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Autofocusing in digital holographic microscopy

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3D Research

Abstract

Many applications in non-destructive testing at a microscopic level and in live cell imaging require automated focusing due to unstable environmental conditions, moving specimen or the limited depth of field of the applied optical imaging systems. Digital holography permits the recording and the numerical reconstruction of optical wave fields in amplitude and phase. This enables imaging of multiple focal planes from a single recorded hologram without mechanical realignment. The combination of numerical refocusing with image sharpness quantification algorithms yields subsequent autofocusing. With calibrated optical imaging systems this feature can be used also to determine the position and axial displacements of a sample. In order to show the application potential of digital holographic autofocusing in microscopy the method and results from investigations on several amplitude and phase objects are reviewed. This includes a demonstration of the reliability of automated refocusing, multi-focus quantitative phase contrast imaging of suspended cells, refocusing of quantitative phase contrast images during the analysis of the temporal dependency of cell spreading on surfaces and the quantification of toxin mediated morphological cell alterations during long-term observations. It is also shown for the example of sedimenting red blood cells that the method can be applied for minimally-invasive tracking of multiple particles. Finally, the usage of numerical autofocus for quantitative migration analysis of arbitrary shaped cells in a three-dimensional collagen matrix is demonstrated.

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References

  1. J. W. Goodmann, R. W. Lawrence (1967) Digital image formation from electronically detected holograms, Appl. Phys. Lett. 11: 77–79

    Article  Google Scholar 

  2. U. Schnars, W. Jüptner (1994) Direct recording of holograms by a CCD target and numerical reconstruction, Appl. Opt. 33: 179–181

    Article  Google Scholar 

  3. W. S. Haddad, D. Cullen, J. C. Solem, J. W. Longworth, A. McPherson, K. Boyer, C. K. Rhodes (1992) Fourier-transform holographic microscope, Appl. Opt. 31: 4973–4978

    Article  Google Scholar 

  4. E. Cuche, F. Bevilacqua, C. Depeursinge (1999) Digital holography for quantitative phase-contrast imaging, Opt. Lett. 24(5): 291–93

    Article  Google Scholar 

  5. Y. Takaki, H. Ohzu (1999) Fast numerical reconstruction technique for high-resolution hybrid holographic microscopy, Appl. Opt. 38: 2204–2211

    Article  Google Scholar 

  6. P. Pedrini, S. Schedin, H. J. Tiziani (2000) Spatial filtering in digital holographic microscopy, J. Mod. Opt. 47: 1447–1454

    Article  Google Scholar 

  7. W. Xu, M. H. Jericho, I. A. Meinertzhagen, H. J. Kreuzer (2001) Digital in-line holography for biological applications, PNAS 98: 11301–11305

    Article  Google Scholar 

  8. M. Kanka, R. Riesenberg, H. J. Kreuzer (2009) Reconstruction of high-resolution holographic microscopic images, Opt. Lett. 34: 1162–1164

    Article  Google Scholar 

  9. W. Yang, A. B. Kostinski, R. A. Shaw (2006) Phase signature for particle detection with digital in-line holography, Opt. Lett. 31: 1399–1401

    Article  Google Scholar 

  10. E. Cuche, P. Marquet, C. Depeursinge (1999) Simultaneous amplitude contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms, Appl. Opt. 38: 6694–7001

    Article  Google Scholar 

  11. D. Carl, B. Kemper, G. Wernicke, G. von Bally (2004) Parameteroptimized digital holographic microscope for high-resolution livingcell analysis, Appl. Opt. 43: 6536–6544

    Article  Google Scholar 

  12. P. Marquet, B. Rappaz, P. J. Magistretti, E. Cuche, Y. Emery, T. Colomb, C. Depeursinge (2005) Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy, Opt. Lett. 30: 468–470

    Article  Google Scholar 

  13. C. J. Mann, L. Yu, C.-M. Lo, M. K. Kim (2005) High-resolution quantitative phase-contrast microscopy by digital holography, Opt. Express. 13: 8693–8698

    Article  Google Scholar 

  14. P. Ferraro, S. Grilli, D. Alfieri, S. De Nicola, A. Finizio, G. Pierattini, B. Javidi, G. Coppola, V. Striano (2005) Extended focused image in microscopy by digital Holography, Opt. Express. 13: 6738–6749

    Article  Google Scholar 

  15. M. Antkowiak, N- Callens, C. Yourassowsky, F. Dubois (2008) Extended focused imaging of a microparticle field with digital holographic microscopy, Opt. Lett. 33: 1626–1628

    Article  Google Scholar 

  16. T. Colomb, N. Pavillon, J. Kühn, E. Cuche, C. Depeursinge, Y. Emery (2010) Extended depth-of-focus by digital holographic microscopy, Opt. Lett. 35: 1840–1842

