Abstract

Using the color selectivity of a spatial light modulator (SLM) for both, tailoring the excitation beam at one wavelength, and multiplexing the image at the red-shifted fluorescence wavelength, it is possible to parallelize confocal microscopy, i.e. to simultaneously detect an axial stack (z-stack) of a sample. For this purpose, two diffractive patterns, one steering the excitation light, and the other manipulating the emission light, are combined within the same area of the SLM, which acts as a pure phase modulator. A recently demonstrated technique allows one to combine the patterns with high diffraction efficiency and low crosstalk, using the extended phase shifting capability of the SLM, which covers multiples of 2π at the respective wavelengths. For a first demonstration we compare standard confocal imaging with simultaneous image acquisition in two separate sample planes, which shows comparable results.

© 2016 Optical Society of America

Full Article  |  PDF Article
OSA Recommended Articles
Resolution enhancement in standing-wave total internal reflection microscopy: a point-spread-function engineering approach

Peter T. C. So, Hyuk-Sang Kwon, and Chen Y. Dong
J. Opt. Soc. Am. A 18(11) 2833-2845 (2001)

Optimized pupil-plane filters for confocal microscope point-spread function engineering

M. A. A. Neil, R. Juškaitis, T. Wilson, Z. J. Laczik, and V. Sarafis
Opt. Lett. 25(4) 245-247 (2000)

3D image scanning microscopy with engineered excitation and detection

Clemens Roider, Rafael Piestun, and Alexander Jesacher
Optica 4(11) 1373-1381 (2017)

References

  • View by:
  • |
  • |
  • |

  1. M. Liang, R. L. Stehr, and A. W. Krause, “Confocal pattern period in multiple-aperture confocal imaging systems with coherent illumination,” Opt. Lett. 22, 751–753 (1997).
    [Crossref] [PubMed]
  2. M. A. A. Neil, T. Wilson, and R. Juskaitis, “A light efficient optically sectioning microscope,” J. of Microsc. 189114–117 (1998).
    [Crossref]
  3. R. GrÃd’f, J. Rietdorf, and T. Zimmermann, “Live cell spinning disk microscopy,” Advances in Biochemical Engineering/Biotechnology 95, 57–75 (2005).
    [Crossref]
  4. D. Faklis and G. M. Morris, “Spectral properties of multiorder diffractive lenses,” Appl. Opt. 34, 2462–2468 (1995).
    [Crossref] [PubMed]
  5. D. W. Sweeney and G. E. Sommargren, “Harmonic diffractive lenses,” Appl. Opt. 34, 2469–2475 (1995).
    [Crossref] [PubMed]
  6. S. Noach, A. Lewis, Y. Arieli, and N. Eisenberg, “Integrated diffractive and refractive elements for spectrum shaping,” Appl. Opt. 35, 3635–3639 (1996).
    [Crossref] [PubMed]
  7. I. M. Barton, P. Blair, and M. Taghizadeh, “Dual-wavelength operation diffractive phase elements for pattern formation,” Opt. Exp. 1, 54–59 (1997).
    [Crossref]
  8. J. Bengtsson, “Kinoforms designed to produce different fan-out patterns for two wavelengths,” Appl. Opt. 37, 2011–2020 (1998).
    [Crossref]
  9. T. R. M. Sales and D. H. Raguin, “Multiwavelength operation with thin diffractive elements,” Appl. Opt. 38, 3012–3018 (1999).
    [Crossref]
  10. Y. Ogura, N. Shirai, J. Tanida, and Y. Ichioka, “Wavelength-multiplexing diffractive phase elements: design, fabrication, and performance evaluation,” JOSA A 18, 1082–1092 (2001).
    [Crossref] [PubMed]
  11. A. J. Caley, A. J. Waddie, and M. R. Taghizadeh, “A novel algorithm for designing diffractive optical elements for two colour far-field pattern formation,” J. Opt. A: Pure Appl. Opt. 7, 276–279 (2005).
    [Crossref]
  12. V. Calero, P. García-Martínez, J. Albero, M. M. Sánchez-López, and I. Moreno, “Liquid crystal spatial light modulator with very large phase modulation operating in high harmonic orders,” Opt. Lett. 38, 4663–4666 (2013).
    [Crossref] [PubMed]
  13. A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Colour hologram projection with an SLM by exploiting its full phase modulation range,” Opt. Exp. 22, 20530–20541 (2014).
    [Crossref]
  14. A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Combined holographic optical trapping and optical image processing using a single diffractive pattern displayed on a spatial light modulator,” Opt. Lett. 39, 5337–5340 (2014).
    [Crossref]
  15. Y. Shechtman, L. E. Weiss, A. S. Backer, M. Y. Lee, and W. E. Moerner, “Multicolour localization microscopy by point-spread-function engineering,” Nat. Photon. 10, 590–594 (2016).
    [Crossref]
  16. C. J. R. Sheppard, “Super-resolution in Confocal Imaging,” Optik 80, 53–54 (1988).
  17. C. Müller and J. Enderlein, “Image Scanning Microscopy,” Phys. Rev. Lett. 104, 198101 (2010).
    [Crossref] [PubMed]
  18. C. J. R. Sheppard, S. B. Mehta, and R. Heintzmann, “Superresolution by image scanning microscopy using pixel reassignment,” Opt. Lett. 38, 2889–2892 (2013).
    [Crossref] [PubMed]
  19. Marc Guillon and Marcel A. Lauterbach, “Quantitative confocal spiral phase contrast,” J. Opt. Soc. Am. A 31, 1215–1225 (2014).
    [Crossref]
  20. R. Piestun, Y. Y. Schechner, and J. Shamir, “Propagation-invariant wave fields with finite energy,” J. Opt. Soc. Am. A 17, 294–303 (2000).
    [Crossref]
  21. S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function,” PNAS 106, 2995–2999 (2009).
    [Crossref]

