High-speed, low-light holography and flagellar dynamics

该提案的目的是开发一种新的光学显微镜系统来捕获单细胞，并以高速和三维方式对其进行成像。计算能力的进步允许快速处理数字图像，并从每个图像中提取更多信息。我使用一个计算密集型处理方案-数字内联全息显微镜（DIHM）-图像快速移动的微观主体。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来获得的。图案是二维的，但包含关于三维样品体积的所有信息。在典型的DIHM实验中，电子传感器（CMOS或CCD）用于捕获二维图像的视频序列，其被存储用于分析。每个帧都可以使用自定义处理算法进行任意“重新聚焦”，从而在每个时间步长显示样品的完整三维配置。为了获得高质量的图像数据，能够捕获和抑制单个细胞是有帮助的。这将通过使用微量移液管系统来实现，在该微量移液管系统中，采用非常精细的玻璃注射器作为捕集器，该注射器具有直径为10微米的数量级的尖端。通过构建并将微量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。一个特别的例子是研究快速移动的生物结构和微生物。我建议把重点放在一个特殊的结构，鞭毛，作为一个证明的原则，该文书。这些鞭状结构存在于游动的藻类和精子尾部，以及附着在人类肺部和大脑中的静止细胞上，在那里它们起到泵送液体的作用。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将使我们能够在三维空间中对测试生物体（一种绿色藻类）上的鞭毛进行成像，并回答有关其机械操作原理的长期问题。具体来说，关于鞭毛中央脊柱的机械作用的数据，以及鞭毛是否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，并直接投入理论建模工作。由于没有合适的成像方案，因此以前无法获得此信息。

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.