Figure 4.

Using the pseudo-coloring option in the ZEN software (Carl Zeiss) according to the characteristics of different intensities, we could compare the green-endogenous autofluorescence and second harmonic generation (SHG) with intravital multiphoton microscopy. (A) (i) SHG is directional depending on the distribution and orientation of the non-linear dipoles, whereas general fluorescence is isotropically emitted. (ii) A diagram illustrating the different reactions in the absorption of two photons to excite the fluorescent molecule and the visible fluorescence emitted during relaxation. (iii) Detection of spectral ranges for excitation at 415–686 nm: half the excitation wavelength for SHG and two spectral bands for two-photon-excited fluorescence (TPEF). (B) Combined SHG/TPEF images of the cochlea clearly show the borders and even the nuclei of cells in the stria ligaments with pseudo-coloring; however, it is not easy to discriminate the borders between the stria vascularis, spiral ligament, and cochlear bone. SHG (violet), cochlear bone; TPEF (green), stria ligaments.