2016 (1)

Y. Shechtman, L. E. Weiss, A. S. Backer, M. Y. Lee, and W. E. Moerner, “Multicolour localization microscopy by point-spread-function engineering,” Nat. Photon. 10, 590–594 (2016).
[Crossref]

2014 (3)

2013 (2)

2010 (1)

C. Müller and J. Enderlein, “Image Scanning Microscopy,” Phys. Rev. Lett. 104, 198101 (2010).
[Crossref] [PubMed]

2009 (1)

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function,” PNAS 106, 2995–2999 (2009).
[Crossref]

2005 (2)

A. J. Caley, A. J. Waddie, and M. R. Taghizadeh, “A novel algorithm for designing diffractive optical elements for two colour far-field pattern formation,” J. Opt. A: Pure Appl. Opt. 7, 276–279 (2005).
[Crossref]

R. GrÃd’f, J. Rietdorf, and T. Zimmermann, “Live cell spinning disk microscopy,” Advances in Biochemical Engineering/Biotechnology 95, 57–75 (2005).
[Crossref]

2001 (1)

Y. Ogura, N. Shirai, J. Tanida, and Y. Ichioka, “Wavelength-multiplexing diffractive phase elements: design, fabrication, and performance evaluation,” JOSA A 18, 1082–1092 (2001).
[Crossref] [PubMed]

2000 (1)

1999 (1)

1998 (2)

J. Bengtsson, “Kinoforms designed to produce different fan-out patterns for two wavelengths,” Appl. Opt. 37, 2011–2020 (1998).
[Crossref]

M. A. A. Neil, T. Wilson, and R. Juskaitis, “A light efficient optically sectioning microscope,” J. of Microsc. 189114–117 (1998).
[Crossref]

1997 (2)

I. M. Barton, P. Blair, and M. Taghizadeh, “Dual-wavelength operation diffractive phase elements for pattern formation,” Opt. Exp. 1, 54–59 (1997).
[Crossref]

M. Liang, R. L. Stehr, and A. W. Krause, “Confocal pattern period in multiple-aperture confocal imaging systems with coherent illumination,” Opt. Lett. 22, 751–753 (1997).
[Crossref] [PubMed]

1996 (1)

1995 (2)

1988 (1)

C. J. R. Sheppard, “Super-resolution in Confocal Imaging,” Optik 80, 53–54 (1988).

Albero, J.

Arieli, Y.

Backer, A. S.

Y. Shechtman, L. E. Weiss, A. S. Backer, M. Y. Lee, and W. E. Moerner, “Multicolour localization microscopy by point-spread-function engineering,” Nat. Photon. 10, 590–594 (2016).
[Crossref]

Barton, I. M.

I. M. Barton, P. Blair, and M. Taghizadeh, “Dual-wavelength operation diffractive phase elements for pattern formation,” Opt. Exp. 1, 54–59 (1997).
[Crossref]

Bengtsson, J.

Bernet, S.

A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Combined holographic optical trapping and optical image processing using a single diffractive pattern displayed on a spatial light modulator,” Opt. Lett. 39, 5337–5340 (2014).
[Crossref]

A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Colour hologram projection with an SLM by exploiting its full phase modulation range,” Opt. Exp. 22, 20530–20541 (2014).
[Crossref]

Biteen, J. S.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function,” PNAS 106, 2995–2999 (2009).
[Crossref]

Blair, P.

