The innate resistance of Gram-negative bacteria to antibiotics is a consequence of the combinatorial effects of two permeability barriers: the outer and inner bacterial cell membranes, their ability to efflux antibiotics out of the cell and their capacity to form antibiotic tolerant biofilms that are up to 100 times more resistant than planktonic bacterial cells. The objectives of this project are to advance the measurement capability by providing the urgently needed essential metrology to quantitatively measure and image the localisation of antibiotics and to understand the penetration and efflux processes in bacteria and biofilms. It is universally acknowledged that the threat of antimicrobial resistance (AMR) to the health and prosperity of Europe and the world is real. The European Union has a major initiative to fight AMR. The Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) is developing a strategic research agenda and is co-ordinating European research in Horizon 2020, in the Innovative Medicines Initiative (IMI) and in the EC’s ERA-NET funding scheme. For example, the New Drugs for Bad Bugs (ND4BB) programme of the IMI involves nine large pharmaceutical companies in seven ND4BB projects, with a total committed budget of more than €600 million. They have identified a metrology gap that EMPIR is uniquely placed to fill. The ND4BB project TRANSLOCATE has stated that “At present, there are no reliable and general methods for measuring these [drug] penetration processes in Gram-negative bacteria and this bottleneck substantially hinders the ability of scientists to optimise antimicrobial activity in intact bacterial cells”. Furthermore, they identify the key need to quantify and image the penetration of drugs into bacteria and to measure the efflux processes. MetVBadBugs is directly focused at these metrology challenges. There is clearly no single technique that can deliver all the measurement answers needed by scientists studying AMR and developing new antibiotics. A robust metrology framework is needed, which is built on fundamental studies of the techniques as well as being combined with cross measurement platform validation including pre-normative studies. The objectives of this project are clearly aimed at addressing these needs. Objectives To develop urgently needed new metrological capabilities for: the label‑free 3D imaging of antibacterial agents in bacteria. This requires a new 3D chemical imaging instrument with 100 times better sensitivity and a high‑spatial resolution (100 nm). The instrument will have a mass resolution of >100 000 and the ability to sample from sub‑micron areas, simultaneously the traceable quantification of the vertical concentration profile of antibacterial agents in bacteria and biofilms. Measurements will be performed in liquid and at near ambient pressure imaging surface macromolecules, such as porins or metal‑transport proteins, to study the efflux mechanisms in Gram‑negative bacteria and to give real‑time quantitative measurements of drug‑uptake in bacteria and biofilms. Numerical modelling and algorithms will be developed to support measurements in complex biological environments To develop well‑controlled model systems to allow cross‑platform measurement of penetration, accumulation and efflux of antibacterial agents in single cells, in suspended cellular aggregates, as well as in biofilm communities including binding to biofilm matrix components. The efficacy of novel antibacterial agents and efflux pump inhibitors will be investigated. To develop signal enhancement strategies and advanced sample preparation methods for studying antibacterial agents in bacteria and biofilms including: advanced cryo preparation methods to enable ‘liquid’ (vitrified) water to be present in the vacuum of high‑performance metrology instruments without ultrastructural reorganisation and translocation of exo/endo ‑genous molecules novel methods to nano‑sculpt bacteria for chemical imaging at 50 nm resolution nano structured substrates for enhanced sensitivity To facilitate the take up of the technology and measurement infrastructure developed by the project by healthcare professionals (hospitals and health centres) and industry (pharmaceutical companies), in order to fight the threat from antimicrobial resistance to the health and prosperity of Europe.