Expanding the biologist's toolbox with microfabricated sample holders for single objective SPIM and Single Particle Tracking for the characterization of nanomedicines

The first decade of the 20th century saw the birth of the very first fluorescence microscope, developed by Otto Heimstaedt and Heinrich Lehmann to study the autofluorescence of bacteria, plant and animal tissues. Little they knew that barely one hundred years later fluorescence microscopy would become a cornerstone of modern biological laboratories and one of biologist’s most precious allies. However, progress in fluorescence microscopy didn’t happen gradually. The last twenty years have seen a sudden expansion of the field and a blooming of new technologies and illumination modalities. Such a rapid expansion especially favored the life sciences, which greatly benefitted from these advanced techniques and used them to explore the micro- and nano-world. Suddenly, the invisible became visible – chromosomes, organelles, 3D cellular structures – finally enabling the observation of molecular dynamics within the cellular context. As a consequence, these modern times are characterized by a level of collaborations among different branches of science (biology, chemistry, physics, optics, informatics) never seen before. Considerable progress is achieved whenever a close collaboration between these fields is possible. However, increasingly complex instrumentations and techniques require an adequate level of expertise in such fields from the end user as well. Therefore the implementation of a new advanced microscopy technique in a lab or department may require a substantial amount of resources, in terms of funding/money, time and effort to bring the users to the required level of expertise. Within our work, we aim to bridge the gap between the resources needed to implement a novel, advanced microscopy technique and the resources available to less-specialized laboratories. Light sheet fluorescence microscopy is a recent technique that enables high resolution 3D imaging of large and small samples for extended periods of time with limited phototoxic effects. However, implementation of the technique in less-specialized laboratories is hindered by its peculiar configuration, the special requirements of its sample holders and the expertise required to assembly the optical path. To address the issue, we aim to develop technology to enable single lens light sheet microscopy that would be relatively simple to implement on existing epi-fluorescent microscopes. In particular, we’ll focus on the fabrication of inexpensive mass-producible microfluidic sample holders with integrated optical components that will allow on-chip light sheet illumination. In this approach only a single objective lens is needed to both provide the light sheet illumination and do the fluorescent imaging. In the second part of the thesis, special attention will be given to another well-known, advanced microscopy method: Single Particle Tracking (SPT). In a pharmaceutical context, SPT has proven to be a precious ally in the development of safe and successful nucleic acid-based therapeutics against genetic diseases. Indeed, to be able to express their therapeutic potential, those nucleic acid-based nanomedicines need to cross several extra- and intra-cellular barriers before reaching their target. Highly sensitive light microscopy techniques, like SPT, are thus necessary for a thorough characterization of nanomedicines as as they travel through those various barriers. The most frequent application is the use of SPT to study nanoparticle mobility in cells and tissues. But SPT can also be used to measure nanoparticle size and concentration in complex biological fluids such as blood. As such SPT is an important tool for in vitro bio-barrier studies, thus providing important information on the suitability of a particular nanoformulation for in vivo applications. Here, we propose to expand the biologist’s toolkit by devising a new method to perform additional characterization of nucleic acid-based therapeutics using SPT. We’ll exploit the technique to get a new type of information that was inaccessible before, which is the average number of nucleic acid molecules complexed within a nanocarrier, in order to improve complexation protocols and eventually to better understand the relation between nucleic acid content per nanoparticle and its final biological effect. The first chapter presents a review on light sheet fluorescence microscopy, both in its traditional form and according to the latest developments. Special attention is given to how to implement a light sheet microscope in a research lab, both by pointing out the currently commercially available models, as well as by referencing to websites for do-it-yourself assembly of light sheet microscopes and finally by illustrating methodologies to implement light sheet microscopy on standard epi-fluorescent microscope’s bodies that are commonly available in biology labs. The second chapter will focus on the design, development and characterization of disposables sample holders to enable light sheet microscopy on epi-fluorescent microscopes. Special attention will be given to strategies that will allow a swift integration of the optical set-up on the microscope and to fabrication methods that may lead to mass-production of the sample holders. In the third chapter single particle tracking SPT is reviewed, describing both fundamental aspects and its applications to gene- and drug-delivery. Finally, one of the biggest challenge of nucleic acids delivery is the design of nanomaterials that protect their cargo and overcome the numerous biological barriers our bodies have developed to protect us from external pathogens. A thorough characterization of the interaction between these nanomaterial and their therapeutic cargo is thus of the utmost importance for a rational optimization of the nanomaterial. As such, the fourth chapter will describe a SPT-based method to determine the average number of nucleic acids therapeutics complexed with nanoparticles, aimed at providing further fundamental information that will lead to rational optimization of nanocarriers. Finally, in the fifth chapter the broader context is discussed, briefly addressing the development of life-science microscopes and discussing the relevance of the work in this thesis for the field of live-cell imaging. Further improvements are finally discussed to the micromirror sample holders and the SPT-based method for characterization of nanomedicines.