Quantitative Microbiology with Microscopy: Effects of Projection and Diffraction

Abstract Live cell imaging of microbial cells with microscopy has revolutionised quantitative microbiology. Micrographs are one of the most information-rich data types captured about a microbe, allowing quantification of the size and morphology of individual cells and their gene-expression over time. However, an optical microscope is a diffraction-limited system, and the comparable size of the system’s point spread function to the size of a microbial cell can lead to imaging artefacts which corrupt and bias the data. Additionally, the comparable thickness of a microbe to the microscope’s depth of field means that the 2D image contains compressed, projected 3D information. This makes it difficult to extract the underlying 3D distribution of photon emitters. For unknown distributions, the problem can be as ill-posed as a deconvolution problem, usually not having a unique solution. Together, the diffraction and projection effects affect our ability to accurately quantify the size and shape of microbial cells from their images and their contents from intensity measurements. In this paper, we use a mixture of simulations and experiments of microscopic image formation of microbial cells to illustrate the effects of diffraction and projection on cell segmentation and signal quantification. We use targeted experiments to validate the predictions where possible. Finally, we use the knowledge of these effects to design experiments which can help to reduce the errors and biases in our analysis. Awareness of these effects and the approaches towards alleviating them will help to accurately quantify microbiology from microscopy data.