Motivation

In the field of biological sciences, the observation and investigation of living specimens at the microscopic size-scale are especially invaluable. Hence, optical microscopy plays an unrivalled role in exploring the insight of biological processes by observing living cells, organs or embryos during development, that would otherwise be impossible. Living biological systems are highly complex. It comprises a host of cascading dynamic interactions related to cellular mechanics well as biochemical signals. Due to these tiny spatial dynamics between cells, real-time three-dimensional quantification plays a significant part in revealing the biomechanical/biochemical properties of cells. Consequently, copious amounts of efforts have been devoted to developing volumetric imaging techniques to satisfy various cellular biology studies 1-4. Three-dimensional imaging using laser scanning techniques, such as confocal laser scanning microscopy 5-6, multiphoton microscopy 7-8_{, light sheet microscopy} 9_{, and super-resolution microscopes} 10-11

are the representatives of the frontiers of volumetric imaging techniques. High resolution, depth selectivity and single protein specificity offered by these techniques have accelerated progress in pathological research of diseases at the cellular level 12-13, which complements other biochemical research 14-15, single molecule 16 for last decades. However, one of drawbacks in fluorescence microscopy is requirement to selectively stain samples. Sample preparation often involves exogenous fluorophore which usually need to attach to another molecule before it can be attached to a cell. This alteration could potentially affects cells’ natural state 17-18. On the other hand, there are more detrimental effects such as photobleaching and phototoxicity. Direct laser irradiation for efficient fluorescence excitation have shown to impair cell functions and reduce its viability for long-term investigation 17, 48. Emerging techniques to overcome phototoxicity in fluorescent imaging include light sheet microscopy and confocal spinning disc microscopy. Without fluorescent-labelling, it is also possible to capture sub-cellular structures using phase-based light microscopy. Quantitative phase microscopy (QPM) 19-22 has emerged into a powerful tool for label-free live cells study. It offers a two-dimensional height map of the sample by retrieving the

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phase delay of the light passing through the sample. Based on a 2D array detector (digital camera), the imaging speed of QPM is only limited by the speed of detector (from tens to thousands of Hertz) which is well-suited for real-time monitoring of biological events. Unlikely traditional workhorse technologies such as epifluorescence microscopy and phase contrast microscopy, QPM faces some limitations which needs to be overcome before it can become an universal imaging tool. Primary limitations of QPM are the inability to distinguish heterogeneity along the vertical direction, and additional digital reconstructions. For full axial resolvable QPM, one needs to use optical tomography techniques 23- 24_{. However, optical tomography is laden with multi-frame acquisitions and}

additional digital transforms 25 _{that contributes to a reduction in imaging speed}

and is precluded from its applications on real-time image reconstruction. On the other hand, automation of the QPM reconstruction processes in great demand to make it accessible for biologists to obtain statistically meaningful data.

Understanding dynamic processes and mechanisms of disease-related events is important in biology studies. The blood disease and disorders are one of diseases that remains to be overcome due to complexity in fluidic flows within living vessels 26-27. Red blood cells (RBCs) and platelets contribute to majority of the blood components. The diseases related to morbidity of RBCs and platelet disorders lead to the major lethality, such as malaria 28, haemophilia 29, and thrombosis 27. A number of research tools and methods have been developed in the past two decades for investigating the fundamental mechanism of blood related disorders with dynamic manipulation and cellular response measurement

30-31_{. Microfluidic technique is one of the tools that can realize high-throughput}

measurements on single-cell biophysical properties at a relatively low cost 32-37. Thus, this thesis is primarily motivated to develop an automatic volumetric imaging system to investigate the dynamic response of RBCs and platelets under fluid shear conditions. For RBCs, the focus on parasite-induced alteration in morphology and deformability is motivated by efforts in diagnosing early stages of malaria infection. Study of platelets provides an effective real time measurement for investigating the platelet disorders and thrombosis. Dynamic imaging of platelets aggregation and thrombus formation in microfluidic device has been ongoing for more than ten years 38. However, most quantifications of

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platelet and thrombus formation are accomplished by integrating the fluorescence intensity of images. The morphological characterization of individual thrombus received less attention. One of the main reasons is that the fluorescence imaging techniques usually obtain morphology reconstruction at the cost of sacrificing the imaging speed by vertical laser scanning. However, imaging speed is vital in investigating dynamic events, for example, thrombus formation and embolism in the fluidic. As a result, real-time morphological measurements of thrombus formation and embolism is targeted using QPM system developed in the thesis, which could potentially provide the ability of phenotypic quantifications and therapy tests. Since every single imaging technique has its own merit and shortcoming, the combination of two or more imaging modalities have been studied and employed to provide multifunctional imaging 39-41_{. At the end of the}

thesis, an initial version of a multimodality imaging system was designed and implemented for microfluidic experiments.