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This doctoral research project aims to analyse complex processes of living cells using Digital Holographic Microscopy (DHM) as a three-dimensional (3D) imaging tool. DHM is a real-time, high-throughput, label-free and quantitative phase imaging technique which permits advanced cell analysis in microfluidic environment. In particular, an innovative optofluidic platform is implemented, composed of a DHM modulus and aided by holographic optical tweezers (HOT) for optical manipulation and a fluorescence modulus. This platform has been used for blood disease screening, cell manipulation studies and tracking of migrating cells. In this thesis, three main topics have been investigated. The first topic focuses on diagnostics, which plays several critical roles in healthcare. Here a novel and cost-effective approach for detecting real blood disorders such as iron-deficiency anaemia and thalassemia at lab-on-chip scale is shown. In addition, cell dynamics studies were performed by DHM. In particular, a study regarding the temporal evolution of cell morphology and volume during blue light exposure is reported. The second topic aims to investigate cell mechanics. To this end, the capabilities of HOT were used to enable the generation and the independent high-precision control of an arbitrary number of 3D optical traps. The combination of HOT and DHM provides the possibility to manipulate cells, detect nano-mechanical cell response in the pN range, and reveal cytoskeleton formation. To confirm the formation of the cytoskeleton structures after the stimulation, a fluorescence imaging system was used as control. Finally, the third topic focuses on cell manipulation using an innovative electrode-free dielectrophoretic approach (DEP) for investigating smart but simple strategies for orientation and immobilization of biological samples such as bacteria and fibroblast. In particular, the light-induced DEP is achieved using ferroelectric iron- doped lithium niobate crystal as substrate. In this way, a dynamic platform that can dynamically regulate the cell response has been developed. In this case, DHM is going to be used as a time-lapse imaging tool for the characterization of dynamic cell processes. In conclusion, the results show that DHM is a highly relevant method that allows novel insights into dynamic cell biology, with applications in cancer research and toxicity testing. In addition, this study could pave the way for detecting and quantifying circulating tumor cells and for providing multidimensional information on tumour metastasis. In this framework, the optofluidic platform is a promising tool for both identification and characterization of “foreign” cancer cells in the blood stream in order to achieve an early diagnosis.