Kavanagh K Y, Murphy E J, Harmey M, Farrell M A, Hardimann O, Perryman R, Walsh J E
Optical Sensors and Metrology Laboratory, School of Physics, Dublin Institute of Technology, Ireland.
Physiol Meas. 1999 Aug;20(3):303-11. doi: 10.1088/0967-3334/20/3/307.
A microspectrophotometer system to monitor the reduction of mitochondrial respiratory pigments in cell extracts and permeabilized cells has been developed. The novel optical fibre set-up uses visible spectrophotometry to measure the reduction of mitochondrial electron carriers. The basis of the system is an Ocean Optics S1000 spectrometer, a broadband tungsten based light source, input and output coupling fibre optics and a fibre optic dip-probe which requires less than 20 microl of sample for analysis. The spectral range of the system is from 250 to 850 nm with a spectral resolution of 0.5 nm. Data are presented for the reduction of purified cytochrome c by the reducing agent sodium dithionite and the reduction of cytochrome c by isolated mitochondria using sodium succinate as substrate. Reduction of cytochrome c by digitonin permeabilized cultured mouse cells, C2C12, is also shown. The effect of temperature on cytochrome c reduction in these assays is also demonstrated. The optical design of the probe system is optimized to maintain maximum light throughput and spectral resolution. The key features of the system are small sample size, front-end adaptability, high sensitivity and fast multispectral acquisition which are essential for observing these biological reactions in vivo.
已开发出一种用于监测细胞提取物和透化细胞中线粒体呼吸色素还原情况的显微分光光度计系统。这种新颖的光纤装置利用可见分光光度法来测量线粒体电子载体的还原情况。该系统的基础是海洋光学S1000光谱仪、基于宽带钨的光源、输入和输出耦合光纤以及一根光纤浸入式探头，分析所需样品量不到20微升。该系统的光谱范围为250至850纳米，光谱分辨率为0.5纳米。给出了用连二亚硫酸钠还原剂还原纯化细胞色素c以及以琥珀酸钠为底物用分离的线粒体还原细胞色素c的数据。还展示了用洋地黄皂苷透化培养的小鼠细胞C2C12还原细胞色素c的情况。也证明了温度对这些测定中细胞色素c还原的影响。探头系统的光学设计经过优化，以保持最大光通量和光谱分辨率。该系统的关键特性是样品量小、前端适应性强、灵敏度高和多光谱采集速度快，这些对于在体内观察这些生物反应至关重要。
