Homepage

mmc2021 Abstract Database

Introducing the M⁴ (MultiModal Modular Microscopy) 3D Printed Microscope System for Imaging Macrophage-Pathogen Interactions

Introducing the M⁴ (MultiModal Modular Microscopy) 3D Printed Microscope System for Imaging Macrophage-Pathogen Interactions

Session

Stream 5: Up Close with the Enemy: Imaging Pathogen-host Dynamics

Authors

Miss Gemma Cairns (2, 1), Prof David Dockrell (1), Dr Brian Patton (2)

Affiliations

1. University of Edinburgh
2. University of Strathclyde

Keywords

Macrophages, Streptococcus pneumoniae, Host-Pathogen Interaction, 3D Printing, Open Source Microscopy, Microscope Development, Fluorescence

Abstract text

Mitochondrial reactive oxygen species (mROS) have recently emerged as critical microbicidal factors employed by macrophages^1,2. Our long-term aim is to quantify the role of mROS in the clearance of internalised extracellular bacteria in alveolar macrophages, particularly Streptococcus pneumoniae, the most common cause of the inflammatory condition pneumonia². Recently, colocalisation has been observed between mROS, generated in response to S. pneumoniae, phagolysosomes and the bacteria itself². 

Advances in microscopy and image analysis offer new opportunities to quantify these interactions to allow future examination of our ability to pharmacologically enhance host responses against antimicrobial resistant bacteria. However, there are numerous challenges posed when trying to image mROS in macrophages, particularly when also trying to correlate mROS location and dynamics with specific host responses. 

To address some of these challenges, we have developed the M⁴: MultiModal Modular Microscopy system, a fully open-source 3D-printable optics system which allows users to design and build different setups for different imaging modalities. Advanced microscopy hardware, for techniques such as live cell and super-resolution microscopy, which are key for investigating host-pathogen interactions, can be costly and often inaccessible to research groups. Frugal innovation will therefore allow an increased availability of advanced microscopy technologies, particularly when combined with computational microscopy techniques.  

For colocalisation studies in cells, for example between mitochondria and S. pneumoniae, it is necessary to use multichannel fluorescence microscopy. Here, we present the M⁴ system in combination with the OpenFlexure³ stage, as seen in the figure below, and alignment/calibration tools based on ray transfer matrix analysis for building a multichannel imaging system. We also present initial imaging data of macrophages using a multichannel fluorescence microscope built with the M⁴ system. In future we hope to employ the M⁴ system within a cell culture incubator for live cell imaging of macrophage-pathogen interactions. 

 

References

1. West, A. P. et al. TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature 472, 476-480 (2011).
2. Bewley, M. A. et al. Impaired Mitochondrial Microbicidal Responses in Chronic Obstructive Pulmonary Disease Macrophages. Am. J. Respir. Crit. Care Med. 196, 845-855 (2017).  
3. Sharkey, J. P. et al. A one-piece 3D printed flexure translation stage for open-source microscopy. Rev. Sci. Instrum. 87, 25104 (2016).