OSA's Digital Library

Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 2, Iss. 10 — Oct. 1, 2011
  • pp: 2837–2849
« Show journal navigation

Combined third-harmonic generation and four-wave mixing microscopy of tissues and embryos

Pierre Mahou, Nicolas Olivier, Guillaume Labroille, Louise Duloquin, Jean-Marc Sintes, Nadine Peyriéras, Renaud Legouis, Delphine Débarre, and Emmanuel Beaurepaire  »View Author Affiliations


Biomedical Optics Express, Vol. 2, Issue 10, pp. 2837-2849 (2011)
http://dx.doi.org/10.1364/BOE.2.002837


View Full Text Article

Acrobat PDF (2119 KB)





Browse Journals / Lookup Meetings

Browse by Journal and Year


   


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools

Share
Citations

Abstract

Nonlinear microscopy can be used to probe the intrinsic optical properties of biological tissues. Using femtosecond pulses, third-harmonic generation (THG) and four-wave mixing (FWM) signals can be efficiently produced and detected simultaneously. Both signals probe a similar parameter, i.e. the real part of the third-order nonlinear susceptibility χ(3). However THG and FWM images result from different phase matching conditions and provide complementary information. We analyze this complementarity using calculations, z-scan measurements on water and oils, and THG-FWM imaging of cell divisions in live zebrafish embryos. The two signals exhibit different sensitivity to sample size and clustering in the half-wavelength regime. Far from resonance, THG images reveal spatial variations |Δχ(3)(−3ω;ω,ω,ω)| with remarkable sensitivity while FWM directly reflects the distribution of χ(3)(−2ω1 + ω2;ω1, –ω2, ω1). We show that FWM images provide χ(3) maps useful for proper interpretation of cellular THG signals, and that combined imaging carries additional structural information. Finally we present simultaneous imaging of intrinsic THG, FWM, second-harmonic (SHG) and two-photon-excited fluorescence (2PEF) signals in live Caenorhabditis elegans worms illustrating the information provided by multimodal nonlinear imaging of unstained tissue.

© 2011 OSA

1. Introduction

Nonlinear (or multiphoton) microscopy is an effective method for obtaining 3D-resolved images of biological tissues, and two-photon-excited fluorescence (2PEF) microscopy has found a broad range of applications in the life sciences [1

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

]. In addition to 2PEF imaging of exogenous labels and fluorescent proteins, multiphoton microscopy can be used to probe the intrinsic nonlinear optical properties of tissues. A growing literature shows that physiologically and/or structurally relevant information is obtained through the detection of coherent signals such as second-harmonic generation (SHG) [2

2. W. Mohler, A. C. Millard, and P. J. Campagnola, “Second harmonic imaging of endogenous structural proteins,” Methods 29, 97–109 (2003) [CrossRef] [PubMed]

], third-harmonic generation (THG) [3

3. D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, “Depth-resolved structural imaging by third-harmonic generation microscopy,” J. Struct. Biol. 147, 3–11 (2004). [CrossRef] [PubMed]

, 4

4. C. K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, and H.-J. Tsai, “Higher harmonic generation microscopy for developmental biology,” J. Struct. Biol. 147, 19–30 (2004). [CrossRef] [PubMed]

, 5

5. N. Olivier, M. Luengo-Oroz, L. Duloquin, E. Faure, T. Savy, I. Veilleux, X. Solinas, D. Débarre, P. Bourgine, A. Santos, N. Peyriéras, and E. Beaurepaire, “Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy,” Science 339, 967–71 (2010). [CrossRef]

], or four-wave mixing (FWM) processes [6

6. Y. Wang, C.-Y. Lin, A. Nikolaenko, V. Raghunathan, and E. O. Potma, “Four-wave mixing microscopy of nanostructures,” Advances in optics and photonics 3, 1–52 (2011). [CrossRef]

] such as coherent Raman scattering (CARS, etc) [7

7. J.-X. Cheng and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications,” J. Phys. Chem. B 108, 827–840 (2004). [CrossRef]

, 8

8. A. Volkmer, “Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering microscopy,” J. Phys. D: Appl. Phys. 38, R59–R81 (2005). [CrossRef]

] or stimulated parametric emission (SPE) [9

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, 786–793 (2006). [CrossRef] [PubMed]

]. In particular, THG imaging detects spatial variations of the electronic part of the third-order nonlinear susceptibility χ(3)(–3ω;ω,ω,ω) [10

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

, 11

11. M. Müller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998). [CrossRef] [PubMed]

], such as lipid inclusions in an aqueous environment [12

12. D. Débarre and E. Beaurepaire, “Quantitative characterization of biological liquids for third-harmonic generation microscopy,” Biophys. J. 92, 603–612 (2007). [CrossRef]

], and has proven useful for embryo and tissue imaging applications [3

3. D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, “Depth-resolved structural imaging by third-harmonic generation microscopy,” J. Struct. Biol. 147, 3–11 (2004). [CrossRef] [PubMed]

, 4

4. C. K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, and H.-J. Tsai, “Higher harmonic generation microscopy for developmental biology,” J. Struct. Biol. 147, 19–30 (2004). [CrossRef] [PubMed]

