Abstract

We demonstrate a significant resolution enhancement beyond the conventional limit in multiphoton microscopy (MPM) using saturated excitation of fluorescence. Our technique achieves super-resolved imaging by temporally modulating the excitation laser-intensity and demodulating the higher harmonics from the saturated fluorescence signal. The improvement of the lateral and axial resolutions is measured on a sample of fluorescent microspheres. While the third harmonic already provides an enhanced resolution, we show that a further improvement can be obtained with an appropriate linear combination of the demodulated harmonics. Finally, we present in vitro imaging of fluorescent microspheres incorporated in HeLa cells to show that this technique performs well in biological samples.

© 2015 Optical Society of America

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References

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  1. W. Denk, J.H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
    [Crossref] [PubMed]
  2. J. Pawley and B. R. Masters, Handbook of Confocal Microscopy, 3rd ed. (Optical Engineering, International Society for Optics and Photonics, 2006).
    [Crossref]
  3. W. Denk and K. Svoboda, “Photon upmanship: why multiphoton imaging is more than a gimmick,” Neuron 18, 351–357 (1997).
    [Crossref] [PubMed]
  4. W. R. Zipfel, R. M. Wiliams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotech. 21(11), 1369–1377 (2003).
    [Crossref]
  5. K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
    [Crossref]
  6. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Let. 19(11), 780–782 (1994).
    [Crossref]
  7. G. Moneron and S. W. Hell, “Two-photon excitation STED microscopy,” Opt. Express 17(17), 14567–14573 (2009).
    [Crossref] [PubMed]
  8. D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” Journal of Microsopy 236(1), 35–43 (2009).
    [Crossref]
  9. G. C. Cianci, J. Wu, and K. M. Berland, “Saturation modified point spread function in two-photon microscopy,” Microsc. Res. Tech. 64, 135–141 (2004).
    [Crossref] [PubMed]
  10. K. Visscher, G. J. Brakenhoff, and T. D. Visser, “Fluorescence saturation in confocal microscopy,” J. Microsopy 175(2), 162–165 (1994).
    [Crossref]
  11. M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit: biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13(5), 050507 (2008).
    [Crossref] [PubMed]
  12. M. Yamanaka, Y. Yonemaru, S. Kawano, K. Uegaki, N. I. Smith, S. Kawata, and K. Fujita, “Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters,” J. Biomed. Opt. 1(12), 126002 (2013).
    [Crossref]

2013 (1)

M. Yamanaka, Y. Yonemaru, S. Kawano, K. Uegaki, N. I. Smith, S. Kawata, and K. Fujita, “Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters,” J. Biomed. Opt. 1(12), 126002 (2013).
[Crossref]

2009 (2)

G. Moneron and S. W. Hell, “Two-photon excitation STED microscopy,” Opt. Express 17(17), 14567–14573 (2009).
[Crossref] [PubMed]

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” Journal of Microsopy 236(1), 35–43 (2009).
[Crossref]

2008 (1)

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit: biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13(5), 050507 (2008).
[Crossref] [PubMed]

2007 (1)

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
[Crossref]

2004 (1)

G. C. Cianci, J. Wu, and K. M. Berland, “Saturation modified point spread function in two-photon microscopy,” Microsc. Res. Tech. 64, 135–141 (2004).
[Crossref] [PubMed]

2003 (1)

W. R. Zipfel, R. M. Wiliams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotech. 21(11), 1369–1377 (2003).
[Crossref]

1997 (1)

W. Denk and K. Svoboda, “Photon upmanship: why multiphoton imaging is more than a gimmick,” Neuron 18, 351–357 (1997).
[Crossref] [PubMed]

1994 (2)

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

K. Visscher, G. J. Brakenhoff, and T. D. Visser, “Fluorescence saturation in confocal microscopy,” J. Microsopy 175(2), 162–165 (1994).
[Crossref]

1990 (1)

W. Denk, J.H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Berland, K. M.

G. C. Cianci, J. Wu, and K. M. Berland, “Saturation modified point spread function in two-photon microscopy,” Microsc. Res. Tech. 64, 135–141 (2004).
[Crossref] [PubMed]

Brakenhoff, G. J.

K. Visscher, G. J. Brakenhoff, and T. D. Visser, “Fluorescence saturation in confocal microscopy,” J. Microsopy 175(2), 162–165 (1994).
[Crossref]

Cianci, G. C.

G. C. Cianci, J. Wu, and K. M. Berland, “Saturation modified point spread function in two-photon microscopy,” Microsc. Res. Tech. 64, 135–141 (2004).
[Crossref] [PubMed]

Denk, W.

