Abstract

The reduction of out of focus signal is a general task in fluorescence microscopy and is especially important in the recently developed super-resolution techniques because of the degradation of the final image. Several illumination methods have been developed to provide decreased out of focus signal level relative to the common epifluorescent illumination. In this paper we examine the highly inclined and the total internal reflection illumination techniques using the ray tracing method. Two merit functions were introduced for the quantitative description of the excitation of the selected region. We studied the feasibility of illumination methods, and the required corrections arising from the imperfections of the optical elements.

© 2014 Optical Society of America

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References

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  1. J. Lakovich, Principles of Fluorescence Spectroscopy (Plenum, 1986), Chap. 21.
  2. D. Axelrod, “Total internal reflection fluorescence microscopy,” in Optical Imaging and Microscopy, Vol. 87 of Springer Series in Optical Sciences, P. Torok and F. J. Kao, eds. (Springer Verlag, 2007), Chap. 8.
  3. C. A. Konopka and S. Y. Bednarek, “Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex,” Plant J. 53(1), 186–196 (2008).
    [CrossRef] [PubMed]
  4. M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, “Highly inclined thin illumination enables clear single-molecule imaging in cells,” Nat. Methods 5(2), 159–161 (2008).
    [CrossRef] [PubMed]
  5. C. Dunsby, “Optically sectioned imaging by oblique plane microscopy,” Opt. Express 16(25), 20306–20316 (2008).
    [CrossRef] [PubMed]
  6. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
    [CrossRef] [PubMed]
  7. J. Huisken, J. Swoger, S. Lindek, and E. H. K. Stelzer, “Selective Plane Illumination Microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Springer, 2006), pp. 672–679.
  8. M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
    [CrossRef] [PubMed]
  9. S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
    [CrossRef] [PubMed]
  10. S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
    [CrossRef] [PubMed]
  11. R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
    [CrossRef] [PubMed]
  12. D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981).
    [CrossRef] [PubMed]
  13. A. L. Mattheyses, S. M. Simon, and J. Z. Rappoport, “Imaging with total internal reflection fluorescence microscopy for the cell biologist,” J. Cell Sci. 123(Pt 21), 3621–3628 (2010).
    [CrossRef] [PubMed]
  14. Lambda Research Corp., OSLO optics software, optics reference ver. 6.1.
  15. M. Mandai and K. Yamaguchi, “Immersion microscope objective lens”, US patent US 7,046,451 B2 (2006).
  16. G. Gajdátsy and M. Erdelyi, “Analysis of focus distortion based on birefringence,” J. Opt. A, Pure Appl. Opt. 9(11), 982–987 (2007).
    [CrossRef]
  17. L. M. Bennie, P. T. Starkey, M. Jasperse, C. J. Billington, R. P. Anderson, and L. D. Turner, “A versatile high resolution objective for imaging quantum gases,” Opt. Express 21(7), 9011–9016 (2013).
    [CrossRef] [PubMed]
  18. J. P. Zinter and M. J. Levene, “Maximizing fluorescence collection efficiency in multiphoton microscopy,” Opt. Express 19(16), 15348–15362 (2011).
    [CrossRef] [PubMed]
  19. J. A. Buytaert and J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt. 12(1), 014039 (2007).
    [CrossRef] [PubMed]
  20. Y. Lu, T. Bifano, S. Ünlü, and B. Goldberg, “Aberration compensation in aplanatic solid immersion lens microscopy,” Opt. Express 21(23), 28189–28197 (2013).
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  21. O. Zhernovaya, O. Sydoruk, V. Tuchin, and A. Douplik, “The refractive index of human hemoglobin in the visible range,” Phys. Med. Biol. 56(13), 4013–4021 (2011).
    [CrossRef] [PubMed]
  22. S. van de Linde and M. Sauer, “How to switch a fluorophore: From undesired blinking to controlled photoswitching,” Chem. Soc. Rev. 43(4), 1076–1087 (2014).
    [CrossRef] [PubMed]

2014 (1)

S. van de Linde and M. Sauer, “How to switch a fluorophore: From undesired blinking to controlled photoswitching,” Chem. Soc. Rev. 43(4), 1076–1087 (2014).
[CrossRef] [PubMed]

2013 (2)

2011 (3)

J. P. Zinter and M. J. Levene, “Maximizing fluorescence collection efficiency in multiphoton microscopy,” Opt. Express 19(16), 15348–15362 (2011).
[CrossRef] [PubMed]

O. Zhernovaya, O. Sydoruk, V. Tuchin, and A. Douplik, “The refractive index of human hemoglobin in the visible range,” Phys. Med. Biol. 56(13), 4013–4021 (2011).
[CrossRef] [PubMed]

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[CrossRef] [PubMed]

2010 (1)

A. L. Mattheyses, S. M. Simon, and J. Z. Rappoport, “Imaging with total internal reflection fluorescence microscopy for the cell biologist,” J. Cell Sci. 123(Pt 21), 3621–3628 (2010).
[CrossRef] [PubMed]

2008 (3)

C. A. Konopka and S. Y. Bednarek, “Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex,” Plant J. 53(1), 186–196 (2008).
[CrossRef] [PubMed]

