Abstract

Mechanical drift is a long-standing problem in optical microscopy that occurs in all three dimensions. This drift increasingly limits the resolution of advanced surface-coupled, single-molecule experiments. We overcame this drift and achieved atomic-scale stabilization (0.1  nm) of an optical microscope in 3D. This was accomplished by measuring the position of a fiducial mark coupled to the microscope cover slip using back-focal-plane (BFP) detection and correcting for the drift using a piezoelectric stage. Several significant factors contributed to this experimental realization, including (i) dramatically reducing the low frequency noise in BFP detection, (ii) increasing the sensitivity of BFP detection to vertical motion, and (iii) fabricating a regular array of nanometer-sized fiducial marks that were firmly coupled to the cover slip. With these improvements, we achieved short-term (1  s) stabilities of 0.11, 0.10, and 0.09  nm (rms) and long-term (100  s) stabilities of 0.17, 0.12, and 0.35  nm (rms) in x, y, and z, respectively, as measured by an independent detection laser.

© 2007 Optical Society of America

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  1. K. Svoboda, C. F. Schmidt, B. J. Schnapp, and S. M. Block, "Direct observation of kinesin stepping by optical trapping interferometry," Nature 365, 721-727 (1993).
    [CrossRef] [PubMed]
  2. A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization," Science 300, 2061-2065 (2003).
    [CrossRef] [PubMed]
  3. C. Kural, H. Kim, S. Syed, G. Goshima, V. I. Gelfand, and P. R. Selvin, "Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement?" Science 308, 1469-1472 (2005).
    [CrossRef] [PubMed]
  4. V. Westphal and S. W. Hell, "Nanoscale resolution in the focal plane of an optical microscope," Phys. Rev. Lett. 94, 143903 (2005).
    [CrossRef] [PubMed]
  5. J. T. Finer, R. M. Simmons, and J. A. Spudich, "Single myosin molecule mechanics: piconewton forces and nanometre steps," Nature 368, 113-119 (1994).
    [CrossRef] [PubMed]
  6. A. D. Mehta, R. S. Rock, M. Rief, J. A. Spudich, M. S. Mooseker, and R. E. Cheney, "Myosin-V is a processive actin-based motor," Nature 400, 590-593 (1999).
    [CrossRef] [PubMed]
  7. H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, and J. Gelles, "Transcription against an applied force," Science 270, 1653-1657 (1995).
    [CrossRef] [PubMed]
  8. L. Nugent-Glandorf and T. T. Perkins, "Measuring 0.1-nm motion in 1 ms in an optical microscope with differential back-focal-plane detection," Opt. Lett. 29, 2611-2613 (2004).
    [CrossRef] [PubMed]
  9. K. C. Neuman and S. M. Block, "Optical trapping," Rev. Sci. Instrum. 75, 2787-2809 (2004).
    [CrossRef]
  10. E. A. Abbondanzieri, W. J. Greenleaf, J. W. Shaevitz, R. Landick, and S. M. Block, "Direct observation of base-pair stepping by RNA polymerase," Nature 438, 460-465 (2005).
    [CrossRef] [PubMed]
  11. K. M. Herbert, A. La Porta, B. J. Wong, R. A. Mooney, K. C. Neuman, R. Landick, and S. M. Block, "Sequence-resolved detection of pausing by single RNA polymerase molecules," Cell 125, 1083-1094 (2006).
    [CrossRef] [PubMed]
  12. T. T. Perkins, H. W. Li, R. V. Dalal, J. Gelles, and S. M. Block, "Forward and reverse motion of single RecBCD molecules on DNA," Biophys. J. 86, 1640-1648 (2004).
    [CrossRef] [PubMed]
  13. M. D. Wang, M. J. Schnitzer, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Force and velocity measured for single molecules of RNA polymerase," Science 282, 902-907 (1998).
    [CrossRef] [PubMed]
  14. M. D. Wang, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Stretching DNA with optical tweezers," Biophys. J. 72, 1335-1346 (1997).
    [CrossRef] [PubMed]
  15. W. Steffen, D. Smith, R. Simmons, and J. Sleep, "Mapping the actin filament with myosin," Proc. Natl. Acad. Sci. U.S.A. 98, 14949-14954 (2001).
    [CrossRef] [PubMed]
  16. 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]
  17. K. Visscher, S. P. Gross, and S. M. Block, "Construction of multiple-beam optical traps with nanometer-resolution position sensing," IEEE J. Sel. Top. Quantum Electron. 2, 1066-1076 (1996).
    [CrossRef]
  18. F. Gittes and C. F. Schmidt, "Interference model for back-focal-plane displacement detection in optical tweezers," Opt. Lett. 23, 7-9 (1998).
    [CrossRef]
  19. W. Denk and W. W. Webb, "Optical measurement of picometer displacements of transparent microscopic objects," Appl. Opt. 29, 2382-2391 (1990).
    [CrossRef] [PubMed]
  20. A. Pralle, M. Prummer, E.-L. Florin, E. H. K. Stelzer, and J. K. H. Horber, "Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light," Microsc. Res. Tech. 44, 378-386 (1999).
    [CrossRef] [PubMed]
  21. We note that our differential BFP detection is immune to common mode fluctuations such as air currents and lens motion. However, a large fraction (40%) of the optical path is not common mode, and the common mode optical elements (excluding the objective) are rigidly attached to the microscope frame or the optical table by custom-made, large-diameter (>38 mm) aluminum posts. Vibrational testing suggests that the current limits in the mechanical stability of our system are the fiber launches and the QPDs, which are independent for each laser; therefore, the second, 850 nm laser represents an independent measurement.
  22. The laser diode was driven by custom electronics that stabilized the temperature to ±15 mK/°C ambient temperature variation. The current stability of the driver was 25 ppm/°C. The manufacturer's specification of the laser diode's spectral linewidth is ∼0.5 nm FWHM.
  23. Unexpectedly, our 850 nm laser performed 100-fold better than its intensity specification even after the PBS and did not require intensity stabilization (σI/I=3×10-5). Several identical lasers performed only 10% better than specification after the PBS. In general, intensity stabilization will be required to achieve 0.1 nm vertical resolution.
  24. K. C. Neuman, E. A. Abbondanzieri, and S. M. Block, "Measurement of the effective focal shift in an optical trap," Opt. Lett. 30, 1318-1320 (2005).
    [CrossRef] [PubMed]
  25. H. Namatsu, Y. Takahashi, K. Yamazaki, T. Yamaguchi, M. Nagase, and K. Kurihara, "Three-dimensional siloxane resist for the formation of nanopatterns with minimum linewidth fluctuations," J. Vac. Sci. Technol. B 16, 69-76 (1998).
    [CrossRef]
  26. K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, "Characterization of photodamage to escherichia coli in optical traps," Biophys. J. 77, 2856-2863 (1999).
    [CrossRef] [PubMed]
  27. Increasing the laser power created a drift in the positive z direction. Decreasing the laser power caused a negative z drift. This drift corresponded to a movement of the laser focus (set by the objective) relative to the fiducial mark (set by the sample). Furthermore, drift rates increased linearly with the change in laser power. Finally, after ∼15 min at a particular laser power the drift would settle, indicating a new equilibrium had been reached. Since all other optical components have >97% transmission at 1064 nm, these data are best explained by the thermal expansion (or contraction) of the objective as the main source of this drift since its transmission at 1064 nm is 59%.
  28. K. Svoboda and S. M. Block, "Force and velocity measured for single kinesin molecules," Cell 77, 773-784 (1994).
    [CrossRef] [PubMed]
  29. A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, "Kinesin walks hand-over-hand," Science 303, 676-678 (2004).
    [CrossRef]
  30. L. Finzi and J. Gelles, "Measurement of lactose repressor-mediated loop formation and breakdown in single DNA molecules," Science 267, 378-380 (1995).
    [CrossRef] [PubMed]
  31. M. E. J. Friese, H. Rubinsztein-Dunlop, N. R. Heckenberg, and E. W. Dearden, "Determination of the force constant of a single-beam gradient trap by measurement of backscattered light," Appl. Opt. 35, 7112-7116 (1996).
    [CrossRef] [PubMed]

