Engineering genetic reporters for in vivo photoacoustic imaging of mammalian tissues

在对单个细胞如何工作有了更多的了解之后，生物学家正在将注意力转移到了解组织和器官中有多少不同的细胞一起工作。对活体动物的细胞功能进行成像，让我们对这些复杂系统的工作原理有了深刻的了解。如果成像不仅告诉我们特定细胞的位置，而且还告诉我们随着时间的推移细胞内部发生了什么，这一点尤其正确。有了这笔资金，我们计划使用一种新的技术，称为光声成像沿着基因工程，这样我们就可以成像特定的细胞功能在一个活的动物。目前，我们可以使用医学成像技术对小动物的细胞团块进行成像。磁共振成像（MRI）和计算机断层扫描（CT）可以产生整个小动物的详细成像。然而，很难识别我们特别感兴趣的稀有细胞。在另一个尺度上，研究人员可以使用显微镜观察活体动物的细胞。通过这种工作已经获得了相当多的信息。虽然允许非常详细的图像，但这种成像一次只能观察一小部分组织。此外，通常必须对动物进行手术，以允许感兴趣的组织片适合显微镜设备。我们真正需要的是能够在显微镜和整个动物成像之间的分辨率下成像的东西，这样我们就可以以非侵入性的方式观察整个组织功能。我们目前正在研究一种称为“光声成像”的技术，它可以满足这些要求。这种非侵入性技术是光学成像和超声的独特组合。我们首先将激光照射到动物组织中。这种光的强度不足以造成任何伤害，但当激光击中吸收光的东西时，它会在组织内产生微小的冲击波。例如，充满红细胞的小血管非常好地吸收光。这些小冲击波与超声成像仪接收到的信号非常相似，我们使用超声接收器来生成活体小动物组织切片的3D图像。成像的规模正是我们研究复杂组织的目的。虽然我们可以用这项技术清楚地看到小血管，但直到最近，我们还不得不向动物体内注射化学染料，试图标记某些细胞类型。这很难做到，因为染料并不总是只到达感兴趣的细胞，并且在活的动物中，染料被分解。我们已经想到了我们感兴趣的基因工程细胞的想法，以便它们能够自己制造染料。我们已经从珊瑚中提取了两个基因，并将它们引入细菌。这些基因指示细胞产生蓝色色素，使我们能够对这些特定的细胞进行成像。我们已经进行了许多实验，以表明这些蓝色蛋白质应该是光声成像的理想选择，特别是因为它们吸收的红光可以深入组织，而红细胞不会吸收太多。在这变得实际有用之前，需要做大量的工作来修改这些珊瑚蛋白质，并确定它们在成像时的确切行为。例如，这些珊瑚蛋白必须经过工程改造，使它们更容易由哺乳动物细胞制造，而不是聚集或结块。接下来，再次通过工程，我们可以改变这些蛋白质的颜色，从而使我们能够同时成像许多不同的细胞类型。接下来，我们可以改变这些蛋白质作为传感器，所以它们只有在细胞内发生特定事件时才有颜色。最后，我们需要对小动物中表达这些蛋白质的细胞进行大量工作，以确定如何最好地使用它们。这些新的蛋白质将大大增加光声成像的有用性，并将帮助我们了解复杂的细胞集合如何协同工作。

After gaining much understand about how individual cells work, biologists are moving their attention to understand how many different cells work together in tissues and organs. Imaging cells functioning in a living animal gives us tremendous insight into how these complex systems work. This is particularly true if the imaging not only tells us where a particular cell is but also gives us some information about what is going on inside it over time. With this grant, we plan to use a new technique called photoacoustic imaging along with genetic engineering to so we can image particular cells functioning within a living animal. Currently, we can image clumps of cells in small animals using imaging techniques used in medicine. Magnetic resonance imaging (MRI) and computed tomography (CT) can produce detailed imaging of a whole small animal. However, it is difficult to identify rare cells we are particular interested in. On the other scale, researchers can use microscopes to observe cells in living animals. Considerable information has been gained by this kind of work. Although allowing very detailed images, this kind of imaging can only observe a small portion of tissue at a time. Further, very often surgery must be performed on the animal to allow the piece of tissue of interest to fit into the microscope apparatus. What we really need is something which can image at resolutions between microscopy and whole animal imaging so we can observe a whole section of tissue function in a non-invasive way. We are currently working on a technique called 'photoacoustic imaging' which can meet these requirements. This non-invasive technique is a unique mixture of optical imaging and ultrasound. We first shine laser light into animal tissue. This light is not powerful enough to cause any damage, but it causes tiny shock waves within the tissues whenever the laser hits something which absorbs light. For instance, small blood vessel packed full of red blood cells absorb light very well. These small shock waves are very similar to the signals received by Ultrasound imagers, and we use an ultrasound receiver to generate 3D images of a section of tissue in a living small animal. The scale of the imaging is precisely what we desire to study complex tissues in action. While we can see small blood vessels clearly with this technique, up until recently we have had to inject chemical dyes into the animals to try and label certain cell types. This is difficult to do as the dyes do not always go just to the cells of interest, and in a living animal the dyes are broken down. We have thought of the idea of genetically engineering cells we are interested in studying so that they make their own dyes. We have taken two genes from coral and introduced them into bacteria. These genes instruct the cells to make a blue pigment allowing us to image these particular cells. We have performed many experiments to show that these blue proteins should be ideal for photoacoustic imaging, particularly since the red light they absorb can penetrate deeply into tissues and is not absorbed so much by red blood cells. Before this can become practically useful, a lot of work needs to be done to modify these coral proteins and establish exactly how they behave when imaged. For example, these coral proteins have to be engineered so that they can be more easily made by mammalian cells and not aggregate or clump. Next, again by engineering, we can alter the colour of these proteins potentially allowing us to image many different cell types at the same time. Next, we can alter these proteins to act as sensors, so they only have colour when a particular event is happening within the cell. Finally, we need to perform a lot of work with cells expressing these proteins in small animals to establish how best to use them. These new proteins will greatly increase the usefulness of photoacoustic imaging and will help us understand how complex collections of cells work together.