, 5

5. N. Olivier, M. Luengo-Oroz, L. Duloquin, E. Faure, T. Savy, I. Veilleux, X. Solinas, D. Débarre, P. Bourgine, A. Santos, N. Peyriéras, and E. Beaurepaire, “Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy,” Science 339, 967–71 (2010). [CrossRef]

, 13

13. D. Débarre, W. Supatto, E. Farge, B. Moulia, M. -C. Schanne-Klein, and E. Beaurepaire, “Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos,” Opt. Lett. 29, 2881 (2004). [CrossRef]

, 14

14. D. Débarre, W. Supatto, A.-M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M.-C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3, 47–53 (2006). [CrossRef]

]. When experimentally available, the combination of different nonlinear contrasts usually provides complementary information. Using pulsed excitation provided by a femtosecond Ti:sapphire oscillator and a synchronously pumped optical parametric oscillator (OPO), THG and four-wave mixing (FWM) signals can in principle be efficiently produced and detected simultaneously. In isotropic media exhibiting no electronic nor vibrational resonance, THG and FWM can be viewed as both probing a similar parameter, i.e. the real part of χ(3). However THG and FWM signal levels result from different phase matching conditions. In contrast with FWM, TH coherent signal buildup in the forward direction is frustrated by the Gouy phase shift of the focused excitation beam, resulting in no THG from a homogeneous medium having normal dispersion [10

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

, 15

15. R. W. Boyd, Nonlinear Optics, 2nd ed. (Academic, 2003).

]. Non-resonant FWM has received relatively little attention for nonlinear microscopy applications, apart for being considered as an unwanted “non-resonant background” in CARS microscopy. However, given their different dependence on sample size, combined THG and FWM imaging of the same objects should provide more structural information than either modality alone. In this study we first discuss the contrast mechanisms of THG and FWM imaging as a function of sample size and geometry and we then present imaging examples from model liquids and live tissues. We discuss the properties of the images and some implications for THG and CARS microscopy. THG and FWM are shown to provide complementary information on χ(3) variations, compatible with other femtosecond laser-based imaging modalities.

2. THG and FWM contrast mechanisms

2.1. Excitation fields near focus

Throughout this paper, we assume that the excitation beams entering the objective are Gaussian with linear polarization along the x axis. In this case the vectorial nature of the focused fields can be neglected for parametric processes in isotropic media [16

16. J.-X. Cheng and X. S. Xie, “Green’s function formulation for third harmonic generation microscopy,” J. Opt. Soc. Am. B 19, 1604–1610 (2002). [CrossRef]

, 25

25. J.-X. Cheng, A. Volkmer, and X. S. Xie, “Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy,” J. Opt. Soc. Am. B 19, 1663–1675 (2002). [CrossRef]

, 17

17. N. Olivier and E. Beaurepaire, “Third-harmonic generation microscopy with focus-engineered beams: a numerical study,” Opt. Express , 16(19), 14703–14715 (2008). [CrossRef] [PubMed]

]. The x component of the field near focus can be expressed as [19

19. L. Novotny and B. Hecht, Principles of nano-optics (Cambridge Univ Press, 2006).

]:
Ef(ρ,ϕ,z)=02π0θmaxexp(sin2θf02)sinθcos(θ)[cosθ+sin2ψ(1cosθ)]exp(ikωρsinθcos(ψϕ))exp(ikωzcosθ).dθ.dψ
(1)
where n(ω) (resp. kω) is the index of refraction (resp. wave vector) at the fundamental wavelength, θmax = arcsin(NA/n(ω)), with NA being the numerical aperture of the objective, and f0 is the filling factor of the objective (ratio between the beam waist and the diameter of the back aperture).

2.2. THG-FWM third-order polarization and nonlinear susceptibility

The third-order polarizations induced by the focused fields are described by:
P(THG)=χ(3)(3ω;ω,ω,ω)EEE
(2)
P(FWM)=χ(3)((2ω1ω2);ω1,ω2,ω1)E1E2*E1
(3)
where 1 and 2 denote the two excitation fields in the case of FWM.

For isotropic media the THG and FWM nonlinear susceptiblities can be written as [7

7. J.-X. Cheng and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications,” J. Phys. Chem. B 108, 827–840 (2004). [CrossRef]

, 8

8. A. Volkmer, “Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering microscopy,” J. Phys. D: Appl. Phys. 38, R59–R81 (2005). [CrossRef]

, 15

15. R. W. Boyd, Nonlinear Optics, 2nd ed. (Academic, 2003).

]:
χijkl(3)(3ω;ω,ω,ω)=χ1111(THG)(δijδkl+δikδjl+δilδjk)
(4)
χijkl(3)((2ω1ω2);ω1,ω2,ω1)=χ1111(FWM)(δijδkl+δikδjl)+χ1221(FWM)(δilδjk)
(5)

Several semiempirical models have been proposed for relating the nonresonant component of χ(3) to linear indices [15

15. R. W. Boyd, Nonlinear Optics, 2nd ed. (Academic, 2003).

, 22

22. C. Wang, “Empirical relation between the linear and the thord-order nonlinar optical susceptibilities,” Physssss; Rev. B 2, 2045–2048 (1970). [CrossRef]

]. A generalized version of Miller’s rule valid for some materials [15

15. R. W. Boyd, Nonlinear Optics, 2nd ed. (Academic, 2003).

] proposes that χ(3)(ω4,ω3,ω2,ω1) ≈ n(ω4)n(ω3)n(ω2)n(ω1), so that χ(THG)n(3ω)n(ω)3 and χ(FWM)n(ω)4. Far from resonance, THG and FWM can therefore be expected to probe a similar parameter χ(3)nr. However it is in practice uncommon to find materials that are transparent (i.e. non-resonant) for the frequency range involved in the processes considered here, i.e. encompassing ω and 3ω.

