Grünwald David, Shenoy Shailesh M, Burke Sean, Singer Robert H
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Nat Protoc. 2008;3(11):1809-14. doi: 10.1038/nprot.2008.180.
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.
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束路径、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变得复杂。使用热耦合功率计，可以测量荧光显微镜中施加到样本上的光的精确量，无论光源如何，因为所测量的光功率可以转换为样本处的功率密度。这个广泛使用且简单的工具构成了实验和设置之间新的校准精度和结果可比性的基础。在这里，我们描述了一个易于遵循的方案，该方案允许研究人员使用市售的光机械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体，该方案的总时长为75分钟，包括灯的启动时间。
