Optical Coherence Microscopy in Developmental Biology

发育生物学中的光学相干显微镜

• 批准号: 9612240
• 负责人: Richard Haskell
• 金额: $ 68.21万
• 依托单位: Harvey Mudd College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-15 至 2000-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9612240&HistoricalAwards=false
• 关键词: Optical; Coherence; Microscopy; Developmental; Biology

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.

项目概述将设计和建造一台光学相干显微镜(OCM)，以研究发育生物学的基本问题。该显微镜将能够对位于活组织表面以下一毫米的细胞进行成像，尽管样品中的光散射会使其对传统或共聚焦光学显微镜不透明。深度穿透是利用相干长度为20um的近红外超辐射二极管光源和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~m的深度分辨率。横向分辨率为10微米或更好的是通过将照明光束向下聚焦到一个小光点来实现的。光束光斑的二维横向扫描在样品中的固定深度产生光学部分。通过在不同深度层叠连续的光学切片获得三维图像。这样的三维扫描应该不到一分钟。OCM将被用于研究发育生物学中的两个关键系统--非洲爪哇和植物拟南芥。在青蛙胚胎中，原肠形成和神经形成的过程将被实时监测。具体地说，将跟踪表层细胞(外胚层)和深层细胞(中胚层)的相对运动，以更好地定义在对神经模式至关重要的相互作用中响应细胞和信号细胞之间的空间关系，并更好地表征中胚层划分为轴向和旁轴结构的特征。这些事件发生在胚胎的几百微米处，由于多次散射，共焦显微镜无法到达的深度。在拟南芥中，胚胎的形成发生在母体组织的深处，因此无法用共聚焦显微镜观察。OCM将首次用于观察植物胚胎在体内(在含有种子的角果中)的发育。两个专门的胚胎组织，即茎和根分生组织，以某种方式指导植物随后的胚胎和胚胎后发育。野生型和突变型拟南芥植株将被用来研究顶端分生组织在胚胎发育中的作用。胚后，顶端分生组织指导叶片的分化(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隐藏。有了OCM，第一次有可能实时观察到与叶序相关的最早的形态变化。这些对形态的观察将与遗传分析和其他实验操作相关联，以探索植物发育的基本分子机制。一个由具有生物学、生物物理学、计算机工程、光学科学和物理学背景的教员组成的合作团队将指导研究计划，本科生将在暑期和整个学年广泛参与。除了这项建议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要的研究项目，这些项目将有助于准备和激励他们在科学和技术领域的多学科活动中追求职业生涯。