2.3. Propagation of the harmonic fields and signal creation

Once the polarization distribution near focus has been calculated, fields scattered from all positions r in the focal region are propagated to a position R in the collection optics aperture:
EFF(R)=VP(r)GFF(Rr)dV
(10)
where V spans the excitation volume and GFF is the far-field Green’s function
GFF(R)=exp(ikR)4πR[IRR/R2]
(11)
where R = |R| and I is the third-order identity tensor. Finally the total THG/FWM intensity scattered in the forward direction is estimated by integrating |EFF (R)|2 over the front aperture of a transmission-collecting objective.

2.4. Phase-matching and structure sensitivity of THG and FWM

Fig. 1 Effect of phase matching and geometry on THG-FWM contrast. (a) Calculation of the THG and FWM intensity obtained when scanning a focused Gaussian beam across a transverse interface between vacuum and an isotropic nonlinear medium. The THG intensity is highest when the interface is in focus (z = 0). (b) Calculation of the THG and FWM intensity obtained from a three-dimensional checkerboard-like medium as a function of granularity. This geometry corresponds to a constant average value of < χ(3) > inside the excitation volume, with variable sub-wavelength spatial distribution. In this configuration, THG and FWM exhibit different sensitivities to sample granularity in the half-wavelength regime. The dashed red line on the THG graph indicates the signal level for a transverse interface geometry with similar < χ(3) >, shown for comparison. Simulation conditions: NA = 1.2, Excitation with λ1 = 1.2μm and λ2 = 0.8μm

The case of a structured sample is more intriguing. In the geometry considered here, the average value of χ3 inside the excitation volume is constant and the sample characteristic size varies from λ/20 to 2.5λ. It is seen that FWM does not depend on sample granularity, whereas THG intensity exhibits significant variation for structure sizes in the range λ/3 – λ, and almost no change outside this range. This can be interpreted as follows. For granularity elc,eff(THG) the medium may be seen as homogeneous for the THG process, and therefore THG → 0 and FWM ∝ < χ(3) >2. In contrast for cube sizes in the range λ/3 – λ the medium is not homogeneous at the scale of the THG coherence length, and THG is observed due to partly non-destructive interferences, whereas < χ(3) > and FWM are unchanged. Finally for e >> λ the sample geometry resembles a corner interface with the same average < χ(3) >, and constant THG and FWM signals are predicted. Although the signals can also depend on sample position, these simulations indicate that the association of THG and FWM microscopy may provide more information on the sub-wavelength χ(3) spatial distribution than either modality alone, and motivates this combination.

3. Experimental results

3.1. Experimental setup

THG and FWM are third-order electronic processes, and can be efficiently produced and detected simultaneously using femtosecond pulses. The experimental setup is depicted in Fig. 2. Excitation pulses were provided by a titanium:sapphire (TiS) oscillator (80MHz, Chameleon, Coherent) and by a KTP-based synchronously pumped optical parametric oscillator (OPO) (APE). TiS and OPO pulses were overlapped using a dichroic mirror (Chroma 1000dcxr) and synchronized in the sample using a delay line. Imaging was performed on a lab-built microscope incorporating galvanometer mirrors (GSI Lumonics), photomultiplier tubes (PMT, SensTech), counting electronics, and water immersion objectives (Olympus). TiS and OPO beam polarizations in the microscope were linear and co-aligned. The objectives used for excitation were a 60× 1.2NA coverslip-corrected and a 20× 0.95NA with its pupil underfilled (approximately 0.75 effective excitation NA). For the experiments presented in this study, TiS and OPO pulse durations at the sample were approximately 250fs. Coherent scattering (THG, FWM, and optionally SHG) was detected in the forward direction and selected with dichroics and filters (Semrock).

Fig. 2 Experimental setup. TiS: femtosecond titanium:sapphire laser. OPO: optical parametric oscillator. X-Y: beam scanning system. In this study coherent signals (FWM, THG, SHG) are detected in the transmitted direction and 2PEF signals are epidetected.

3.2. Nonlinear susceptibility measurement of isotropic media with THG and FWM

We performed z-scan measurements to evaluate χ(THG) and χ(FWM) for several isotropic media. We analyzed three types of liquids: water, chlorotrifluoroethylene (Voltalef® immersion oil) and triolein (plant oil). For the measurements, the liquid was sealed between two microscope coverslips (see Fig. 3). THG and FWM radiations were separated using a dichroic mirror and detected simultaneously on two detectors. Shortpass filters (Semrock) were used to isolate the signals from scattered laser noise.

