Calibrating excitation light fluxes for quantitative light microscopy in cell biology
Calibrating excitation light fluxes for quantitative light microscopy in cell biology
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DOI：
10.1038/nprot.2008.180
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Singer, Robert H.
中科院分区：
文献类型：
--
作者：
Gruenwald, David;Shenoy, Shailesh M.;Singer, Robert H.
灯泡的功率输出会随时间变化，所传递的总能量将取决于显微镜的光路、所使用的滤光片组和物镜，因此在不同显微镜上进行的实验之间进行比较是复杂的。使用热电偶功率计，无论光源如何，都有可能测量荧光显微镜中施加到样本上的准确光量，因为所测量的光功率可以转换为样本处的功率密度。这种广泛使用且简单的工具为实验和装置之间新的校准精度和结果可比性奠定了基础。在此我们描述一种易于遵循的操作流程，该流程允许研究人员使用市售的光机组件精确估计物平面中的激发强度。对于一个物镜和六个滤光片立方，包括灯泡启动时间在内，该流程的总时长为75分钟。
Power output of light bulbs changes over time and the total energy delivered will depend on the optical beam path of the microscope, filter sets and objectives used, thus making comparison between experiments performed on different microscopes complicated. Using a thermocoupled power meter, it is possible to measure the exact amount of light applied to a specimen in fluorescence microscopy, regardless of the light source, as the light power measured can be translated into a power density at the sample. This widely used and simple tool forms the basis of a new degree of calibration precision and comparability of results among experiments and setups. Here we describe an easy-to-follow protocol that allows researchers to precisely estimate excitation intensities in the object plane, using commercially available opto-mechanical components. The total duration of this protocol for one objective and six filter cubes is 75 min including start-up time for the lamp.