Abstract

We present a novel calibration technique for determining the shear distance of a Nomarski Differential Interference Contrast prism, which is used in Differential Interference Contrast microscopy as well as for the recently developed dual-focus fluorescence correlation spectroscopy. In both applications, an exact knowledge of the shear distance induced by the Nomarski prism is important for a quantitative data evaluation. In Differential Interference Contrast microscopy, the shear distance determines the spatial resolution of imaging, in dual-focus fluorescence correlation spectroscopy, it represents the extrinsic length scale for determining diffusion coefficients. The presented calibration technique is itself based on a combination of fluorescence correlation spectroscopy and dynamic light scattering. The method is easy to implement and allows for determining the shear distance with nanometer accuracy.

© 2008 Optical Society of America

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References

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  1. D. Murphy, "Differential interference contrast (DIC) microscopy and modulation contrast microscopy," in Fundamentals of Light Microscopy and Digital Imaging (Wiley-Liss, 2001), pp. 153-168.
  2. E. Salmon and P. Tran, "High-resolution video-enhanced differential interference contrast (VE-DIC) light microscope," in Video Microscopy, G. Sluder, and D. Wolf, eds., (Academic Press, 1998), pp. 153-184.
  3. G. Nomarski, "Dispositif Oculaire a Contraste De Phase Pour Microscope," J. Phys. Radium 11, 9-10 (1950).
  4. G. Nomarski, "Interference Microscopy - State of Art and Its Future," J. Opt. Soc. Am. 60, 1575-1575 (1970).
  5. G. Nomarski, "Double-Point-Reference Interference Comparator (DPRIC)," J. Opt. Soc. Am. 61, 1560-1560 (1971).
  6. D. Lessor, J. Hartman, and R. Gordon, "Quantitative Surface Topography Determination by Nomarski Reflection Microscopy. I. Theory," J. Opt. Soc. Am. 69, 357-366 (1979).
    [CrossRef]
  7. T. Holmes and W. Levy, "Signal-Processing Characteristics of Differential Interference Contrast Microscopy," Appl. Opt. 26, 3929-3939 (1987).
    [CrossRef] [PubMed]
  8. C. J. Cogswell and C. Sheppard, "Confocal Differential Interference Contrast (DIC) Microscopy: including a Theoretical Analysis of Conventional and Confocal DIC Imaging," J. Microsc. 165, 81-101 (1992).
    [CrossRef]
  9. C. Preza, D. Snyder, and J. Conchello, "Theoretical development and experimental evaluation of imaging models for Differential-Interference-Contrast Microscopy," J. Opt. Soc. Am. A 16, 2185-2199 (1999).
    [CrossRef]
  10. P. R. T. Munro and P. Török, "Vectorial, High Numerical Aperture Study of Nomarski's Differential Interference Contrast Microscope," Opt. Express 13, 6833-6847 (2005).
    [CrossRef] [PubMed]
  11. D. Magde, W. W. Webb, and E. Elson, "Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
    [CrossRef]
  12. J. Enderlein, I. Gregor, D. Patra, and J. Fitter, "Art and artefacts of fluorescence correlation spectroscopy," Curr. Pharm. Biotechnol. 5, 155-161 (2004).
    [CrossRef] [PubMed]
  13. J. Enderlein, I. Gregor, D. Patra, T. Dertinger, and U. B. Kaupp, "Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration," ChemPhysChem 6, 2324-2336 (2005).
    [CrossRef] [PubMed]
  14. T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements," Chem. PhysChem. 8, 433-443 (2007).
    [CrossRef] [PubMed]
  15. J. Enderlein and I. Gregor, "Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy," Rev. Sci. Instrum. 76, 033102 (2005).
    [CrossRef]
  16. M. Böhmer, F. Pampaloni, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, "Time-resolved confocal scanning device for ultrasensitive fluorescence detection," Rev. Sci. Instrum. 72, 4145-4152 (2001).
    [CrossRef]
  17. B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, "Pulsed interleaved excitation," Biophys. J. 89, 3508-3522 (2005).
    [CrossRef] [PubMed]
  18. D. V. O'Connor and D. Phillips, Time-correlated Single Photon Counting (Academic Press, 1984).
  19. M. Wahl, I. Gregor, M. Patting, and J. Enderlein, "Fast calculation of fluorescence correlation data with asynchronous time-correlated single-photon counting," Opt. Express 11, 3583-3591 (2003).
    [CrossRef] [PubMed]

2007 (1)

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements," Chem. PhysChem. 8, 433-443 (2007).
[CrossRef] [PubMed]

2005 (4)

J. Enderlein and I. Gregor, "Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy," Rev. Sci. Instrum. 76, 033102 (2005).
[CrossRef]

B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, "Pulsed interleaved excitation," Biophys. J. 89, 3508-3522 (2005).
[CrossRef] [PubMed]

