A modified confocal laser scanning microscope allows fast ultraviolet ratio imaging of intracellular Ca2+ activity using Fura-2

A confocal, ultraviolet laser scanning microscope (LSM) for reliable ratio measurements of localized intracellular Ca2+ gradients using the Ca2+-sensitive dye Fura-2 was developed. In a commercial LSM, the filter wheels for the excitation band-pass filters and the grey filters were replaced by acousto-optic tunable filters (AOTF) for rapid switching (less than or equal to 1.5 mu s) of the ultraviolet (351 and 364 nm) and the visible (457, 476, 488, 514 nm) excitation light. This enabled dual wavelength excitation of Fura-2, or 2'7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) for pH measurements. Changing to a transmitted-light detector of high sensitivity allowed for simultaneous recording of differential interference contrast images of the preparation with the excitation light. The AOTF fine control of the intensity of the excitation light and improvements in the emission detector sensitivity enabled the acquisition of up to 120 ratio pairs of high-quality images from a single cell. The optical capabilities and limitations of the instrument were evaluated with fluorescent beads and dye-loaded cultured cells. Agonist-induced intracellular Ca2+ transients in HT29 cells were recorded to test for the instrument's ability to measure changes in [Ca2+](i). Ratio z-sections from Fura-2-loaded cells showed an inhomogeneity of the Fura-2 loading with an accumulation of the dye mostly in the mitochondria. We show, as an example of the microscope's achievable resolution, the spatial and temporal heterogeneity of [Ca2+](i) signals in mitochondria and the cytosol in response to agonist-evoked stimulation of HT29 cells. In addition. we show that the lipophilic, membrane-bound Fura-2 derivative Fura-C-18, for measurements of near-membrane Ca2+ changes, can be used with this confocal microscope. This new LSM is expected to deepen our understanding of localized [Ca2+](i) signals; for example, the nuclear Ca2+ signalling or the [Ca2+](i) changes that occur during stimulation of ion secretion in polarized epithelial cells.