使用荧光指示剂Indo-1在三维钙成像中比较双光子激发激光扫描显微镜与紫外共聚焦激光扫描显微镜。

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.

作者信息

Sako Y, Sekihata A, Yanagisawa Y, Yamamoto M, Shimada Y, Ozaki K, Kusumi A

机构信息

Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Japan.

出版信息

J Microsc. 1997 Jan;185(Pt 1):9-20. doi: 10.1046/j.1365-2818.1997.1480707.x.

DOI:10.1046/j.1365-2818.1997.1480707.x

PMID:9057318

Abstract

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 2.6-phi pinhole (in normalized optical coordinate) gives better signal-to-noise ratio, contrast and resolution than 0.09-mW laser power with a 4.9-phi 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.

摘要

在培养的活细胞三维（3-D）钙成像方面，对双光子激发激光扫描荧光显微镜（2p-LSM）与紫外激发共聚焦激光扫描荧光显微镜（UV-CLSM）进行了比较。使用 Indo-1 作为钙指示剂。由于 2p-LSM 的激发体积比 UV-CLSM 更受限且激发波长更长，2p-LSM 相对于 UV-CLSM 表现出几个优点：（1）背景信号水平低 6 - 17 倍，对比度提高 6 - 21 倍；（2）光漂白率低 2 - 4 倍；（3）光毒性略低。当重复采集 3-D 图像时，UV-CLSM 测定的钙浓度强烈依赖于数据采集次数，并且核区域错误地显示出低钙浓度，这可能归因于 Indo-1 不同程度的光漂白与自发荧光的相互作用，而 2p-LSM 评估的钙浓度在整个细胞质中是稳定且均匀的。由于激发波长较长，2p-LSM 在焦平面上的空间分辨率差 10%，沿光轴差 30%。通过添加共聚焦针孔（双光子激发共聚焦激光扫描荧光显微镜）可以克服这一缺点，使其分辨率与 UV-CLSM 相似。这些结果表明，2p-LSM 更适合对活细胞中钙浓度进行重复的 3-D 重建。在 UV-CLSM 中，与使用 4.9-phi 针孔的 0.09-mW 激光功率相比，使用 2.6-phi 针孔（在归一化光学坐标中）的 0.18-mW 激光功率能提供更好的信噪比、对比度和分辨率。然而，由于在前一种条件下对细胞的损伤和光漂白率要大得多，它不适合重复采集 3-D 图像。