Third harmonic generation microscopy for lipid deposition imaging.

<p>(<b>a</b>) Schematic representation of the non-linear microscope configuration. Two paths are used for detection, one in reflection (for TPEF signals) and the other in transmission mode (for THG signals). ND: Neutral Density filters, GM: Galvanometric mirrors, PMT: photomultiplier tube, L1, L2: telescope lenses, DM: Dichroic mirror, F1, F2: Filters, O: Objective lens, C: Condenser lens, M: Mirror. (<b>b</b>) Geometry of third harmonic generation (fundamental frequency ω), via non-linear optical interaction with a medium (). (<b>c</b>) The respective energy-level diagram describing the interaction of three photons of angular frequency ω with a non-linear material, to form one photon of triple frequency (and thus energy) compared to initial incident photons. (<b>d</b>) When a Gaussian beam is focused within the volume of a birefringent medium that perfectly compensates dispersion for the fundamental and third harmonic frequencies, THG waves symmetrical to the beam waist position (blue and dotted line respectively) interfere destructively due to a phase shift by π radians, and no signal is obtained. Destructive interference taking place in such a birefringent material can be avoided if the regions before and after the beam waist position possess unequal third order susceptibility values, since the generated third harmonic waves differ in amplitude. (<b>e</b>) For a material presenting a perfect dispersion compensation between frequencies ω and 3ω (implying that the phase matching condition Δk = 3k_ω - k_3ω = 0 is satisfied), the momentum conservation of the respective photons can be met. However, no signal is generated due to destructive interference of THG waves (shown in panel d). For common normal dispersive material (n_3ω>n_ω), momentum conservation condition cannot be met in any manner; and therefore, effective THG is not possible. If n_3ωω, efficient THG can occur since the angularly spread wave vectors of the focused fundamental beam can be added effectively so that the total momentum before and after the nonlinear interaction is conserved.</p