J. Enderlein, I. Gregor, D. Patra, T. Dertinger, and U. B. Kaupp, "Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration," ChemPhysChem 6, 2324-2336 (2005).
[CrossRef] [PubMed]

P. R. T. Munro and P. Török, "Vectorial, High Numerical Aperture Study of Nomarski's Differential Interference Contrast Microscope," Opt. Express 13, 6833-6847 (2005).
[CrossRef] [PubMed]

2004 (1)

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, "Art and artefacts of fluorescence correlation spectroscopy," Curr. Pharm. Biotechnol. 5, 155-161 (2004).
[CrossRef] [PubMed]

2003 (1)

2001 (1)

M. Böhmer, F. Pampaloni, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, "Time-resolved confocal scanning device for ultrasensitive fluorescence detection," Rev. Sci. Instrum. 72, 4145-4152 (2001).
[CrossRef]

1999 (1)

1992 (1)

C. J. Cogswell and C. Sheppard, "Confocal Differential Interference Contrast (DIC) Microscopy: including a Theoretical Analysis of Conventional and Confocal DIC Imaging," J. Microsc. 165, 81-101 (1992).
[CrossRef]

1987 (1)

1979 (1)

1972 (1)

D. Magde, W. W. Webb, and E. Elson, "Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

1971 (1)

G. Nomarski, "Double-Point-Reference Interference Comparator (DPRIC)," J. Opt. Soc. Am. 61, 1560-1560 (1971).

1970 (1)

G. Nomarski, "Interference Microscopy - State of Art and Its Future," J. Opt. Soc. Am. 60, 1575-1575 (1970).

1950 (1)

G. Nomarski, "Dispositif Oculaire a Contraste De Phase Pour Microscope," J. Phys. Radium 11, 9-10 (1950).

Böhmer, M.

M. Böhmer, F. Pampaloni, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, "Time-resolved confocal scanning device for ultrasensitive fluorescence detection," Rev. Sci. Instrum. 72, 4145-4152 (2001).
[CrossRef]

Bräuchle, C.

B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, "Pulsed interleaved excitation," Biophys. J. 89, 3508-3522 (2005).
[CrossRef] [PubMed]

Cogswell, C. J.

C. J. Cogswell and C. Sheppard, "Confocal Differential Interference Contrast (DIC) Microscopy: including a Theoretical Analysis of Conventional and Confocal DIC Imaging," J. Microsc. 165, 81-101 (1992).
[CrossRef]

Conchello, J.

Dertinger, T.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements," Chem. PhysChem. 8, 433-443 (2007).
[CrossRef] [PubMed]

J. Enderlein, I. Gregor, D. Patra, T. Dertinger, and U. B. Kaupp, "Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration," ChemPhysChem 6, 2324-2336 (2005).
[CrossRef] [PubMed]

Elson, E.

D. Magde, W. W. Webb, and E. Elson, "Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

Enderlein, J.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements," Chem. PhysChem. 8, 433-443 (2007).
[CrossRef] [PubMed]

J. Enderlein and I. Gregor, "Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy," Rev. Sci. Instrum. 76, 033102 (2005).
[CrossRef]

J. Enderlein, I. Gregor, D. Patra, T. Dertinger, and U. B. Kaupp, "Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration," ChemPhysChem 6, 2324-2336 (2005).
[CrossRef] [PubMed]

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, "Art and artefacts of fluorescence correlation spectroscopy," Curr. Pharm. Biotechnol. 5, 155-161 (2004).
[CrossRef] [PubMed]

M. Wahl, I. Gregor, M. Patting, and J. Enderlein, "Fast calculation of fluorescence correlation data with asynchronous time-correlated single-photon counting," Opt. Express 11, 3583-3591 (2003).
[CrossRef] [PubMed]

M. Böhmer, F. Pampaloni, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, "Time-resolved confocal scanning device for ultrasensitive fluorescence detection," Rev. Sci. Instrum. 72, 4145-4152 (2001).
[CrossRef]

Erdmann, R.

M. Böhmer, F. Pampaloni, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, "Time-resolved confocal scanning device for ultrasensitive fluorescence detection," Rev. Sci. Instrum. 72, 4145-4152 (2001).
[CrossRef]

Fitter, J.

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, "Art and artefacts of fluorescence correlation spectroscopy," Curr. Pharm. Biotechnol. 5, 155-161 (2004).
[CrossRef] [PubMed]

Gordon, R.