2006 (1)

K. M. Herbert, A. La Porta, B. J. Wong, R. A. Mooney, K. C. Neuman, R. Landick, and S. M. Block, "Sequence-resolved detection of pausing by single RNA polymerase molecules," Cell 125, 1083-1094 (2006).
[CrossRef] [PubMed]

2005 (5)

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]

C. Kural, H. Kim, S. Syed, G. Goshima, V. I. Gelfand, and P. R. Selvin, "Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement?" Science 308, 1469-1472 (2005).
[CrossRef] [PubMed]

V. Westphal and S. W. Hell, "Nanoscale resolution in the focal plane of an optical microscope," Phys. Rev. Lett. 94, 143903 (2005).
[CrossRef] [PubMed]

E. A. Abbondanzieri, W. J. Greenleaf, J. W. Shaevitz, R. Landick, and S. M. Block, "Direct observation of base-pair stepping by RNA polymerase," Nature 438, 460-465 (2005).
[CrossRef] [PubMed]

K. C. Neuman, E. A. Abbondanzieri, and S. M. Block, "Measurement of the effective focal shift in an optical trap," Opt. Lett. 30, 1318-1320 (2005).
[CrossRef] [PubMed]

2004 (4)

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, "Kinesin walks hand-over-hand," Science 303, 676-678 (2004).
[CrossRef]

L. Nugent-Glandorf and T. T. Perkins, "Measuring 0.1-nm motion in 1 ms in an optical microscope with differential back-focal-plane detection," Opt. Lett. 29, 2611-2613 (2004).
[CrossRef] [PubMed]

K. C. Neuman and S. M. Block, "Optical trapping," Rev. Sci. Instrum. 75, 2787-2809 (2004).
[CrossRef]

T. T. Perkins, H. W. Li, R. V. Dalal, J. Gelles, and S. M. Block, "Forward and reverse motion of single RecBCD molecules on DNA," Biophys. J. 86, 1640-1648 (2004).
[CrossRef] [PubMed]

2003 (1)

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

2001 (1)

W. Steffen, D. Smith, R. Simmons, and J. Sleep, "Mapping the actin filament with myosin," Proc. Natl. Acad. Sci. U.S.A. 98, 14949-14954 (2001).
[CrossRef] [PubMed]

1999 (3)

A. D. Mehta, R. S. Rock, M. Rief, J. A. Spudich, M. S. Mooseker, and R. E. Cheney, "Myosin-V is a processive actin-based motor," Nature 400, 590-593 (1999).
[CrossRef] [PubMed]

A. Pralle, M. Prummer, E.-L. Florin, E. H. K. Stelzer, and J. K. H. Horber, "Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light," Microsc. Res. Tech. 44, 378-386 (1999).
[CrossRef] [PubMed]

K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, "Characterization of photodamage to escherichia coli in optical traps," Biophys. J. 77, 2856-2863 (1999).
[CrossRef] [PubMed]

1998 (3)

