Spectrophotometric Analysis of Pigments: A Critical Assessment of a High-Throughput Method for Analysis of Algal Pigment Mixtures by Spectral Deconvolution.
Spectrophotometric Analysis of Pigments: A Critical Assessment of a High-Throughput Method for Analysis of Algal Pigment Mixtures by Spectral Deconvolution.
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DOI：
10.1371/journal.pone.0137645
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Andersen T
中科院分区：
文献类型：
--
作者：
Thrane JE;Kyle M;Striebel M;Haande S;Grung M;Rohrlack T;Andersen T
高斯峰光谱（GPS）方法表示为高斯函数的加权和个别色素光谱，并使用这些模型的吸收光谱浮游植物色素混合物。我们在这里提出了对此类方法的几项改进，包括适应读板器技术和通过开源软件进行高效模型拟合。我们使用一个一步建模的色素吸收和背景衰减与非负最小二乘法，一次特定的仪器校准。拟合的背景被示出为高于溶剂空白，具有反映来自散射和非颜料吸收的贡献的特征。我们通过Monte Carlo模拟评估了由于吸收光谱相似性而导致的色素混叠，并使用这些信息来选择一组稳健的可识别色素，这些色素预计在天然样品中也很常见。为了测试该方法的性能，我们分析了从沉积物芯，75个天然湖泊样品，和四个浮游植物培养物的色素提取物的吸收光谱，并比较了估计的色素浓度与使用高效液相色谱法（HPLC）获得的浓度。观察到的光谱和拟合光谱之间的偏差一般是非常低的，表明测量光谱可以成功地重建为加权和的颜料和背景成分。总叶绿素和总类胡萝卜素的浓度可以准确地估计沉积物和湖泊样品，但个别色素浓度（特别是类胡萝卜素）证明难以解决，由于它们的吸收光谱之间的相似性。在一般情况下，我们的改进的GPS方法提供了一个改进的GPS方法，是一个快速，廉价，高通量的替代品，用于筛选浮游植物材料样品中的色素成分。
The Gauss-peak spectra (GPS) method represents individual pigment spectra as weighted sums of Gaussian functions, and uses these to model absorbance spectra of phytoplankton pigment mixtures. We here present several improvements for this type of methodology, including adaptation to plate reader technology and efficient model fitting by open source software. We use a one-step modeling of both pigment absorption and background attenuation with non-negative least squares, following a one-time instrument-specific calibration. The fitted background is shown to be higher than a solvent blank, with features reflecting contributions from both scatter and non-pigment absorption. We assessed pigment aliasing due to absorption spectra similarity by Monte Carlo simulation, and used this information to select a robust set of identifiable pigments that are also expected to be common in natural samples. To test the method’s performance, we analyzed absorbance spectra of pigment extracts from sediment cores, 75 natural lake samples, and four phytoplankton cultures, and compared the estimated pigment concentrations with concentrations obtained using high performance liquid chromatography (HPLC). The deviance between observed and fitted spectra was generally very low, indicating that measured spectra could successfully be reconstructed as weighted sums of pigment and background components. Concentrations of total chlorophylls and total carotenoids could accurately be estimated for both sediment and lake samples, but individual pigment concentrations (especially carotenoids) proved difficult to resolve due to similarity between their absorbance spectra. In general, our modified-GPS method provides an improvement of the GPS method that is a fast, inexpensive, and high-throughput alternative for screening of pigment composition in samples of phytoplankton material.