Gregor, I.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements," Chem. PhysChem. 8, 433-443 (2007).
[CrossRef] [PubMed]

J. Enderlein and I. Gregor, "Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy," Rev. Sci. Instrum. 76, 033102 (2005).
[CrossRef]

J. Enderlein, I. Gregor, D. Patra, T. Dertinger, and U. B. Kaupp, "Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration," ChemPhysChem 6, 2324-2336 (2005).
[CrossRef] [PubMed]

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, "Art and artefacts of fluorescence correlation spectroscopy," Curr. Pharm. Biotechnol. 5, 155-161 (2004).
[CrossRef] [PubMed]

M. Wahl, I. Gregor, M. Patting, and J. Enderlein, "Fast calculation of fluorescence correlation data with asynchronous time-correlated single-photon counting," Opt. Express 11, 3583-3591 (2003).
[CrossRef] [PubMed]

Hartman, J.

Hartmann, R.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements," Chem. PhysChem. 8, 433-443 (2007).
[CrossRef] [PubMed]

Holmes, T.

Kaupp, U. B.

J. Enderlein, I. Gregor, D. Patra, T. Dertinger, and U. B. Kaupp, "Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration," ChemPhysChem 6, 2324-2336 (2005).
[CrossRef] [PubMed]

Lamb, D. C.

B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, "Pulsed interleaved excitation," Biophys. J. 89, 3508-3522 (2005).
[CrossRef] [PubMed]

Lessor, D.

Levy, W.

Magde, D.

D. Magde, W. W. Webb, and E. Elson, "Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

Müller, B. K.

B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, "Pulsed interleaved excitation," Biophys. J. 89, 3508-3522 (2005).
[CrossRef] [PubMed]

Munro, P. R. T.

Nomarski, G.

G. Nomarski, "Double-Point-Reference Interference Comparator (DPRIC)," J. Opt. Soc. Am. 61, 1560-1560 (1971).

G. Nomarski, "Interference Microscopy - State of Art and Its Future," J. Opt. Soc. Am. 60, 1575-1575 (1970).

G. Nomarski, "Dispositif Oculaire a Contraste De Phase Pour Microscope," J. Phys. Radium 11, 9-10 (1950).

Pacheco, V.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements," Chem. PhysChem. 8, 433-443 (2007).
[CrossRef] [PubMed]

Pampaloni, F.

M. Böhmer, F. Pampaloni, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, "Time-resolved confocal scanning device for ultrasensitive fluorescence detection," Rev. Sci. Instrum. 72, 4145-4152 (2001).
[CrossRef]

Patra, D.

J. Enderlein, I. Gregor, D. Patra, T. Dertinger, and U. B. Kaupp, "Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration," ChemPhysChem 6, 2324-2336 (2005).
[CrossRef] [PubMed]

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, "Art and artefacts of fluorescence correlation spectroscopy," Curr. Pharm. Biotechnol. 5, 155-161 (2004).
[CrossRef] [PubMed]

Patting, M.

Preza, C.

Rahn, H. J.

M. Böhmer, F. Pampaloni, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, "Time-resolved confocal scanning device for ultrasensitive fluorescence detection," Rev. Sci. Instrum. 72, 4145-4152 (2001).
[CrossRef]

Sheppard, C.

C. J. Cogswell and C. Sheppard, "Confocal Differential Interference Contrast (DIC) Microscopy: including a Theoretical Analysis of Conventional and Confocal DIC Imaging," J. Microsc. 165, 81-101 (1992).
[CrossRef]

Snyder, D.

Török, P.

von der Hocht, I.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements," Chem. PhysChem. 8, 433-443 (2007).
[CrossRef] [PubMed]

Wahl, M.

M. Wahl, I. Gregor, M. Patting, and J. Enderlein, "Fast calculation of fluorescence correlation data with asynchronous time-correlated single-photon counting," Opt. Express 11, 3583-3591 (2003).
[CrossRef] [PubMed]

M. Böhmer, F. Pampaloni, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, "Time-resolved confocal scanning device for ultrasensitive fluorescence detection," Rev. Sci. Instrum. 72, 4145-4152 (2001).
[CrossRef]

Webb, W. W.

D. Magde, W. W. Webb, and E. Elson, "Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

Zaychikov, E.

B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, "Pulsed interleaved excitation," Biophys. J. 89, 3508-3522 (2005).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biophys. J. (1)

B. K. Müller, E. Zaychikov, C. Bräuchle, and D. C. Lamb, "Pulsed interleaved excitation," Biophys. J. 89, 3508-3522 (2005).
[CrossRef] [PubMed]

Chem. PhysChem. (1)

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, "Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements," Chem. PhysChem. 8, 433-443 (2007).
[CrossRef] [PubMed]

ChemPhysChem (1)

J. Enderlein, I. Gregor, D. Patra, T. Dertinger, and U. B. Kaupp, "Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration," ChemPhysChem 6, 2324-2336 (2005).
[CrossRef] [PubMed]

Curr. Pharm. Biotechnol. (1)

