Many biological structures of interest are beyond the diffraction limit of conventional microscopes and their visualization requires application of super-resolution techniques. Such techniques have found remarkable success in surpassing the diffraction limit to achieve sub-diffraction limited resolution; however, they are predominantly limited to fluorescent samples. Here, we introduce a non-fluorescent analogue to structured illumination microscopy, termed structured oblique illumination microscopy (SOIM), where we use simultaneous oblique illuminations of the sample to multiplex high spatial-frequency content into the frequency support of the system. We introduce a theoretical framework describing how to demodulate this multiplexed information to reconstruct an image with a spatial-frequency support exceeding that of the system’s classical diffraction limit. This approach allows enhanced-resolution imaging of non-fluorescent samples. Experimental confirmation of the approach is obtained in a reflection test target with moderate numerical aperture.

© 2012 OSA

1. J. Pawley, Handbook of Biological Confocal Microscopy (Springer Science+Business Media, New York, 1989).
2. M. Born and E. Wolf, Principles of Optics (Cambridge University Press, Cambridge, UK, 1959).
3. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006). [CrossRef] [PubMed]
4. M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006). [CrossRef] [PubMed]
5. M. Bates, B. Huang, and X. Zhuang, “Super-resolution microscopy by nanoscale localization of photo-switchable fluorescent probes,” Curr. Opin. Chem. Biol.12(5), 505–514 (2008). [CrossRef] [PubMed]
6. X. Zhuang, “Nano-imaging with Storm,” Nat. Photonics3(7), 365–367 (2009). [CrossRef] [PubMed]
7. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett.19(11), 780–782 (1994). [CrossRef] [PubMed]
8. S. W. Hell and M. Kroug, “Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit,” Appl. Phys. (Berl.)60(5), 495–497 (1995). [CrossRef]
9. M. Gustafsson, D. Agard, and J. Sedat, “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination,” Proc. SPIE3919, 141–150 (2000). [CrossRef]
10. M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc.198(2), 82–87 (2000). [CrossRef] [PubMed]
11. S. A. Shroff, J. R. Fienup, and D. R. Williams, “Phase-shift estimation in sinusoidally illuminated images for lateral superresolution,” J. Opt. Soc. Am. A26(2), 413–424 (2009). [CrossRef] [PubMed]
12. R. Heintzmann, T. M. Jovin, and C. Cremer, “Saturated patterned excitation microscopy--a concept for optical resolution improvement,” J. Opt. Soc. Am. A19(8), 1599–1609 (2002). [CrossRef] [PubMed]
13. M. G. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A.102(37), 13081–13086 (2005). [CrossRef] [PubMed]
14. E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A.109(3), E135–E143 (2012). [CrossRef] [PubMed]
15. W. Lukosz, “Optical systems with resolving powers exceeding the classical limit,” J. Opt. Soc. Am.56(11), 1463–1471 (1966). [CrossRef]
16. P. C. Sun and E. N. Leith, “Superresolution by spatial-temporal encoding methods,” Appl. Opt.31(23), 4857–4862 (1992). [CrossRef] [PubMed]
17. B. Littleton, K. Lai, D. Longstaff, V. Sarafis, P. Munroe, N. Heckenberg, and H. Rubinsztein-Dunlop, “Coherent super-resolution microscopy via laterally structured illumination,” Micron38(2), 150–157 (2007). [CrossRef] [PubMed]
18. K. M. Hajek, B. Littleton, D. Turk, T. J. McIntyre, and H. Rubinsztein-Dunlop, “A method for achieving super-resolved widefield CARS microscopy,” Opt. Express18(18), 19263–19272 (2010). [CrossRef] [PubMed]
19. A. Mudassar, A. R. Harvey, A. H. Greenaway, and J. D. C. Jones, “Resolution beyond classical limites with spatial frequency heterodyning,” Chin. Opt. Lett.4(3), 148–151 (2006).
20. J. Goodman, Introduction to Fourier Optics (Roberts & Company, Greenwood Village, CO, 2005).
21. F. Orieux, E. Sepulveda, V. Loriette, B. Dubertret, and J. C. Olivo-Marin, “Bayesian estimation for optimized structured illumination microscopy,” IEEE Trans. Image Process.21(2), 601–614 (2012). [CrossRef] [PubMed]
22. J. Prince and J. Links, Medical Imaging Signals and Systems (Pearson Prentice Hall, Upper Saddle River, NJ, 2006).

