OSA's Digital Library

Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 4, Iss. 10 — Oct. 1, 2013
  • pp: 1937–1945
« Show journal navigation

Implementation of spatial overlap modulation nonlinear optical microscopy using an electro-optic deflector

Keisuke Isobe, Hiroyuki Kawano, Akiko Kumagai, Atsushi Miyawaki, and Katsumi Midorikawa  »View Author Affiliations


Biomedical Optics Express, Vol. 4, Issue 10, pp. 1937-1945 (2013)
http://dx.doi.org/10.1364/BOE.4.001937


View Full Text Article

Acrobat PDF (3150 KB)





Browse Journals / Lookup Meetings

Browse by Journal and Year


   


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools

Share
Citations

Abstract

A spatial overlap modulation (SPOM) technique is a nonlinear optical microscopy technique which enhances the three-dimensional spatial resolution and rejects the out-of-focus background limiting the imaging depth inside a highly scattering sample. Here, we report on the implementation of SPOM in which beam pointing modulation is achieved by an electro-optic deflector. The modulation and demodulation frequencies are enhanced to 200 kHz and 400 kHz, respectively, resulting in a 200-fold enhancement compared with the previously reported system. The resolution enhancement and suppression of the out-of-focus background are demonstrated by sum-frequency-generation imaging of pounded granulated sugar and deep imaging of fluorescent beads in a tissue-like phantom, respectively.

© 2013 OSA

1. Introduction

Nonlinear optical microscopy based on a variety of nonlinear optical techniques, such as two-photon excitation fluorescence (TPEF) [1

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

3

3. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003). [CrossRef] [PubMed]

], second-harmonic generation (SHG) [4

4. P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21(11), 1356–1360 (2003). [CrossRef] [PubMed]

], third-harmonic generation [5

5. J. Squier and M. Müller, “High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum. 72(7), 2855–2867 (2001). [CrossRef]

, 6

6. Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70(8), 922–924 (1997). [CrossRef]

], four-wave mixing [7

7. M. D. Duncan, J. Reintjes, and T. J. Manuccia, “Scanning coherent anti-Stokes Raman microscope,” Opt. Lett. 7(8), 350–352 (1982). [CrossRef] [PubMed]

10

10. K. Isobe, T. Kawasumi, T. Tamaki, S. Kataoka, Y. Ozeki, and K. Itoh, “Three-dimensional profiling of refractive index distribution inside transparent materials by use of nonresonant four-wave mixing microscopy,” Appl. Phys. Express 1, 022006 (2008). [CrossRef]

], two-photon absorption (TPA) [11

11. D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007). [CrossRef] [PubMed]

, 12

12. D. Fu, T. Ye, T. E. Matthews, B. J. Chen, G. Yurtserver, and W. S. Warren, “High-resolution in vivo imaging of blood vessels without labeling,” Opt. Lett. 32(18), 2641–2643 (2007). [CrossRef] [PubMed]

], stimulated Raman scattering (SRS) [13

13. C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science 322(5909), 1857–1861 (2008). [CrossRef] [PubMed]

15

15. Y. Ozeki, F. Dake, S. Kajiyama, K. Fukui, and K. Itoh, “Analysis and experimental assessment of the sensitivity of stimulated Raman scattering microscopy,” Opt. Express 17(5), 3651–3658 (2009). [CrossRef] [PubMed]

] and cross-phase modulation [16

16. P. Samineni, B. Li, J. W. Wilson, W. S. Warren, and M. C. Fischer, “Cross-phase modulation imaging,” Opt. Lett. 37(5), 800–802 (2012). [CrossRef] [PubMed]

, 17

17. J. W. Wilson, P. Samineni, W. S. Warren, and M. C. Fischer, “Cross-phase modulation spectral shifting: nonlinear phase contrast in a pump-probe microscope,” Biomed. Opt. Express 3(5), 854–862 (2012). [CrossRef] [PubMed]

