Quantitative fluorescence microscopy for studying peptide transport and optimizing localization with structured illumination

There is an increasing interest in oral administered biopharmaceuticals due to the high customer compliance. However, the bioavailability is only 1 – 2 % due to natural barriers, such as the intestinal barrier, protecting us from foreign molecules entering the cardiovascular system. Understanding the transport of drugs across barriers could help design novel drugs with higher bioavailability. However, cellular interaction with a drug can be complex and happen differently. For example, the transport can happen across a cell layer through active transportation involving biological processes, such as endocytosis. Alternatively, the drug can undergo passive transportation where diffusion dictates the transport.
Often, the investigation of drug transport through a cell layer is in systems that do not allow for imaging or tracking the drug, limiting the mechanistic insight into the transport of the drug. However, fluorescence microscopy offers an alternative way of investigating several biological problems by linking fluorescent dyes to the drug and possibly to cellular organelles. Here, two types of experiments can gain a mechanistic understanding of drug transport.
One type of experiment focuses on tracking single molecules over time. Here it is crucial to localize the molecule with the best precision possible to more accurately determine the interactions between the molecule and, e.g., cellular compartments. A recent approach to improve the localization precision is structured illumination microscopy. A second type of experiment quantifies drug amounts in various cellular compartments using single-cell analysis. A preferred microscope for single-cell analysis is a spinning disk confocal microscope.
The first part of the thesis deals with a novel method to improve the localization of single molecules. I first build an experimental setup that, combined with data analysis from collaborators, can localize fluorophores more precisely than conventional methods, such as camera-based localization microscopy. The structured illumination came from using a digital micro-mirror device as a spatial light modulator. As a proof-of-concept, single-bead and single-molecule samples were used to calibrate the structured illumination, achieving a structured illumination with a sinusoidal pattern with a period of approximately 230 nm and a modulation depth of 0.9. An alternative setup with non-harmonic structured illumination gave a period of roughly 400 nm and a modulation depth of 0.75. Ultimately, this led to an increase in precision localization of 2.1x and 1.3x, respectively.
The second part of the thesis addresses the challenges of quantifying peptide transport through a monolayer of Caco-2 cells by developing a method to quantify peptides’ transport by single-cell analysis by deconvoluting the axial intensity signal obtained by a spinning disk confocal microscope. The method was used to show that the lipidation of salmon calcitonin with two C8 lipid chains increases the transport significantly by a factor of 2.6x. The method was also used to gain mechanistic insight showing that the lipidation with two C4 lipid chains alters the transport mechanism of salmon calcitonin to a non-dynamin dependent process.
In the future perspective, combining the two types of experiments would extract the most amount of information possible in one experiment. A lattice light-sheet microscope would be able to facilitate that as it has structured illumination and is very suitable for live-cell imaging.
In conclusion, the results showed that the lipidation of salmon calcitonin significantly increases the transport of the peptide across a monolayer. Furthermore, we showed that optimal structured illumination could achieve a more than two-fold precision on localization.