Single-cell measurement of microbial growth rate with Raman microspectroscopy

Author:

Caro Tristan A.ORCID,Kashyap SrishtiORCID,Brown George,Chen Claudia,Kopf Sebastian H.ORCID,Templeton Alexis S.ORCID

Abstract

AbstractRates of microbial activity and growth are fundamental to understanding environmental geochemistry and ecology. However, measuring the heterogeneity of microbial activity at the single-cell level, especially within complex populations and environmental matrices, remains a forefront challenge. Stable Isotope Probing (SIP) is a standard method for assessing microbial activity and involves measuring the incorporation of an isotopically labeled compound into microbial biomass. Here, we assess the utility of Raman microspectroscopy as a SIP technique, specifically focusing on the measurement of deuterium (2H), a tracer of microbial biomass production. We generate calibrations of microbial biomass2H values and find that Raman microspectroscopy reliably quantifies2H incorporation ranging between 0 and 40 at. %. Applying the results of this calibration to a SIP model, we explicitly parameterize the factors controlling microbial growth quantification, demonstrating how Raman-SIP can measure the growth of microorganisms with doubling times ranging from hours to years. Furthermore, we correlatively compare our Raman-derived measurements with those of nanoscale secondary ion mass spectrometry (nanoSIMS) to compare the relative strengths of nanoSIMS- and Raman-based SIP approaches. We find that Raman microspectroscopy is a robust, accessible methodology that can readily differentiate and quantify the growth of individual microbial cells in complex samples.ImportanceGrowth rate, the rate at which organisms grow and reproduce, is a key metric with which to evaluate microbial physiology and contributions to system-level processes. The heterogeneity of microbial growth across space, time, and populations is often difficult to capture with bulk-scale techniques. Single-cell methods hold promise for measuring the heterogeneity of microbial growth rates and responses to changing conditionsin situ, without the need for cultivation of microbial isolates. In this study, we evaluated the ability of Raman microspectroscopy, a non-destructive and rapid technique, to measure the assimilation of isotopically labeled water into individual microbial cells and thereby calculate their rates of growth. We explicitly parameterize the factors controlling the quantification of microbial growth rate and compare this technique to standard methods. The framework we report allows researchers to couple single-cell and aggregate rate measurements to functional or system-level properties, a forefront challenge in microbiology.

Publisher

Cold Spring Harbor Laboratory

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