Three dimensional, third harmonic microscopy of living systems

Summary form only given. Recently Barad et al. (1997) showed that the third harmonic generated at the interface of a transparent sample can be used to map the surface of optical glass fibers in an index matching fluid. In the paper, we extend the technique and produce three-dimensional, third harmonic volume images of live biological specimens. Third-harmonic generation (THG) imaging has several advantages for biological imaging: (1) it is a background-free imaging technique requiring no additional staining, (2) it is non-fading in nature, and (3), it can be used with low phase contrast specimens. Because the third harmonic production is localized at interfaces, where there is a change in refractive index or nonlinear susceptibility, the technique as applied to microscopy inherently produces optically sectioned data sets. This allows three-dimensional reconstruction as in traditional confocal microscopy. We present a three dimensional image of a spiral algae formation taken using THG in this manner. In the paper, we demonstrate for the first time to our knowledge, dynamic imaging of live specimens, under moderate NA (0.6) and high NA (1.3) conditions. While significant work remains to fully develop THG microscopy for biological systems, this first demonstration is critical in that it establishes the relevance of the technique to living specimens.