*4.4. Fluorescence Microscopy Analysis*

For the fluorescence observation, the nematodes were placed on a 2% agar pad on a microscope slide and anesthetized with 4 μL NaN3 (1M). The images were taken with the aid of the Axioskop fluorescence microscope (Carl Zeiss, Oberkochen, Germany) and filter set 13 from the Zeiss 4880 series (Carl Zeiss, Oberkochen, Germany). Nematodes with a ruptured vulva phenotype were excluded from analysis. The images were captured and analysed on the 3rd, 7th or 12th day of adulthood, respectively.

Wild type nematodes were analysed to determine and quantify ageing-related pigment accumulation. The images were captured at 100x magnification and a red filterset (TRITC, 545/30 nm ex, 610/70 nm em) was used. It was necessary to acquire additional images in a bright field, because of the poor visibility of the body contour in the dark field. The images in the bright field were used to delineate the perimeter of the worm to which the dark field image was overlayed by the CellProfiler Software (Version 3.1.9; Broad Institute, Cambridge, MA, USA) [122,123]. The quantification of age-related pigment accumulation was expressed by the mean intensity of the red fluorescence per total worm body.

The OW13 transgenic strain features yellow fluorescent protein linked to α-synuclein in the body wall muscle cells. Therefore, the nematodes were monitored using a yellow barrier filter with 100x magnification to quantify the light emission proportional to the amount of accumulation of the pathological protein. The images were processed using the CellProfiler software, and the yellow fluorescence intensity emitted per total worm body was calculated.

The UA44 transgenic strain features GFP linked to the dopamine transporter in the six dopaminergic neurons of the head and two in the tail as well as harmful α-synuclein in dopaminergic neurons. The green fluorescence intensity represents the vitality of the neurons, therefore, the green barrier filter was used for the analysis. The number of detectable anterior neurons was counted at the microscope with 200x magnification. In addition, individual images of the head were captured. The nematodes were assayed for patterns of degeneration at indicated time points, as described previously from Harrington, et al. [68].
