Multi-laser Total Internal Reflection Fluorescence Microscopy

全内反射荧光显微术是荧光成像领域的一项新技术。研究人员现在可以检查单个粒子的行为，例如运动蛋白在丝状轨道上的运动，或者通过用荧光蛋白标记感兴趣的对象来测量活细胞中细胞成分的活性。使用TIRF，只有非常薄的样品层暴露于导致荧光输出的光，从而更好地区分单个荧光物体的能力，以及在长时间内以低激发对样品进行成像的能力。这项建议将使最近购买的基本TIRF系统通过购买3个额外的激光器和一个摄像机，使其能够同时对多个荧光物体进行成像，从而达到全面运作的能力。具有广泛应用和背景的研究人员将受益于这一新设施。CRC Geoffrey Corneys正在揭示驱动动态细丝（称为微管）生长和收缩的机制，微管形成了对细胞过程至关重要的微型机器。他的团队将使用TIRF系统来观察与这些微管聚合物相关并控制其生长和收缩的蛋白质的行为。数学家Eric Cytrynbaum将使用TIRF数据作为计算模拟的输入，以数学术语模拟产生亚细胞组织模式的自组织过程。Lacey Samuels的实验室将使用TIRF系统来研究植物细胞中脂质的分泌和回收，这是一个对植物生长和发育至关重要的过程。对于Jay Kizhakkedathu的研究小组来说，TIRF是唯一已知的方法，通过这种方法，他们可以监测细胞如何粘附在他们正在开发的生物活性表面上并与之增殖，这一过程将优化药物输送和植入物。旧金山州立大学的生物制药学家南希·福尔德是一个国际团队的成员，该团队试图制造第一个自主的、基于蛋白质的合成分子马达。对于他们来说，TIRF对于可视化和量化原型发动机的活动至关重要。

Total Internal Reflection Fluorescence (TIRF) Microscopy is a recent innovation in the field of fluoresence imaging. Researchers can now examine the behaviour of single particles, such as the movement of motor proteins on filamentous tracks or to measure the activity of cellular components in living cells by tagging their objects of interest with fluorescent proteins. With TIRF, only a very thin layer of the sample is exposed to the light that causes fluorescent output, resulting in a better ability to distinguish individual fluorescent objects, and the capacity to image specimens with low excitation over long periods of time. This proposal will bring a recently purchased basic TIRF system to full operating capacity through the acquisition of 3 additional lasers and a camera that will enable simultaneous imaging of multiple fluorescent objects. Researchers with a wide range of applications and backgound will benefit from this new facility. CRC Geoffrey Wasteneys is uncovering the mechanisms that drive the growth and shrinkage of dynamic filaments known as microtubules, which form form micro-machines critical for cellular processes. His team will use the TIRF system to observe the behaviour of proteins that associate with and control the growth and shrinkage of these microtubule polymers. Mathematician Eric Cytrynbaum will use TIRF data as inputs for computational simulations, to model in mathematical terms the processes of self organization that generate patterns of subcellular organization. Lacey Samuels' laboratory will use the TIRF system to study how lipids are secreted and recycled in plant cells, a little understood process critical for plant growth and development. For Jay Kizhakkedathu's research team, TIRF is the only known method by which they can monitor how cells adhere to and proliferate with the bioactive surfaces they are developing, a process that will optimizing drug delivery and implants. Nancy Forde, a biophysicist at SFU is part of an international team attempting to produce the first autonomous, protein-based synthetic molecular motor. For them, TIRF is critical for visualising and quantifying the activity of their prototype motors.