The causative agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb), is an intracellular pathogen infecting nearly 10 million people world-wide. Its cousin, Mycobacterium abscessus (MABC), is a major cause of death in immunocompromised individuals. However, despite the availability of multi-drug chemotherapy, the majority of deaths in both are due to drug-sensitive strains. This is often due to the presence of a phenotypically-resistant sub-population of bacteria termed antibiotic persisters in TB, which increase upon intracellular exposure, or antibiotic-tolerant biofilms within the lung airways for MABC. Determining antibiotic penetration and targeting persisters and biofilms can be challenging in highly heterogenous mycobacteria. We aim to use single cell techniques, such as microfluidics, nano-scale secondary ion mass spectrometry and atomic force microscopy, to evaluate antibiotic penetration and survival and to identify novel strategies to curtail these global health threats.