Adaptive optics and wavefront shaping
Focus engineering can be used to control phase-matching conditions within the sample. Most notably, in coherent nonlinear microscopy, the phase and amplitude distribution of the excitation beam critically influences the signal created when interacting with the 3D distribution of nonlinear scatterers in the sample.
A simple example is the influence of the size of the excitation beam at the entrance pupil of the objective on THG images : because the coherence length of the focused beam depends on the excitation numerical aperture (NA, controlled by the beam size before the objective) and because the coherent build up of the signal occurs on a scale of the order of this coherence length, the relative contrast of structures of different sizes can be changed by changing the effective excitation NA (Debarre et al., 2005, Olivier et al., 2010).
THG image of a Drosophila embryo(inverted contrast) with tight (top) and weaker (bottom) focussing. dz is the axial resolution of the image.
In addition to amplitude and phase shaping, polarization control can also be used to modulate the image contrast in organized samples. For example, no THG is obtained from an isotropic sample excited with circular polarization. Therefore THG with circular polarization can be used to detect anisotropic media, as illustrated in the human cornea images below. Click here for more information
Polarization-senstive THG imaging of a Human cornea excited with linear (left) and circular (right) polarization. Adapted from Olivier et al, Opt Express 2010.
In coherent nonlinear microscopy (such as THG), signal level and emission directionality are governed by phase matching, i.e. by the good overlap of excitation and scattered waves. In particular, phase matching conditions govern the ratio of backward-to-forward coherent radiation. This efficiency depends both on the spatial distribution of the focused beam and on the sample strucure. This is illustrated by the calculation below: in a periodic sample, quasi-phase-matching may occur in the backward direction. Coherent backward emission may be obtained from certain organized structures or from metallic probes (Debarre et al., 2007).
Forward- and backward emitted THG power for a periodic object with period de and various excitation NAs. B, scattering patterns.
Spatial beam shaping therefore provides a means to probe the sample structure by measuring its response to the modulation of the focal volume. Perspectives include the detection of specific sub-wavelength geometries (Olivier et al., 2008).
Forward- and backward emitted THG power for a periodic object with period de and various excitation excitation beam shapes (HG, Hermite-Gaussian, LG, Laguerre-Gaussian) and polarizations (lin, linear; rad, radial, az, azimuthal).
"Probing ordered lipid assemblies with polarized third-harmonic-generation microscopy"
M. Zimmerley, P. Mahou, D. Débarre, M.-C. Schanne-Klein & E. Beaurepaire
Phys. Rev. X 3, 011002 (2013).
"Third-harmonic generation microscopy with Bessel beams: a numerical study"
N. Olivier, D. Débarre, P. Mahou, and E. Beaurepaire
Opt. Express 20(22), 24886-24902 (2012).
"Harmonic microscopy of isotropic and anisotropic microstructure of the human cornea"
N. Olivier, F. Aptel, K. Plamann, M.-C. Schanne-Klein & E. Beaurepaire
Opt. Express 18(5), 5028-40 (2010).
"Third-harmonic generation microscopy with focus-engineered beams: a numerical study"
N. Olivier & E. Beaurepaire
Opt. Express 16(19), 14703-14715 (2008).
"Signal epidetection in third-harmonic generation microscopy of turbid media"
D. Débarre, N. Olivier & E. Beaurepaire
Opt. Express 15(14), 8913-8924 (2007).
"Structure sensitivity in third-harmonic generation microscopy"
D. Débarre, W. Supatto & E. Beaurepaire
Opt. Lett. 30(16), 2134-2136 (2005). PDF