Mechanochemical study of single living cells and biomolecules combining optical tweezers and raman spectroscopy

This thesis is devoted to the study of mechanochemistry of single cells and single biomolecules. Mechanochemistry relates to the association of mechanical and chemical occurrence at the molecular level. Using a combination of microrheological measurements and Raman spectroscopy based on optical tweezers we have shown how the molecular structures of single living cells and biomolecules are coupled with mechanical deformations. The basic questions to respond in this work are: (i) How does a cell or a biomolecule detect an external force? (ii) How do the viscoelastic properties of living cells and biomolecules depend on its environment? (iii) How does the structure of the cell or biomolecules change because of the external force? We use microrheology to detect the response of single cells (or biomolecules) to an external force. Two point microrheology is applied to study these samples - both in their relax configuration and in a stress situation when a known external force is applied. We are able to find non-linearities in mechanical response of RBC and DNA molecules subjected to an external load. In both the cases, an increase in elastic stiffness of the system is observed with increase in the force. To detect changes in the molecular structures of DNA or a cell as a result of an applied mechanical load, Raman spectroscopy is used. Raman signals from biomolecules are rich and complex in nature and are accompanied with several noises. Hence processing of data is an important aspect in Raman spectroscopy. For gaining in-depth information regarding the behavior of the molecule under study, we used statistical techniques such as principal component analysis and 2D correlation spectroscopy. Raman measurement of single DNA, with excitation optical beam focused by conventional optics, is almost impossible owing to very small thickness of DNA and inherent low efficiency of Raman scattering. To overcome this difficulty, we used additional optical phenomena - Surface Enhanced Raman Scattering (SERS) technique. With implementation of SERS, we were able to detect signals from single DNA molecule. Our study presents substantial findings that describe the structural changes of the phosphate backbone when DNA is extended in the entropic force regime. The current belief is that such forces are balanced by lowering DNA entropy via unfolding as the molecule is extended. While this is accurate, we observe structural movement at the bond level when the single biomolecule is extended at these low forces. This revelation has never been measured directly or predicted theoretically. The result gives a different view of DNA mechanical properties. We confirm and substantiate our experimental results with molecular dynamics simulations.Postprint (published version

New optical techniques and hardware for studying live cell dynamics

This thesis was previously held under moratorium from 12/02/2019 to 21/05/2021.Fluorescence optical microscopy has become an integral technique in the life sciences and has opened the door to investigating live biological specimens non-invasively at sub-cellular spatial resolutions with high specificity and temporal resolutions. One of the limiting factors of optical microscopy is that the spatial resolution is dictated by the diffraction limit of light.;This work shows the first use of LEDs to carry out widefield axial super-resolution standing wave microscopy with high temporal resolution. The technique was used to image red blood cell membrane dynamics in real time with no increase in photobleaching or toxicity rates compared to standard widefield imaging. This work also presents 3D computational reconstructions of the data allowing for easier visualisation and the possibility of carrying out further quantitative analysis.;Following on from Chapter 2, is an investigation into the development and application of multi-wavelength standing wave microscopy on live specimens in both emission and excitation modalities. These techniques are henceforth referred to in this thesis as TartanSW. This investigation found that using multiple excitation wavelengths allowed for a reduction in the nodal contribution of the images resulting in obtaining 32.3 % more spatial information about the structure of the specimen. It is also shown that by taking the difference images between each excitation channel the standing wave antinodal planes could be reduced in thickness enabling axial resolutions on the order of 55 nm when imaging live cell experiments.;The multi-emission technique was shown that it could be applied to be applied to imaging biological specimens using both widefield and confocal microscopy. However, the widefield data was not in line with the expected theoretical structure. There is the possibility of using plane ordering though to infer the directionality of a specimen structure and extract height maps though further work to develop computational tools to enable this will have to be implemented.;Finally, this thesis describes the work carried out making use of a new high-brightness 340 nm LED to develop a fast switching 340/380 nm illuminator and demonstrate its application for ratiometric Fura-2 Ca2+ imaging of live cell specimens with sub-5 nM precision that supports full frame video-rate temporal resolutions.Fluorescence optical microscopy has become an integral technique in the life sciences and has opened the door to investigating live biological specimens non-invasively at sub-cellular spatial resolutions with high specificity and temporal resolutions. One of the limiting factors of optical microscopy is that the spatial resolution is dictated by the diffraction limit of light.;This work shows the first use of LEDs to carry out widefield axial super-resolution standing wave microscopy with high temporal resolution. The technique was used to image red blood cell membrane dynamics in real time with no increase in photobleaching or toxicity rates compared to standard widefield imaging. This work also presents 3D computational reconstructions of the data allowing for easier visualisation and the possibility of carrying out further quantitative analysis.;Following on from Chapter 2, is an investigation into the development and application of multi-wavelength standing wave microscopy on live specimens in both emission and excitation modalities. These techniques are henceforth referred to in this thesis as TartanSW. This investigation found that using multiple excitation wavelengths allowed for a reduction in the nodal contribution of the images resulting in obtaining 32.3 % more spatial information about the structure of the specimen. It is also shown that by taking the difference images between each excitation channel the standing wave antinodal planes could be reduced in thickness enabling axial resolutions on the order of 55 nm when imaging live cell experiments.;The multi-emission technique was shown that it could be applied to be applied to imaging biological specimens using both widefield and confocal microscopy. However, the widefield data was not in line with the expected theoretical structure. There is the possibility of using plane ordering though to infer the directionality of a specimen structure and extract height maps though further work to develop computational tools to enable this will have to be implemented.;Finally, this thesis describes the work carried out making use of a new high-brightness 340 nm LED to develop a fast switching 340/380 nm illuminator and demonstrate its application for ratiometric Fura-2 Ca2+ imaging of live cell specimens with sub-5 nM precision that supports full frame video-rate temporal resolutions