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, “Highly inclined thin illumination enables clear single-molecule imaging in cells,” Nat. Methods 5(2), 159–161 (2008).
[CrossRef] [PubMed]

C. Dunsby, “Optically sectioned imaging by oblique plane microscopy,” Opt. Express 16(25), 20306–20316 (2008).
[CrossRef] [PubMed]

2007 (2)

G. Gajdátsy and M. Erdelyi, “Analysis of focus distortion based on birefringence,” J. Opt. A, Pure Appl. Opt. 9(11), 982–987 (2007).
[CrossRef]

J. A. Buytaert and J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt. 12(1), 014039 (2007).
[CrossRef] [PubMed]

2006 (2)

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

2004 (1)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

2002 (1)

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[CrossRef] [PubMed]

1981 (1)

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981).
[CrossRef] [PubMed]

Anderson, R. P.

Axelrod, D.

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981).
[CrossRef] [PubMed]

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Bednarek, S. Y.

C. A. Konopka and S. Y. Bednarek, “Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex,” Plant J. 53(1), 186–196 (2008).
[CrossRef] [PubMed]

Bennie, L. M.

Bifano, T.

Billington, C. J.

Buytaert, J. A.

J. A. Buytaert and J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt. 12(1), 014039 (2007).
[CrossRef] [PubMed]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Dirckx, J. J.

J. A. Buytaert and J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt. 12(1), 014039 (2007).
[CrossRef] [PubMed]

Douplik, A.

O. Zhernovaya, O. Sydoruk, V. Tuchin, and A. Douplik, “The refractive index of human hemoglobin in the visible range,” Phys. Med. Biol. 56(13), 4013–4021 (2011).
[CrossRef] [PubMed]

Dunsby, C.

Erdelyi, M.

G. Gajdátsy and M. Erdelyi, “Analysis of focus distortion based on birefringence,” J. Opt. A, Pure Appl. Opt. 9(11), 982–987 (2007).
[CrossRef]

Gajdátsy, G.

G. Gajdátsy and M. Erdelyi, “Analysis of focus distortion based on birefringence,” J. Opt. A, Pure Appl. Opt. 9(11), 982–987 (2007).
[CrossRef]

Girirajan, T. P. K.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Goldberg, B.

Heidbreder, M.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[CrossRef] [PubMed]

Heilemann, M.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[CrossRef] [PubMed]

Hess, S. T.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Huisken, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Imamoto, N.

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, “Highly inclined thin illumination enables clear single-molecule imaging in cells,” Nat. Methods 5(2), 159–161 (2008).
[CrossRef] [PubMed]

Jasperse, M.

Klein, T.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[CrossRef] [PubMed]

Konopka, C. A.

C. A. Konopka and S. Y. Bednarek, “Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex,” Plant J. 53(1), 186–196 (2008).
[CrossRef] [PubMed]

Larson, D. R.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[CrossRef] [PubMed]

Levene, M. J.

Löschberger, A.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[CrossRef] [PubMed]

Lu, Y.

Mason, M. D.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Mattheyses, A. L.

A. L. Mattheyses, S. M. Simon, and J. Z. Rappoport, “Imaging with total internal reflection fluorescence microscopy for the cell biologist,” J. Cell Sci. 123(Pt 21), 3621–3628 (2010).
[CrossRef] [PubMed]

Rappoport, J. Z.

A. L. Mattheyses, S. M. Simon, and J. Z. Rappoport, “Imaging with total internal reflection fluorescence microscopy for the cell biologist,” J. Cell Sci. 123(Pt 21), 3621–3628 (2010).
[CrossRef] [PubMed]

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Sakata-Sogawa, K.

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, “Highly inclined thin illumination enables clear single-molecule imaging in cells,” Nat. Methods 5(2), 159–161 (2008).
[CrossRef] [PubMed]

Sauer, M.

S. van de Linde and M. Sauer, “How to switch a fluorophore: From undesired blinking to controlled photoswitching,” Chem. Soc. Rev. 43(4), 1076–1087 (2014).
[CrossRef] [PubMed]

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[CrossRef] [PubMed]

Simon, S. M.

A. L. Mattheyses, S. M. Simon, and J. Z. Rappoport, “Imaging with total internal reflection fluorescence microscopy for the cell biologist,” J. Cell Sci. 123(Pt 21), 3621–3628 (2010).
[CrossRef] [PubMed]

Starkey, P. T.

Stelzer, E. H. K.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Swoger, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Sydoruk, O.

O. Zhernovaya, O. Sydoruk, V. Tuchin, and A. Douplik, “The refractive index of human hemoglobin in the visible range,” Phys. Med. Biol. 56(13), 4013–4021 (2011).
[CrossRef] [PubMed]

Thompson, R. E.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[CrossRef] [PubMed]

Tokunaga, M.

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, “Highly inclined thin illumination enables clear single-molecule imaging in cells,” Nat. Methods 5(2), 159–161 (2008).
[CrossRef] [PubMed]

Tuchin, V.