I. M. Barton, P. Blair, and M. Taghizadeh, “Dual-wavelength operation diffractive phase elements for pattern formation,” Opt. Exp. 1, 54–59 (1997).
[Crossref]

Calero, V.

Caley, A. J.

A. J. Caley, A. J. Waddie, and M. R. Taghizadeh, “A novel algorithm for designing diffractive optical elements for two colour far-field pattern formation,” J. Opt. A: Pure Appl. Opt. 7, 276–279 (2005).
[Crossref]

Eisenberg, N.

Enderlein, J.

C. Müller and J. Enderlein, “Image Scanning Microscopy,” Phys. Rev. Lett. 104, 198101 (2010).
[Crossref] [PubMed]

Faklis, D.

García-Martínez, P.

GrÃd’f, R.

R. GrÃd’f, J. Rietdorf, and T. Zimmermann, “Live cell spinning disk microscopy,” Advances in Biochemical Engineering/Biotechnology 95, 57–75 (2005).
[Crossref]

Guillon, Marc

Heintzmann, R.

Ichioka, Y.

Y. Ogura, N. Shirai, J. Tanida, and Y. Ichioka, “Wavelength-multiplexing diffractive phase elements: design, fabrication, and performance evaluation,” JOSA A 18, 1082–1092 (2001).
[Crossref] [PubMed]

Jesacher, A.

A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Colour hologram projection with an SLM by exploiting its full phase modulation range,” Opt. Exp. 22, 20530–20541 (2014).
[Crossref]

A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Combined holographic optical trapping and optical image processing using a single diffractive pattern displayed on a spatial light modulator,” Opt. Lett. 39, 5337–5340 (2014).
[Crossref]

Juskaitis, R.

M. A. A. Neil, T. Wilson, and R. Juskaitis, “A light efficient optically sectioning microscope,” J. of Microsc. 189114–117 (1998).
[Crossref]

Krause, A. W.

Lauterbach, Marcel A.

Lee, M. Y.

Y. Shechtman, L. E. Weiss, A. S. Backer, M. Y. Lee, and W. E. Moerner, “Multicolour localization microscopy by point-spread-function engineering,” Nat. Photon. 10, 590–594 (2016).
[Crossref]

Lewis, A.

Liang, M.

Liu, N.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function,” PNAS 106, 2995–2999 (2009).
[Crossref]

Lord, S. J.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function,” PNAS 106, 2995–2999 (2009).
[Crossref]

Mehta, S. B.

Moerner, W. E.

Y. Shechtman, L. E. Weiss, A. S. Backer, M. Y. Lee, and W. E. Moerner, “Multicolour localization microscopy by point-spread-function engineering,” Nat. Photon. 10, 590–594 (2016).
[Crossref]

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function,” PNAS 106, 2995–2999 (2009).
[Crossref]

Moreno, I.

Morris, G. M.

Müller, C.

C. Müller and J. Enderlein, “Image Scanning Microscopy,” Phys. Rev. Lett. 104, 198101 (2010).
[Crossref] [PubMed]

Neil, M. A. A.

M. A. A. Neil, T. Wilson, and R. Juskaitis, “A light efficient optically sectioning microscope,” J. of Microsc. 189114–117 (1998).
[Crossref]

Noach, S.

Ogura, Y.

Y. Ogura, N. Shirai, J. Tanida, and Y. Ichioka, “Wavelength-multiplexing diffractive phase elements: design, fabrication, and performance evaluation,” JOSA A 18, 1082–1092 (2001).
[Crossref] [PubMed]

Pavani, S. R. P.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function,” PNAS 106, 2995–2999 (2009).
[Crossref]

Piestun, R.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function,” PNAS 106, 2995–2999 (2009).
[Crossref]

R. Piestun, Y. Y. Schechner, and J. Shamir, “Propagation-invariant wave fields with finite energy,” J. Opt. Soc. Am. A 17, 294–303 (2000).
[Crossref]

Raguin, D. H.

Rietdorf, J.

R. GrÃd’f, J. Rietdorf, and T. Zimmermann, “Live cell spinning disk microscopy,” Advances in Biochemical Engineering/Biotechnology 95, 57–75 (2005).
[Crossref]

Ritsch-Marte, M.

A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Combined holographic optical trapping and optical image processing using a single diffractive pattern displayed on a spatial light modulator,” Opt. Lett. 39, 5337–5340 (2014).
[Crossref]

A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Colour hologram projection with an SLM by exploiting its full phase modulation range,” Opt. Exp. 22, 20530–20541 (2014).
[Crossref]

Sales, T. R. M.

