Department of Stem Cell Biology, Center for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg, Germany.
Plant J. 2011 Oct;68(2):377-85. doi: 10.1111/j.1365-313X.2011.04692.x. Epub 2011 Aug 4.
Most plant growth occurs post-embryonically and is characterized by the constant and iterative formation of new organs. Non-invasive time-resolved imaging of intact, fully functional organisms allows studies of the dynamics involved in shaping complex organisms. Conventional and confocal fluorescence microscopy suffer from limitations when whole living organisms are imaged at single-cell resolution. We applied light sheet-based fluorescence microscopy to overcome these limitations and study the dynamics of plant growth. We designed a special imaging chamber in which the plant is maintained vertically under controlled illumination with its leaves in the air and its root in the medium. We show that minimally invasive, multi-color, three-dimensional imaging of live Arabidopsis thaliana samples can be achieved at organ, cellular and subcellular scales over periods of time ranging from seconds to days with minimal damage to the sample. We illustrate the capabilities of the method by recording the growth of primary root tips and lateral root primordia over several hours. This allowed us to quantify the contribution of cell elongation to the early morphogenesis of lateral root primordia and uncover the diurnal growth rhythm of lateral roots. We demonstrate the applicability of our approach at varying spatial and temporal scales by following the division of plant cells as well as the movement of single endosomes in live growing root samples. This multi-dimensional approach will have an important impact on plant developmental and cell biology and paves the way to a truly quantitative description of growth processes at several scales.
大多数植物生长发生在胚胎后时期，其特征是不断地、迭代地形成新的器官。对完整的、功能齐全的活体进行非侵入性的、时间分辨的成像，可以研究参与塑造复杂生物体的动力学。传统的共聚焦荧光显微镜在对整个活体进行单细胞分辨率成像时存在局限性。我们应用基于光片的荧光显微镜来克服这些限制，并研究植物生长的动力学。我们设计了一个特殊的成像室，在这个成像室中，植物在受控光照下垂直保持，其叶片在空气中，根部在培养基中。我们表明，可以在器官、细胞和亚细胞尺度上以最小的侵入性、多色、三维方式对活体拟南芥样本进行成像，成像时间从几秒钟到几天不等，对样本的损伤最小。我们通过记录几个小时内主根根尖和侧根原基的生长，说明了该方法的能力。这使我们能够量化细胞伸长对侧根原基早期形态发生的贡献，并揭示侧根的昼夜生长节律。我们通过跟踪植物细胞的分裂以及活体生长的根样本中单个内体的运动，在不同的空间和时间尺度上展示了我们方法的适用性。这种多维方法将对植物发育和细胞生物学产生重要影响，并为在几个尺度上对生长过程进行真正的定量描述铺平道路。