], has been developed for applications in physics, chemistry, and biology. Nonlinear optical microscopy offers several advantages over linear optical microscopy, which include three-dimensional resolution without a confocal pinhole, high penetration depth with near-infrared light excitation, less out-of-focus photon-induced damage and photobleaching. In particular, multi-photon excited fluorescence microscopy benefits from the ability to image deeper inside samples than confocal fluorescence microscopy when near-IR excitation that allows maximum optical transparency in biological systems is employed [18

18. P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 μm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett. 28(12), 1022–1024 (2003). [CrossRef] [PubMed]

22

22. N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013). [CrossRef]

]. Nonetheless, deep imaging is intrinsically difficult because the excitation light is attenuated by scattering and absorption in the sample. To maintain sufficient excitation intensity at the focus at significant depths in scattering media, the energy of the excitation pulse must be increased. A regenerative amplifier producing 150-fs pulses at the μJ level has been used to image green-fluorescent-protein-labelled neurons at a depth of up to 1 mm within the neocortex [18

18. P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 μm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett. 28(12), 1022–1024 (2003). [CrossRef] [PubMed]

]. However, increasing the pulse energy causes an increase in background signals, which include fluorescent signals generated in the out-of-focus regions. This background limits the maximum imaging depth inside a highly scattering sample [19

19. P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A 23(12), 3139–3149 (2006). [CrossRef] [PubMed]

].

Extensive efforts have been made to suppress out-of-focus signals [20

20. D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009). [CrossRef] [PubMed]

29

29. Z. Chen, L. Wei, X. Zhu, and W. Min, “Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores,” Opt. Express 20(17), 18525–18536 (2012). [CrossRef] [PubMed]

]. A decrease in scattering of the excitation light in the sample, which was achieved by using TPEF at 1280 nm [20

20. D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009). [CrossRef] [PubMed]

, 21

21. D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt. 16(10), 106014 (2011). [CrossRef] [PubMed]

] and three-photon excited fluorescence at 1700 nm [22

22. N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013). [CrossRef]

], has extended the maximum imaging depth. Temporal focusing suppresses out-of-focus signals in wide-field TPEF microscopy [23

23. G. Zhu, J. van Howe, M. Durst, W. Zipfel, and C. Xu, “Simultaneous spatial and temporal focusing of femtosecond pulses,” Opt. Express 13(6), 2153–2159 (2005). [CrossRef] [PubMed]

, 24

24. D. Oron, E. Tal, and Y. Silberberg, “Scanningless depth-resolved microscopy,” Opt. Express 13(5), 1468–1476 (2005). [CrossRef] [PubMed]

]. Structured illumination microscopy [25

25. M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997). [CrossRef] [PubMed]

], focal modulation microscopy [26

26. N. Chen, C.-H. Wong, and C. J. R. Sheppard, “Focal modulation microscopy,” Opt. Express 16(23), 18764–18769 (2008). [CrossRef] [PubMed]

] and differential aberration imaging techniques [27

27. A. Leray and J. Mertz, “Rejection of two-photon fluorescence background in thick tissue by differential aberration imaging,” Opt. Express 14(22), 10565–10573 (2006). [CrossRef] [PubMed]

], which employ spatial intensity modulation near the focal point, provide out-of-focus background rejection. Adaptive optics can be used to recover diffraction-limited performance by compensating for wavefront distortion and have been applied to TPEF imaging [28

28. N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7(2), 141–147 (2010). [CrossRef] [PubMed]

]. The suppression of the background fluorescence in TPEF microscopy has been also achieved by utilizing photoactivatable fluorophores, which remain in a non-fluorescent state until optically triggered [29

29. Z. Chen, L. Wei, X. Zhu, and W. Min, “Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores,” Opt. Express 20(17), 18525–18536 (2012). [CrossRef] [PubMed]

]. This technique is similar to cyclic-sequential-multiphoton excitation microscopy using reversible photoswitchable fluorophores [30

30. K. Isobe, A. Suda, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “High-resolution fluorescence microscopy based on a cyclic sequential multiphoton process,” Biomed. Opt. Express 1(3), 791–797 (2010). [CrossRef] [PubMed]

].

