Spining Disk Confocal Microscopy of Calcium Signalling in Blood Vessel Walls

Abstract Spinning disk confocal laser microscopy systems can be used for observing fast events occurringin a small volume when they include a sensitive electron-multiplying CCD camera. Such aconfocal system was recently used to capture the first pictures of intracellular calcium signallingwithin the projections of endothelial cells to the adjacent smooth muscle cells in the blood vesselwall. Detection of these calcium signals required high spatial and temporal resolution. A newlydeveloped calcium ion (Ca 2+ ) biosensor was also used. This exclusively expressed in theendothelium and fluoresced when Ca 2+ concentrations increased during signalling. This workgives insights into blood vessel disease because Ca 2+ signalling is critical for blood flow andpressure regulation. Keywords confocal microscopy; laser spinning disk; electron-multiplying CCD camera; endothelial cells;intracellular signals; blood vessels INTRODUCTION With conventional confocal laser scanning microscopy (CLSM), there is a trade-off betweenimage resolution and speed. The laser beam is scanned point by point in a raster pattern andsignal is detected sequentially from each point. If the array consists of a 512×512 pixel arrayand each point is illuminated for 1 microsecond, then each scan will take about 262milliseconds. The signal from each point must be acquired in that 1 µs and there is time‘skew’ of 0.26 second between the first and last points in the scan. To compensate for thebrief illumination of each pixel, an intense laser beam is required, and if the specimen isdynamic the time skew can lead to errors in observation.Spinning disk confocal laser microscopy overcomes this problem by exploiting themultiplex principle. This was originally proven by Felgett in spectroscopy and shows thatusing parallel detection delivers enhanced sensitivity [1]. A recent publication by Wang [2]