| dc.description.abstract | Advances in single-molecule imaging and super-resolution microscopy have revolutionized the field of cell biology, offering unprecedented insights into complex biological processes at the nanoscale. In this thesis, we present a comprehensive exploration of single-molecule imaging techniques and their applications in various biological contexts. The ability to study individual molecules in real-time over a large area has proven invaluable in understanding intracellular biophysical processes, cell-cell interactions, and the mechanisms underlying diseases like Influenza A and dengue type 2 (Denv-2) infection. Our research encompasses the development of cutting-edge imaging systems, multimodal clustering approaches, orientation analysis, and the investigation of drug-induced cellular changes. By integrating advanced microscopy techniques and sophisticated data analysis methods, we aim to shed light on the intricate workings of biological systems and pave the way for novel discoveries in the fields of cell biology and cancer therapy.These thesis comprises of seven chapter
The first chapter provides an overview of the fundamental concepts in fluorescence imaging. A brief history of emergence of the file is provided in this chapter along with the evolution of different super - resolution microscopes. Tehse techniques have brought about a huge transformation in the field of biology by providing access to the sub-cellular system to single molecule systems of a micro-organism. The discovery of the concept of fluorescence and the invention of different type of super resolution along with the fluorescence microscopy is discussed in this chapter. We also provide a brief explanation of different fluorescence imaging techniques and some trending techniques that have come to use widely in the area of fluorescence imaging. This chapter provide a thorough foundation for the research work presented in the thesis.
The chapter 2 introduces a novel technique called autotunable widefield non- scanning system (aSMLM) for single-molecule imaging over a large area. The primary aim of this system is to enable real-time adaptability of the field-of-view
(FOV) for improved imaging capabilities. The system incorporates a 4f-autotunable optical sub-system, which includes an auto-tunable lens and an objective lens, allowing for rapid changes in focus at the specimen plane.By converging and diverging the combined incident parallel beams of different wavelengths (405 nm and 561 nm) through the 4f sub-system, the focal spot at the working distance of the objective lens is effectively altered. This results in a defocused field with an increased FOV, ranging from 14.79 μm2 to 316.31 μm2. The most significant advantage of aSMLM lies in its tunability of FOV within this broad range, which facilitates adaptability to various experimental needs. A dedicated control unit is employed to achieve rapid focus shifts at a rate of 200Hz, ensuring the desired spot size (FOV) is achieved efficiently. The detection subsystem, a 4f-system, collects emitted light from the specimen plane and generates an image at the focus of the tube-lens. This system is demonstrated to offer near-uniform illumination over the desired FOV and exhibits a threefold increase in the number of detected single molecules. The potential applications of aSMLM are exemplified through the study of single-molecule (Dendra2-HA) clusters in transfected NIH3T3 cells. By demonstrating enhanced FOV and improved imaging capabilities, aSMLM shows promise for applications in the emerging field of single-molecule biophysics and fluorescence microscopy.
Chapter 3 introduces a novel technique called "Orientation Single-Molecule Localization Microscopy" (oSMLM) that enables the real-time decoding of the orientation of single molecules in super-resolution imaging. Standard single-molecule localization microscopy (SMLM) provides high-resolution maps of the locations and localization precision of individual molecules. However, it lacks information about the orientation of these molecules, which is crucial for understanding their behavior and catalytic activity.
The oSMLM method is based on the interaction between field dipoles (flu- orophores) and the polarization of light. The distribution of emitted photons strongly depends on the orientation of the dipole with respect to the polarization of light. By analyzing the emitted photons and their resulting point spread function
(PSF) distribution, the orientation of single molecules can be decoded in real-time. Computational studies reveal three distinct distributions (Gaussian, bivariate- Gaussian, and skewed-Gaussian) of the recorded PSF, each corresponding to a specific orientation of the molecules. Experiments conducted on Dendra2-Actin and Dendra2-HA transfected cells validate the emission model and demonstrate a localization precision of approximately 20 nm and an orientation precision of
±5°.The study shows that the oSMLM technique provides valuable information about the orientation and conformational changes of single molecules in cellular sub- domains/partitions. This additional feature expands the capabilities of localization microscopy and opens up new possibilities for investigating the link between molecular orientation and cellular function. Overall, oSMLM has wide-ranging applications in cell biology, biophysics, and fluorescence imaging. It enhances our understanding of the behavior of macromolecules and their role in cellular processes. The technique may offer valuable insights into various biological phenomena and could potentially lead to the discovery of new drug targets or mechanisms of action for disease treatment.
