The non-linear techniques Second and Third Harmonic Generation (SHG, THG) have been employed simultaneously to record three dimensional (3D) imaging and localize the lipid content of the muscular areas (ectopic fat) of Caenorhabditis elegans (C. elegans). Simultaneously, Two-Photon Fluorescence (TPEF) was used initially to localize the stained lipids with Nile Red, but also to confirm the THG potential to image lipids successfully. In addition, GFP labelling of the somatic muscles, proves the initial suggestion of the existence of ectopic fat on the muscles and provides complementary information to the SHG imaging of the pharynx. The ectopic fat may be related to a complex of pathological conditions including type-2 diabetes, hypertension and cardiovascular diseases. The elucidation of the molecular path leading to the development of metabolic syndrome is a vital issue with high biological significance and necessitates accurate methods competent of monitoring lipid storage distribution and dynamics in vivo. THG microscopy was employed as a quantitative tool to monitor the lipid accumulation in non-adipose tissues in the pharyngeal muscles of 12 unstained specimens while the SHG imaging revealed the anatomical structure of the muscles. The ectopic fat accumulation on the pharyngeal muscles increases in wild type (N2) C. elegans between 1 and 9 days of adulthood. This suggests a correlation of the ectopic fat accumulation with the aging. Our results can provide new evidence relating the deposition of ectopic fat with aging, but also validate SHG and THG microscopy modalities as new, non-invasive tools capable of localizing and quantifying selectively lipid accumulation and distribution.
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.
Second-harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) are relatively new and promising tools for the detailed imaging of biological samples and processes at the microscopic level. By exploiting these nonlinear phenomena phototoxicity and photobleaching effects on the specimens are reduced dramatically. The main target of this work was the development of a compact inexpensive and reliable experimental apparatus for nonlinear microscopy measurements. Femtosecond laser pulses were utilized for excitation. We achieved high-resolution imaging and mapping of Caenorhabditis elegans (C. elegans) neurons and muscular structures of the pharynx, at the microscopic level by performing SHG and TPEF measurements. By detecting nonlinear phenomena such as SHG and TPEF it is feasible to extract valuable information concerning the structure and the function of nematode neurons.
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