CONFOCAL CALCIUM IMAGING WITH ULTRAVIOLET LASER-SCANNING MICROSCOPY AND INDO-1

We developed a newly designed ultraviolet laser-scanning confocal microscopy (UV-LSCM) system and applied it for quantitative confocal imaging of intracellular calcium concentration ([Ca2+]i) with the dual-emission wavelength indicator indo-1. The resolution and contrast of the biological sample images obtained using the current UV system were found to be comparable to those obtained with the conventional visible LSM optics and hence the UV-transmittable optics in our LSM system employing an achromatic objective lens provides appreciable confocality in the UV range. When indo-1 is used with this UV-LSCM system, dual-imaging ratiometric measurement of [Ca2+]i can be easily performed without requiring time-consuming geometrical decalibration of the two simultaneously obtained images. The resulting confocal images allow quantitative analysis of [Ca2+]i in rapidly contractile cardiac cells at a high temporal resolution in line-scan and fast frame-scan modes. The combined use of the UV-LSCM and dual-emission ratiometric indicator is now practical and we anticipate its widespread application in physiological and pathological studies in living cells.