Spinning Disk Confocal System

Abstract

Abstract : Radiofrequency (RF) radiation is in the frequency range in which the outer membrane of mammalian cells is no longer a barrier to electric field penetration, allowing access of the RF to subcellular structures. The biological effect of RF radiation, in particular high power, wideband radiation or ultrashort electrical pulses, is the topic of an AFOSR-administered MURI research program with the PI of this proposal serving as Pi of the MURI. Experimental studies in which human cells were exposed to pulsed electric fields of up to 300 kV/cm amplitude, with durations as short as 10 ns, have confirmed that these pulses have strong effects on subcellular structures. In order to identify all of the intracellular targets of these short-pulse electric fields, we must conduct live cell imaging with very high temporal resolution. An instrument has been developed for exactly this type of live-cell imaging. This new instrument scans 1000 microbeams across the cell simultaneously using a spinning disk and can generate images at a rate of 350-1000 frames per second. Reality limits the speed images can be captured to the readout speed from the camera to the computer, but image acquisition at over 120 frames per second is still possible for a 300X250 pixel image. Using organelle-specific fluorescent dyes, we are able to image the live cell response of specific organelles, as well as calcium concentration and membrane potential at a resolution of 8 milliseconds avoiding the phototoxicity problems of conventional laser-scanning confocal microscopy. First experiments on the effects of pulsed electric fields on the generation of reactive oxygen species and calcium release have demonstrated the importance of this new, high resolution of this diagnostic system.