F. Gittes and C. F. Schmidt, "Interference model for back-focal-plane displacement detection in optical tweezers," Opt. Lett. 23, 7-9 (1998).
[CrossRef]

H. Namatsu, Y. Takahashi, K. Yamazaki, T. Yamaguchi, M. Nagase, and K. Kurihara, "Three-dimensional siloxane resist for the formation of nanopatterns with minimum linewidth fluctuations," J. Vac. Sci. Technol. B 16, 69-76 (1998).
[CrossRef]

M. D. Wang, M. J. Schnitzer, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Force and velocity measured for single molecules of RNA polymerase," Science 282, 902-907 (1998).
[CrossRef] [PubMed]

1997 (1)

M. D. Wang, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Stretching DNA with optical tweezers," Biophys. J. 72, 1335-1346 (1997).
[CrossRef] [PubMed]

1996 (2)

K. Visscher, S. P. Gross, and S. M. Block, "Construction of multiple-beam optical traps with nanometer-resolution position sensing," IEEE J. Sel. Top. Quantum Electron. 2, 1066-1076 (1996).
[CrossRef]

M. E. J. Friese, H. Rubinsztein-Dunlop, N. R. Heckenberg, and E. W. Dearden, "Determination of the force constant of a single-beam gradient trap by measurement of backscattered light," Appl. Opt. 35, 7112-7116 (1996).
[CrossRef] [PubMed]

1995 (2)

L. Finzi and J. Gelles, "Measurement of lactose repressor-mediated loop formation and breakdown in single DNA molecules," Science 267, 378-380 (1995).
[CrossRef] [PubMed]

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, and J. Gelles, "Transcription against an applied force," Science 270, 1653-1657 (1995).
[CrossRef] [PubMed]

1994 (2)

J. T. Finer, R. M. Simmons, and J. A. Spudich, "Single myosin molecule mechanics: piconewton forces and nanometre steps," Nature 368, 113-119 (1994).
[CrossRef] [PubMed]

K. Svoboda and S. M. Block, "Force and velocity measured for single kinesin molecules," Cell 77, 773-784 (1994).
[CrossRef] [PubMed]

1993 (1)

K. Svoboda, C. F. Schmidt, B. J. Schnapp, and S. M. Block, "Direct observation of kinesin stepping by optical trapping interferometry," Nature 365, 721-727 (1993).
[CrossRef] [PubMed]

1990 (1)

Abbondanzieri, E. A.

E. A. Abbondanzieri, W. J. Greenleaf, J. W. Shaevitz, R. Landick, and S. M. Block, "Direct observation of base-pair stepping by RNA polymerase," Nature 438, 460-465 (2005).
[CrossRef] [PubMed]

K. C. Neuman, E. A. Abbondanzieri, and S. M. Block, "Measurement of the effective focal shift in an optical trap," Opt. Lett. 30, 1318-1320 (2005).
[CrossRef] [PubMed]

Bergman, K.

K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, "Characterization of photodamage to escherichia coli in optical traps," Biophys. J. 77, 2856-2863 (1999).
[CrossRef] [PubMed]

Block, S. M.

K. M. Herbert, A. La Porta, B. J. Wong, R. A. Mooney, K. C. Neuman, R. Landick, and S. M. Block, "Sequence-resolved detection of pausing by single RNA polymerase molecules," Cell 125, 1083-1094 (2006).
[CrossRef] [PubMed]

K. C. Neuman, E. A. Abbondanzieri, and S. M. Block, "Measurement of the effective focal shift in an optical trap," Opt. Lett. 30, 1318-1320 (2005).
[CrossRef] [PubMed]

E. A. Abbondanzieri, W. J. Greenleaf, J. W. Shaevitz, R. Landick, and S. M. Block, "Direct observation of base-pair stepping by RNA polymerase," Nature 438, 460-465 (2005).
[CrossRef] [PubMed]

K. C. Neuman and S. M. Block, "Optical trapping," Rev. Sci. Instrum. 75, 2787-2809 (2004).
[CrossRef]

T. T. Perkins, H. W. Li, R. V. Dalal, J. Gelles, and S. M. Block, "Forward and reverse motion of single RecBCD molecules on DNA," Biophys. J. 86, 1640-1648 (2004).
[CrossRef] [PubMed]

K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, "Characterization of photodamage to escherichia coli in optical traps," Biophys. J. 77, 2856-2863 (1999).
[CrossRef] [PubMed]

M. D. Wang, M. J. Schnitzer, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Force and velocity measured for single molecules of RNA polymerase," Science 282, 902-907 (1998).
[CrossRef] [PubMed]

M. D. Wang, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Stretching DNA with optical tweezers," Biophys. J. 72, 1335-1346 (1997).
[CrossRef] [PubMed]

K. Visscher, S. P. Gross, and S. M. Block, "Construction of multiple-beam optical traps with nanometer-resolution position sensing," IEEE J. Sel. Top. Quantum Electron. 2, 1066-1076 (1996).
[CrossRef]

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, and J. Gelles, "Transcription against an applied force," Science 270, 1653-1657 (1995).
[CrossRef] [PubMed]

K. Svoboda and S. M. Block, "Force and velocity measured for single kinesin molecules," Cell 77, 773-784 (1994).
[CrossRef] [PubMed]

K. Svoboda, C. F. Schmidt, B. J. Schnapp, and S. M. Block, "Direct observation of kinesin stepping by optical trapping interferometry," Nature 365, 721-727 (1993).
[CrossRef] [PubMed]

Capitanio, M.

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]

Chadd, E. H.