    Article  Google Scholar 

  17. J. Kühn, T. Colomb, F. Montfort, F. Charrière, Y. Emery, E. Cuche, P. Marquet, C. Depeursinge (2007) Real-time dual-wavelength digital holographic microscopy with a single hologram acquisition, Opt. Express. 15: 7231–7242

    Article  Google Scholar 

  18. B. Kemper, G. von Bally (2008) Digital holographic microscopy for live cell applications and technical inspection, Appl. Opt. 47: A52–A61

    Article  Google Scholar 

  19. F. Charrière, J. Kühn, T. Colomb, F. Montfort, E. Cuche, Y. Emery, K. Weible, P. Marquet, C. Depeursinge (2006) Characterization of microlenses by digital holographic microscopy, Appl. Opt. 45: 829–835

    Article  Google Scholar 

  20. B. Kemper, D. Carl, J. Schnekenburger, I. Bredebusch, M. Schäfer, W. Domschke, G. von Bally (2006) Investigation of living pancreas tumor cells by digital holographic microscopy, J. Biomed. Opt. 11: 034005

    Article  Google Scholar 

  21. P. Ferraro, G. Coppola, S. De Nicola, A. Finizio, G. Pierattini (2003) Digital holographic microscope with automatic focus tracking by detecting sample displacement in real time, Opt. Lett. 28: 1257–1259

    Article  Google Scholar 

  22. M. Liebling, M. Unser (2004) Autofocus for digital Fresnel holograms by use of a Fresnelet-sparsity criterion, J. Opt. Soc. Am. A 21: 2424–2430

    Article  MathSciNet  Google Scholar 

  23. F. Dubois, C. Schockaert, N. Callens, C. Yourassowsky (2006) Focus plane detection criteria in digital holography microscopy by amplitude analysis, Opt. Express. 14: 5895–5908

    Article  Google Scholar 

  24. P. Langehanenberg, B. Kemper, G. von Bally (2007) Autofocus algorithms for digital-holographic microscopy, Proc. SPIE 6633, 66330E

  25. W. Li, N. C. Loomis, Q. Hu, C. S. Davis (2007) Focus detection, from digital in-line holograms based on spectral l1 norms, J. Opt. Soc. Am. A 24: 3054–3062

    Article  Google Scholar 

  26. P. Langehanenberg, B. Kemper, D. Dirksen, G. von Bally (2008) Autofocusing in digital holographic phase contrast microscopy on pure phase objects for live cell imaging, Appl. Opt. 47: D176–D182

    Article  Google Scholar 

  27. Y. Yang, B. Kang, Y. Choo (2008) Application of the correlation coefficient method for determination of the focal plane to digital particle holography, Appl. Opt. 47: 817–824

    Article  Google Scholar 

  28. M. L. Tachiki, M. Itoh, T. Yatagai (2008) Simultaneous depth determination of multiple objects by focus analysis in digital holography, Appl. Opt. 47: D144–D153

    Article  Google Scholar 

  29. F. Dubois, C. Yourassowsky, O. Monnom, J.-C. Legros (2006) Digital holographic microscopy for the three-dimensional dynamic analysis of in vitro cancer cell migration, J. Biomed. Opt. 11: 054032

    Article  Google Scholar 

  30. P. Langehanenberg, L. Ivanova, I. Bernhardt, S. Ketelhut, A. Vollmer, D. Dirksen, G. Georgiev, G. von Bally, B. Kemper (2009) Automated three-dimensional tracking of living cells by digital holographic microscopy, J. Biomed. Opt. 14: 014018

    Article  Google Scholar 

  31. U. Schnars, W. Jüptner (2002) Digital recording and numerical reconstruction of holograms, Meas. Sci. Technol. 13: R85–R101

    Article  Google Scholar 

  32. L. Yaroslavsky (2004) Digital Holography and Digital Image Processing: Principles, Methods, Algorithms, Kluwer Academic Publishers

  33. T. Kreis (2005) Handbook of Holographic Interferometry: Optical and Digital Methods, Wiley-VCH

  34. M.K. Kim, L. Yu, C.J. Mann (2006) Interference techniques in digital holography, J. Opt. A 8: S518–523

    Article  Google Scholar 

  35. T.-C. Poon (2006) Digital Holography and Three-Dimensional Display, Springer

  36. M. Liebling, T. Blu, M. Unser (2004) Complex-wave retrieval from a single off-axis hologram, J. Opt. Soc. Am. A 21: 367–377

    Article  Google Scholar 

  37. B. Kemper, D. Carl, J. Schnekenburger, I. Bredebusch, M. Schäfer, W. Domschke, G. von Bally (2006) Investigation of living pancreas tumor cells by digital holographic microscopy, J. Biomed. Opt. 11: 034005

