Quantitative microbiology with widefield microscopy: navigating optical artefacts for accurate interpretations

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 point spread function of the microscope 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 depth of field of the microscope 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.

Transertion and cell geometry organize theEscherichia colinucleoid during rapid growth

“…This arrangement was not seen in the 2D projection ( Fig. 1A i ), highlighting that projection effects in widefield and 2D SMLM images might not reveal the actual organization of the target structure 7,39 . In the 3D image, the nucleoid appears positioned close to the cell membrane, as we have previously shown using STED microscopy 40 .…”

“…RNL describes the fraction of cell length that is populated by chromosomal DNA. This metric is similar to the relative nucleoid size used by Cabrera and colleagues 47 , yet we prefer RNL in diffraction-limited images due to the 2D projection effects shown in Figure 1A and literature 7,39 . To monitor reorganization of MreB following antibiotic treatment, we quantified its relative distribution at the cell cylinder and cell poles (Fig.…”