Chapter 4 focuses on studying molecular clustering in a complex cellular environment using super-resolution imaging and advanced clustering methods. The research aims to estimate critical biophysical parameters related to single-molecule clusters, such as cluster density, cluster area, pairwise distance, and the number of molecules per cluster. The study specifically investigates Hemaglutinin (HA) molecules in an Influenza type A disease model. The paper employs powerful clustering techniques, including K-means, Gaussian mixture, and point clustering, to estimate the biophysical parameters associated with both dense and sparse molecular clusters. The results show a significant difference among existing clustering techniques, indicating that a single method may not be adequate for accurately estimating all relevant parameters. Therefore, the authors propose a multimodal approach to characterizing molecular clusters and determining critical parameters, as the dynamics of single-molecules in a cellular system are driven by interlinked biophysical processes. By analyzing HA clusters in transfected NIH3T3 cells, the study demonstrates the efficiency of the advanced clustering methods over a variable field-of-view (FOV). The research contributes to the understanding of molecular clustering in cellular systems and the importance of accurate parameter estimation for studying biological mechanisms. The combination of super-resolution imaging and advanced clustering methods holds potential for applications in cell biology, biophysics, and fluorescence imaging. By developing reliable clustering techniques, this work may facilitate a better understanding of single-molecule clusters and their role in cellular processes, aiding in the investigation of various diseases and providing valuable insights for biomedical research.
Chapter 5 investigates the role of the NS2B3 protein complex during the early onset of dengue type 2 (Denv-2) viral infection using single-molecule-based super-resolution microscopy. NS2B3 is a crucial protein complex responsible for proteolytic activity and processing of viral polyprotein during Denv-2 infection. The study focuses on understanding the mechanism of NS2B3 clustering and its impact on the mitochondrial network in NIH3T3 cells. To achieve this, two distinct photoactivable fusion plasmid DNAs (mEos3.2-NS2B3 and PAGFP - NS2B3) were transfected into the cells. Super-resolution microscopy was employed to visualize the formation of NS2B3 clusters on the mitochondrial network. The statistical analysis of the super-resolution data estimates critical parameters related to the NS2B3 clusters, including an average cluster area of ≈ 0.005μm2, a density of ≈ 3500mol./μm2, and an average of ≈ 120 molecules per cluster. The findings of the study suggest that the formation of NS2B3 clusters induces fragmentation of the mitochondrial network. The NS2B3 complex acts as a protease, clipping specific sites of mitofusin (MFN1/2) proteins responsible for mitochondrial fusion. This disruption leads to the fragmentation of the mitochondrial network. Understanding the biophysical mechanism of NS2B3 clustering at the single-molecule level is critical in elucidating the early stages of dengue viral infection. The study offers insights into potential drug targets and mechanisms of action to disrupt the NS2B3 clusters, which could have significant implications for containing and treating dengue viral infection. Overall, the single-molecule-based super-resolution study provides valuable information about the clustering dynamics of NS2B3 during viral infection. This knowledge could aid in the development of targeted therapeutic approaches to combat dengue and other related viral infections. Additionally, the use of super- resolution microscopy allows for a deeper understanding of the cellular events during viral infection, contributing to the advancement of virology and antiviral research.
Chapter 6 presents a comprehensive investigation into the effects of the anticancer drug paclitaxel on Hela cells at varying concentrations (10, 25, 75, 100, and 250 nM). The primary focus of the study is to analyze the drug’s impact on the integrity of the mitochondrial membrane and the generation of reactive oxygen species (ROS). To gain deeper insights into these cellular changes, the researchers employed a combination of confocal microscopy and super-resolution microscopy, allowing them to explore the drug’s effects at both the cellular and nanoscale levels. The study first assessed the drug-induced alterations in mitochondrial membrane potential using the JC1 staining technique. They observed a remarkable increase in ROS levels and a concurrent decrease in mitochondrial membrane potential following paclitaxel treatment, signifying the induction of oxidative stress and impairment of mitochondrial function. Next, confocal microscopy was used to investigate mitochondrial morphology and dynamics using Mitotracker and Meos tom20. With increasing drug concentration, they observed a significant fragmentation of the mitochondria, indicating substantial changes in the organelle’s structure and distribution. To further explore these cellular changes at the nanoscale, super-resolution microscopy experiments were conducted. These high-resolution imaging techniques revealed the aggregation of molecules, leading to the formation of distinct clusters within the Hela cells. Notably, as the drug concentration increased, the number of clusters also increased, and the perimeter of these clusters expanded considerably. The findings from both confocal and super-resolution microscopy support the observation that the increasing drug concentration is linked to mitochondrial fragmentation. The formation of molecular clusters and their growing perimeter suggest a possible mechanism behind the disruption of mitochondrial morphology at higher drug concentrations. In conclusion, this comprehensive investigation sheds light on the effects of paclitaxel on Hela cells, specifically focusing on mitochondrial membrane disruption and ROS generation. The integration of confocal microscopy and super-resolution microscopy provided valuable insights into drug-induced changes in mitochondrial morphology and dynamics. These findings contribute significantly to our understanding of paclitaxel’s mechanisms of action and its potential implications for cancer therapy. The use of super-resolution microscopy offers a powerful tool to study cellular events at the nanoscale, providing valuable information for the development of targeted cancer treatments and advancing cancer research.
Finally we conclude the thesis with a brief section on the contribution of the thesis and the future scope the work presented. | en_US |