Fig. 3 Effect of χ(3) on THG-FWM contrast. (a) z-scan geometry used for estimating χ(THG) and χ(FWM) of various liquids relative to glass. The focus of a coverslip-corrected objective is scanned through a water-glass and a glass-liquid interface. (b) Top, THG (left) and FWM (right) z-scan profiles for different liquids (immersion oil, water, plant oil). The displayed profiles are normalized to their values at the first interface. Bottom, estimated values of χ(THG) (left) and χ(FWM) (right) relative to glass (see text), for different OPO wavelengths and TiS-OPO frequency differences. Deviation of the water χ(FWM) curve near 3600cm−1 is due to the presence of CARS at this frequency shift. It can be seen that χwater(3)<χoil(3) in all the conditions explored here. (c) Simultaneously recorded images of immersion oil droplets in water for λOPO = 1140nm and ωTiSωOPO = 3890cm−1. Water/immersion oil interfaces are visible in the THG images, and immersion oil produces a stronger FWM signal than water. Scale bar: 50μm.

In the absence of resonance, the value of χ(FWM) of a pure liquid relative to glass can be estimated from a z-scan profile such as the ones displayed in Fig. 3 using the relation:
χliq(FWM)χglass(FWM)=IliqInoiseIglassInoise
(12)
where Iliq (resp. Iglass) is the FWM intensity measured in liquid (resp. glass) just after (resp. before) the glass-liquid interface, and Inoise is noise principally due to imperfectly rejected TiS light, measured in air and corrected by the transmission of the setup. Measuring Iliq and Iglass close to the interface minimizes aberration variations between different experiments and the subsequent measurement errors [23

23. R. S. Pillai, G. J. Brakenhoff, and M. Müller, “Analysis of the influence of spherical aberration from focusing through a dielectric slab in quantitative nonlinear optical susceptibility measurements using third-harmonic generation,” Opt. Express 14, 260–269 (2006). [CrossRef] [PubMed]

].

The THG χ(3) of a liquid relative to glass can be estimated from two z-scan experiments: (i) a water-glass-liquid scan as shown in Fig. 3, and (ii) a water-glass-air reference scan (not shown). Following [20

20. G. Clay, A. Millard, C. Schaffer, J. Aus-der-Au, P. Tsai, J. Squier, and D. Kleinfeld, “Spectroscopy of third-harmonic generation: evidence for resonances in model compounds and ligated hemoglobin,” J. Opt. Soc. Am. B 23, 932–950 (2006). [CrossRef]

, 24

24. R. Barille, L. Canioni, L. Sarger, and G. Rivoire, “Nonlinearity measurements of thin films by third-harmonic generation microscopy,” Phys. Rev. E 66, 067602 (2002). [CrossRef]

], the χ(3) relative to glass can be estimated as:
χliq(THG)χglass(THG)=(1±Iglass/liqIglass/air×C)bliqJ(bliqΔkliq)bglassJ(bglassΔkglass)
(13)
where b is the confocal parameter: b=2n(ω)λπ(n(ω)2NA2NA2),

J is the phase matching integral: J(bΔk)=0+exp(ibΔkϕ)(1+2iϕ)2dϕ,

Δk = 3kωk3ω is the phase mismatch, and C is a normalization factor that takes into account power fluctuation and transmission changes between the first and second z-scan. Iglass/liq and Iglass/air are the THG signals measured at the glass/liquid and glass/air interface in the first and second z-scan. Under moderate numerical aperture excitation, the above equation can simplified as follows:
χliq(THG)χglass(THG)=(1±Iglass/liqIglass/air×C)
(14)

As seen from this expression an ambiguity remains in the determination of χ(THG) due to the two possible signs. This ambiguity was resolved using additional relative measurements at the interfaces between the different liquids. [12

12. D. Débarre and E. Beaurepaire, “Quantitative characterization of biological liquids for third-harmonic generation microscopy,” Biophys. J. 92, 603–612 (2007). [CrossRef]

, 20

20. G. Clay, A. Millard, C. Schaffer, J. Aus-der-Au, P. Tsai, J. Squier, and D. Kleinfeld, “Spectroscopy of third-harmonic generation: evidence for resonances in model compounds and ligated hemoglobin,” J. Opt. Soc. Am. B 23, 932–950 (2006). [CrossRef]

]

We estimated χ(FWM) as a function of the frequency difference between the Ti:S and the OPO for frequency shifts ranging from 3550cm−1 to 3890cm−1, and χ(THG) as a function of the OPO wavelength between 1140nm and 1200nm. The measured values relative to glass are displayed in Fig. 3.

These experiments indicate that χ(THG) and χ(FWM) have comparable values, and that their relative values for the materials considered here have similar relative order, i.e. χwater(3)<χimmersionoil(3)<χglass(3)<χplantoil(3) holds true for both processes.

