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Project: Disposable sample holder with integrated optics for enabling Light Sheet Microscopy on standard light microscopes

2013-01-01 – 2016-12-31

Abstract

The human body is a true wonder. It is composed by an average of A.G trillion cells, subdivided in more than MNN different cell types, each with its function and its own specialization. Yet, life is possible only if those cells manage to communicate and closely collaborate with each other, to reach their common goal. Clearly, collaboration is the key to evolution and progress. This is no different in science. The technological progresses of this era, such as in the field of optical microscopy, have given an inestimable contribution to the progresses recently achieved in the life sciences. In turn, the challenges encountered while studying those microscopic, remarkably complex biological systems have been a fertile ground for the development of new, specialized technological systems. Such a close collaboration across scientific fields clearly is a driver for innovation and scientific progress. However, increased technological capabilities comes with increased complexity as well. Hence, to implement a novel stateof- the-art technique, such as LSFM and SPT in this work, research groups must commit an increasing amount of resources, in terms of time, funding and efforts to properly use those techniques to their full capabilities and in training its main users. In this thesis we aim to increase the accessibility of a recently developed technique to the general biology laboratory. In first instance, in chapter " the implementation of the various existing light sheet fluorescence microscopes (LSFM) is described in detail. The review at first focuses on the advantages and disadvantages of multiview imaging, the benefits of using selfhealing beams compared to traditional beams, and suggests how to improve the performance of the microscope by modifying the detection path. The second part of the review discusses the various possibilities to get started with LSFM as a non-expert. Commercially available instruments are hence presented, together with the tutorials, available on-line, that describe step-by-step how to build a LSFM with basic optical components. Special attention is given to those solutions that allow a rapid implementation of a LSFM system on epi-fluorescent microscopes, since those kind of microscopes are commonly present in biological research labs. One of those solution is presented in chapter two. To increase access to the technique, we designed, developed and replicated special microfluidic sample holders with integrated optical components to allow single lens light sheet microscopy directly on-chip. The sample holders make use of a micromirror to reflect in the channel the light that comes from the objective lens, while the same objective lens is used for imaging of the sample in the sample holder. This clever design eliminates de facto the need for (at least) two objective lenses as is needed for traditional LSFM. Two designs have been proposed, tested and replicated in an inexpensive UV-curable epoxy. The quality of the beam generated in the microchannels has been dutifully assessed. The light sheet in the channel presents a beam thickness of approximately M μm, comparable to the light sheet thickness of most LSFM. Also, the sample holders perform well in terms of contrast achieved in the presence of a high background noise and proved to be adapt to acquire high-quality images of spheroids. The second part of the thesis focused on a different sort of advanced microscopy technique, Single Particle Tracking. SPT is described in chapter three, explaining its fundamentals and its application to study drug- and gene-delivery oriented biocomplexes in extra- and intra-cellular environments. SPT is a highly sensitive, non-invasive optical technique that is able to study the behavior of the nanoparticles at a single particle level. It has proven to be particularly beneficial in studies where monitoring the size distribution, degree of aggregation and concentration of the nanoparticle is crucial. Therefore, in chapter four we exploited the unique characteristic of the technique to devise a novel method for the characterization of nucleic acid-loaded nanocomplexes. Nucleic acid molecules (NAms) based therapeutics show great potential for the treatment of genetic disorders and cancer. However, the many environments the therapeutics need to cross to reach its desired target alter the physicochemical properties of the nanocomplex and more often than not result into degradation of the complex and of its precious cargo. Moreover, the nanocomplex itself, if poorly designed, may trigger acute inflammatory responses in vivo and be toxic for its target cells. Consequently, a thorough characterization of NAms based complexes is of the utmost importance to properly engineer safe and effective NAms based therapeutics. One property of the nanocomplexes that has been mostly overlooked is the actual number of NAms complexed per nanocarrier. We focused on DOTAP:DOPE liposomes as a model carrier and studied which conditions affect the number of pDNA molecules that are incorporated per lipoplex. We discovered that the pDNA/carrier complexation is highly influenced by their chance on collision when the carrier and cargo are mixed. Thus the average number of plasmid per nanocomplex depends on the concentration of carrier and cargo, on the size of the pDNA and whether or not the carrier is PEGylated. In future research it will be of interest to apply the SPT method to different types of cargo and carriers and to see how the number of cargo molecules per complex influences the final biological effect.