Microfluidic devices for automated analysis of ingested bacteria in the Caenorhabditis elegans intestine

In this thesis, we explored on-chip high-resolution imaging of the fate of intestinal bacteria and bacterial products in Caenorhabditis elegans (C. elegans). In the first part, we carried out high-resolution z-stack fluorescent imaging of Red Fluorescent Protein (RFP)-labelled Escherichia coli (E. coli) OP50 in the intestine of C. elegans using a spinning disk confocal microscope (SDCM) equipped with a 60x oil immersion objective. To enable z-stack fluorescence imaging, the worms were loaded and fixed on microfluidic chips based on a thin cover glass substrate. The obtained z-stack fluorescence images were afterwards analysed using IMARIS software, whereby a 3D representation of the intestine of each worm was constructed. Subsequently, bacterial spots were detected by IMARIS software and were later analysed in an automated fashion through bivariate histograms that displayed the intensities and the volumes of these spots.In the second part, we investigated the bacterial food digestion and accumulation in adult C. elegans worms that have fed on either non-pathogenic RFP-labelled E. coli OP50 or pathogenic RFP-labelled Pseudomonas aeruginosa (P. aeruginosa) PAO1 during the first 4 days of adulthood. We discovered that the pattern of the bacteria-derived fluorescence signal in the intestine of the worms depended on the bacteria they were fed. For both cases, the quantitative analysis showed that the intestinal bacterial load generally increases with age of the worm, albeit at a faster rate for the RFP-labelled P. aeruginosa PAO1-fed worms. The qualitative observations and the quantitative results suggest a reduced metabolism in the RFP-labelled P. aeruginosa PAO1-fed worms compared to those fed on RFP-labelled E. coli OP50.In the third part, we made efforts to replicate the same level of high-resolution imaging and analysis through in vivo and reversible immobilization on a microfluidic chip. We thus designed a number of microfluidics devices featuring valves for the immobilization of the worms. While we could sufficiently immobilize the worms for high-resolution imaging of intestinal bacteria, we could not reliably control the positions where the worms were immobilized. More importantly, due to intestinal peristalsis, the bacteria are regularly displaced within the intestinal lumen and therefore it was not possible to acquire z-stack fluorescence images, which are necessary for constructing 3D representations of intestinal bacteria. As a result, performing in vivo automated high-resolution z-stack fluorescence imaging was out of reach.Lastly, in a collaborative work with the lab of Prof. Gönczy, we designed several Polydimethylsiloxane (PDMS) microwell arrays for investigating symmetry breaking and polarity establishment in one-cell stage C. elegans embryos. We designed and fabricated arrays of PDMS microwells with different shapes and sizes to facilitate the placement of the ellipsoid embryos in the vertical orientation and thereby enable vertical imaging of symmetry breaking and polarity establishment from the posterior pole. We evaluated the fabricated microwells in terms of the ease of embryo insertion and the verticality of the placed embryos. We also checked the embryos for potential deformations or viability issues.