J. Enderlein, I. Gregor, D. Patra, and J. Fitter, "Art and artefacts of fluorescence correlation spectroscopy," Curr. Pharm. Biotechnol. 5, 155-161 (2004).
[CrossRef] [PubMed]

J. Microsc. (1)

C. J. Cogswell and C. Sheppard, "Confocal Differential Interference Contrast (DIC) Microscopy: including a Theoretical Analysis of Conventional and Confocal DIC Imaging," J. Microsc. 165, 81-101 (1992).
[CrossRef]

J. Opt. Soc. Am. (3)

G. Nomarski, "Interference Microscopy - State of Art and Its Future," J. Opt. Soc. Am. 60, 1575-1575 (1970).

G. Nomarski, "Double-Point-Reference Interference Comparator (DPRIC)," J. Opt. Soc. Am. 61, 1560-1560 (1971).

D. Lessor, J. Hartman, and R. Gordon, "Quantitative Surface Topography Determination by Nomarski Reflection Microscopy. I. Theory," J. Opt. Soc. Am. 69, 357-366 (1979).
[CrossRef]

J. Opt. Soc. Am. A (1)

J. Phys. Radium (1)

G. Nomarski, "Dispositif Oculaire a Contraste De Phase Pour Microscope," J. Phys. Radium 11, 9-10 (1950).

Opt. Express (2)

Phys. Rev. Lett. (1)

D. Magde, W. W. Webb, and E. Elson, "Thermodynamic Fluctuations in a Reacting System - Measurement by Fluorescence Correlation Spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

Rev. Sci. Instrum. (2)

J. Enderlein and I. Gregor, "Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy," Rev. Sci. Instrum. 76, 033102 (2005).
[CrossRef]

M. Böhmer, F. Pampaloni, M. Wahl, H. J. Rahn, R. Erdmann, and J. Enderlein, "Time-resolved confocal scanning device for ultrasensitive fluorescence detection," Rev. Sci. Instrum. 72, 4145-4152 (2001).
[CrossRef]

Other (3)

D. V. O'Connor and D. Phillips, Time-correlated Single Photon Counting (Academic Press, 1984).

D. Murphy, "Differential interference contrast (DIC) microscopy and modulation contrast microscopy," in Fundamentals of Light Microscopy and Digital Imaging (Wiley-Liss, 2001), pp. 153-168.

E. Salmon and P. Tran, "High-resolution video-enhanced differential interference contrast (VE-DIC) light microscope," in Video Microscopy, G. Sluder, and D. Wolf, eds., (Academic Press, 1998), pp. 153-184.

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Figures (3)

Fig. 1.
Fig. 1.

Confocal setup with PicoQuant MicroTime 200, equipped with: 1a+b.) Laserhead 637 nm, 1c.) Laserhead 532 nm, 1d & e.) Laserhead 470 nm, 2.) Mirror, 3.) Adjustable zero order half-waveplate, 4.) Polarizing cube, 5.) Beamdisplacer, 6.) Fiber coupler, 7a-c.) Single mode fiber, 8.) Dichroic, 9.) Lens, 10.) Shutter, 11.) Confocal aperture, 12.) Fluorescence filter, 13.) 50/50 Mirror, 14.) Single photon avalanche diode

Fig. 2.
Fig. 2.

Main panel: DLS at 90° of mono disperse TetraSpeck 100 latex particles, fitted with a 2nd order cumulant fit. Inset: standard plot of ACF.

Fig. 3.
Fig. 3.

Main picture: Wavelength dependent determination of Nomarski-DIC-prism shear distance, by comparison of DLS and 2f-FCS measurements, obtained from enhanced model for multi labelled particles. Inset: 2fFCS measurement of TetraSpec 100 latex particles. Autocorrelation (ACF) and cross-correlation (CCF) functions, fitted with 2fFCS model.

Tables (1)

Tables Icon

Table 1. Wavelength dependent shear distances obtained from comparison of DLS and 2fFCS experiments.

Equations (5)

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g ~ ( t , δ , v ) = c 4 π Dt d z 1 d z 2
κ ( z 1 ) κ ( z 2 ) 8 Dt + w 2 ( z 1 ) + w 2 ( z 2 ) exp [ ( z 2 z 1 v z t ) 2 4 Dt 2 ( δ v x t ) 2 + v y 2 t 2 8 D t + w 2 ( z 1 ) + w 2 ( z 2 ) ] ,
w ( z ) = w 0 [ 1 + ( λ ex z π w 0 2 n ) 2 ] 1 2 ,
κ ( z ) = 2 0 a d ρρ R 2 ( z ) exp ( 2 ρ 2 R 2 ( z ) ) = 1 exp ( 2 a 2 R 2 ( z ) ) ,
R ( z ) = R 0 [ 1 + ( λ em z π R 0 2 n ) 2 ] 1 2 ,

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