K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, "Characterization of photodamage to escherichia coli in optical traps," Biophys. J. 77, 2856-2863 (1999).
[CrossRef] [PubMed]

Cheney, R. E.

A. D. Mehta, R. S. Rock, M. Rief, J. A. Spudich, M. S. Mooseker, and R. E. Cheney, "Myosin-V is a processive actin-based motor," Nature 400, 590-593 (1999).
[CrossRef] [PubMed]

Cicchi, R.

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]

Dalal, R. V.

T. T. Perkins, H. W. Li, R. V. Dalal, J. Gelles, and S. M. Block, "Forward and reverse motion of single RecBCD molecules on DNA," Biophys. J. 86, 1640-1648 (2004).
[CrossRef] [PubMed]

Dearden, E. W.

Denk, W.

Finer, J. T.

J. T. Finer, R. M. Simmons, and J. A. Spudich, "Single myosin molecule mechanics: piconewton forces and nanometre steps," Nature 368, 113-119 (1994).
[CrossRef] [PubMed]

Finzi, L.

L. Finzi and J. Gelles, "Measurement of lactose repressor-mediated loop formation and breakdown in single DNA molecules," Science 267, 378-380 (1995).
[CrossRef] [PubMed]

Florin, E.-L.

A. Pralle, M. Prummer, E.-L. Florin, E. H. K. Stelzer, and J. K. H. Horber, "Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light," Microsc. Res. Tech. 44, 378-386 (1999).
[CrossRef] [PubMed]

Forkey, J. N.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

Friese, M. E. J.

Gelfand, V. I.

C. Kural, H. Kim, S. Syed, G. Goshima, V. I. Gelfand, and P. R. Selvin, "Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement?" Science 308, 1469-1472 (2005).
[CrossRef] [PubMed]

Gelles, J.

T. T. Perkins, H. W. Li, R. V. Dalal, J. Gelles, and S. M. Block, "Forward and reverse motion of single RecBCD molecules on DNA," Biophys. J. 86, 1640-1648 (2004).
[CrossRef] [PubMed]

M. D. Wang, M. J. Schnitzer, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Force and velocity measured for single molecules of RNA polymerase," Science 282, 902-907 (1998).
[CrossRef] [PubMed]

M. D. Wang, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Stretching DNA with optical tweezers," Biophys. J. 72, 1335-1346 (1997).
[CrossRef] [PubMed]

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, and J. Gelles, "Transcription against an applied force," Science 270, 1653-1657 (1995).
[CrossRef] [PubMed]

L. Finzi and J. Gelles, "Measurement of lactose repressor-mediated loop formation and breakdown in single DNA molecules," Science 267, 378-380 (1995).
[CrossRef] [PubMed]

Gittes, F.

Goldman, Y. E.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

Goshima, G.

C. Kural, H. Kim, S. Syed, G. Goshima, V. I. Gelfand, and P. R. Selvin, "Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement?" Science 308, 1469-1472 (2005).
[CrossRef] [PubMed]

Greenleaf, W. J.

E. A. Abbondanzieri, W. J. Greenleaf, J. W. Shaevitz, R. Landick, and S. M. Block, "Direct observation of base-pair stepping by RNA polymerase," Nature 438, 460-465 (2005).
[CrossRef] [PubMed]

Gross, S. P.

K. Visscher, S. P. Gross, and S. M. Block, "Construction of multiple-beam optical traps with nanometer-resolution position sensing," IEEE J. Sel. Top. Quantum Electron. 2, 1066-1076 (1996).
[CrossRef]

Ha, T.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

Heckenberg, N. R.

Hell, S. W.

V. Westphal and S. W. Hell, "Nanoscale resolution in the focal plane of an optical microscope," Phys. Rev. Lett. 94, 143903 (2005).
[CrossRef] [PubMed]

Herbert, K. M.

K. M. Herbert, A. La Porta, B. J. Wong, R. A. Mooney, K. C. Neuman, R. Landick, and S. M. Block, "Sequence-resolved detection of pausing by single RNA polymerase molecules," Cell 125, 1083-1094 (2006).
[CrossRef] [PubMed]

Horber, J. K. H.

A. Pralle, M. Prummer, E.-L. Florin, E. H. K. Stelzer, and J. K. H. Horber, "Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light," Microsc. Res. Tech. 44, 378-386 (1999).
[CrossRef] [PubMed]

Kim, H.

C. Kural, H. Kim, S. Syed, G. Goshima, V. I. Gelfand, and P. R. Selvin, "Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement?" Science 308, 1469-1472 (2005).
[CrossRef] [PubMed]

Kural, C.

C. Kural, H. Kim, S. Syed, G. Goshima, V. I. Gelfand, and P. R. Selvin, "Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement?" Science 308, 1469-1472 (2005).
[CrossRef] [PubMed]

Kurihara, K.

H. Namatsu, Y. Takahashi, K. Yamazaki, T. Yamaguchi, M. Nagase, and K. Kurihara, "Three-dimensional siloxane resist for the formation of nanopatterns with minimum linewidth fluctuations," J. Vac. Sci. Technol. B 16, 69-76 (1998).
[CrossRef]

La Porta, A.

K. M. Herbert, A. La Porta, B. J. Wong, R. A. Mooney, K. C. Neuman, R. Landick, and S. M. Block, "Sequence-resolved detection of pausing by single RNA polymerase molecules," Cell 125, 1083-1094 (2006).
[CrossRef] [PubMed]

Landick, R.

