One Cell at a Time: Advances in Single-Cell Methods and Instrumentation for Discovery in Aquatic Microbiology

Abstract

Studying microbes from a single-cell perspective has become a major theme and interest within the field of aquatic microbiology. One emerging trend is the unfailing observation of heterogeneity in activity levels within microbial populations. Wherever researchers have looked, intra-population variability in biochemical composition, growth rates, and responses to varying environmental conditions has been evident and probably reflect coexisting genetically distinct strains of the same species. Such observations of heterogeneity require a shift away from bulk analytical approaches and development of new methods or adaptation of existing techniques, many of which were first pioneered in other, unrelated fields, e.g., material, physical, and biomedical sciences. Many co-opted approaches were initially optimized using model organisms. In a field with so few cultivable models, method development has been challenging but has also contributed tremendous insights, breakthroughs, and stimulated curiosity. In this perspective, we present a subset of methods that have been effectively applied to study aquatic microbes at the single-cell level. Opportunities and challenges for innovation are also discussed. We suggest future directions for aquatic microbiological research that will benefit from open access to sophisticated instruments and highly interdisciplinary collaborations.

Exploring environmental microfungal diversity through serial single cell screening

“…Microbial ecologists have recently been able to adapt and combine a variety of tools previously developed in far-ranging fields [Grujcic et al 2022] for fungal identification, such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [Cornut et al 2019]; single-spore isolation, followed by marker gene (rDNA) barcoding sequencing [Bärlocher et al 2010; Karpov et al 2020; Kagami et al 2021; Strassert et al 2021; van den Wyngaert et al 2022; Nakanishi et al 2023] and even single-spore isolation, genome amplification and whole-genome sequencing [Ciobanu et al 2022]. While the single cell analysis using flow cytometry is an established method, it is limited by the size of the cells and only works in an automated manner, which usually excludes manual inspections by cells.…”

Targeted Cell Labeling and Sorting of Prokaryotes for Cultivation and Omics Approaches

“…As a result, mass spectrometric imaging 3D mapping of complex microbiological forms (e.g., aggregates or biofilms) can be difficult. Additional measures are required for successful sample preparation, demanding further stabilization or freezing, which generally results in loss of live cells [Watrous and Dorrestein, 2011;Grujcic et al, 2022]. Moreover, SIMS imaging employs a harsh and destructive ionization technique leaving cells in a nonviable state after analysis [Watrous and Dorrestein, 2011].…”

“…Most NanoSIMS analysis efforts utilize isotope and/or rare element labeling to enable the detection of processes or species of interest inside biological samples [Nuñez et al, 2018]. NanoSIMS is now frequently used in combination with other measures such as fluorescent in situ hybridization (FISH) or bioorthogonal noncanonical amino acid tagging (BONCAT) [Grujcic et al, 2022] (discussed below). Although the method is destructive and downstream applications such as cultivation and omics approaches are not possible, its application in a variety of studies shows a valuable information gain in the detection of MDM and description of prokaryotic communities on single-cell level (reviewed in, e.g., [Orphan and House, 2009;Gao et al, 2016;Musat et al, 2016;Nuñez et al, 2018]).…”

MALDI Mass Spectrometry Imaging: A Potential Game-Changer in a Modern Microbiology

“…Therefore, by adapting the laser geometry or optimizing the laser focusing, MALDI-MSI seems to be in a position to get closer to microscopic techniques. Additionally, the potential combination of single-cell imaging and single-cell sequencing was underlined as being able to give important insights regarding aquatic microbes and their environment [ 131 ]. Secondly, the limit of detection of microorganisms per pixel needs to be established.…”