2. SPOM-NOM using an EOD

3. Sample preparation

To demonstrate the background suppression by SPOM-NOM, we prepared a tissue-like phantom as a highly scattering sample. We added 45 μL of a yellow-green (505/515) fluorescent polystyrene bead solution (Molecular Probes F8877) with a concentration of 4.55 × 109 beads/mL to 160 μL of low-melting-point agarose gel, then pipetted it onto a 35-mm Petri dish with a cover glass bottom and covered it with another cover glass. The phantom contained fluorescent polystyrene beads with a diameter of 2 μm at a concentration of 1.0 × 109 beads/mL as scatterers and tracers. To demonstrate the resolution enhancement by SPOM-NOM, we also prepared samples for SFG imaging. We pounded granulated sugar in a mortar and sandwiched it between a glass slide and a cover glass.

4. Results and discussion

We investigated SNRs at demodulation frequencies of 400 kHz and 2 kHz by changing the generated signal intensity. As the signal, we used the SFG signal from a 10-μm-thick β-barium borate (BBO) crystal. The SHG signal generated only by either the Ti:sapphire laser pulse or the OPO pulse was not removed by the BPF but was employed as the background. The integration time of the lock-in amplifier was set to 3 ms. As shown in Fig. 2
Fig. 2 SNRs at demodulation frequencies of (red square) 400 kHz and (black circk) 2 kHz.
, we can see that SNR at 400 kHz is improved at the higher signal level by 10 dB compared with that at 2 kHz. At the higher signal level and the lower frequency, the laser 1/f noise dominates over other noise sources. The improved SNR is attributed to the reduction of the laser 1/f noise because of the increase of the demodulation frequency. By increasing the demodulation frequency to the MHz range, the SNR could be more improved.

To quantify the background suppression, the TPEF intensity of the beads in the focal plane (signal) and the background TPEF intensity are plotted as a function of penetration depth in Figs. 4(a) and 4(b), and the contrast ratio between the signal and background intensities is plotted as a function of penetration depth in Fig. 4(c). We obtained the signal and background intensities by taking the averages of TPEF intensities at 49-neighboring pixels (7 × 7 pixels) around the center of a bead, and at 49-neighboring pixels (7 × 7 pixels) around a dark pixel in each image, respectively. We found that even though the penetration depth is increased, the signal intensity in the focal region can be maintained by compensating for the reduced excitation intensity at the focus by increasing the excitation power at the sample surface. On the other hand, the background TPEF intensity increased with increasingpenetration depth. As a result, the signal-to-background ratio (SBR) decreased with increasing penetration depth by a factor of e-az. However, the rate of increase of the background TPEF intensity is reduced by the SPOM technique. Thus, the rate of decrease of the SBR is lower using the SPOM technique. We performed a least squares fit to the SBR, S/B, using the function log(S/B) = -az + b. We obtained a = 0.00597 μm−1 and b = 2.41 for conventional TPEF microscopy, and a = 0.00291 μm−1 and b = 2.86 for SPOM-NOM. This result represents that the rate of decrease of the SBR of the SPOM-NOM can be improved by a factor of 2.1. The demodulated signal intensity in SPOM-NOM is proportional to the square of the maximum focus displacement [31

31. K. Isobe, H. Kawano, T. Takeda, A. Suda, A. Kumagai, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Background-free deep imaging by spatial overlap modulation nonlinear optical microscopy,” Biomed. Opt. Express 3(7), 1594–1608 (2012). [CrossRef] [PubMed]

31. K. Isobe, H. Kawano, T. Takeda, A. Suda, A. Kumagai, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Spatial overlap modulation nonlinear optical microscopy,” in Conference on Lasers and Electro-Optics 2012, OSA Technical Digest (online) (Optical Society of America, 2012), paper JW3G.4. [CrossRef]

]. Therefore, by increasing the maximum focus displacement, the SBR can be more improved.