3.3. THG-FWM microscopy of a zebrafish embryo during early divisions

THG microscopy has proven effective for studying cell divisions in the early zebrafish embryo [5

5. N. Olivier, M. Luengo-Oroz, L. Duloquin, E. Faure, T. Savy, I. Veilleux, X. Solinas, D. Débarre, P. Bourgine, A. Santos, N. Peyriéras, and E. Beaurepaire, “Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy,” Science 339, 967–71 (2010). [CrossRef]

]. In particular, THG images highlight the boundaries of cells, nuclei, and yolk platelets with remarkable contrast [4

4. C. K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, and H.-J. Tsai, “Higher harmonic generation microscopy for developmental biology,” J. Struct. Biol. 147, 19–30 (2004). [CrossRef] [PubMed]

, 5

5. N. Olivier, M. Luengo-Oroz, L. Duloquin, E. Faure, T. Savy, I. Veilleux, X. Solinas, D. Débarre, P. Bourgine, A. Santos, N. Peyriéras, and E. Beaurepaire, “Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy,” Science 339, 967–71 (2010). [CrossRef]

]. However the interpretation of these images is not immediate, since a strong THG signal can originate from different sample geometries (interfaces or inclusions), and from either a local increase or a local decrease in χ(3). To gain insight into the origin of these THG signals, we recorded time-lapse THG-FWM images of zebrafish embryos during early cell divisions. The embryos were mechanically dechorionated, and maintained in the observation chamber using low-density agarose as described in [4

4. C. K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, and H.-J. Tsai, “Higher harmonic generation microscopy for developmental biology,” J. Struct. Biol. 147, 19–30 (2004). [CrossRef] [PubMed]

, 5

5. N. Olivier, M. Luengo-Oroz, L. Duloquin, E. Faure, T. Savy, I. Veilleux, X. Solinas, D. Débarre, P. Bourgine, A. Santos, N. Peyriéras, and E. Beaurepaire, “Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy,” Science 339, 967–71 (2010). [CrossRef]

]. Representative images are shown in Fig. 4.

Fig. 4 THG-FWM microscopy of a zebrafish embryo during early divisions. (a) Imaging geometry. (b) Simultaneous THG and FWM images showing the different contrasts. THG highlights the spatial variations Δχ(3). FWM reveals χ(3) distribution, which is not homogeneous. THG contrast from cells, nuclei contours, vesicles, and yolk structures can be understood from the FWM image. (c,d, and Media 1) Dividing cells before (c) and after (d) disruption of the nuclear envelope. THG and FWM images provide different information about the structural reorganisation of the cytoplasm and the nucleus (see text). Pixel accumulation time: 5μs. Scale bars: 30μm.

Another observation is that THG and FWM images are significantly different. As discussed above, THG images highlight the spatial variations Δχ(3), and FWM can be qualitatively used as a χ(3) map to interpret THG images. In the zebrafish embryo during early divisions, THG signals produce contrasted images of cell contours. Such a contrast could result either from a χ(3) difference between adjacent cells, or from the presence of a large inter-cellular space at this stage of development. The latter interpretation was proposed in [5

5. N. Olivier, M. Luengo-Oroz, L. Duloquin, E. Faure, T. Savy, I. Veilleux, X. Solinas, D. Débarre, P. Bourgine, A. Santos, N. Peyriéras, and E. Beaurepaire, “Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy,” Science 339, 967–71 (2010). [CrossRef]

] and is here confirmed by combined THG-FWM imaging: a local FWM signal decrease is consistently detected around cells (figure 4b). This indicates that the cytoplasm has higher χ(3) than the extracellular medium, and that it is the inter-cellular spacing rather than optical differences between cells or a local lipid accumulation near membranes which is responsible from the high THG signal around cells. //

Signals in the yolk region are also remarkable. In the early zebrafish embryo, the vitelline stores form a foam-like arrangement of packed yolk globules (or platelets) separated by canaliculi filled with ooplasm (see e.g. [26

26. R. Fuentes and J. Fernández, “Ooplasmic segregation in the zebrafish zygote and early embryo: pattern of ooplasmic movements and transport pathways,” Dev. Dyn. 2392172–2189 (2010). [CrossRef] [PubMed]

]). The yolk generally produces a strong FWM signal, and the contours of the yolk globules are readily visible in THG microscopy images. From the ratio between xy and xz oriented yolk interfaces in THG images [5

5. N. Olivier, M. Luengo-Oroz, L. Duloquin, E. Faure, T. Savy, I. Veilleux, X. Solinas, D. Débarre, P. Bourgine, A. Santos, N. Peyriéras, and E. Beaurepaire, “Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy,” Science 339, 967–71 (2010). [CrossRef]

], the spacing (ooplasm) between globules can be estimated to be less than 500nm wide, in agreement with morphological observations [26

26. R. Fuentes and J. Fernández, “Ooplasmic segregation in the zebrafish zygote and early embryo: pattern of ooplasmic movements and transport pathways,” Dev. Dyn. 2392172–2189 (2010). [CrossRef] [PubMed]

]. Since signal in coherent nonlinear microscopy scales as the squared volume of the probed structure, this small size explains why these contours do not clearly emerge against the strong coherent signal from the yolk globules in the FWM images (figure 4b). This observation again illustrates the complementarity of the two images. THG exhibits superior sensitivity to χ(3) spatial variations, but images must be interpreted with care (different geometries and different Δχ(3) signs give the same signal), while FWM signal level provide clearer information about the geometry and media being observed. //

3.4. Label-free multimodal THG-FWM-SHG-2PEF imaging of live Caenorhabditis elegans worms

In addition to THG and FWM, other nonlinear signals can be produced with femtosecond pulses in biological tissues. We recorded simultaneous THG-FWM-SHG-2PEF images of live unlabeled C. elegans worms, shown in Fig. 5. Young adult worms from the wild type strain N2 were transferred to a microscope slide coated with a thin layer of agarose gel and were immobilized using sodium azide and levamisole. Coherent THG, FWM, and SHG signals were detected in the forward direction and separated with two dichroics, while 2PEF was epidetected. Signals were detected using standard pixel accumulation times, i.e. 5–10μs. We note that coherent nonlinear signals (THG, SHG, FWM) are emitted over spectrally separated narrow bandwidths (a few nanometers), so that independent detection of multiple coherent signals is relatively simpler than multicolor fluorescence detection.

