Abstract

We describe a novel second harmonic generation (SHG) microscope that employs heterodyne detection by interfering the epidirected SHG from a sample with SHG from a reference crystal. In addition, the microscope provides complementary reflectance information based on optical coherence microscopy (OCM). The instrument features dual balanced detection to minimize the effect of source fluctuations, and polarization-sensitive detection to measure the nonlinear susceptibility of the sample. Interferometric detection can potentially improve the sensitivity and thus extend the imaging depth as compared with direct detection of SHG.

© 2004 Optical Society of America

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References

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Annu. Rev. Biomed. Eng.

P.T.C. So, C.Y. Dong, B.R. Masters and K.M.Berland,"Two-photon excitation fluorescence microscopy,�?? Annu. Rev. Biomed. Eng. 2, 399-429 (2000)
[CrossRef]

Appl. Phys. B

R. Stolle, G. Marowsky, E. Schwarzberg and G. Berkovic, "Phase measurements in nonlinear optics," Appl. Phys. B 63, 491-498 (1996)

Biophys J

P. J. Campagnola, M.-D. Wei, A. Lewis and L. M. Loew, "High-resolution nonlinear optical imaging of live cells by second harmonic generation," Biophys J. 77, 3341-3349 (1999)
[CrossRef] [PubMed]

Biophys. J.

I. Freund, M. Deutsch and A. Sprecher, "Connective tissue polarity: optical second-harmonic micrscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon," Biophys. J. 50, 693-712 (1986)
[CrossRef] [PubMed]

Chem. Phys. Lett.

K. Kemnitz, K. Bhattacharyya, J. M. Hicks, G. R. Pinto and K. B. Eisenthal, "The phase of second-harmonic light generated at an interface and its relation to absolute molecular orientation," Chem. Phys. Lett. 131, 285-290 (1986)
[CrossRef]

Chem. Rev.

R. M. Corn and D. A. Higgins, "Optical second harmonic generation as a probe of surface chemistry," Chem. Rev. 94, 107-125 (1994)
[CrossRef]

J. Opt. Soc. Am. B

Nature

Y. R. Shen, "Surface properties probed by second-harmonic and sum-frequency generation," Nature 337, 519-525 (1989)
[CrossRef]

Opt. Express

Opt. Lett.

Phys. Rev. Lett.

R. K. Chang, J. Ducuing and N. Bloembergen, "Relative phase measurement between fundamental and second-harmonic light," Phys. Rev. Lett. 15, 6-8 (1965)
[CrossRef]

Phys. Solid State

Y. Uesu and N. Kato, "Multi-purpose nonlinear optical microscope it's principles and applications to polar thin-film observation," Phys. Solid State 41, 688-692 (1999)
[CrossRef]

Proc. Nat. Acad. Sci.

A. Zoumi, A. Yeh and B. J. Tromberg, "Imaging cells and extra cellular matrix in vivo by using second harmonic generation and two-photon excited fluorescence," Proc. Nat. Acad. Sci. USA 99, 11014-11019 (2002)
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman and W. W. Webb, "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," Proc. Nat. Acad. Sci. USA 100, 7075-7080 (2003)
[CrossRef] [PubMed]

Science

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito and J. G. Fujimoto, "Optical coherence tomography," Science 254, 1178-1181 (1991)
[CrossRef] [PubMed]

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Figures (6)

Fig. 1.
Fig. 1.

Schematic of interferometric second harmonic generation (SHG) microscope. The fundamental wavelength is shown in black and the second harmonic is shown in gray. BS1-BS3, 50/50 beam splitters; λ/2, half-wave plate; PD, photodiode; PMT, photo-multiplier tube; LBO, lithium triborate crystal; GRIN, graded-index collimating lens; BPF, bandpass filter; NA, numerical aperture; TIA, transimpedance amplifier, A/D, analog-to-digital conversion.

Fig. 2.
Fig. 2.

Interferometric detector output as a function of reference arm position for the (a) OCM and (b) SHG channels, using a 1 mm thick BBO crystal as the sample.

Fig. 3.
Fig. 3.

Detail of interferometric detector outputs indicates a fringe spacing of 400 nm for the OCM channel and 200 nm for the ISHG channel.

Fig. 4.
Fig. 4.

(a) Peak ISHG amplitude as a function of power incident on the sample, indicating the quadratic nature of the nonlinear process. The OCM signal amplitude scaled linearly with incident power (not shown.) (b) Variation of ISHG signal as a function of the entrance waveplate angle ψ, and cos2 (2ψ) fit.

Fig. 5.
Fig. 5.

Simultaneous OCM (left) and ISHG (right) images from BBO microcrystals at two different depths. The image area is 100×100 µm2, containing 128×128 pixels. The signal at each pixel is the average amplitude over a 5µm depth. Inset: 40×40 µm2 image of a single microcrystal fragment shows greater detail. The bright background in the bottom OCM image resulted from focusing near the glass slide.

Fig. 6.
Fig. 6.

Polarization sensitive detection in ISHG microscopy. The polarization state of the reference arm was held perpendicular (⊥)and parallel (‖) to the incident wave. A single lateral scan through both images reveals structural detail of the crystals and may potentially be used for mapping the complex nonlinear susceptibility of the sample. Arrows indicate a region polarized along one of the axes, with high extinction in the orthogonal axis.

Equations (3)

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P = ε 0 ( χ ( 1 ) E + χ ( 2 ) EE * + )
i dn E rn E sn cos ( 2 k n δ l )
i d β χ r ( 2 ) χ s ( 2 ) I 0 2 cos ( 2 k 2 δ l )

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