DEVELOPMENT AND BIOLOGICAL APPLICATIONS OF HIGH-RESOLUTION ION BEAM INDUCED FLUORESCENCE MICROSCOPY

Ph.DDOCTOR OF PHILOSOPH

Automatic Segmentation and Classification of Red and White Blood cells in Thin Blood Smear Slides

In this work we develop a system for automatic detection and classification of cytological images which plays an increasing important role in medical diagnosis. A primary aim of this work is the accurate segmentation of cytological images of blood smears and subsequent feature extraction, along with studying related classification problems such as the identification and counting of peripheral blood smear particles, and classification of white blood cell into types five. Our proposed approach benefits from powerful image processing techniques to perform complete blood count (CBC) without human intervention. The general framework in this blood smear analysis research is as follows. Firstly, a digital blood smear image is de-noised using optimized Bayesian non-local means filter to design a dependable cell counting system that may be used under different image capture conditions. Then an edge preservation technique with Kuwahara filter is used to recover degraded and blurred white blood cell boundaries in blood smear images while reducing the residual negative effect of noise in images. After denoising and edge enhancement, the next step is binarization using combination of Otsu and Niblack to separate the cells and stained background. Cells separation and counting is achieved by granulometry, advanced active contours without edges, and morphological operators with watershed algorithm. Following this is the recognition of different types of white blood cells (WBCs), and also red blood cells (RBCs) segmentation. Using three main types of features: shape, intensity, and texture invariant features in combination with a variety of classifiers is next step. The following features are used in this work: intensity histogram features, invariant moments, the relative area, co-occurrence and run-length matrices, dual tree complex wavelet transform features, Haralick and Tamura features. Next, different statistical approaches involving correlation, distribution and redundancy are used to measure of the dependency between a set of features and to select feature variables on the white blood cell classification. A global sensitivity analysis with random sampling-high dimensional model representation (RS-HDMR) which can deal with independent and dependent input feature variables is used to assess dominate discriminatory power and the reliability of feature which leads to an efficient feature selection. These feature selection results are compared in experiments with branch and bound method and with sequential forward selection (SFS), respectively. This work examines support vector machine (SVM) and Convolutional Neural Networks (LeNet5) in connection with white blood cell classification. Finally, white blood cell classification system is validated in experiments conducted on cytological images of normal poor quality blood smears. These experimental results are also assessed with ground truth manually obtained from medical experts

Intramolecular Hydrogen Bonding 2021

This book describes the results of both theoretical and experimental research on many topical issues in intramolecular hydrogen bonding. Its great advantage is that the presented research results have been obtained using many different techniques. Therefore, it is an excellent review of these methods, while showing their applicability to the current scientific issues regarding intramolecular hydrogen bonds. The experimental techniques used include X-ray diffraction, infrared and Raman spectroscopy (IR), nuclear magnetic resonance spectroscopy (NMR), nuclear quadrupole resonance spectroscopy (NQR), incoherent inelastic neutron scattering (IINS), and differential scanning calorimetry (DSC). The solvatochromic and luminescent studies are also described. On the other hand, theoretical research is based on ab initio calculations and the Car–Parrinello Molecular Dynamics (CPMD). In the latter case, a description of nuclear quantum effects (NQE) is also possible. This book also demonstrates the use of theoretical methods such as Quantum Theory of Atoms in Molecules (QTAIM), Interacting Quantum Atoms (IQA), Natural Bond Orbital (NBO), Non-Covalent Interactions (NCI) index, Molecular Tailoring Approach (MTA), and many others