Imaging & Spectroscopy

Offworld Life Science Instrumentation: Single-cell Measurement Of Microbial Growth Rate With Raman Microspectroscopy

By Keith Cowing

Status Report

January 8, 2024

Filed under biorxiv.org, imaging, microbiology, microscopy, Nanotechnology, Raman microspectroscopy

Offworld Life Science Instrumentation: Single-cell Measurement Of Microbial Growth Rate With Raman Microspectroscopy — Representative Raman and nanoSIMS data collected in this study. Across all panels data from two representative cells, “1” and “2”, are noted. Representative nanoSIMS isotopic images of are shown in Panel A, with pixel counts labeled on the x and y axes. From left to right, the pixel intensity in each panel corresponds to direct ion counts for 1H-, 2H- , and the fractional abundance of deuterium (2F), respectively. The top row shows cells grown in 0% 2H2O (natural abundance); the bottom row displays cells grown in 50 at. % 2H2O. Representative single-cell fitted Raman spectra are shown in Panel B, with the characteristic C-H band between 2800 and 3000 cm-1 clearly visible. For cells grown in deuterated media (Cell 2), the C-D band emerges between 2040 and 2300 cm-1. Panel C displays a typical micrograph captured through a confocal Raman microspectroscope. The scale bar represents 2µm. Cells in all panels represent T. hydrogeniphilus. The yellow boxes in the top-left facet of Panel A, and Panel C, represent a correlated region imaged with nanoSIMS and Raman (reflected light), respectively. — biorxiv.org

Rates 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 biomass 2H values and find that Raman microspectroscopy reliably quantifies 2H 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.

Tristan A. Caro, Srishti Kashyap, George Brown, Claudia Chen, Sebastian H. Kopf, Alexis S. Templeton
doi: https://doi.org/10.1101/2023.12.16.571966

Single-cell Measurement Of Microbial Growth Rate With Raman Microspectroscopy

Astrobiology, Tricorder

Keith Cowing

Explorers Club Fellow, ex-NASA Space Station Payload manager/space biologist, Away Teams, Journalist, Lapsed climber, Synaesthete, Na’Vi-Jedi-Freman-Buddhist-mix, ASL, Devon Island and Everest Base Camp veteran, (he/him) 🖖🏻

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