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

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.

Third harmonic generation microscopy of cells and tissue organization

“…In Drosophila embryos for example, tightening the focus enhances the signal from the external membrane (a scenario comparable to that in Fig. 1E, example 6), whereas, when using a weakly focused beam, signals predominantly originate from densely packed internal micrometer-sized organelles (Débarre et al, 2005;Mahou et al, 2011) (a scenario comparable to that in Fig. 1E, example 4).…”

Accumulating advantages, reducing limitations: Multimodal nonlinear imaging in biomedical sciences – The synergy of multiple contrast mechanisms

“…However, for most molecules this quantity is equal to zero due to the molecular symmetry. Hence, the in general lowest order nonlinear quantity in matter is c (3) or the second order hyperpolarizability g. Nonlinear effects described by this quantity include two photon absorption (TPA), two photon excited fluorescence (2PF), three photon excited fluorescence (3PF), third harmonic generation (THG), four wave mixing [19] (FWM) and multiple coherent Raman scattering (CRS) processes, e.g., stimulated Raman scattering [20], coherent antiStokes Raman scattering (CARS) [21], coherent Stokes Raman scattering (CSRS) and Raman induced Kerr effect (RIKE) [22]. Recently a variety of additional nonlinear contrast mechanisms has been proposed, e.g., cross phase modulation microscopy [23,24], or nonlinear phase dispersion microscopy [25].…”

“…stretching vibration [27]. On the other hand, processes relying on very general optical properties, e.g., refractive index changes in FWM and THG, are powerful for label-free visualization of the overall tissue structure, but provide only unspecific molecular information [19]. Here coherent Raman processes are very powerful enabling both the visualization of the tissue structure, e.g., by utilizing abundant vibrational resonances for imaging, but also highly selective visualization, when a molecule or structure specific vibrational resonance is probed, e.g., for drug delivery studies with isotope labeling [28,29].…”

Development and Application of Optical Coherence Tomography (OCT)

“…(i,j) Birefringence in the retinal nerve fiber layer (RNFL) imaged by PS-OCT (adapted with permission from [18], Optical Society of America, 2014). [167], ARVO, 2014). Photographs (red lines indicate locations of OCT scans), phase retardation and intensity images with PS-OCT at one day, one week, and one month after surgery.…”

“…Finally, we can add that all these techniques are based on non-linear processes that require similar kinds of sources, i.e., femtosecond lasers, and can be ingeniously combined all together from a single laser source [167]. However, such non-linear processes are quite weak.…”