K. M. Herbert, A. La Porta, B. J. Wong, R. A. Mooney, K. C. Neuman, R. Landick, and S. M. Block, "Sequence-resolved detection of pausing by single RNA polymerase molecules," Cell 125, 1083-1094 (2006).
[CrossRef] [PubMed]

E. A. Abbondanzieri, W. J. Greenleaf, J. W. Shaevitz, R. Landick, and S. M. Block, "Direct observation of base-pair stepping by RNA polymerase," Nature 438, 460-465 (2005).
[CrossRef] [PubMed]

M. D. Wang, M. J. Schnitzer, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Force and velocity measured for single molecules of RNA polymerase," Science 282, 902-907 (1998).
[CrossRef] [PubMed]

M. D. Wang, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Stretching DNA with optical tweezers," Biophys. J. 72, 1335-1346 (1997).
[CrossRef] [PubMed]

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, and J. Gelles, "Transcription against an applied force," Science 270, 1653-1657 (1995).
[CrossRef] [PubMed]

Li, H. W.

T. T. Perkins, H. W. Li, R. V. Dalal, J. Gelles, and S. M. Block, "Forward and reverse motion of single RecBCD molecules on DNA," Biophys. J. 86, 1640-1648 (2004).
[CrossRef] [PubMed]

Liou, G. F.

K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, "Characterization of photodamage to escherichia coli in optical traps," Biophys. J. 77, 2856-2863 (1999).
[CrossRef] [PubMed]

McKinney, S. A.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

Mehta, A. D.

A. D. Mehta, R. S. Rock, M. Rief, J. A. Spudich, M. S. Mooseker, and R. E. Cheney, "Myosin-V is a processive actin-based motor," Nature 400, 590-593 (1999).
[CrossRef] [PubMed]

Mooney, R. A.

K. M. Herbert, A. La Porta, B. J. Wong, R. A. Mooney, K. C. Neuman, R. Landick, and S. M. Block, "Sequence-resolved detection of pausing by single RNA polymerase molecules," Cell 125, 1083-1094 (2006).
[CrossRef] [PubMed]

Mooseker, M. S.

A. D. Mehta, R. S. Rock, M. Rief, J. A. Spudich, M. S. Mooseker, and R. E. Cheney, "Myosin-V is a processive actin-based motor," Nature 400, 590-593 (1999).
[CrossRef] [PubMed]

Nagase, M.

H. Namatsu, Y. Takahashi, K. Yamazaki, T. Yamaguchi, M. Nagase, and K. Kurihara, "Three-dimensional siloxane resist for the formation of nanopatterns with minimum linewidth fluctuations," J. Vac. Sci. Technol. B 16, 69-76 (1998).
[CrossRef]

Namatsu, H.

H. Namatsu, Y. Takahashi, K. Yamazaki, T. Yamaguchi, M. Nagase, and K. Kurihara, "Three-dimensional siloxane resist for the formation of nanopatterns with minimum linewidth fluctuations," J. Vac. Sci. Technol. B 16, 69-76 (1998).
[CrossRef]

Neuman, K. C.

K. M. Herbert, A. La Porta, B. J. Wong, R. A. Mooney, K. C. Neuman, R. Landick, and S. M. Block, "Sequence-resolved detection of pausing by single RNA polymerase molecules," Cell 125, 1083-1094 (2006).
[CrossRef] [PubMed]

K. C. Neuman, E. A. Abbondanzieri, and S. M. Block, "Measurement of the effective focal shift in an optical trap," Opt. Lett. 30, 1318-1320 (2005).
[CrossRef] [PubMed]

K. C. Neuman and S. M. Block, "Optical trapping," Rev. Sci. Instrum. 75, 2787-2809 (2004).
[CrossRef]

K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, "Characterization of photodamage to escherichia coli in optical traps," Biophys. J. 77, 2856-2863 (1999).
[CrossRef] [PubMed]

Nugent-Glandorf, L.

Pavone, F. S.

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]

Perkins, T. T.

L. Nugent-Glandorf and T. T. Perkins, "Measuring 0.1-nm motion in 1 ms in an optical microscope with differential back-focal-plane detection," Opt. Lett. 29, 2611-2613 (2004).
[CrossRef] [PubMed]

T. T. Perkins, H. W. Li, R. V. Dalal, J. Gelles, and S. M. Block, "Forward and reverse motion of single RecBCD molecules on DNA," Biophys. J. 86, 1640-1648 (2004).
[CrossRef] [PubMed]

Pralle, A.

A. Pralle, M. Prummer, E.-L. Florin, E. H. K. Stelzer, and J. K. H. Horber, "Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light," Microsc. Res. Tech. 44, 378-386 (1999).
[CrossRef] [PubMed]

Prummer, M.

A. Pralle, M. Prummer, E.-L. Florin, E. H. K. Stelzer, and J. K. H. Horber, "Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light," Microsc. Res. Tech. 44, 378-386 (1999).
[CrossRef] [PubMed]

Rief, M.

A. D. Mehta, R. S. Rock, M. Rief, J. A. Spudich, M. S. Mooseker, and R. E. Cheney, "Myosin-V is a processive actin-based motor," Nature 400, 590-593 (1999).
[CrossRef] [PubMed]

Rock, R. S.

A. D. Mehta, R. S. Rock, M. Rief, J. A. Spudich, M. S. Mooseker, and R. E. Cheney, "Myosin-V is a processive actin-based motor," Nature 400, 590-593 (1999).
[CrossRef] [PubMed]

Rubinsztein-Dunlop, H.

Schmidt, C. F.

