Hematopoietic stem cells (HSC) are responsible for the life-long maintenance of our blood system. Their long-term capacity to both self-renew and differentiate and the ability to efficiently ‘home’ to their bone marrow niches when injected in the blood stream, makes these rare cells ideal candidates for transplantation for the treatment of various blood cancers. However, countless attempts to expand HSC in vitro without losing their stem cell potential have failed, which is, to a large extent, linked to our poor understanding of the mechanisms that control HSC fate. Recent discoveries have revealed a crucial role of metabolism in controlling HSC function in vivo. However, because of major technical difficulties, we do not yet know the underlying mechanisms behind the metabolic control of HSC fate. Traditional, population-level experimental approaches link the fate of a stem cell to a cell surface protein expression phenotype. This method does not take into account the large heterogeneity of metabolic states in HSC. Consequently, to get mechanistic insights on HSC fate decision-making, metabolism must be studied at single cell level. Therefore, the overall goal of this thesis is to establish novel tools to study metabolic regulation of HSC at single cell level. Here we specifically focused on two functional metabolic read-outs, protein turnover and mitochondrial pH. Mitochondrial matrix pH is one of the most important functional parameters of mitochondria, as it is directly related to the efficiency of ATP production and thus reflects the metabolic state of the mitochondria. Here, a new ratiometric fluorescent probe, 5FA-PIP-Cy5-DA, able to reliably quantify mitochondrial pH, has been developed. It selectively accumulates in mitochondria of live cells and can be used to sensitively detect mitochondrial pH fluctuations upon various external stimuli. Spectral properties of 5FA-PIP-Cy5-DA allow it to be combined with the total mitochondrial membrane potential-dependent probes, enabling simultaneous imaging of total mitochondrial potential and mitochondrial pH. To complement the data on mitochondrial pH in live cells with an additional independent parameter of mitochondrial metabolism, a method of correlated TEM and nanoscale secondary ion mass-spectrometry (NanoSIMS) imaging was developed, that for the first time allows to study the subcellular protein turnover in single stem cells. Application of these novel tools to study HSC metabolism revealed a striking correlation between the levels of mitochondrial protein turnover and mitochondrial pH. This correlation reflects the burst in mitochondrial metabolism upon HSC activation. Intriguingly, an extreme burst in both mitochondrial pH alkalinization and protein turnover rate was observed for the most potent long-term HSC (LT-HSC) upon their activation and induction of differentiation. A new model is proposed to explain the link between the observed activation of mitochondrial protein structure reorganization and increase in mitochondrial pH level of LT-HSC undergoing differentiation. Finally, to demonstrate the broad range of applicability of the new mitochondrial pH probe, we applied it to measure the impact of anticancer treatment on mitochondrial metabolism in ovarian cancer cells exposed to two anticancer drugs, RAPTA-T and cis-platin. Distinctly different mitochondrial pH responses were measured for these two drugs, revealing alternative mechanisms behind their impact on mitochondria.