Sánchez-López, M. M.

Schechner, Y. Y.

Shamir, J.

Shechtman, Y.

Y. Shechtman, L. E. Weiss, A. S. Backer, M. Y. Lee, and W. E. Moerner, “Multicolour localization microscopy by point-spread-function engineering,” Nat. Photon. 10, 590–594 (2016).
[Crossref]

Sheppard, C. J. R.

Shirai, N.

Y. Ogura, N. Shirai, J. Tanida, and Y. Ichioka, “Wavelength-multiplexing diffractive phase elements: design, fabrication, and performance evaluation,” JOSA A 18, 1082–1092 (2001).
[Crossref] [PubMed]

Sommargren, G. E.

Stehr, R. L.

Sweeney, D. W.

Taghizadeh, M.

I. M. Barton, P. Blair, and M. Taghizadeh, “Dual-wavelength operation diffractive phase elements for pattern formation,” Opt. Exp. 1, 54–59 (1997).
[Crossref]

Taghizadeh, M. R.

A. J. Caley, A. J. Waddie, and M. R. Taghizadeh, “A novel algorithm for designing diffractive optical elements for two colour far-field pattern formation,” J. Opt. A: Pure Appl. Opt. 7, 276–279 (2005).
[Crossref]

Tanida, J.

Y. Ogura, N. Shirai, J. Tanida, and Y. Ichioka, “Wavelength-multiplexing diffractive phase elements: design, fabrication, and performance evaluation,” JOSA A 18, 1082–1092 (2001).
[Crossref] [PubMed]

Thompson, M. A.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function,” PNAS 106, 2995–2999 (2009).
[Crossref]

Twieg, R. J.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function,” PNAS 106, 2995–2999 (2009).
[Crossref]

Waddie, A. J.

A. J. Caley, A. J. Waddie, and M. R. Taghizadeh, “A novel algorithm for designing diffractive optical elements for two colour far-field pattern formation,” J. Opt. A: Pure Appl. Opt. 7, 276–279 (2005).
[Crossref]

Weiss, L. E.

Y. Shechtman, L. E. Weiss, A. S. Backer, M. Y. Lee, and W. E. Moerner, “Multicolour localization microscopy by point-spread-function engineering,” Nat. Photon. 10, 590–594 (2016).
[Crossref]

Wilson, T.

M. A. A. Neil, T. Wilson, and R. Juskaitis, “A light efficient optically sectioning microscope,” J. of Microsc. 189114–117 (1998).
[Crossref]

Zimmermann, T.

R. GrÃd’f, J. Rietdorf, and T. Zimmermann, “Live cell spinning disk microscopy,” Advances in Biochemical Engineering/Biotechnology 95, 57–75 (2005).
[Crossref]

Advances in Biochemical Engineering/Biotechnology (1)

R. GrÃd’f, J. Rietdorf, and T. Zimmermann, “Live cell spinning disk microscopy,” Advances in Biochemical Engineering/Biotechnology 95, 57–75 (2005).
[Crossref]

Appl. Opt. (5)

J. of Microsc. (1)

M. A. A. Neil, T. Wilson, and R. Juskaitis, “A light efficient optically sectioning microscope,” J. of Microsc. 189114–117 (1998).
[Crossref]

J. Opt. A: Pure Appl. Opt. (1)

A. J. Caley, A. J. Waddie, and M. R. Taghizadeh, “A novel algorithm for designing diffractive optical elements for two colour far-field pattern formation,” J. Opt. A: Pure Appl. Opt. 7, 276–279 (2005).
[Crossref]

J. Opt. Soc. Am. A (2)

JOSA A (1)

Y. Ogura, N. Shirai, J. Tanida, and Y. Ichioka, “Wavelength-multiplexing diffractive phase elements: design, fabrication, and performance evaluation,” JOSA A 18, 1082–1092 (2001).
[Crossref] [PubMed]

Nat. Photon. (1)

Y. Shechtman, L. E. Weiss, A. S. Backer, M. Y. Lee, and W. E. Moerner, “Multicolour localization microscopy by point-spread-function engineering,” Nat. Photon. 10, 590–594 (2016).
[Crossref]

Opt. Exp. (2)

I. M. Barton, P. Blair, and M. Taghizadeh, “Dual-wavelength operation diffractive phase elements for pattern formation,” Opt. Exp. 1, 54–59 (1997).
[Crossref]

A. Jesacher, S. Bernet, and M. Ritsch-Marte, “Colour hologram projection with an SLM by exploiting its full phase modulation range,” Opt. Exp. 22, 20530–20541 (2014).
[Crossref]

