Kohli Vikram, Rehn Kira, Sumanas Saulius
Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, USA.
J Vis Exp. 2011 Oct 5(56):3172. doi: 10.3791/3172.
The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos, PAmCherry, Kaede, pKindling, and KikGR have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system.
对单个细胞进行差异标记的能力在发育生物学中具有重要意义。例如，确定造血、淋巴和血管谱系在发育胚胎中如何产生，需要对未分化的前体细胞进行命运图谱绘制和谱系追踪。最近，包括Eos、PAmCherry、Kaede、pKindling和KikGR在内的光激活蛋白作为细胞追踪探针受到了广泛关注。这些光敏蛋白的荧光光谱可以通过紫外线激发轻松转换，从而使一群细胞能够与相邻细胞区分开来。然而，激活蛋白的光效率可能会限制长期细胞追踪。作为光激活蛋白的替代方法，笼形荧光素-葡聚糖已在胚胎模型系统中广泛使用。传统上，为了解笼荧光素-葡聚糖，使用了来自荧光灯箱或单光子紫外激光的紫外线激发；然而，这些光源限制了光激活的空间分辨率。在这里，我们报告了一种用于命运图谱绘制、谱系追踪和检测单个标记细胞的方案。注射了笼形荧光素-葡聚糖的胚胎中的单个细胞用钛宝石飞秒激光产生的近红外激光脉冲进行光激活。这种激光在所有双光子共聚焦显微镜中都很常见，例如本文中使用的LSM 510 META NLO显微镜。由于生物组织对近红外辐射是透明的，激光脉冲可以聚焦到胚胎深处，而不会使选定焦平面上方或下方的细胞解笼。因此，仅在几何焦点处诱导非线性双光子吸收，以在单个细胞中解笼荧光素-葡聚糖。为了检测含有解笼荧光素-葡聚糖的细胞，我们描述了一种简单的免疫组织化学方案，以快速可视化激活的细胞。本文介绍的激活和检测方案具有通用性，可应用于任何模型系统。