W. Denk and K. Svoboda, “Photon upmanship: why multiphoton imaging is more than a gimmick,” Neuron 18, 351–357 (1997).
[Crossref] [PubMed]

W. Denk, J.H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Fujita, K.

M. Yamanaka, Y. Yonemaru, S. Kawano, K. Uegaki, N. I. Smith, S. Kawata, and K. Fujita, “Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters,” J. Biomed. Opt. 1(12), 126002 (2013).
[Crossref]

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit: biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13(5), 050507 (2008).
[Crossref] [PubMed]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
[Crossref]

Hell, S. W.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” Journal of Microsopy 236(1), 35–43 (2009).
[Crossref]

G. Moneron and S. W. Hell, “Two-photon excitation STED microscopy,” Opt. Express 17(17), 14567–14573 (2009).
[Crossref] [PubMed]

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

Kastrup, L.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” Journal of Microsopy 236(1), 35–43 (2009).
[Crossref]

Kawano, S.

M. Yamanaka, Y. Yonemaru, S. Kawano, K. Uegaki, N. I. Smith, S. Kawata, and K. Fujita, “Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters,” J. Biomed. Opt. 1(12), 126002 (2013).
[Crossref]

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit: biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13(5), 050507 (2008).
[Crossref] [PubMed]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
[Crossref]

Kawata, S.

M. Yamanaka, Y. Yonemaru, S. Kawano, K. Uegaki, N. I. Smith, S. Kawata, and K. Fujita, “Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters,” J. Biomed. Opt. 1(12), 126002 (2013).
[Crossref]

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit: biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13(5), 050507 (2008).
[Crossref] [PubMed]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
[Crossref]

Kobayashi, M.

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
[Crossref]

Masters, B. R.

J. Pawley and B. R. Masters, Handbook of Confocal Microscopy, 3rd ed. (Optical Engineering, International Society for Optics and Photonics, 2006).
[Crossref]

Medda, R.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” Journal of Microsopy 236(1), 35–43 (2009).
[Crossref]

Moneron, G.

Pawley, J.

J. Pawley and B. R. Masters, Handbook of Confocal Microscopy, 3rd ed. (Optical Engineering, International Society for Optics and Photonics, 2006).
[Crossref]

Smith, N. I.

M. Yamanaka, Y. Yonemaru, S. Kawano, K. Uegaki, N. I. Smith, S. Kawata, and K. Fujita, “Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters,” J. Biomed. Opt. 1(12), 126002 (2013).
[Crossref]

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit: biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13(5), 050507 (2008).
[Crossref] [PubMed]

Strickler, J.H.

W. Denk, J.H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Svoboda, K.

W. Denk and K. Svoboda, “Photon upmanship: why multiphoton imaging is more than a gimmick,” Neuron 18, 351–357 (1997).
[Crossref] [PubMed]

Uegaki, K.

M. Yamanaka, Y. Yonemaru, S. Kawano, K. Uegaki, N. I. Smith, S. Kawata, and K. Fujita, “Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters,” J. Biomed. Opt. 1(12), 126002 (2013).
[Crossref]

Visscher, K.

K. Visscher, G. J. Brakenhoff, and T. D. Visser, “Fluorescence saturation in confocal microscopy,” J. Microsopy 175(2), 162–165 (1994).
[Crossref]

Visser, T. D.

K. Visscher, G. J. Brakenhoff, and T. D. Visser, “Fluorescence saturation in confocal microscopy,” J. Microsopy 175(2), 162–165 (1994).
[Crossref]

Webb, W. W.

W. R. Zipfel, R. M. Wiliams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotech. 21(11), 1369–1377 (2003).
[Crossref]

W. Denk, J.H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Wichmann, J.

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

Wildanger, D.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” Journal of Microsopy 236(1), 35–43 (2009).
[Crossref]

Wiliams, R. M.

W. R. Zipfel, R. M. Wiliams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotech. 21(11), 1369–1377 (2003).
[Crossref]

Wu, J.

G. C. Cianci, J. Wu, and K. M. Berland, “Saturation modified point spread function in two-photon microscopy,” Microsc. Res. Tech. 64, 135–141 (2004).
[Crossref] [PubMed]

Yamanaka, M.

M. Yamanaka, Y. Yonemaru, S. Kawano, K. Uegaki, N. I. Smith, S. Kawata, and K. Fujita, “Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters,” J. Biomed. Opt. 1(12), 126002 (2013).
[Crossref]

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit: biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13(5), 050507 (2008).
[Crossref] [PubMed]

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
[Crossref]

Yonemaru, Y.

M. Yamanaka, Y. Yonemaru, S. Kawano, K. Uegaki, N. I. Smith, S. Kawata, and K. Fujita, “Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters,” J. Biomed. Opt. 1(12), 126002 (2013).
[Crossref]

Zipfel, W. R.