To demonstrate the resolution enhancement by SPOM-NOM, we acquired SFG images of pounded sugar. The input power from the Ti:sapphire oscillator and the OPO was 1 mW. The full results for the stack of images in the yz plane can be seen in an online movie (Media 1) in the supplementary material. Figures 5(a)
Fig. 5 Enhancement of the spatial resolution in SFG imaging by SPOM-NOM. (a, b) SFG images of granulated sugar pounded in a mortar obtained by (a) conventional microscopy and (b) SPOM-NOM. (c-e) Normalized signal profiles along the lateral and axial directions indicated by (c) blue, (d) green, (e) red, (f) yellow, (g) pink arrows in (a) and (b).
and 5(b) respectively show single frames from Media 1 for conventional SFG microscopy and SPOM-NOM with a maximum focus displacement of 200 nm in the lateral (y) direction, which corresponds to the modulation depth of 21%. Because we can achieve a sufficiently high enhancement of the spatial resolution by setting the maximum focus displacement to half the radius of the focal spot [31

31. K. Isobe, H. Kawano, T. Takeda, A. Suda, A. Kumagai, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Background-free deep imaging by spatial overlap modulation nonlinear optical microscopy,” Biomed. Opt. Express 3(7), 1594–1608 (2012). [CrossRef] [PubMed]

31. K. Isobe, H. Kawano, T. Takeda, A. Suda, A. Kumagai, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Spatial overlap modulation nonlinear optical microscopy,” in Conference on Lasers and Electro-Optics 2012, OSA Technical Digest (online) (Optical Society of America, 2012), paper JW3G.4. [CrossRef]

], we adjusted the maximum focus displacement to 200 nm. The movie was reconstructed from 41 xy images (128 × 128 pixels) obtained at depth increments of 0.5 μm. Figures 5(c)-5(g) illustrate the normalized signal profiles along the lateral (y) and axial (z) directions indicated by blue, green, red, yellow, and pink arrows in Figs. 5(a) and 5(b). From the profiles in Figs. 5(c)-5(g), we can observe that smaller structures are clearly visible and distinctive from each other in the SPOM-NOM image, and that the background is significantly reduced. This result indicates that our SPOM-NOM system can provide better lateral and axial resolutions, and higher image contrast.

5. Conclusions

Acknowledgment

This work was supported by the Konica Minolta Imaging Science Foundation.

References and links

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.

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200(2), 83–104 (2000). [CrossRef] [PubMed]

3.

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

4.

P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol. 21(11), 1356–1360 (2003). [CrossRef] [PubMed]

5.

J. Squier and M. Müller, “High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum. 72(7), 2855–2867 (2001). [CrossRef]

6.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett. 70(8), 922–924 (1997). [CrossRef]

7.

M. D. Duncan, J. Reintjes, and T. J. Manuccia, “Scanning coherent anti-Stokes Raman microscope,” Opt. Lett. 7(8), 350–352 (1982). [CrossRef] [PubMed]

8.

A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett. 82(20), 4142–4145 (1999). [CrossRef]

9.

K. Isobe, S. Kataoka, R. Murase, W. Watanabe, T. Higashi, S. Kawakami, S. Matsunaga, K. Fukui, and K. Itoh, “Stimulated parametric emission microscopy,” Opt. Express 14(2), 786–793 (2006). [CrossRef] [PubMed]

10.

K. Isobe, T. Kawasumi, T. Tamaki, S. Kataoka, Y. Ozeki, and K. Itoh, “Three-dimensional profiling of refractive index distribution inside transparent materials by use of nonresonant four-wave mixing microscopy,” Appl. Phys. Express 1, 022006 (2008). [CrossRef]

11.

D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt. 12(5), 054004 (2007). [CrossRef] [PubMed]

12.

D. Fu, T. Ye, T. E. Matthews, B. J. Chen, G. Yurtserver, and W. S. Warren, “High-resolution in vivo imaging of blood vessels without labeling,” Opt. Lett. 32(18), 2641–2643 (2007). [CrossRef] [PubMed]

13.

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science 322(5909), 1857–1861 (2008). [CrossRef] [PubMed]

14.

P. Nandakumar, A. Kovalev, and A. Volkmer, “Vibrational imaging based on stimulated Raman scattering microscopy,” New J. Phys. 11(3), 033026 (2009). [CrossRef]

15.