Fig. 5 Simultaneous THG-FWM-SHG-2PEF imaging of live unstained C. elegans worms. The four signals were detected simultaneously from the pharynx (a–e) and the midbody (f–j) of young adults. THG/FWM provide complementary morphological information from the pharynx, intestinal cells, embryos, and lipid stores. Additionally, SHG reveals pharynx, vulva, and crawling muscles, and 2PEF reveals fluorescent gut vesicles. pha: pharynx; int: intestine; bwm: body wall muscles; emb: embryo; vul: vulva; vm: vulva muscles. Scale bars: 20μm. See also Media 2.

4. Discussion

Far from resonance, THG and FWM probe a similar parameter (the real part of the third-order nonlinear susceptibility, even if χ(THG) and χ(FWM) are generally different). However the two signals result from different phase-matching conditions and therefore provide different information. Practical implementation of THG imaging is much simpler because it requires a single excitation beam and no spatio-temporal synchronization. As such, it is easily combined with e.g. SHG imaging. Moreover, THG highlights heterogeneities and produce contrasted, background-free images amenable to algorithmic analysis [5

5. N. Olivier, M. Luengo-Oroz, L. Duloquin, E. Faure, T. Savy, I. Veilleux, X. Solinas, D. Débarre, P. Bourgine, A. Santos, N. Peyriéras, and E. Beaurepaire, “Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy,” Science 339, 967–71 (2010). [CrossRef]

]. However THG image interpretation is complex, so that when working on a new application, THG contrast often needs to be characterized. FWM images provide χ(3) maps and are therefore useful for THG image interpretation. Moreover, we have shown that combined THG-FWM imaging has the potential to provide additional size information in the sub-wavelength range.

We also point out that wave-mixing processes such as FWM are sensitive to chromatic aberration induced by the microscope and the sample. Any mismatch between the foci results in efficiency drop and dark artefacts in the images - a difficulty common to imaging techniques combining two laser beams.

Our experiments show that, despite these technical difficulties, efficient multimodal imaging combining simultaneous 2PEF, THG, FWM and SHG signals is possible in live organisms, and illustrate the complementary information provided by these modalities.

Acknowledgments

We thank M. Zimmerley, M. Joffre, M.-C. Schanne-Klein, W. Supatto and J.-L. Martin for discussions and comments. This work was supported by Fondation Louis D de l’Institut de France, by Agence Nationale de la Recherche (ANR-RIB) and by ERC COST Action MP0603.

References and links

1.

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

2.

W. Mohler, A. C. Millard, and P. J. Campagnola, “Second harmonic imaging of endogenous structural proteins,” Methods 29, 97–109 (2003) [CrossRef] [PubMed]

3.

D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, “Depth-resolved structural imaging by third-harmonic generation microscopy,” J. Struct. Biol. 147, 3–11 (2004). [CrossRef] [PubMed]

4.

C. K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, and H.-J. Tsai, “Higher harmonic generation microscopy for developmental biology,” J. Struct. Biol. 147, 19–30 (2004). [CrossRef] [PubMed]

5.

N. Olivier, M. Luengo-Oroz, L. Duloquin, E. Faure, T. Savy, I. Veilleux, X. Solinas, D. Débarre, P. Bourgine, A. Santos, N. Peyriéras, and E. Beaurepaire, “Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy,” Science 339, 967–71 (2010). [CrossRef]

6.

Y. Wang, C.-Y. Lin, A. Nikolaenko, V. Raghunathan, and E. O. Potma, “Four-wave mixing microscopy of nanostructures,” Advances in optics and photonics 3, 1–52 (2011). [CrossRef]

7.

J.-X. Cheng and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications,” J. Phys. Chem. B 108, 827–840 (2004). [CrossRef]

8.

A. Volkmer, “Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering microscopy,” J. Phys. D: Appl. Phys. 38, R59–R81 (2005). [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, 786–793 (2006). [CrossRef] [PubMed]

10.

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

11.

M. Müller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc. 191, 266–274 (1998). [CrossRef] [PubMed]

12.

D. Débarre and E. Beaurepaire, “Quantitative characterization of biological liquids for third-harmonic generation microscopy,” Biophys. J. 92, 603–612 (2007). [CrossRef]

13.

D. Débarre, W. Supatto, E. Farge, B. Moulia, M. -C. Schanne-Klein, and E. Beaurepaire, “Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos,” Opt. Lett. 29, 2881 (2004). [CrossRef]

14.

D. Débarre, W. Supatto, A.-M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M.-C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3, 47–53 (2006). [CrossRef]

15.

R. W. Boyd, Nonlinear Optics, 2nd ed. (Academic, 2003).

16.