F. Gittes and C. F. Schmidt, "Interference model for back-focal-plane displacement detection in optical tweezers," Opt. Lett. 23, 7-9 (1998).
[CrossRef]

K. Svoboda, C. F. Schmidt, B. J. Schnapp, and S. M. Block, "Direct observation of kinesin stepping by optical trapping interferometry," Nature 365, 721-727 (1993).
[CrossRef] [PubMed]

Schnapp, B. J.

K. Svoboda, C. F. Schmidt, B. J. Schnapp, and S. M. Block, "Direct observation of kinesin stepping by optical trapping interferometry," Nature 365, 721-727 (1993).
[CrossRef] [PubMed]

Schnitzer, M. J.

M. D. Wang, M. J. Schnitzer, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Force and velocity measured for single molecules of RNA polymerase," Science 282, 902-907 (1998).
[CrossRef] [PubMed]

Selvin, P. R.

C. Kural, H. Kim, S. Syed, G. Goshima, V. I. Gelfand, and P. R. Selvin, "Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement?" Science 308, 1469-1472 (2005).
[CrossRef] [PubMed]

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, "Kinesin walks hand-over-hand," Science 303, 676-678 (2004).
[CrossRef]

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

Shaevitz, J. W.

E. A. Abbondanzieri, W. J. Greenleaf, J. W. Shaevitz, R. Landick, and S. M. Block, "Direct observation of base-pair stepping by RNA polymerase," Nature 438, 460-465 (2005).
[CrossRef] [PubMed]

Simmons, R.

W. Steffen, D. Smith, R. Simmons, and J. Sleep, "Mapping the actin filament with myosin," Proc. Natl. Acad. Sci. U.S.A. 98, 14949-14954 (2001).
[CrossRef] [PubMed]

Simmons, R. M.

J. T. Finer, R. M. Simmons, and J. A. Spudich, "Single myosin molecule mechanics: piconewton forces and nanometre steps," Nature 368, 113-119 (1994).
[CrossRef] [PubMed]

Sleep, J.

W. Steffen, D. Smith, R. Simmons, and J. Sleep, "Mapping the actin filament with myosin," Proc. Natl. Acad. Sci. U.S.A. 98, 14949-14954 (2001).
[CrossRef] [PubMed]

Smith, D.

W. Steffen, D. Smith, R. Simmons, and J. Sleep, "Mapping the actin filament with myosin," Proc. Natl. Acad. Sci. U.S.A. 98, 14949-14954 (2001).
[CrossRef] [PubMed]

Spudich, J. A.

A. D. Mehta, R. S. Rock, M. Rief, J. A. Spudich, M. S. Mooseker, and R. E. Cheney, "Myosin-V is a processive actin-based motor," Nature 400, 590-593 (1999).
[CrossRef] [PubMed]

J. T. Finer, R. M. Simmons, and J. A. Spudich, "Single myosin molecule mechanics: piconewton forces and nanometre steps," Nature 368, 113-119 (1994).
[CrossRef] [PubMed]

Steffen, W.

W. Steffen, D. Smith, R. Simmons, and J. Sleep, "Mapping the actin filament with myosin," Proc. Natl. Acad. Sci. U.S.A. 98, 14949-14954 (2001).
[CrossRef] [PubMed]

Stelzer, E. H. K.

A. Pralle, M. Prummer, E.-L. Florin, E. H. K. Stelzer, and J. K. H. Horber, "Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light," Microsc. Res. Tech. 44, 378-386 (1999).
[CrossRef] [PubMed]

Svoboda, K.

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, and J. Gelles, "Transcription against an applied force," Science 270, 1653-1657 (1995).
[CrossRef] [PubMed]

K. Svoboda and S. M. Block, "Force and velocity measured for single kinesin molecules," Cell 77, 773-784 (1994).
[CrossRef] [PubMed]

K. Svoboda, C. F. Schmidt, B. J. Schnapp, and S. M. Block, "Direct observation of kinesin stepping by optical trapping interferometry," Nature 365, 721-727 (1993).
[CrossRef] [PubMed]

Syed, S.

C. Kural, H. Kim, S. Syed, G. Goshima, V. I. Gelfand, and P. R. Selvin, "Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement?" Science 308, 1469-1472 (2005).
[CrossRef] [PubMed]

Takahashi, Y.

H. Namatsu, Y. Takahashi, K. Yamazaki, T. Yamaguchi, M. Nagase, and K. Kurihara, "Three-dimensional siloxane resist for the formation of nanopatterns with minimum linewidth fluctuations," J. Vac. Sci. Technol. B 16, 69-76 (1998).
[CrossRef]

Tomishige, M.

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, "Kinesin walks hand-over-hand," Science 303, 676-678 (2004).
[CrossRef]

Vale, R. D.

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, "Kinesin walks hand-over-hand," Science 303, 676-678 (2004).
[CrossRef]

Visscher, K.

K. Visscher, S. P. Gross, and S. M. Block, "Construction of multiple-beam optical traps with nanometer-resolution position sensing," IEEE J. Sel. Top. Quantum Electron. 2, 1066-1076 (1996).
[CrossRef]

Wang, M. D.

M. D. Wang, M. J. Schnitzer, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Force and velocity measured for single molecules of RNA polymerase," Science 282, 902-907 (1998).
[CrossRef] [PubMed]

M. D. Wang, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Stretching DNA with optical tweezers," Biophys. J. 72, 1335-1346 (1997).
[CrossRef] [PubMed]

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, and J. Gelles, "Transcription against an applied force," Science 270, 1653-1657 (1995).
[CrossRef] [PubMed]

Webb, W. W.

Westphal, V.

