Res. Tech. 65, 139-149 (2004).
[CrossRef] [PubMed]

Nature

C. Bustamante, Z. Bryant, and S. B. Smith, "Ten years of tension: single-molecule DNA mechanics," Nature 421, 423-427(2003).
[CrossRef] [PubMed]

Nucleic Acids Res.

T. Lionnet, A. Dawid, S. Bigot, F.-X. Barre, O. A. Saleh, F. Heslot, J.-F. Allemand, D. Bensimon, and V. Croquette, "DNA mechanics as a tool to probe helicase and translocase activity," Nucleic Acids Res. 34, 4232-4244 (2006).
[CrossRef] [PubMed]

Opt. Express

Phys. Rev. Lett.

A. Zidovska and E. Sackmann, "Brownian motion of nucleated cell envelopes impedes adhesion," Phys. Rev. Lett. 96, 048103 (2006).
[CrossRef] [PubMed]

Prog. Biophys. Mol. Biol.

T. Strick, J.-F. Allemand, V. Croquette, and D. Bensimon, "Twisting and stretching single DNA molecules," Prog. Biophys. Mol. Biol. 74, 115-140 (2000).
[CrossRef] [PubMed]

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Figures (7)

Fig. 1
Fig. 1

Schematic diagrams of the illumination geometry for RICM: L, Hg Lamp; AD, aperture diaphragm; FD, field diaphragm; F1, incident bandpass filter; F2, imaging bandpass filter; P, parfocal lens; M, beam splitter; PI, piezo-objective positioner; Obj, objective lens. n o , n L , and n G are refractive indices of the object, water, and cover glass, respectively, and CCD represents a CCD camera with a 60 Hz frame rate. The inset shows the optical geometry of reflecting beams near the object. R is the radius of the spherical object and h is the separation distance from the water–cover glass interface.

Fig. 2
Fig. 2

(a) Schematic diagram of reference (left) and testing (right) objects. z t is the objective position when properly focused. (b) Image acquired near proper focus. The left and right interference images are formed from the reference and testing objects, respectively. The shadow image of the field diaphragm can be seen in the bottom-left corner.

Fig. 3
Fig. 3

(a) Radial intensity profiles I ( r ) calculated from interference images of the reference object in Fig. 2. The dashed box illustrates the region that is eventually used for the focus function. I ( r ) values correspond to 8 bit intensities per CCD pixel. (b) Focus functions calculated from the intensity profiles in (a); note that F r is calculated as a unitless relative intensity [Eq. (2)].

Fig. 4
Fig. 4

Comparison of optimal focus functions measured with different methods. F * ( z ) was calculated by use of either 100 nm objective steps (filled circles) or 10 nm steps (open circles). A cubic-spline interpolation (solid line) between the 100 nm steps matches the data taken using 10 nm steps.

Fig. 5
Fig. 5

Optimal focus function selected from those in Fig. 3b ( r = 7 ). z t was calculated by interpolating F * ( z ) at F target * .

Fig. 6
Fig. 6

Focal position versus time during feedback control. The standard deviation of the focal position from raw data acquired at 60 Hz was 9.8 nm .

Fig. 7
Fig. 7

(a) Change of the apparent separation distance h eff calculated from the testing object (a 4.5 μm bead). Raw data acquired at 60 Hz (the frame rate of the CCD camera) are shown; no filtering was applied to these traces. The feedback routine was turned off around t = 45   min . The inset shows a magnification of the boxed area. (b) The probability distribution of the change of h eff for 45 min. The distribution is well fit by a Gaussian function (solid line).

Equations (4)

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

I ( x , y ) = I 1 + I 2 + 2 I 1 I 2 cos [ k Δ ( x , y ) + ϕ ] ,
F r = f r / f r > > R ,
f r = I ( r 1 ) + I ( r ) + I ( r + 1 ) 3 ,
Λ = { α ( z c z t ) , | z c z t | < δ z max sign [ z c z t ] δ z max , | z c z t | δ z max ,

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