Appl. Opt.

• P. C. Sun and E. N. Leith, “Superresolution by spatial-temporal encoding methods,” Appl. Opt.31(23), 4857–4862 (1992). [CrossRef] [PubMed]

Appl. Phys. (Berl.)

• S. W. Hell and M. Kroug, “Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit,” Appl. Phys. (Berl.)60(5), 495–497 (1995). [CrossRef]

Chin. Opt. Lett.

• A. Mudassar, A. R. Harvey, A. H. Greenaway, and J. D. C. Jones, “Resolution beyond classical limites with spatial frequency heterodyning,” Chin. Opt. Lett.4(3), 148–151 (2006).

Curr. Opin. Chem. Biol.

• M. Bates, B. Huang, and X. Zhuang, “Super-resolution microscopy by nanoscale localization of photo-switchable fluorescent probes,” Curr. Opin. Chem. Biol.12(5), 505–514 (2008). [CrossRef] [PubMed]

IEEE Trans. Image Process.

• F. Orieux, E. Sepulveda, V. Loriette, B. Dubertret, and J. C. Olivo-Marin, “Bayesian estimation for optimized structured illumination microscopy,” IEEE Trans. Image Process.21(2), 601–614 (2012). [CrossRef] [PubMed]

J. Microsc.

• M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc.198(2), 82–87 (2000). [CrossRef] [PubMed]

J. Opt. Soc. Am.

• W. Lukosz, “Optical systems with resolving powers exceeding the classical limit,” J. Opt. Soc. Am.56(11), 1463–1471 (1966). [CrossRef]

J. Opt. Soc. Am. A

• S. A. Shroff, J. R. Fienup, and D. R. Williams, “Phase-shift estimation in sinusoidally illuminated images for lateral superresolution,” J. Opt. Soc. Am. A26(2), 413–424 (2009). [CrossRef] [PubMed]
• R. Heintzmann, T. M. Jovin, and C. Cremer, “Saturated patterned excitation microscopy--a concept for optical resolution improvement,” J. Opt. Soc. Am. A19(8), 1599–1609 (2002). [CrossRef] [PubMed]

Micron

• B. Littleton, K. Lai, D. Longstaff, V. Sarafis, P. Munroe, N. Heckenberg, and H. Rubinsztein-Dunlop, “Coherent super-resolution microscopy via laterally structured illumination,” Micron38(2), 150–157 (2007). [CrossRef] [PubMed]

Nat. Methods

• M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006). [CrossRef] [PubMed]

Nat. Photonics

• X. Zhuang, “Nano-imaging with Storm,” Nat. Photonics3(7), 365–367 (2009). [CrossRef] [PubMed]

Opt. Express

• K. M. Hajek, B. Littleton, D. Turk, T. J. McIntyre, and H. Rubinsztein-Dunlop, “A method for achieving super-resolved widefield CARS microscopy,” Opt. Express18(18), 19263–19272 (2010). [CrossRef] [PubMed]

Opt. Lett.

• S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett.19(11), 780–782 (1994). [CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A.

• M. G. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A.102(37), 13081–13086 (2005). [CrossRef] [PubMed]
• E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A.109(3), E135–E143 (2012). [CrossRef] [PubMed]

Proc. SPIE

• M. Gustafsson, D. Agard, and J. Sedat, “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination,” Proc. SPIE3919, 141–150 (2000). [CrossRef]

Science

• E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006). [CrossRef] [PubMed]

Other

• J. Pawley, Handbook of Biological Confocal Microscopy (Springer Science+Business Media, New York, 1989).
• M. Born and E. Wolf, Principles of Optics (Cambridge University Press, Cambridge, UK, 1959).
• J. Goodman, Introduction to Fourier Optics (Roberts & Company, Greenwood Village, CO, 2005).
• J. Prince and J. Links, Medical Imaging Signals and Systems (Pearson Prentice Hall, Upper Saddle River, NJ, 2006).

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