V. Westphal and S. W. Hell, "Nanoscale resolution in the focal plane of an optical microscope," Phys. Rev. Lett. 94, 143903 (2005).
[CrossRef] [PubMed]

Wong, B. J.

K. M. Herbert, A. La Porta, B. J. Wong, R. A. Mooney, K. C. Neuman, R. Landick, and S. M. Block, "Sequence-resolved detection of pausing by single RNA polymerase molecules," Cell 125, 1083-1094 (2006).
[CrossRef] [PubMed]

Yamaguchi, T.

H. Namatsu, Y. Takahashi, K. Yamazaki, T. Yamaguchi, M. Nagase, and K. Kurihara, "Three-dimensional siloxane resist for the formation of nanopatterns with minimum linewidth fluctuations," J. Vac. Sci. Technol. B 16, 69-76 (1998).
[CrossRef]

Yamazaki, K.

H. Namatsu, Y. Takahashi, K. Yamazaki, T. Yamaguchi, M. Nagase, and K. Kurihara, "Three-dimensional siloxane resist for the formation of nanopatterns with minimum linewidth fluctuations," J. Vac. Sci. Technol. B 16, 69-76 (1998).
[CrossRef]

Yildiz, A.

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, "Kinesin walks hand-over-hand," Science 303, 676-678 (2004).
[CrossRef]

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

Yin, H.

M. D. Wang, M. J. Schnitzer, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Force and velocity measured for single molecules of RNA polymerase," Science 282, 902-907 (1998).
[CrossRef] [PubMed]

M. D. Wang, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Stretching DNA with optical tweezers," Biophys. J. 72, 1335-1346 (1997).
[CrossRef] [PubMed]

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, and J. Gelles, "Transcription against an applied force," Science 270, 1653-1657 (1995).
[CrossRef] [PubMed]

Appl. Opt. (2)

Biophys. J. (3)

K. C. Neuman, E. H. Chadd, G. F. Liou, K. Bergman, and S. M. Block, "Characterization of photodamage to escherichia coli in optical traps," Biophys. J. 77, 2856-2863 (1999).
[CrossRef] [PubMed]

T. T. Perkins, H. W. Li, R. V. Dalal, J. Gelles, and S. M. Block, "Forward and reverse motion of single RecBCD molecules on DNA," Biophys. J. 86, 1640-1648 (2004).
[CrossRef] [PubMed]

M. D. Wang, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Stretching DNA with optical tweezers," Biophys. J. 72, 1335-1346 (1997).
[CrossRef] [PubMed]

Cell (2)

K. M. Herbert, A. La Porta, B. J. Wong, R. A. Mooney, K. C. Neuman, R. Landick, and S. M. Block, "Sequence-resolved detection of pausing by single RNA polymerase molecules," Cell 125, 1083-1094 (2006).
[CrossRef] [PubMed]

K. Svoboda and S. M. Block, "Force and velocity measured for single kinesin molecules," Cell 77, 773-784 (1994).
[CrossRef] [PubMed]

Eur. Phys. J. B (1)

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]

IEEE J. Sel. Top. Quantum Electron. (1)

K. Visscher, S. P. Gross, and S. M. Block, "Construction of multiple-beam optical traps with nanometer-resolution position sensing," IEEE J. Sel. Top. Quantum Electron. 2, 1066-1076 (1996).
[CrossRef]

J. Vac. Sci. Technol. B (1)

H. Namatsu, Y. Takahashi, K. Yamazaki, T. Yamaguchi, M. Nagase, and K. Kurihara, "Three-dimensional siloxane resist for the formation of nanopatterns with minimum linewidth fluctuations," J. Vac. Sci. Technol. B 16, 69-76 (1998).
[CrossRef]

Microsc. Res. Tech. (1)

A. Pralle, M. Prummer, E.-L. Florin, E. H. K. Stelzer, and J. K. H. Horber, "Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light," Microsc. Res. Tech. 44, 378-386 (1999).
[CrossRef] [PubMed]

Nature (4)

E. A. Abbondanzieri, W. J. Greenleaf, J. W. Shaevitz, R. Landick, and S. M. Block, "Direct observation of base-pair stepping by RNA polymerase," Nature 438, 460-465 (2005).
[CrossRef] [PubMed]

K. Svoboda, C. F. Schmidt, B. J. Schnapp, and S. M. Block, "Direct observation of kinesin stepping by optical trapping interferometry," Nature 365, 721-727 (1993).
[CrossRef] [PubMed]

J. T. Finer, R. M. Simmons, and J. A. Spudich, "Single myosin molecule mechanics: piconewton forces and nanometre steps," Nature 368, 113-119 (1994).
[CrossRef] [PubMed]

A. D. Mehta, R. S. Rock, M. Rief, J. A. Spudich, M. S. Mooseker, and R. E. Cheney, "Myosin-V is a processive actin-based motor," Nature 400, 590-593 (1999).
[CrossRef] [PubMed]

Opt. Lett. (3)

Phys. Rev. Lett. (1)

V. Westphal and S. W. Hell, "Nanoscale resolution in the focal plane of an optical microscope," Phys. Rev. Lett. 94, 143903 (2005).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (1)

W. Steffen, D. Smith, R. Simmons, and J. Sleep, "Mapping the actin filament with myosin," Proc. Natl. Acad. Sci. U.S.A. 98, 14949-14954 (2001).
[CrossRef] [PubMed]

Rev. Sci. Instrum. (1)

K. C. Neuman and S. M. Block, "Optical trapping," Rev. Sci. Instrum. 75, 2787-2809 (2004).
[CrossRef]

Science (6)

