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后，生物学家们正在转移他们的注意力，以了解在组织和器官中有多少不同的细胞共同工作。在活体动物中对细胞进行成像使我们对这些复杂的系统如何工作有了极大的了解。如果成像不仅告诉我们特定细胞在哪里，而且还给我们一些关于随着时间的推移它内部发生了什么的信息，这一点就尤其正确。有了这笔赠款，我们计划使用一种名为光声成像的新技术，并结合基因工程，这样我们就可以成像在活动物体内发挥作用的特定细胞。目前，我们可以使用医学上使用的成像技术对小动物的细胞团进行成像。磁共振成像(MRI)和计算机断层扫描(CT)可以对整个小动物进行详细的成像。然而，很难识别我们特别感兴趣的稀有细胞。在另一种尺度上，研究人员可以使用显微镜观察活动物的细胞。通过这种工作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，以使感兴趣的组织块适合显微镜设备。我们真正需要的是一种可以在显微镜和整个动物成像之间进行分辨率成像的设备，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名为“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波的独特结合。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损害，但每当激光击中吸收光线的物体时，它就会在组织内产生微小的冲击波。例如，装满红血球的小血管吸收光线非常好。这些小冲击波与超声成像仪接收到的信号非常相似，我们使用超声波接收器生成活着的小动物组织切片的3D图像。成像的规模正是我们在研究复杂组织时所希望的。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，以尝试标记某些细胞类型。这很难做到，因为染料并不总是直接进入感兴趣的细胞，而且在活着的动物体内染料是被分解的。我们已经想到了对我们感兴趣的细胞进行基因工程的想法，这样它们就可以制造自己的染料。我们从珊瑚中提取了两个基因，并将它们引入细菌中。这些基因指示细胞产生一种蓝色色素，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，不会被红细胞吸收那么多。在这项技术变得实用之前，需要做大量的工作来修饰这些珊瑚蛋白质，并准确地确定它们在成像时的行为。例如，这些珊瑚蛋白质必须经过改造，以便它们可以更容易地由哺乳动物细胞制造，而不是聚集或聚集。接下来，同样是通过工程，我们可以改变这些蛋白质的颜色，潜在地允许我们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，这样它们只在细胞内发生特定事件时才有颜色。最后，我们需要对在小动物中表达这些蛋白质的细胞进行大量工作，以确定如何最好地利用它们。这些新的蛋白质将大大增加光声成像的实用性，并将帮助我们了解复杂的细胞集合是如何协同工作的。

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.