    Article  Google Scholar 

  38. T. Colomb, F. Montfort, C. Depeursinge (2008) Small Reconstruction Distance in Convolution Formalism, in Digital Holography and Three-Dimensional Imaging, OSA Technical Digest (Optical Society of America), paper DMA4

  39. T. Kreis (1996) Holographic Interferometry: Principles and Methods, Akademie Publishing

  40. P. Marquet, B. Rappaz, F. Charrière, Y. Emery, C. Depeursinge, P. Magistretti (2007) Analysis of cellular structure and dynamics with digital holographic microscopy, Proc SPIE 6633, 66330F

  41. G. Nomarski (1955) Differential microinterferometer with polarized waves, J. Phys. Radium. 16: 9–13

    Google Scholar 

  42. Y. Sun, S. Duthaler, B. J. Nelson (2004) Autofocusing in computer microscopy: selecting the optimal focus algorithm, Microsc. Res. Tech. 65: 139–149

    Article  Google Scholar 

  43. F. C. Groen, I. T. Young, G. Ligthart (1985) A comparison of different focus functions for use in autofocus algorithms, Cytometry 6: 81–91

    Article  Google Scholar 

  44. L. Firestone, K. Cook, K. Culp, N. Talsania, K. Preston Jr (1991) Comparison of autofocus methods for automated microscopy, Cytometry 12: 195–206

    Article  Google Scholar 

  45. M. Bravo-Zanoguera, B. v. Massenbach, A. L. Kellner, J. H. Price (1998) High-performance autofocus circuit for biological microscopy, Rev. Sci. Instrum. 69: 3966–3977

    Article  Google Scholar 

  46. J. He, R. Zhou, Z. Hong (2003) Modified fast climbing search auto-focus algorithm with adaptive step size searching technique for digital camera, IEEE Transactions on Consumer Electronics 49: 257–262

    Article  Google Scholar 

  47. B. Kemper, D. Carl, A. Höink, G. von Bally, I. Bredebusch, J. Schnekenburger (2006) Modular digital holographic microscopy system for marker free quantitative phase contrast imaging of living cells, Proc. SPIE 6191, 61910T

  48. M. Bielaszewska, A. Bauwens, L. Greune, B. Kemper, U. Dobrindt, J. M. Geelen, K. S. Kim, A. Schmidt, H. Karch (2009) Vacuolization of human microvascular endothelial cells by enterohaemorrhagic Escherichia coli, Thrombosis and Haemostasis 102: 1080–1092

    Google Scholar 

  49. B. Kemper, S. Kosmeier, P. Langehanenberg, G. von Bally, I. Bredebusch, W. Domschke, J. Schnekenburger (2007) Integral refractive index determination of living suspension cells by multifocus digital holographic phase contrast microscopy, J. Biomed. Opt. 12: 054009

    Article  Google Scholar 

  50. B. Kemper, P. Langehanenberg, Gert von Bally (2007) Methods and applications for marker-free quantitative digital holographic phase contrast imaging in life cell analysis, Proc. SPIE 6796, 67960E

  51. B. Kemper, S. Kosmeier, P. Langehanenberg, S. Przibilla, C. Remmersmann, S. Stürwald, G. von Bally (2009) Application of 3D tracking, LED illumination and multi-wavelength techniques for quantitative cell analysis in digital holographic microscopy, Proc SPIE 7184, 71840R

  52. B. Kemper, A. Bauwens, A. Vollmer, S. Ketelhut, P. Langehanenberg, J. Müthing, H. Karch, G. von Bally (2010) Label-free Quantitative Cell Division Monitoring of Endothelial Cells by Digital Holographic Microscopy, J. Biomed. Opt. 15: 036009

    Article  Google Scholar 

  53. B. Kemper, P. Langehanenberg, A. Vollmer, S. Ketelhut, G. von Bally (2010) Digital Holographic Microscopy — Label-free 3D Migration Monitoring of Living Cells, Imaging and Microscopy. 4: 26–28

    Google Scholar 

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Authors and Affiliations

  1. Center for Biomedical Optics and Photonics, University of Muenster, Robert-Koch-Str. 45, D-48149, Muenster, Germany

    Patrik Langehanenberg, Gert von Bally & Björn Kemper

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  1. Patrik Langehanenberg
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  2. Gert von Bally
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  3. Björn Kemper
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Correspondence to Björn Kemper.

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Langehanenberg, P., von Bally, G. & Kemper, B. Autofocusing in digital holographic microscopy. 3D Res 2, 4 (2011). https://doi.org/10.1007/3DRes.01(2011)4

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  • DOI: https://doi.org/10.1007/3DRes.01(2011)4

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