W. R. Zipfel, R. M. Wiliams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotech. 21(11), 1369–1377 (2003).
[Crossref]

J. Biomed. Opt. (2)

M. Yamanaka, S. Kawano, K. Fujita, N. I. Smith, and S. Kawata, “Beyond the diffraction-limit: biological imaging by saturated excitation microscopy,” J. Biomed. Opt. 13(5), 050507 (2008).
[Crossref] [PubMed]

M. Yamanaka, Y. Yonemaru, S. Kawano, K. Uegaki, N. I. Smith, S. Kawata, and K. Fujita, “Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters,” J. Biomed. Opt. 1(12), 126002 (2013).
[Crossref]

J. Microsopy (1)

K. Visscher, G. J. Brakenhoff, and T. D. Visser, “Fluorescence saturation in confocal microscopy,” J. Microsopy 175(2), 162–165 (1994).
[Crossref]

Journal of Microsopy (1)

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” Journal of Microsopy 236(1), 35–43 (2009).
[Crossref]

Microsc. Res. Tech. (1)

G. C. Cianci, J. Wu, and K. M. Berland, “Saturation modified point spread function in two-photon microscopy,” Microsc. Res. Tech. 64, 135–141 (2004).
[Crossref] [PubMed]

Nat. Biotech. (1)

W. R. Zipfel, R. M. Wiliams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotech. 21(11), 1369–1377 (2003).
[Crossref]

Neuron (1)

W. Denk and K. Svoboda, “Photon upmanship: why multiphoton imaging is more than a gimmick,” Neuron 18, 351–357 (1997).
[Crossref] [PubMed]

Opt. Express (1)

Opt. Let. (1)

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

Phys. Rev. Lett. (1)

K. Fujita, M. Kobayashi, S. Kawano, M. Yamanaka, and S. Kawata, “High-resolution confocal microscopy by saturated excitation of fluorescence,” Phys. Rev. Lett. 99(22), 228105 (2007).
[Crossref]

Science (1)

W. Denk, J.H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Other (1)

J. Pawley and B. R. Masters, Handbook of Confocal Microscopy, 3rd ed. (Optical Engineering, International Society for Optics and Photonics, 2006).
[Crossref]

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Figures (6)

Fig. 1
Fig. 1

Apparition of higher harmonics in the saturation regime. (a) Spectrum of the signal of the detected fluorescence at a modulation frequency of νm = 5kHz. (b) Power in the n-th harmonic of the detected fluorescence signal (Semission) for increasing power on the sample.

Fig. 2
Fig. 2

The experimental setup is based on a conventional multi-photon microscope with an acousto-optic modulator to modulate the fluorescence excitation intensity, and a lock-in amplifier to extract the harmonics of the fluorescence signal detected by the PMT.

Fig. 3
Fig. 3

Harmonics in the detected fluorescence signal from 0.19μm microspheres on a coverslip. (a) Frequency spectra of the fluorescence signal emitted by the microspheres for increasing average power. (b) Spectral intensities of the three first harmonics in the fluorescence signal. Linear fits of the data are shown as black lines. The particle concentration was 10mg/mL and the frequency of the excitation modulation νm was 17kHz.

Fig. 4
Fig. 4

Characterization of the lateral resolution improvement using 0.19μm microspheres on a coverslip. (a–d) Images in the (x,y) plane reconstructed with the first, second, and third harmonics and the linear combination of these three harmonics. The (x,y)-scan size is 48.36 × 56.54µm2 (390 × 456 pixels) resulting in a total acquisition time of ~35s. (e) Signal profiles along the orange line in (a–d). The background intensity was measured in an empty area and subtracted everywhere.

Fig. 5
Fig. 5

Characterization of the axial resolution improvement using 0.19μm microspheres on a coverslip. (a–d) Images in the (y,z) plane reconstructed with the first, second, and third harmonics and the linear combination of these three harmonics. The (y,z)-sections are taken from (x,y,z)-stacks consisting of 19 (x,y)-slices of 49.10×58.53µm2 (192 × 236 pixels), resulting in a total acquisition time of 3 minutes per 3D stack. (e) Signal profiles along the yellow line in (a–d). The background intensity was measured in an empty area and subtracted everywhere.

Fig. 6
Fig. 6

Microspheres of 0.51μm in HeLa cells. (a–d) Images in the (y,z) plane show a clear improvement of the resolution in the lateral and axial dimensions. (e–h) 3D renderings for the first, second and third harmonics and the linear combination of the three first harmonics. The (x,y,z)-stacks consisted of 17 (x,y)-slices of 169.6 × 169.6µm2 (800 × 800 pixels), resulting in a total acquisition time of 36 minutes per 3D stack.

Equations (4)

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

P e x c = 1 e α σ 2 P A P 2 P S F 2 ( x , y , z ) ,
P e x c = 1 e α σ 2 P A P 2 [ 1 + cos ( ω t ) ] 2 P S F 2 ( x , y , z ) ,
{ P ν m s i g n a l : 2 P S F 2 7 2 P S F 4 + 33 8 P S F 6 , P 2 ν m s i g n a l : 1 2 P S F 2 7 4 P S F 4 + 495 192 P S F 6 , P 3 ν m s i g n a l : 1 2 P S F 4 55 48 P S F 6 .
Linear combination signal = P ν m s i g n a l 4 P 2 ν m s i g n a l 7 P 3 ν m s i g n a l .

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