Y. Ozeki, F. Dake, S. Kajiyama, K. Fukui, and K. Itoh, “Analysis and experimental assessment of the sensitivity of stimulated Raman scattering microscopy,” Opt. Express 17(5), 3651–3658 (2009). [CrossRef] [PubMed]

16.

P. Samineni, B. Li, J. W. Wilson, W. S. Warren, and M. C. Fischer, “Cross-phase modulation imaging,” Opt. Lett. 37(5), 800–802 (2012). [CrossRef] [PubMed]

17.

J. W. Wilson, P. Samineni, W. S. Warren, and M. C. Fischer, “Cross-phase modulation spectral shifting: nonlinear phase contrast in a pump-probe microscope,” Biomed. Opt. Express 3(5), 854–862 (2012). [CrossRef] [PubMed]

18.

P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 μm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett. 28(12), 1022–1024 (2003). [CrossRef] [PubMed]

19.

P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A 23(12), 3139–3149 (2006). [CrossRef] [PubMed]

20.

D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express 17(16), 13354–13364 (2009). [CrossRef] [PubMed]

21.

D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt. 16(10), 106014 (2011). [CrossRef] [PubMed]

22.

N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics 7(3), 205–209 (2013). [CrossRef]

23.

G. Zhu, J. van Howe, M. Durst, W. Zipfel, and C. Xu, “Simultaneous spatial and temporal focusing of femtosecond pulses,” Opt. Express 13(6), 2153–2159 (2005). [CrossRef] [PubMed]

24.

D. Oron, E. Tal, and Y. Silberberg, “Scanningless depth-resolved microscopy,” Opt. Express 13(5), 1468–1476 (2005). [CrossRef] [PubMed]

25.

M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997). [CrossRef] [PubMed]

26.

N. Chen, C.-H. Wong, and C. J. R. Sheppard, “Focal modulation microscopy,” Opt. Express 16(23), 18764–18769 (2008). [CrossRef] [PubMed]

27.

A. Leray and J. Mertz, “Rejection of two-photon fluorescence background in thick tissue by differential aberration imaging,” Opt. Express 14(22), 10565–10573 (2006). [CrossRef] [PubMed]

28.

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7(2), 141–147 (2010). [CrossRef] [PubMed]

29.

Z. Chen, L. Wei, X. Zhu, and W. Min, “Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores,” Opt. Express 20(17), 18525–18536 (2012). [CrossRef] [PubMed]

30.

K. Isobe, A. Suda, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “High-resolution fluorescence microscopy based on a cyclic sequential multiphoton process,” Biomed. Opt. Express 1(3), 791–797 (2010). [CrossRef] [PubMed]

31.

K. Isobe, H. Kawano, T. Takeda, A. Suda, A. Kumagai, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Background-free deep imaging by spatial overlap modulation nonlinear optical microscopy,” Biomed. Opt. Express 3(7), 1594–1608 (2012). [CrossRef] [PubMed]

K. Isobe, H. Kawano, T. Takeda, A. Suda, A. Kumagai, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Spatial overlap modulation nonlinear optical microscopy,” in Conference on Lasers and Electro-Optics 2012, OSA Technical Digest (online) (Optical Society of America, 2012), paper JW3G.4. [CrossRef]

32.

J. Miyazu, T. Imai, S. Toyoda, M. Sasaura, S. Yagi, K. Kato, Y. Sasaki, and K. Fujiura, “New beam scanning model for high-speed operation using KTa1-xNbxO3 Crystals,” Appl. Phys. Express 4(11), 111501 (2011). [CrossRef]

33.