Opt. Lett. (4)

Optik (1)

C. J. R. Sheppard, “Super-resolution in Confocal Imaging,” Optik 80, 53–54 (1988).

Phys. Rev. Lett. (1)

C. Müller and J. Enderlein, “Image Scanning Microscopy,” Phys. Rev. Lett. 104, 198101 (2010).
[Crossref] [PubMed]

PNAS (1)

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function,” PNAS 106, 2995–2999 (2009).
[Crossref]

Supplementary Material (1)

NameDescription
» Visualization 1: AVI (140 KB)      avi-movie visualization 1 related to Fig5

Cited By

OSA participates in Crossref's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (5)

Fig. 1
Fig. 1

Principle of combining diffractive patterns for two-color operation used for parallel imaging. (a): The two diffractive patterns (top, and bottom) are individually calculated for operation at their respective wavelengths of 460 nm (top), and 515 nm (bottom), each covering a phase range of 2π (gray levels correspond to the voltage levels applied at the respective SLM pixels) at their respective wavelengths. Note that the voltage covers only a range between 0 and 72 at 460 nm, and between 0 and 85 at 515 nm, out of 256 accessible voltage levels. With a method described in the text, the two diffractive phase patterns are combined into a single one (middle), which now covers the entire accessible voltage range. (b): The SLM displaying the combined phase pattern is placed in a plane conjugate to the back aperture of the microscope objective. By illumination with the excitation beam at 460 nm, the first diffractive pattern (top pattern in (a)) is accessed, producing a PSF with two diffraction limited spots in two different z-planes. The corresponding fluorescence light at 515 nm passing back through the objective to the SLM accesses the second diffractive pattern (bottom of (a)), which produces a PSF with two spots, focusing at separate positions of the camera chip, and again with a depth separation corresponding to the first one. As a result, the camera detects two sharp in-focus images of the two excitation spots in the sample.

Fig. 2
Fig. 2

Experimental setup for simultaneous confocal imaging in two different z-planes. Details are explained in the text.

Fig. 3
Fig. 3

Comparison of two simultaneously acquired images of different depths (left column) in a GFP-labelled mouse brain-slice with a control experiment where the same planes have been sequentially acquired (next row). The axial interspacing between the planes is 4 μm. The insets give the total number of pixel counts, which are proportional to the respective image intensities. For comparison, a wide-field fluorescence image of the same sample is shown at the right.

Fig. 4
Fig. 4

Example for colored PSF engineering using a more complex excitation and filtering pattern of 5 × 5 spots. (a) shows a diffractive pattern which is designed only for the excitation wavelength (460 nm), i.e. it covers a phase range of 2π at that wavelength, corresponding to a voltage range between 0 and 72. Camera 1 directly images the excitation pattern in the sample plane, showing the corresponding pattern of fluorescent spots. The image of camera 2 is acquired after the light passing the SLM in both, excitation and detection paths, and thus records a convolution of the excitation and detection PSFs (of different colors), which is in this case (with non-controlled detection PSF) a diffuse pattern of multiplexed spots. (b) shows the same experiment done with a combined diffractive pattern, which is designed to create a 5 × 5 spot array for the excitation light (at 460 nm, see cam 1), and just a plane mirror function at the emission wavelength of 515 nm. As before, the image of camera 2 shows the convolution between excitation and imaging PSFs, which in this case corresponds to the almost undisturbed spot array.

Fig. 5
Fig. 5

In (a) the diffractive pattern for the excitation wavelength (at 460 nm) was designed as the 5 × 5 spot array shown in Fig. 4, whereas the DP in the imaging path (515 nm) was designed to produce a doughnut beam of order 5. The corresponding light pattern seen by camera 2 is a cross-correlation of both, and thus corresponds to an array of doughnut rings. (b) shows the result of a similar experiment where the DP for the excitation beam was calculated to produce a 5 × 5 spot array, which is projected at the surface of the sample. In this case the spots were programmed to focus in different (randomly distributed) z-planes. The DP in the imaging path was designed to produce a double-helix PSF. The corresponding image seen by camera 2 thus shows an array of double-helices, which however stem from excitation spots that are in focus and out of focus at the fluorescent surface. An attached movie shows the effect of scanning the z-position of the sample, resulting in a rotation of the helical PSFs. During the axial scan other helical PSFs which then focus at the sample surface have a changed orientation with respect to ones which focus in other planes (see Visualization 1).

Equations (1)

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

ϕ ( U , λ ) = 2 π d λ n ( U ) ,

Metrics