Coltelli Primo, Barsanti Laura, Evangelista Valter, Frassanito Anna Maria, Gualtieri Paolo
Istituto Scienza e Tecnologie dell'Informazione, CNR, Pisa, Italy.
Istituto di Biofisica, CNR, Pisa, Italy.
J Microsc. 2016 Dec;264(3):311-320. doi: 10.1111/jmi.12445. Epub 2016 Jul 18.
A novel procedure for deriving the absorption spectrum of an object spot from the colour values of the corresponding pixel(s) in its image is presented. Any digital image acquired by a microscope can be used; typical applications are the analysis of cellular/subcellular metabolic processes under physiological conditions and in response to environmental stressors (e.g. heavy metals), and the measurement of chromophore composition, distribution and concentration in cells. In this paper, we challenged the procedure with images of algae, acquired by means of a CCD camera mounted onto a microscope. The many colours algae display result from the combinations of chromophores whose spectroscopic information is limited to organic solvents extracts that suffers from displacements, amplifications, and contraction/dilatation respect to spectra recorded inside the cell. Hence, preliminary processing is necessary, which consists of in vivo measurement of the absorption spectra of photosynthetic compartments of algal cells and determination of spectra of the single chromophores inside the cell. The final step of the procedure consists in the reconstruction of the absorption spectrum of the cell spot from the colour values of the corresponding pixel(s) in its digital image by minimization of a system of transcendental equations based on the absorption spectra of the chromophores under physiological conditions.
本文提出了一种从物体斑点图像中相应像素的颜色值推导其吸收光谱的新方法。可以使用通过显微镜获取的任何数字图像；典型应用包括在生理条件下以及对环境应激源（如重金属）作出反应时对细胞/亚细胞代谢过程的分析，以及对细胞中发色团组成、分布和浓度的测量。在本文中，我们用安装在显微镜上的电荷耦合器件（CCD）相机获取的藻类图像对该方法进行了验证。藻类呈现出的多种颜色是由发色团组合产生的，其光谱信息仅限于有机溶剂提取物，与细胞内记录的光谱相比存在位移、放大以及收缩/扩张现象。因此，需要进行预处理，包括对藻类细胞光合区室吸收光谱的体内测量以及细胞内单个发色团光谱的测定。该方法的最后一步是通过基于生理条件下发色团吸收光谱的超越方程组的最小化，从其数字图像中相应像素的颜色值重建细胞斑点的吸收光谱。