S. Yagi, K. Naganuma, T. Imai, Y. Shibata, S. Ishibashi, Y. Sasaki, M. Sasaura, K. Fujiura, and K. Kato, “A mechanical-free 150-kHz repetition swept light source incorporated a KTN electro-optic deflector,” Proc. SPIE 7889, 78891J, 78891J-6 (2011). [CrossRef]

OCIS Codes
(180.2520) Microscopy : Fluorescence microscopy
(190.4180) Nonlinear optics : Multiphoton processes
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:
Microscopy

History
Original Manuscript: June 11, 2013
Revised Manuscript: July 17, 2013
Manuscript Accepted: August 12, 2013
Published: September 4, 2013

Virtual Issues
Novel Techniques in Microscopy (2013) Biomedical Optics Express

Citation
Keisuke Isobe, Hiroyuki Kawano, Akiko Kumagai, Atsushi Miyawaki, and Katsumi Midorikawa, "Implementation of spatial overlap modulation nonlinear optical microscopy using an electro-optic deflector," Biomed. Opt. Express 4, 1937-1945 (2013)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-4-10-1937


Sort:  Author  |  Year  |  Journal  |  Reset  

References

  1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990). [CrossRef] [PubMed]
  2. K. König, “Multiphoton microscopy in life sciences,” J. Microsc.200(2), 83–104 (2000). [CrossRef] [PubMed]
  3. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol.21(11), 1369–1377 (2003). [CrossRef] [PubMed]
  4. P. J. Campagnola and L. M. Loew, “Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms,” Nat. Biotechnol.21(11), 1356–1360 (2003). [CrossRef] [PubMed]
  5. J. Squier and M. Müller, “High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum.72(7), 2855–2867 (2001). [CrossRef]
  6. Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett.70(8), 922–924 (1997). [CrossRef]
  7. M. D. Duncan, J. Reintjes, and T. J. Manuccia, “Scanning coherent anti-Stokes Raman microscope,” Opt. Lett.7(8), 350–352 (1982). [CrossRef] [PubMed]
  8. A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett.82(20), 4142–4145 (1999). [CrossRef]
  9. K. Isobe, S. Kataoka, R. Murase, W. Watanabe, T. Higashi, S. Kawakami, S. Matsunaga, K. Fukui, and K. Itoh, “Stimulated parametric emission microscopy,” Opt. Express14(2), 786–793 (2006). [CrossRef] [PubMed]
  10. K. Isobe, T. Kawasumi, T. Tamaki, S. Kataoka, Y. Ozeki, and K. Itoh, “Three-dimensional profiling of refractive index distribution inside transparent materials by use of nonresonant four-wave mixing microscopy,” Appl. Phys. Express1, 022006 (2008). [CrossRef]
  11. D. Fu, T. Ye, T. E. Matthews, G. Yurtsever, and W. S. Warren, “Two-color, two-photon, and excited-state absorption microscopy,” J. Biomed. Opt.12(5), 054004 (2007). [CrossRef] [PubMed]
  12. D. Fu, T. Ye, T. E. Matthews, B. J. Chen, G. Yurtserver, and W. S. Warren, “High-resolution in vivo imaging of blood vessels without labeling,” Opt. Lett.32(18), 2641–2643 (2007). [CrossRef] [PubMed]
  13. C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science322(5909), 1857–1861 (2008). [CrossRef] [PubMed]
  14. P. Nandakumar, A. Kovalev, and A. Volkmer, “Vibrational imaging based on stimulated Raman scattering microscopy,” New J. Phys.11(3), 033026 (2009). [CrossRef]
  15. Y. Ozeki, F. Dake, S. Kajiyama, K. Fukui, and K. Itoh, “Analysis and experimental assessment of the sensitivity of stimulated Raman scattering microscopy,” Opt. Express17(5), 3651–3658 (2009). [CrossRef] [PubMed]
  16. P. Samineni, B. Li, J. W. Wilson, W. S. Warren, and M. C. Fischer, “Cross-phase modulation imaging,” Opt. Lett.37(5), 800–802 (2012). [CrossRef] [PubMed]
  17. J. W. Wilson, P. Samineni, W. S. Warren, and M. C. Fischer, “Cross-phase modulation spectral shifting: nonlinear phase contrast in a pump-probe microscope,” Biomed. Opt. Express3(5), 854–862 (2012). [CrossRef] [PubMed]
