Long-term real-time imaging of a voltage sensitive dye in cultured hippocampal neurons using the silver plasmonic dish

[Display omitted] •Long-term real-time VSD imaging was performed on the Ag plasmonic dish.•Larger fluorescence enhancement was obtained in the neurons on Ag surface.•Photobleaching rate decreased with reduction of illumination intensity and noise level on Ag dish.•Ag plasmonic dish was suitable for the long-term VSD imaging. We have demonstrated the real-time voltage sensitive dye (VSD) imaging of a neuronal network using Di-4-ANEPPS at a 1 ms-time resolution on a silver (Ag) plasmonic dish to improve the signals to noise (S/N) ratio. The plasmonic dish is composed of a culture dish and a metal-coated substrate with a wavelength-order periodical structure on its surface that can enhance excitation field due to the surface plasmon resonance (SPR)-field. In our group, spontaneous neural spikes that are undetectable on a conventional culture dish were frequently detected on a gold (Au) plasmonic dish during real-time VSD imaging. In the present study, Ag and Au plasmonic chips were prepared and tested. Larger fluorescence enhancement of the VSD was obtained on the Ag plasmonic dish compared with on the Au. Number of neural spikes were frequently detected on the Ag plasmonic dish compared to the Au plasmonic dish in the control neurons and after application picrotoxin (PTX), because of the photobleaching rate was suppressed and the noise level decreased by the reduction of illumination light on the Ag surface. Therefore, the use of an Ag plasmonic dish was considered to be suitable for the long-term and real-time observation by VSD imaging.