Abstract
A new method to measure growth rates of individual photoautotrophic cells by combining stable isotope probing (SIP) and single-cell resonance Raman microspectrometry is introduced. This report explores optimal experimental design and the theoretical underpinnings for quantitative responses of Raman spectra to cellular isotopic composition. Resonance Raman spectra of isogenic cultures of the cyanobacterium, Synechococcus sp., grown in 13C-bicarbonate revealed linear covariance between wavenumber (cm−1) shifts in dominant carotenoid Raman peaks and a broad range of cellular 13C fractional isotopic abundance. Single-cell growth rates were calculated from spectra-derived isotopic content and empirical relationships. Growth rates among any 25 cells in a sample varied considerably; mean coefficient of variation, CV, was 29 ± 3% (σ/), of which only ~2% was propagated analytical error. Instantaneous population growth rates measured independently by in vivo fluorescence also varied daily (CV ≈ 53%) and were statistically indistinguishable from single-cell growth rates at all but the lowest levels of cell labeling. SCRR censuses of mixtures prepared from Synechococcus sp. and T. pseudonana (a diatom) populations with varying 13C-content and growth rates closely approximated predicted spectral responses and fractional labeling of cells added to the sample. This approach enables direct microspectrometric interrogation of isotopically- and phylogenetically-labeled cells and detects as little as 3% changes in cellular fractional labeling. This is the first description of a non-destructive technique to measure single-cell photoautotrophic growth rates based on Raman spectroscopy and well-constrained assumptions, while requiring few ancillary measurements.
Highlights
Ecological theory suggests that predictions about population dynamics in planktonic systems have limited skill if intraspecific trait variation and heterogeneous resource distributions are ignored
We present a refinement of the stable isotope probing (SIP)-SCRR-fluorescent in situ hybridization (FISH) approach (Li et al, 2012) that enables quantitative Raman spectrometric measurement of growth rates in individual photoautotrophic cells
For SIP experimental design, it was initially critical to establish whether dissolved inorganic carbon (DIC) replacement or augmentation is preferable, to determine how much 13C-bicarbonate tracer is required, and to accurately establish the value of f media
Summary
Ecological theory suggests that predictions about population dynamics in planktonic systems have limited skill if intraspecific trait variation and heterogeneous resource distributions are ignored. Even clonal populations (isogenic) in apparently homogenous cultures exhibit varying genetics, biochemistry, physiology, and behavior, all of which can produce a range of “growth phenotypes” (Lidstrom and Konopka, 2010; Damodaran et al, 2015; Kopf et al, 2015) This recognition has given rise to agent-based or individual-based models which don’t assume that cell attributes within a population are uniformly or normally distributed around their mean values (e.g., Hellweger and Kianirad, 2007). The vast majority of measurements have overlooked this microspatial heterogeneity and cryptic material exchanges (e.g., Canfield et al, 2010), and have provided net biogeochemical transformations at best They fail to unequivocally link key players to particular processes. Single-cell techniques are the only way forward to empirically determine how intraspecific phenotypic/genotypic variations and microspatial architecture (the aquascape) determine individual ecophysiologies and translate into collective population responses
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