Optical Coherence Microscopy in Developmental Biology

项目摘要 将设计和建造一台光学相干显微镜（OCM），用于研究发育生物学中的基本问题。该显微镜将能够成像细胞位于活组织表面以下一毫米，即使在标本中的光散射将使其不透明的传统或共聚焦光学显微镜。深度穿透是通过使用具有20 μ m的相干长度的近红外超辐射发光二极管光源以及基于迈克尔逊干涉仪的相干门来实现的。这种组合排除了从焦外平面反向散射的光，给出了10 μ m的深度分辨率。通过将照明光束聚焦到一个小光斑，可以实现10 μ m或更好的横向分辨率。束斑的二维横向扫描在样品中的固定深度处产生光学截面。通过在不同深度处堆叠连续的光学切片来获得三维图像。这种三维扫描应该不到一分钟。 OCM将用于研究发育生物学中的两个关键系统，青蛙非洲爪蟾和植物拟南芥。在青蛙胚胎中，原肠胚形成和神经胚形成的过程将被真实的实时监测。具体而言，表面细胞（外胚层）和更深的细胞（中胚层）的相对运动将被遵循，以更好地定义响应和信号细胞之间的空间关系，在相互作用的关键神经模式，并更好地表征分割成轴向和近轴结构的中胚层。这些事件发生在胚胎几百微米的深度，由于多重散射，共聚焦显微镜无法到达。 在拟南芥中，胚胎形成发生在母体组织深处，因此共聚焦显微镜无法观察到。OCM将首次用于活体观察植物胚胎发育（含种子角果）。两个专门的胚胎组织，茎和根分生组织，以某种方式指导植物随后的胚胎和胚后发育。 野生型和突变体拟南芥植物将用于研究芽顶端分生组织在胚胎发育中的作用。 胚后，茎顶端分生组织指导叶的发生模式（叶序）。分生组织在这种模式的启动中的作用被覆盖的叶子所掩盖。利用OCM，将有可能第一次在真实的时间内观察到与叶序相关的最早的形态变化。这些形态学观察将与遗传分析和其他实验操作相关，以探索植物发育的基本分子机制。 具有生物学，生物物理学，计算机工程，光学科学和物理学背景的教师合作团队将指导研究计划，本科生在夏季和整个学年都广泛参与。除了本提案中描述的具体研究目标外，另一个关键目标是让本科生参与重大研究项目，这将有助于准备和激励他们从事科学和技术多学科活动的职业。

PROJECT SUMMARY An optical coherence microscope (OCM) will be designed and constructed to study fundamental problems in developmental biology. The microscope will be capable of imaging cells located up to one millimeter below the surface of living tissue, even though light scattering in the specimen would render it opaque to a conventional or confocal light microscope. Depth penetration is achieved by use of a near infrared superluminescent diode light source with a coherence length of 20 um together with a coherence gate based on a Michelson interferometer. This combination excludes light back-scattered from out-of-focus planes, giving a depth resolution of 10 ~m. Lateral resolution of 10 um or better is achieved by focusing the illuminating beam down to a small spot. Two-dimensional lateral scanning of the beam spot produces an optical section at a fixed depth in the sample. A three-dimensional image is obtained by stacking successive optical sections at different depths. Such three-dimensional scans should take less than a minute. OCM will be used to study two key systems in developmental biology, the frog Xenopus laevis and the plant Arabidopsis thaliana. In the frog embryo, the processes of gastrulation and neurulation will be monitored in real time. Specifically, the relative motions of the surface cells (ectoderm) and deeper cells (mesoderm) will be followed to better define the spatial relationships between the responding and signaling cells during interactions critical to neural patterning and to better characterize the segmentation of the mesoderm into axial and paraxial structures. These events occur several hundred micrometers into the embryo, at a depth inaccessible to a confocal microscope because of multiple scattering. In Arabidopsis, embryo formation o ccurs deep within maternal tissues and is therefore inaccessible to confocal microscopy. OCM will be used to observe plant embryo development in vivo (in the seed-containing silique) for the first time. Two specialized embryonic tissues, the shoot and root meristems, somehow direct the plant's subsequent embryonic and post-embryonic development. Wild-type and mutant Arabidopsis plants will be used to study the role of the shoot apical meristem in embryonic development. Post-embryonically, the shoot apical meristem directs the pattern of leaf initiation (phyllotaxy). The role of the meristem in initiation of such patterns is hidden by overlying leaves. With OCM it will be possible for the first time to observe in real time the earliest morphological changes associated with phyllotaxis. These observations of morphology will be correlated with genetic analysis and other experimental manipulations to probe the fundamental molecular mechanisms underlying plant development. A collaborative team of faculty with backgrounds in biology, biophysics, computer engineering, optical sciences, and physics will direct the research program, with extensive participation of undergraduate students both during the summer and throughout the academic year. In addition to the specific research goals described in this proposal, another key objective is to engage undergraduates in significant research projects that will help prepare and motivate them to pursue careers in multidisciplinary activities in science and technology.