J.-X. Cheng and X. S. Xie, “Green’s function formulation for third harmonic generation microscopy,” J. Opt. Soc. Am. B 19, 1604–1610 (2002). [CrossRef]

17.

N. Olivier and E. Beaurepaire, “Third-harmonic generation microscopy with focus-engineered beams: a numerical study,” Opt. Express , 16(19), 14703–14715 (2008). [CrossRef] [PubMed]

18.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanetic system.,” Proc. Royal Soc. A 253, 358–379 (1959). [CrossRef]

19.

L. Novotny and B. Hecht, Principles of nano-optics (Cambridge Univ Press, 2006).

20.

G. Clay, A. Millard, C. Schaffer, J. Aus-der-Au, P. Tsai, J. Squier, and D. Kleinfeld, “Spectroscopy of third-harmonic generation: evidence for resonances in model compounds and ligated hemoglobin,” J. Opt. Soc. Am. B 23, 932–950 (2006). [CrossRef]

21.

X. Liu, W. Rudolph, and J. L. Thomas, “Characterization and application of femtosecond infrared stimulated parametric emission microscopy,” J. Opt. Soc. Am. B 27, 787–795 (2010). [CrossRef]

22.

C. Wang, “Empirical relation between the linear and the thord-order nonlinar optical susceptibilities,” Physssss; Rev. B 2, 2045–2048 (1970). [CrossRef]

23.

R. S. Pillai, G. J. Brakenhoff, and M. Müller, “Analysis of the influence of spherical aberration from focusing through a dielectric slab in quantitative nonlinear optical susceptibility measurements using third-harmonic generation,” Opt. Express 14, 260–269 (2006). [CrossRef] [PubMed]

24.

R. Barille, L. Canioni, L. Sarger, and G. Rivoire, “Nonlinearity measurements of thin films by third-harmonic generation microscopy,” Phys. Rev. E 66, 067602 (2002). [CrossRef]

25.

J.-X. Cheng, A. Volkmer, and X. S. Xie, “Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy,” J. Opt. Soc. Am. B 19, 1663–1675 (2002). [CrossRef]

26.

R. Fuentes and J. Fernández, “Ooplasmic segregation in the zebrafish zygote and early embryo: pattern of ooplasmic movements and transport pathways,” Dev. Dyn. 2392172–2189 (2010). [CrossRef] [PubMed]

27.

T. Hellerer, C. Axäng, C. Brackmann, P. Hillertz, M. Pilon, and A. Enejder, “Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy,” Proc. Nat. Acad. Sci. USA 37, 14658–14663 (2007). [CrossRef]

28.

T. T. Le, H. M. Duren, M. N. Slipchenko, C.-D. Hu, and J.-X. Cheng, “Label-free quantitative analysis of lipid metabolism in living Caenorhabditis elegans,” J. Lipid Res. 51, 672–677 (2010). [CrossRef]

29.

M. C. Wang, W. Min, C. W. Freudiger, G. Ruvkun, and X. S. Xie , “RNAi screening for fat regulatory genes with SRS microscopy,” Nat. Methods 8, 135–138 (2011). [CrossRef] [PubMed]

30.

G. V. Clokey and L. A. Jacobson, “The autofluorescent lipofuscin granules in the intestinal cells of Caenorhabditis elegans are secondary lysosomes,” Mech. Ageing Dev. 35, 79–94 (1986). [CrossRef] [PubMed]

OCIS Codes
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(190.4160) Nonlinear optics : Multiharmonic generation
(190.4380) Nonlinear optics : Nonlinear optics, four-wave mixing
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:
Microscopy

History
Original Manuscript: August 19, 2011
Revised Manuscript: September 19, 2011
Manuscript Accepted: September 19, 2011
Published: September 26, 2011

Citation
Pierre Mahou, Nicolas Olivier, Guillaume Labroille, Louise Duloquin, Jean-Marc Sintes, Nadine Peyriéras, Renaud Legouis, Delphine Débarre, and Emmanuel Beaurepaire, "Combined third-harmonic generation and four-wave mixing microscopy of tissues and embryos," Biomed. Opt. Express 2, 2837-2849 (2011)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-2-10-2837


