High-speed, low-light holography and flagellar dynamics

这项提议的目的是开发一种新的光学显微镜系统来捕获单细胞，并以高速和三维的方式对它们进行成像。计算能力的进步使数字图像能够快速处理，并从每张图像中提取更多信息。我使用一种计算密集型的处理方案-数字内联全息显微镜(DIHM) -对快速移动的微观对象进行成像。全息图像是通过用相干光（在我的例子中是激光）照射物体，并记录散射光和非散射光之间形成的干涉图案而获得的。该模式是二维的，但包含了三维样本体积的所有信息。在典型的DIHM实验中，使用电子传感器（CMOS或CCD）来捕获二维图像的视频序列，并将其存储以供分析。每个帧都可以使用自定义处理算法任意“重新聚焦”，从而在每个时间步显示样品的完整三维结构。为了获得高质量的图像数据，能够捕获和抑制单个细胞是很有帮助的。这将使用微移液器系统来实现，其中非常精细的玻璃注射器，其尖端直径约为10微米，被用作陷阱。通过将微移液器系统集成到全息显微镜中，我将确保成像系统可以充分发挥其潜力。全息显微镜非常适合解决生物物理学中的许多挑战，目前尚未得到充分利用。一个特别的例子是对快速移动的生物结构和微生物的研究。我建议把重点放在一个特殊的结构上，鞭毛，作为该仪器的原理证明。这些鞭状结构存在于游动的藻类和精子尾部，也附着在人类肺部和大脑的固定细胞上，用于泵送液体。它们对许多寄生虫的生存也至关重要，包括疟疾和昏睡病的病原体。DIHM将使我们能够在测试生物体（一种绿藻）上三维成像鞭毛，并回答有关其机械操作原理的长期问题。具体地说，关于鞭毛中央脊柱的机械作用的数据，以及鞭毛是否会围绕其长轴扭曲作为其跳动动作的一部分，将大大增强我们的理解，并直接为理论建模工作提供信息。由于没有合适的成像方案，以前没有获得这些信息。

The aim of this proposal is to develop a new optical microscopy system to trap single cells, and image them at high speeds and in three dimensions. Advances in computing power allow digital images to be processed rapidly, and for more information to be extracted from each image. I use a computationally-intensive processing scheme - digital inline holographic microscopy (DIHM) - to image rapidly moving microscopic subjects. Holographic images are obtained by illuminating a subject with coherent light (a laser in my case), and recording the interference pattern formed between scattered and unscattered light. The pattern is two-dimensional, but contains all of the information about the three-dimensional sample volume. In a typical DIHM experiment, an electronic sensor (CMOS or CCD) is used to capture a video sequence of two-dimensional images, which are stored for analysis. Each frame can be 'refocused' arbitrarily using custom processing algorithms, revealing the fully three-dimensional configuration of the sample at each time step. In order to obtain high-quality image data, it is helpful to be able to capture and restrain single cells. This will be achieved using a micropipette system in which very fine glass syringes, with tips on the order of 10 micrometres in diameter, are employed as traps. By building and integrating a micropipette system into a holographic microscope, I will ensure that the imaging system can be used to its full potential.Holographic microscopes are perfectly suited to addressing a number of challenges in biophysics, and are under-utilised at present. A particular example is the study of fast-moving biological structures and microorganisms. I propose to focus on one particular structure, the flagellum, as a proof-of-principle for the instrument. These whip-like structures are found on swimming algae and in sperm tails, as well as attached to stationary cells in the human lungs and brain, where they serve to pump fluid. They are also critical to the survival of many parasites, including the causative agents of malaria and sleeping sickness. DIHM will allow us to image flagella on a test organism (a species of green algae) in three dimensions, and answer long-standing questions about their mechanical operating principles. Specifically, data about the mechanical role of the flagellum's central spine, and whether flagella twist about their long axis as part of their beating action, will greatly enhance our understanding and feed directly into theoretical modelling efforts. This information has not been available before because no suitable imaging scheme has been available.