In vivo imaging of anatomical features of the nematode Caenorhabditis elegans using non-linear (TPEF-SHG-THG) microscopy.

Abstract

In this study, we present the detailed imaging of the nematode Caenorhabditis elegans (C. elegans) at microscopic level by performing Two-Photon Excitation Fluorescence (TPEF), Second-Harmonic Generation (SHG) and Third Harmonic Generation (THG) measurements. Due to their inherent advantages in comparison with the conventional microscopy (increased resolution, ability to section deep within tissues, minimization of photodamage and photobleaching effects), the non-linear microscopy techniques comprise a unique and extremely powerful tool for the extraction of valuable and unique information from biological samples. We developed a compact, reliable, inexpensive non-linear imaging system, utilizing femtosecond laser pulses (1028nm) for the excitation of biological samples. The use of 1028nm wavelength as excitation source minimizes photodamage effects and unwanted heating (due to the water absorption) of the biological specimens. The emitted THG signal lies in the near UV part of the spectrum (343nm). Detailed and specific structural and anatomical features of the worm were collected by recording THG signals. Consummative, unique information concerning the morphology and the functions of the nematode was obtained by implementing the combination of THG, SHG and TPEF image contrast modalities on the same microscope.