Sort:  Author  |  Year  |  Journal  |  Reset  

References

  1. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol.21, 1369–1377 (2003). [CrossRef] [PubMed]
  2. W. Mohler, A. C. Millard, and P. J. Campagnola, “Second harmonic imaging of endogenous structural proteins,” Methods29, 97–109 (2003) [CrossRef] [PubMed]
  3. D. Oron, D. Yelin, E. Tal, S. Raz, R. Fachima, and Y. Silberberg, “Depth-resolved structural imaging by third-harmonic generation microscopy,” J. Struct. Biol.147, 3–11 (2004). [CrossRef] [PubMed]
  4. C. K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, and H.-J. Tsai, “Higher harmonic generation microscopy for developmental biology,” J. Struct. Biol.147, 19–30 (2004). [CrossRef] [PubMed]
  5. N. Olivier, M. Luengo-Oroz, L. Duloquin, E. Faure, T. Savy, I. Veilleux, X. Solinas, D. Débarre, P. Bourgine, A. Santos, N. Peyriéras, and E. Beaurepaire, “Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy,” Science339, 967–71 (2010). [CrossRef]
  6. Y. Wang, C.-Y. Lin, A. Nikolaenko, V. Raghunathan, and E. O. Potma, “Four-wave mixing microscopy of nanostructures,” Advances in optics and photonics3, 1–52 (2011). [CrossRef]
  7. J.-X. Cheng and X. S. Xie, “Coherent anti-Stokes Raman scattering microscopy: instrumentation, theory, and applications,” J. Phys. Chem. B108, 827–840 (2004). [CrossRef]
  8. A. Volkmer, “Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering microscopy,” J. Phys. D: Appl. Phys.38, R59–R81 (2005). [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, 786–793 (2006). [CrossRef] [PubMed]
  10. Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser microscopy by third harmonic generation,” Appl. Phys. Lett.70, 922–924 (1997). [CrossRef]
  11. M. Müller, J. Squier, K. R. Wilson, and G. J. Brakenhoff, “3D-microscopy of transparent objects using third-harmonic generation,” J. Microsc.191, 266–274 (1998). [CrossRef] [PubMed]
  12. D. Débarre and E. Beaurepaire, “Quantitative characterization of biological liquids for third-harmonic generation microscopy,” Biophys. J.92, 603–612 (2007). [CrossRef]
  13. D. Débarre, W. Supatto, E. Farge, B. Moulia, M. -C. Schanne-Klein, and E. Beaurepaire, “Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos,” Opt. Lett.29, 2881 (2004). [CrossRef]
  14. D. Débarre, W. Supatto, A.-M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M.-C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods3, 47–53 (2006). [CrossRef]
  15. R. W. Boyd, Nonlinear Optics, 2nd ed. (Academic, 2003).
  16. J.-X. Cheng and X. S. Xie, “Green’s function formulation for third harmonic generation microscopy,” J. Opt. Soc. Am. B19, 1604–1610 (2002). [CrossRef]
  17. N. Olivier and E. Beaurepaire, “Third-harmonic generation microscopy with focus-engineered beams: a numerical study,” Opt. Express, 16(19), 14703–14715 (2008). [CrossRef] [PubMed]
  18. B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanetic system.,” Proc. Royal Soc. A253, 358–379 (1959). [CrossRef]
  19. L. Novotny and B. Hecht, Principles of nano-optics (Cambridge Univ Press, 2006).
  20. G. Clay, A. Millard, C. Schaffer, J. Aus-der-Au, P. Tsai, J. Squier, and D. Kleinfeld, “Spectroscopy of third-harmonic generation: evidence for resonances in model compounds and ligated hemoglobin,” J. Opt. Soc. Am. B23, 932–950 (2006). [CrossRef]
  21. X. Liu, W. Rudolph, and J. L. Thomas, “Characterization and application of femtosecond infrared stimulated parametric emission microscopy,” J. Opt. Soc. Am. B27, 787–795 (2010). [CrossRef]
  22. C. Wang, “Empirical relation between the linear and the thord-order nonlinar optical susceptibilities,” Physssss; Rev. B2, 2045–2048 (1970). [CrossRef]
  23. R. S. Pillai, G. J. Brakenhoff, and M. Müller, “Analysis of the influence of spherical aberration from focusing through a dielectric slab in quantitative nonlinear optical susceptibility measurements using third-harmonic generation,” Opt. Express14, 260–269 (2006). [CrossRef] [PubMed]
  24. R. Barille, L. Canioni, L. Sarger, and G. Rivoire, “Nonlinearity measurements of thin films by third-harmonic generation microscopy,” Phys. Rev. E66, 067602 (2002). [CrossRef]
  25. J.-X. Cheng, A. Volkmer, and X. S. Xie, “Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy,” J. Opt. Soc. Am. B19, 1663–1675 (2002). [CrossRef]
  26. R. Fuentes and J. Fernández, “Ooplasmic segregation in the zebrafish zygote and early embryo: pattern of ooplasmic movements and transport pathways,” Dev. Dyn.2392172–2189 (2010). [CrossRef] [PubMed]
  27. T. Hellerer, C. Axäng, C. Brackmann, P. Hillertz, M. Pilon, and A. Enejder, “Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy,” Proc. Nat. Acad. Sci. USA37, 14658–14663 (2007). [CrossRef]
  28. T. T. Le, H. M. Duren, M. N. Slipchenko, C.-D. Hu, and J.-X. Cheng, “Label-free quantitative analysis of lipid metabolism in living Caenorhabditis elegans,” J. Lipid Res.51, 672–677 (2010). [CrossRef]
  29. M. C. Wang, W. Min, C. W. Freudiger, G. Ruvkun, and X. S. Xie , “RNAi screening for fat regulatory genes with SRS microscopy,” Nat. Methods8, 135–138 (2011). [CrossRef] [PubMed]
  30. G. V. Clokey and L. A. Jacobson, “The autofluorescent lipofuscin granules in the intestinal cells of Caenorhabditis elegans are secondary lysosomes,” Mech. Ageing Dev.35, 79–94 (1986). [CrossRef] [PubMed]

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
 

Multimedia

Multimedia FilesRecommended Software
» Media 1: AVI (8724 KB)      QuickTime
» Media 2: AVI (3106 KB)      QuickTime

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited