*4.4. Fluorescence Microscopy Analysis*

For the fluorescence observation, several 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 sets from the Zeiss 4880 series.

To determine the autofluorescence in wild type nematodes, the images were captured at 100× magnification with a red filter set (TRITC, 545/30 nm ex, 610/70 nm em). In addition, bright field images were used to define the shape of each worm. The CellProfiler Software [112,113] was used to determine the mean red fluorescence intensity 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 captured by using a yellow barrier filter with 100× magnification. 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 α-synuclein expression in dopaminergic neurons. To analyse the vitality of the neurons, the green barrier filter was used with a 200× magnification. The number of detectable anterior neurons were finally counted and assayed for patterns of degeneration, as described previously from Harrington et al. [55].
