Improving accuracy of cell and chromophore concentration measurements using optical density.
Improving accuracy of cell and chromophore concentration measurements using optical density.
复制标题
DOI：
10.1186/2046-1682-6-4
复制
发表时间：
2013-04-22
期刊：
影响因子：
--
通讯作者：
Curtis WR
中科院分区：
文献类型：
--
作者：
Myers JA;Curtis BS;Curtis WR
紫外-可见分光光度法光密度（OD）是液体培养中最常用的估计发色团形成和细胞浓度的技术。选择外径波长时往往很少考虑其对测量质量的影响。分析吸收和散射对测量光密度的贡献为理解分光光度计之间的变异性提供了基础，并能够定量评估比尔-朗伯定律的适用性。这为提高作为直接干重（DW）、细胞计数和色素水平代表的外径测量的准确性提供了一种合理的方法。对于有色素的生物，OD波长的选择是测量鲁棒性和灵敏度之间的权衡。在强波长处的OD主要是光散射的结果，不随培养条件的变化而变化；而在敏感波长处的OD则取决于生物体色素对光的吸收。通过将其光谱与染料的真实吸收光谱进行比较，可以为广泛的生物体确定适当的鲁棒和敏感波长。可以通过在较高OD下测量或加入牛血清白蛋白来降低相对散射贡献。减少散射或与非峰光衰减的相关性，可以更准确地评估细胞内的发色团水平。本文列出了17种不同生物的DW、OD和菌落形成单位密度之间的转换因子，以说明这些相关性的可变性范围。最后，展示了一种廉价的短路径长度led流池，用于在线监测培养浓度大于5克干重/升的生物反应器中的生长，否则需要离线稀释以获得非饱和OD测量。当光衰减以散射为主时，OD作为一种省时的生物质浓度替代测量方法是最准确的。然而，基于od相关性的适用性高度依赖于测量规格（分光光度计型号和波长）和培养条件（培养基类型、生长阶段、培养胁迫、细胞/菌落几何形状、分泌化合物的存在和浓度）。这些变化强调了将文献转换因子视为粗略近似值而不是具体常数的重要性。通过考虑OD光谱的散射和吸收依赖波长，有机会优化细胞色素水平的测量。
UV–vis spectrophotometric optical density (OD) is the most commonly-used technique for estimating chromophore formation and cell concentration in liquid culture. OD wavelength is often chosen with little thought given to its effect on the quality of the measurement. Analysis of the contributions of absorption and scattering to the measured optical density provides a basis for understanding variability among spectrophotometers and enables a quantitative evaluation of the applicability of the Beer-Lambert law. This provides a rational approach for improving the accuracy of OD measurements used as a proxy for direct dry weight (DW), cell count, and pigment levels. For pigmented organisms, the choice of OD wavelength presents a tradeoff between the robustness and the sensitivity of the measurement. The OD at a robust wavelength is primarily the result of light scattering and does not vary with culture conditions; whereas, the OD at a sensitive wavelength is additionally dependent on light absorption by the organism’s pigments. Suitably robust and sensitive wavelengths are identified for a wide range of organisms by comparing their spectra to the true absorption spectra of dyes. The relative scattering contribution can be reduced either by measurement at higher OD, or by the addition of bovine serum albumin. Reduction of scattering or correlation with off-peak light attenuation provides for more accurate assessment of chromophore levels within cells. Conversion factors between DW, OD, and colony-forming unit density are tabulated for 17 diverse organisms to illustrate the scope of variability of these correlations. Finally, an inexpensive short pathlength LED-based flow cell is demonstrated for the online monitoring of growth in a bioreactor at culture concentrations greater than 5 grams dry weight per liter which would otherwise require off-line dilutions to obtain non-saturated OD measurements. OD is most accurate as a time-saving proxy measurement for biomass concentration when light attenuation is dominated by scattering. However, the applicability of OD-based correlations is highly dependent on the measurement specifications (spectrophotometer model and wavelength) and culture conditions (media type; growth stage; culture stress; cell/colony geometry; presence and concentration of secreted compounds). These variations highlight the importance of treating literature conversion factors as rough approximations as opposed to concrete constants. There is an opportunity to optimize measurements of cell pigment levels by considering scattering and absorption-dependent wavelengths of the OD spectrum.
登录
查看更多内容
影响因子：
4.4
作者：
Lin, Hsiu-Li;Lin, Chien-Chung;Kuo, Tai-Chih
通讯作者：
Kuo, Tai-Chih
DOI：
10.1016/j.jqsrt.2009.04.005
发表时间：
2009-11-01
影响因子：
2.3
作者：
Berberoglu, Halil;Gomez, Pedro S.;Pilon, Laurent
通讯作者：
Pilon, Laurent
影响因子：
3.2
作者：
Sezonov, Guennadi;Joseleau-Petit, Daniele;D'Ari, Richard
通讯作者：
D'Ari, Richard
影响因子：
64.8
作者：
BERG, HC;BROWN, DA
通讯作者：
BROWN, DA
影响因子：
64.5
作者：
SGANGA, MW;BAUER, CE
通讯作者：
BAUER, CE