normalization of voltage-sensitive dye signal with functional activity measures归一化电压特异性染色信号与功能性活动的措施.pdfVIP

Normalization of Voltage-Sensitive Dye Signal with Functional Activity Measures Kentaroh Takagaki1,2., Michael Thomas Lippert1,3,5., Benjamin Dann1,4,5, Tim Wanger1,5, Frank W. Ohl1,5* 1 Leibniz Institute for Neurobiology, Magdeburg, Germany, 2 School of Medicine, Georgetown University, Washington, D. C., United States of America, 3 Max Planck ¨ Institute for Biological Cybernetics, Tubingen, Germany, 4 Max Planck Institute for Brain Research, Frankfurt/Main, Germany, 5 Institute of Biology, Otto-von-Guericke- University, Magdeburg, Germany Abstract In general, signal amplitude in optical imaging is normalized using the well-established DF/F method, where functional activity is divided by the total fluorescent light flux. This measure is used both directly, as a measure of population activity, and indirectly, to quantify spatial and spatiotemporal activity patterns. Despite its ubiquitous use, the stability and accuracy of this measure has not been validated for voltage-sensitive dye imaging of mammalian neocortex in vivo. In this report, we find that this normalization can introduce dynamic biases. In particular, the DF/F is influenced by dye staining quality, and the ratio is also unstable over the course of experiments. As methods to record and analyze optical imaging signals become more precise, such biases can have an increasingly pernicious impact on the accuracy of findings, especially in the comparison of cytoarchitechtonic areas, in area-of-activation measurements, and in plasticity or developmental experiments. These dynamic biases of the DF/F method may, to an extent, be mitigated by a novel method of normalization, DF/DFepileptiform. This normalization uses as a reference the measured activ