O. Zhernovaya, O. Sydoruk, V. Tuchin, and A. Douplik, “The refractive index of human hemoglobin in the visible range,” Phys. Med. Biol. 56(13), 4013–4021 (2011).
[CrossRef] [PubMed]

Turner, L. D.

Ünlü, S.

van de Linde, S.

S. van de Linde and M. Sauer, “How to switch a fluorophore: From undesired blinking to controlled photoswitching,” Chem. Soc. Rev. 43(4), 1076–1087 (2014).
[CrossRef] [PubMed]

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[CrossRef] [PubMed]

Webb, W. W.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[CrossRef] [PubMed]

Wittbrodt, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Wolter, S.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[CrossRef] [PubMed]

Zhernovaya, O.

O. Zhernovaya, O. Sydoruk, V. Tuchin, and A. Douplik, “The refractive index of human hemoglobin in the visible range,” Phys. Med. Biol. 56(13), 4013–4021 (2011).
[CrossRef] [PubMed]

Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Zinter, J. P.

Biophys. J. (2)

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[CrossRef] [PubMed]

Chem. Soc. Rev. (1)

S. van de Linde and M. Sauer, “How to switch a fluorophore: From undesired blinking to controlled photoswitching,” Chem. Soc. Rev. 43(4), 1076–1087 (2014).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

J. A. Buytaert and J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt. 12(1), 014039 (2007).
[CrossRef] [PubMed]

J. Cell Biol. (1)

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981).
[CrossRef] [PubMed]

J. Cell Sci. (1)

A. L. Mattheyses, S. M. Simon, and J. Z. Rappoport, “Imaging with total internal reflection fluorescence microscopy for the cell biologist,” J. Cell Sci. 123(Pt 21), 3621–3628 (2010).
[CrossRef] [PubMed]

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

G. Gajdátsy and M. Erdelyi, “Analysis of focus distortion based on birefringence,” J. Opt. A, Pure Appl. Opt. 9(11), 982–987 (2007).
[CrossRef]

Nat. Methods (2)

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, “Highly inclined thin illumination enables clear single-molecule imaging in cells,” Nat. Methods 5(2), 159–161 (2008).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Nat. Protoc. (1)

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[CrossRef] [PubMed]

Opt. Express (4)

Phys. Med. Biol. (1)

O. Zhernovaya, O. Sydoruk, V. Tuchin, and A. Douplik, “The refractive index of human hemoglobin in the visible range,” Phys. Med. Biol. 56(13), 4013–4021 (2011).
[CrossRef] [PubMed]

Plant J. (1)

C. A. Konopka and S. Y. Bednarek, “Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex,” Plant J. 53(1), 186–196 (2008).
[CrossRef] [PubMed]

Science (1)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Other (5)

J. Huisken, J. Swoger, S. Lindek, and E. H. K. Stelzer, “Selective Plane Illumination Microscopy,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Springer, 2006), pp. 672–679.

J. Lakovich, Principles of Fluorescence Spectroscopy (Plenum, 1986), Chap. 21.

D. Axelrod, “Total internal reflection fluorescence microscopy,” in Optical Imaging and Microscopy, Vol. 87 of Springer Series in Optical Sciences, P. Torok and F. J. Kao, eds. (Springer Verlag, 2007), Chap. 8.

Lambda Research Corp., OSLO optics software, optics reference ver. 6.1.

M. Mandai and K. Yamaguchi, “Immersion microscope objective lens”, US patent US 7,046,451 B2 (2006).

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Figures (6)

Fig. 1
Fig. 1

Possible illumination methods in wide-field microscopy for imaging ROIs are (a) near the cover plate and (b) deeper inside the sample.

Fig. 2
Fig. 2

(a) The schematic view of the optical system with the tilted mirrors in the conjugate planes, perfect lenses and the objective, (b) Illuminated volumes within and out of ROI.

Fig. 3
Fig. 3

(a)-(d) The EPI-TIRF transition with a tilted INCL mirror and (e) centralizing process by tilting the CORR mirror.

Fig. 4
Fig. 4

(a) VAEM illumination of focal plane located at 1µm deep in the sample at INCL angle 0.32205°, (b) calibration method for CORR angle in case of 0.3862° INCL angle and (c) the realized optimal illumination in case of HILO illumination, (d) calibration method for CORR angle in case of 0.3864° INCL angle and (e)-(g) the illuminations at several CORR angles.

Fig. 5
Fig. 5

(a) Calibration method for the CORR angle in case of an INCL angle of 0.385° and HILO illumination (focal plane at 5 µm), (b-d) actual illuminations at several CORR angles, (e) calibration curve for CORR angle with the quality factor ILLOUT and (f) intensity distribution in the focal plane in case of optimum HILO illumination at an INCL angle of 0.385°.

Fig. 6
Fig. 6

(a) Positioning the focal plane with a moving objective and the effect of additional correction and (b) a calibration curve for additional correction in CORR angle tilting, the initial focal plane was located at 5 µm where the illumination was optimized.

Equations (2)

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

IL L ROI = Illuminated volume within the ROI size of ROI
IL L OUT = Illuminated volume out of ROI size of ROI

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