Development of Instrumentation for Digital Imaging Fluorescence Microscopy

卡林顿，沃尔特9724611项目总结：提出了一项计划，以开发硬件、算法和软件来增强和扩展数字成像显微镜(DIM)的能力，DIM是一种将宽视场显微镜与高速ccd相机、快速焦点改变、去卷积算法以及可视化和分析软件相结合的三维荧光显微镜方法。该计划是为了应对即使是最先进的当前系统对生物实验施加的限制而制定的。将对现有的超高速三维显微镜进行增强，包括增加在需要缓慢读出之前可能获得的高速图像的数量。将开发和测试新的三维图像恢复(反卷积)的计算方法，使用非负的L^p平滑，以获得75 nm的横向分辨率和250 nm的轴向分辨率。将编写新的软件来自动计算这种超分辨率图像恢复方法中使用的次采样点扩展函数。我们将使用SGI的软件开发工具，在单处理器的Silicon Graphics，Inc.(SGI)Origin 200计算机上开发这些算法的新的并行实现。将开发在低端SGI 02工作站上使用硬件纹理映射将3D渲染速度提高100倍的可视化工具。最后，将开发新的方法来分析图像中空间和时间模式的统计意义。将在这一新仪器上开展若干生物学项目，以解决目前现有仪器无法解决的关键问题。例如，GLUT4葡萄糖转运体在被送到质膜上响应胰岛素时通过细胞的途径将被直接可视化，并将确定中心体蛋白的组装动力学以及细胞周期中对中心体功能的组装和拆解的后果。生物学家和工程师将通过参与生物项目和仪器开发，分别接受光学方法和数字成像显微镜方面的培训。我们提出的工作将提高数字成像荧光显微镜的灵敏度、分辨率、数值精度以及可视化和分析能力。因此，它将扩大生物学家可以用它回答的问题的范围。

Carrington, Walter 9724611 Project Summary: A plan is presented to develop hardware, algorithms and software to enhance and extend the capabilities of the digital imaging microscope (DIM), an approach to 3-D fluorescence microscopy which combines wide field microscopy with high speed CCD cameras, fast focus change, deconvolution algorithms and visualization and analysis software. The plan has been developed in response to the limitations imposed on biological experiments by even the most advanced current systems. Enhancements to an existing ultra-fast 3-D microscope will be implemented, including increasing the number of high speed images that may be obtained before slow readout is necessary. New computational methods for 3-D image restoration(deconvolution) will be developed and tested that use L^p smoothing with non-negativity to achieve a lateral resolution of 75nm and axial resolution of 250nm. New software will be written to automate the calculation of a sub-sampled point spread function used in this super- resolution image restoration approach. We will develop a new parallel implemention of these algorithms on a single 4- processor Silicon Graphics, Inc. (SGI) Origin 200 computer using SGI's software development tools. Visualization tools that increase 3-D rendering speeds by 100x using hardware texture mapping on low end SGI 02 workstations will be developed. Finally, new methods for analyzing the statistical significance of spatial and temporal patterns in images will be developed. A number of biological projects will be carried out on this new instrument addressing key problems which are not soluble by currently existing instruments. For example, the pathway through the cell that glut4 glucose transporters take when they are sent to the plasma membrane in response to insulin will be directly visualized and the dynamics of assembly of centrosomal proteins and consequences of assembly and disassembly during the cell cycle for centros ome function will be determined. Biologists and engineers will be trained in optical methods and digital imaging microscopy through involvement in biological projects and instrument development respectively. The work we propose will enhance the sensitivity, resolution, numerical accuracy and visualization and analysis capabilities of the digital imaging fluorescence microscope. It will thus extend the range of questions that biologists can answer with it.