  18. P. Theer, M. T. Hasan, and W. Denk, “Two-photon imaging to a depth of 1000 μm in living brains by use of a Ti:Al2O3 regenerative amplifier,” Opt. Lett.28(12), 1022–1024 (2003). [CrossRef] [PubMed]
  19. P. Theer and W. Denk, “On the fundamental imaging-depth limit in two-photon microscopy,” J. Opt. Soc. Am. A23(12), 3139–3149 (2006). [CrossRef] [PubMed]
  20. D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong, C. B. Schaffer, and C. Xu, “Deep tissue multiphoton microscopy using longer wavelength excitation,” Opt. Express17(16), 13354–13364 (2009). [CrossRef] [PubMed]
  21. D. Kobat, N. G. Horton, and C. Xu, “In vivo two-photon microscopy to 1.6-mm depth in mouse cortex,” J. Biomed. Opt.16(10), 106014 (2011). [CrossRef] [PubMed]
  22. N. G. Horton, K. Wang, D. Kobat, C. G. Clark, F. W. Wise, C. B. Schaffer, and C. Xu, “In vivo three-photon microscopy of subcortical structures within an intact mouse brain,” Nat. Photonics7(3), 205–209 (2013). [CrossRef]
  23. G. Zhu, J. van Howe, M. Durst, W. Zipfel, and C. Xu, “Simultaneous spatial and temporal focusing of femtosecond pulses,” Opt. Express13(6), 2153–2159 (2005). [CrossRef] [PubMed]
  24. D. Oron, E. Tal, and Y. Silberberg, “Scanningless depth-resolved microscopy,” Opt. Express13(5), 1468–1476 (2005). [CrossRef] [PubMed]
  25. M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett.22(24), 1905–1907 (1997). [CrossRef] [PubMed]
  26. N. Chen, C.-H. Wong, and C. J. R. Sheppard, “Focal modulation microscopy,” Opt. Express16(23), 18764–18769 (2008). [CrossRef] [PubMed]
  27. A. Leray and J. Mertz, “Rejection of two-photon fluorescence background in thick tissue by differential aberration imaging,” Opt. Express14(22), 10565–10573 (2006). [CrossRef] [PubMed]
  28. N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods7(2), 141–147 (2010). [CrossRef] [PubMed]
  29. Z. Chen, L. Wei, X. Zhu, and W. Min, “Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores,” Opt. Express20(17), 18525–18536 (2012). [CrossRef] [PubMed]
  30. K. Isobe, A. Suda, H. Hashimoto, F. Kannari, H. Kawano, H. Mizuno, A. Miyawaki, and K. Midorikawa, “High-resolution fluorescence microscopy based on a cyclic sequential multiphoton process,” Biomed. Opt. Express1(3), 791–797 (2010). [CrossRef] [PubMed]
  31. K. Isobe, H. Kawano, T. Takeda, A. Suda, A. Kumagai, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Background-free deep imaging by spatial overlap modulation nonlinear optical microscopy,” Biomed. Opt. Express3(7), 1594–1608 (2012).K. Isobe, H. Kawano, T. Takeda, A. Suda, A. Kumagai, H. Mizuno, A. Miyawaki, and K. Midorikawa, “Spatial overlap modulation nonlinear optical microscopy,” in Conference on Lasers and Electro-Optics 2012, OSA Technical Digest (online) (Optical Society of America, 2012), paper JW3G.4. [CrossRef] [PubMed]
  32. J. Miyazu, T. Imai, S. Toyoda, M. Sasaura, S. Yagi, K. Kato, Y. Sasaki, and K. Fujiura, “New beam scanning model for high-speed operation using KTa1-xNbxO3 Crystals,” Appl. Phys. Express4(11), 111501 (2011). [CrossRef]
  33. S. Yagi, K. Naganuma, T. Imai, Y. Shibata, S. Ishibashi, Y. Sasaki, M. Sasaura, K. Fujiura, and K. Kato, “A mechanical-free 150-kHz repetition swept light source incorporated a KTN electro-optic deflector,” Proc. SPIE7889, 78891J, 78891J-6 (2011). [CrossRef]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

Figures

Fig. 1 Fig. 2 Fig. 3
 
Fig. 4 Fig. 5
 

Supplementary Material


» Media 1: AVI (17307 KB)     

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited