Axially-confined in vivo single-cell labeling by primed conversion using blue and red lasers with conventional confocal microscopes
Axially-confined in vivo single-cell labeling by primed conversion using blue and red lasers with conventional confocal microscopes
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使用蓝色和红色激光与传统共焦显微镜通过引物转换进行轴向限制的体内单细胞标记
DOI：
10.1111/dgd.12412
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Higashijima Shin-ichi
中科院分区：
文献类型：
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作者：
Taniguchi Atsushi;Kimura Yukiko;Mori Ikue;Nonaka Shigenori;Higashijima Shin-ichi
已经发现绿色至红色光转换荧光蛋白通过称为引发转换的新方法进行有效的光转换，该方法使用蓝色和红色/近红外光的双波长照明。通过修改共焦激光扫描显微镜（CLSM），使两个激光束仅在焦平面相遇，实现了轴向尺寸的受限光转换。然而，对CLSM进行这种自定义修改的必要性排除了这种方法的广泛使用。在这里，我们研究了空间受限的启动转换是否可以在没有任何硬件修改的情况下用CLSM实现。我们发现，使用具有两个可见激光器（473 nm和635 nm）和高NA物镜透镜（NA，1.30）的常规CLSM的Dendra 2的引发转换导致光转换体积的显著限制：轴向尺寸的半高半宽低于5 μm，这与使用显微镜修改的原始方法的结果相当。为了证明这种方法的有效性，我们在活体斑马鱼胚胎中使用了这种技术，并成功地揭示了相邻细胞之间单个神经元的复杂解剖结构。由于未修饰的CLSM广泛可用，因此该方法可广泛适用于标记具有单细胞分辨率的细胞。
Green‐to‐red photoconvertible fluorescent proteins have been found to undergo efficient photoconversion by a new method termed primed conversion that uses dual wave‐length illumination with blue and red/near‐infrared light. By modifying a confocal laser‐scanning microscope (CLSM) such that two laser beams only meet at the focal plane, confined photoconversion at the axial dimension has been achieved. The necessity of this custom modification to the CLSM, however, has precluded the wide‐spread use of this method. Here, we investigated whether spatially‐restricted primed conversion could be achieved with CLSM without any hardware modification. We found that the primed conversion of Dendra2 using a conventional CLSM with two visible lasers (473 nm and 635 nm) and a high NA objective lens (NA, 1.30) resulted in dramatic restriction of photoconversion volume: half‐width half‐maximum for the axial dimension was below 5 μm, which is comparable to the outcome of the original method that used the microscope modification. As a proof of this method's effectiveness, we used this technique in living zebrafish embryos and succeeded in revealing the complex anatomy of individual neurons packed between neighboring cells. Because unmodified CLSMs are widely available, this method can be widely applicable for labeling cells with single‐cell resolution.