Taylor Gordon T, Suter Elizabeth A, Li Zhuo Q, Chow Stephanie, Stinton Dallyce, Zaliznyak Tatiana, Beaupré Steven R
School of Marine and Atmospheric Sciences, Stony Brook UniversityStony Brook, NY, United States.
Front Microbiol. 2017 Aug 3;8:1449. doi: 10.3389/fmicb.2017.01449. eCollection 2017.
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, sp., grown in C-bicarbonate revealed linear covariance between wavenumber (cm) shifts in dominant carotenoid Raman peaks and a broad range of cellular C 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/[Formula: see text]), of which only ~2% was propagated analytical error. Instantaneous population growth rates measured independently by 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 sp. and (a diatom) populations with varying C-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.
介绍了一种通过结合稳定同位素探测（SIP）和单细胞共振拉曼显微光谱法来测量单个光合自养细胞生长速率的新方法。本报告探讨了最佳实验设计以及拉曼光谱对细胞同位素组成定量响应的理论基础。在碳酸氢盐碳源中生长的蓝藻等基因培养物的共振拉曼光谱显示，主要类胡萝卜素拉曼峰的波数（cm）位移与广泛的细胞碳分数同位素丰度之间存在线性协方差。根据光谱衍生的同位素含量和经验关系计算单细胞生长速率。样品中任意25个细胞的生长速率差异很大；平均变异系数CV为29±3%（σ/[公式：见正文]），其中只有约2%是传播分析误差。通过荧光独立测量的瞬时群体生长速率每天也有所变化（CV≈53%），并且在除最低细胞标记水平外的所有水平上，与单细胞生长速率在统计学上没有区别。对由不同碳含量和生长速率的蓝藻和硅藻（一种硅藻）群体制备的混合物进行的SCRR普查，紧密近似于预测的光谱响应和添加到样品中的细胞的分数标记。这种方法能够对同位素标记和系统发育标记的细胞进行直接的显微光谱询问，并能检测到细胞分数标记低至3%的变化。这是首次描述一种基于拉曼光谱和严格假设来测量单细胞光合自养生长速率的非破坏性技术，同时几乎不需要辅助测量。
