Comparison of two‐photon excitation laser scanning microscopy with UV‐confocal laser scanning microscopy in three‐dimensional calcium imaging using the fluorescence indicator Indo‐1

Two‐photon excitation laser scanning fluorescence microscopy (2p‐LSM) was compared with UV‐excitation confocal laser scanning fluorescence microscopy (UV‐CLSM) in terms of three‐dimensional (3‐D) calcium imaging of living cells in culture. Indo‐1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p‐LSM than in UV‐CLSM, 2p‐LSM exhibited several advantages over UV‐CLSM: (1) a lower level of background signal by a factor of 6–17, which enhances the contrast by a factor of 6–21; (2) a lower rate of photobleaching by a factor of 2–4; (3) slightly lower phototoxicity. When 3‐D images were repeatedly acquired, the calcium concentration determined by UV‐CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations, probably due to an interplay of different levels of photobleaching of Indo‐1 and autofluorescence, while the calcium concentration evaluated by 2p‐LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p‐LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two‐photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV‐CLSM. These results indicate that 2p‐LSM is preferable for repeated 3‐D reconstruction of calcium concentration in living cells. In UV‐CLSM, 0.18‐mW laser power with a 2.φ pinhole (in normalized optical coordinate) gives better signal‐to‐noise ratio, contrast and resolution than 0.09‐mW laser power with a 4.9‐φ pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3‐D images.