Digital holographic microscopy for tracking micro-organisms in 3D

大多数单细胞生物体积极地在环境中导航。例如，许多种类的细菌使用微小的旋转马达来驱动螺旋细丝来产生推进力。就细菌而言，需要主动导航以获取营养和避免毒素，彼此之间形成共生关系，并向病原体入侵的地点移动。目前，我们研究细菌游泳的唯一方法是观察传统显微镜的2D图像，并推断细菌游进和游出显微镜焦点时的3D性质。对细菌游动的全面3D了解对于探索细菌如何移动的许多悬而未决的问题至关重要。该项目的目的是开发一种新的显微镜技术，能够高速记录游泳细菌的3D位置。这将通过开发一种名为数字全息显微镜的技术来实现。全息图是样品发出的光和已知参考光束之间的干涉图案。事实上，我们知道这个“参考光束”是先验的，这意味着我们可以在计算机中处理干涉图案，以生成关于我们所成像的样品的三维信息。数字全息显微镜在形成全息图之前使用显微镜物镜将样品大大放大。通过这种方式，我们可以获得像细菌这样小到1-2微米长的生物的3D位置。最近的两项技术发展使我们能够以3D和高速研究这些微生物。首先是数码相机技术的进步。新的相机芯片现在有足够的像素分辨率以非常快的帧速率(高达每秒2000帧)捕捉这些放大的全息图的精细细节。第二是图形处理单元(GPU)用于图像处理的可用性。GPU最初是为计算机游戏行业发明的，但现在已经在数字处理应用程序中找到了一席之地，因为它们在处理大量数据方面比传统CPU效率高得多。我们的3D全息显微镜必然会产生大量数据(典型的3D全息图每帧1 GB)；因此，图形处理器是处理这些信息不可或缺的一部分。解释为什么数字全息成像还没有以这种方式使用的最大绊脚石之一是缺乏可用的与GPU兼容的软件来有效处理这些数据量。我们建议的技术将把显微镜、高速、高分辨率数码相机和GPU的处理能力结合在一起，以实现对细菌和其他微生物的三维研究。它将涉及一种特殊形式的全息术，称为“离轴全息术”。这种技术在更高的放大倍数下更难在光学上实现，但它提供了比该技术的另一个主要分支--在线全息术--更高的分辨率。在开发了3D显微成像之后，我们提出了两种方法，用于研究细菌如何游泳。第一种是使用相对较低的放大倍率显微镜(40倍)来捕捉在一个视野中游泳的许多细菌的全息图。我们将使用计算机软件以3D方式跟踪这些细菌的运动。通过这种方式，我们可以研究一群生物的集体游泳行为。第二种方法使用更高的放大倍数(225倍)光学元件来研究单个细菌如何与它们的流体环境相互作用。这个例子将捕捉到微小的“示踪剂”粒子的运动，使我们能够可视化由单个细菌游泳经过所引起的流体流动。我们预计，这项技术将对许多对微生物和其他细胞--如精子--如何运动感兴趣的科学分支具有更广泛的吸引力。

The majority of single-celled organisms actively navigate their environment. Many species of bacteria, for instance, use a tiny rotary motor to drive a helical filament to generate propulsion. In the case of bacteria, active navigation is required to source nutrients and to avoid toxins, to form symbiotic relationships with each other, and to move towards sites for pathogenic invasion. Currently, the only method we have of studying bacterial swimming is to look at 2D images from a conventional microscope and to infer the 3D nature of the swimming as the bacteria swim in and out of the microscope focus. A full 3D understanding of bacterial swimming is vital to probe many of the unanswered questions about how they move.The aim of this project is to develop a new microscopy technique that is capable of recording the 3D positions of swimming bacteria at high speed. This will be achieved through developing a technique known as digital holographic microscopy. A hologram is an interference pattern between light emanating from a sample and a known reference beam. The fact that we know what this 'reference beam' is a priori means we can process the interference pattern in a computer to generate three-dimensional information about the sample we have imaged. A digital holographic microscope uses a microscope objective to greatly magnify the sample before forming the hologram. In this way we can obtain the 3D positions of organisms as small as bacteria, which are 1-2 microns long.Two recent technological developments allow us to study these microorganisms in 3D, and at high speeds. The first is the advance in digital camera technology. New camera chips now have sufficient pixel resolution to capture the fine detail of these magnified holograms at very fast frame-rates (up to 2,000 frames per second). The second is the availability of graphical processing units (GPUs) for image processing. GPUs were originally invented for the computer gaming industry, but have now found a place in digital processing applications as they are far more efficient at processing large volumes of data than conventional CPUs. Our 3D holographic microscope will necessarily generate huge amounts of data (1GB per frame for a typical 3D hologram); hence, GPUs are integral to the handling of that information. One of the largest stumbling blocks that explains why digital holographic imaging has not yet been used in this manner is the lack of available GPU-compatible software to effectively process these volumes of data.Our proposed technology will bring together microscopy, high-speed, high-resolution digital cameras and the processing power of GPUs to enable the three-dimensional study of bacteria and other microorganisms. It will involve a specific form of holography called 'off-axis holography'. This technique is more difficult to implement optically at higher magnifications, yet it offers improved resolution over 'inline holography' - the other main branch of the technology. Having developed 3D microscopic imaging, we propose two methods in which it will be used to study how bacteria swim. The first uses a relatively low magnification microscope (40x) to capture holograms of many bacteria swimming in one field of view. We will use computer software to track the motion of these bacteria in 3D. In this way, we can investigate the collective swimming behaviour of a population of organisms. The second method uses higher magnification (225x) optics to investigate how individual bacteria interact with their fluid environment. This example would capture the motion of tiny 'tracer' particles that allow us to visualize the fluid flow caused by a single bacterium swimming past. We anticipate that this technology would have a wider appeal to many branches of science interested in how microorganisms and other cells - such as sperm - move.