Development of microscopy hardware & imaging techniques for macrophage host defence research

Mitochondrial reactive oxygen species (mROS) have recently emerged as critical microbicidal factors employed by macrophages in the clearance of internalised Streptococcus pneumoniae [1]. S. pneumoniae is a type of bacteria found in the upper respiratory tract, which when colonised can spread and lead to life threatening invasive pneumococcal diseases (IPD) [2]. Infants, the elderly and immunocompromised individuals are more susceptible to IPD [3]. A study has shown alveolar macrophages from patient with chronic obstructive pulmonary disease were unable to increase mROS production after bacterial challenge with S. pneumoniae, which correlated with impaired bacterial clearance [1]. They also observed colocalization between mROS, phagolysosomes and S. pneumoniae in human monocyte derived macrophages from healthy donors. There is a growing need to develop alternatives to conventional antimicrobials, for example pharmacologically enhancing the responses of macrophages, due to the growing worldwide problem of antimicrobial resistance, which has already been identified in S. pneumoniae to penicillin [4]. Furthermore, the number of S. pneumoniae serotypes included in currently available vaccinations is limited [5, 6]. Advances in microscopy and image analysis offer new opportunities to quantify interactions between mROS and S. pneumoniae. This thesis presents early studies which showed aberrations were present in confocal images of mitochondria labelled within human monocyte derived macrophages and the types and magnitudes of aberrations present varied across the sample, which could be problematic for colocalization imaging studies. Also, as prevalence of IPD is higher in some developing countries in Africa and Asia [7, 8] where resources are lower, we developed M4All: MultiModal Modular Microscopy for All [9], a 3D printable microscopy system, to increase accessibility to advanced microscopy techniques for macrophage host defence research. We also carried out initial studies implementing open-source computational microscopy techniques such as super-resolution radial fluctuations [10] and the transport of intensity equation [11].Mitochondrial reactive oxygen species (mROS) have recently emerged as critical microbicidal factors employed by macrophages in the clearance of internalised Streptococcus pneumoniae [1]. S. pneumoniae is a type of bacteria found in the upper respiratory tract, which when colonised can spread and lead to life threatening invasive pneumococcal diseases (IPD) [2]. Infants, the elderly and immunocompromised individuals are more susceptible to IPD [3]. A study has shown alveolar macrophages from patient with chronic obstructive pulmonary disease were unable to increase mROS production after bacterial challenge with S. pneumoniae, which correlated with impaired bacterial clearance [1]. They also observed colocalization between mROS, phagolysosomes and S. pneumoniae in human monocyte derived macrophages from healthy donors. There is a growing need to develop alternatives to conventional antimicrobials, for example pharmacologically enhancing the responses of macrophages, due to the growing worldwide problem of antimicrobial resistance, which has already been identified in S. pneumoniae to penicillin [4]. Furthermore, the number of S. pneumoniae serotypes included in currently available vaccinations is limited [5, 6]. Advances in microscopy and image analysis offer new opportunities to quantify interactions between mROS and S. pneumoniae. This thesis presents early studies which showed aberrations were present in confocal images of mitochondria labelled within human monocyte derived macrophages and the types and magnitudes of aberrations present varied across the sample, which could be problematic for colocalization imaging studies. Also, as prevalence of IPD is higher in some developing countries in Africa and Asia [7, 8] where resources are lower, we developed M4All: MultiModal Modular Microscopy for All [9], a 3D printable microscopy system, to increase accessibility to advanced microscopy techniques for macrophage host defence research. We also carried out initial studies implementing open-source computational microscopy techniques such as super-resolution radial fluctuations [10] and the transport of intensity equation [11]