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Abstract

Nucleic acid based imaging methods have given rise to high-end quantitative functional imaging for fixed as well as live biological samples (Chakraborty et al. 2016). This has been achieved by virtue of predictable base pairing, high programmability, biocompatibility and development of advanced synthesis methods to synthesize nucleic acids in large quantities with high purity. A few notable applications include endosomal pH sensing using a pH- sensitive DNA nanodevice called the I-switch (Modi et al. 2009; Surana et al. 2011), chloride sensing using a pH- insensitive DNA nanodevice called Clensor (Saha et al. 2015a; Chakraborty et al. 2017), biologically active molecule delivery with spatiotemporal control using light responsive polymer loaded into an icosahedron made from DNA (Veetil et al. 2017), DNA-based super resolution imaging (Jungmann et al. 2014) and molecule counting using quantitative Points Accumulation In Nanoscale Topography (qPAINT) (Jungmann et al. 2016). ,Chloride plays a major role in cellular homeostasis by regulating the lumenal pH of intracellular organelles and its function (Weisz 2003), and its imbalance leads to diseased conditions like cystic fibrosis and lysosomal storage disorders (Di et al. 2006; Stauber and Jentsch 2013). Clensor based chloride measurement was a major landmark in the field of chloride sensing as it enabled first measurement of luminal chloride concentration in lysosomes (Saha et al. 2015b) as well as along the endocytic pathway of a live multicellular organism (Chakraborty et al. 2017). Clensor based chloride measurements have provided insight into intracellular localization of chloride channels as well as their role in lysosomal storage disorders. These measurements were made possible by optimizing the molecular design of Clensor to achieve high sensitivity and specificity while maintaining its robustness and targetability. Fluorescence lifetime based molecular probing has been used extensively to study variation of photophysical properties of fluorophores as a function of their environment (Anon 2006). In chapter I of this thesis, we have given a brief description of existing quantitative bioimaging techniques and what has been our addition to the field. In chapter II of this thesis, we have explored the use of fluorescence lifetime and ratiometric fluorescence intensity to get an insight into general design principles of ion-sensitive nucleic acid reporters based on the sensing strategy of Clensor (Prakash et al. 2016). ,Molecule counting is a technique using which one can count individual molecules of a given species in a sample with high spatial precision and is a growing area of research in the field of quantitative biology. To utilize nucleic acid based sensing and cargo delivery platform maximally, it is indispensable to understand how many such devices are uptaken by a cell or accommodated in an organelle and how are they spatiotemporally processed within a cell. Despite the development of several methods for cargo quantification in the cytosol (Puchner et al. 2013; Jungmann et al. 2016; Lee et al. 2012; Pitchiaya et al. 2012), cargo quantification in subcellular compartments has not yet been achieved. (Jung et al. 2017). In chapter III, we have modified an advanced imaging protocol called intracellular Single-molecule High-Resolution Localization and Counting (iSHiRLoC) (Pitchiaya et al. 2012) in conjunction with LabView and Python based image analysis (Tsekouras et al. 2016) to quantify the number of DNA nanodevices uptaken by receptor mediated endocytosis as a function of time and the effect of DNase II activity in macrophages. This study sets up a general platform for quantification of cargo within intracellular compartments.

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