H. Yin, M. D. Wang, K. Svoboda, R. Landick, S. M. Block, and J. Gelles, "Transcription against an applied force," Science 270, 1653-1657 (1995).
[CrossRef] [PubMed]

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, "Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization," Science 300, 2061-2065 (2003).
[CrossRef] [PubMed]

C. Kural, H. Kim, S. Syed, G. Goshima, V. I. Gelfand, and P. R. Selvin, "Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement?" Science 308, 1469-1472 (2005).
[CrossRef] [PubMed]

M. D. Wang, M. J. Schnitzer, H. Yin, R. Landick, J. Gelles, and S. M. Block, "Force and velocity measured for single molecules of RNA polymerase," Science 282, 902-907 (1998).
[CrossRef] [PubMed]

A. Yildiz, M. Tomishige, R. D. Vale, and P. R. Selvin, "Kinesin walks hand-over-hand," Science 303, 676-678 (2004).
[CrossRef]

L. Finzi and J. Gelles, "Measurement of lactose repressor-mediated loop formation and breakdown in single DNA molecules," Science 267, 378-380 (1995).
[CrossRef] [PubMed]

Other (4)

Increasing the laser power created a drift in the positive z direction. Decreasing the laser power caused a negative z drift. This drift corresponded to a movement of the laser focus (set by the objective) relative to the fiducial mark (set by the sample). Furthermore, drift rates increased linearly with the change in laser power. Finally, after ∼15 min at a particular laser power the drift would settle, indicating a new equilibrium had been reached. Since all other optical components have >97% transmission at 1064 nm, these data are best explained by the thermal expansion (or contraction) of the objective as the main source of this drift since its transmission at 1064 nm is 59%.

We note that our differential BFP detection is immune to common mode fluctuations such as air currents and lens motion. However, a large fraction (40%) of the optical path is not common mode, and the common mode optical elements (excluding the objective) are rigidly attached to the microscope frame or the optical table by custom-made, large-diameter (>38 mm) aluminum posts. Vibrational testing suggests that the current limits in the mechanical stability of our system are the fiber launches and the QPDs, which are independent for each laser; therefore, the second, 850 nm laser represents an independent measurement.

The laser diode was driven by custom electronics that stabilized the temperature to ±15 mK/°C ambient temperature variation. The current stability of the driver was 25 ppm/°C. The manufacturer's specification of the laser diode's spectral linewidth is ∼0.5 nm FWHM.

Unexpectedly, our 850 nm laser performed 100-fold better than its intensity specification even after the PBS and did not require intensity stabilization (σI/I=3×10-5). Several identical lasers performed only 10% better than specification after the PBS. In general, intensity stabilization will be required to achieve 0.1 nm vertical resolution.

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Figures (6)

Fig. 1
Fig. 1

(a) One application of 3D stabilization is optical-trapping experiments where both horizontal and vertical drift are important. The bead is trapped in a focused laser beam (red dashed curve) and its position is detected by a laser (red). A second laser (blue) measures the fiducial mark position, shown here as a post. (b) Schematic of the 3D stabilization system: QPD, quadrant photodiode; CCD, charge-coupled device; PZT Mirror, closed-loop lead-zirconate-titanate mirror. The blue-shaded components are in optically conjugate planes.

Fig. 2
Fig. 2

Low-frequency noise reduction. (a) Optical layout of the intensity feedback loop: OI, optical isolator; PD, photodiode; BS, beam sampler; PBS, polarizing beam splitter; AOM, acousto-optic modulator. (b) Laser intensity prior to stabilization (gray) and after stabilization (black). Data were boxcar averaged to 100   Hz .

Fig. 3
Fig. 3

Vertical BFP detection enhancement. (a) Height calibration signals for an affixed bead that was scanned vertically through the detection laser. The traces represent the inherent signal (purple), offset-amplified signal (black), offset-amplified signal with intensity servo active (blue). (b) Differential BFP detection measurements of vertical motion ( z d i f = z 785 z 850 ) using two lasers and one affixed bead as a fiducial mark. Traces represent the inherent signal (purple), the intensity-stabilized signal (gray), and the offset-amplified signal with intensity servo active (blue). Traces displaced vertically for clarity.

Fig. 4
Fig. 4

Fiducial mark movement. (a) Differential BFP detection in x ( x d i f = x 785 x 850 , green) and y ( y d i f = y 785 y 850 , red) using both lasers to measure the position of one affixed bead as a fiducial mark (inset). (b) Differential BFP detection as in (a) except that the lasers measure the position of different affixed beads (inset). The displayed trace reflects the median short-term noise observed in ten trials. (c) Differential BFP detection as in (a) except that the lasers measure the position of two nanofabricated posts (inset). Traces displaced vertically for clarity.

Fig. 5
Fig. 5

Fiducial mark signals. (a) Horizontal response for posts of varying size. Posts were a constant height h = 600   nm with widths (w) of 350   nm (cyan), 550   nm (green), or 725   nm (red). Inset, geometry of a post. (b) Horizontal response for holes of varying size. Holes were a constant depth d 500   nm with initial widths (w) of 100   nm (cyan), 200   nm (green), or 300   nm (red). Inset, geometry of a hole.

Fig. 6
Fig. 6

Active stabilization. (a) Post position measured by the independent 850   nm detection beam, x 850 (green), y 850 , (red), and z 850 (blue). (b) As in (a), independent position records with active stabilization before and during heating. At 40   s , the objective was heated, leading to significant vertical drift that was compensated for by moving the PZT stage (e.g., z st , purple, vertical stage position). Traces displaced vertically for clarity. Inset, atomic force microscope image of a post with a height of 600   nm .

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