Shaping light for volumetric microscope imaging in the heart

光片荧光显微镜的最新进展使生物医学研究人员能够观察和研究活体动物，如斑马鱼，因为它们从一个单细胞成长为一个功能齐全的有机体。然而，在心脏中获得连续的3D图像是一个特别的挑战（因为它一直在跳动），随着动物变大，图像变得模糊和模糊，有必要通过越来越多的覆盖组织来成像，以看到感兴趣的器官。我们将使用我们将开发的特定的新照明和成像技术获得显微镜图像，使我们能够获得比以前更高质量的活体组织内部图像。这些图像的原始形式与传统图像不同，但在现代计算机强大的成像处理能力的帮助下，我们将能够分析和组合原始图像，以恢复比传统显微镜成像更好的图像。具体来说，我们将研究和实现三种技术：1。散斑光片成像。在这里，我们将用随机散斑场代替均匀光照亮我们的样品。因此，我们的原始图像会出现“斑驳”和不清晰，但经过计算机图像处理后，生成的图像会清晰得多，并且受阴影效果和光线穿过的覆盖组织的影响更小。聚焦不变同步心脏成像的波前编码。当我们拍摄心脏的3D视频图像时，我们必须面对这样一个事实，即心脏跳动的速度比我们通常获得完整的3D图像要快。我们通过使用图像分析和计算机控制来同步我们的图像采集与心跳来克服这个问题。然而，这是特别困难的，因为我们通常会移动样本来拍摄3D图像，这破坏了同步。波前编码使我们获得的图像看起来不再像心脏的清晰图像，但在我们移动样本时仍保持不变，从而使我们能够建立一个更简单、更便宜的同步成像系统。快照体成像的波前编码。我们的同步成像技术假定心脏正常跳动，而根据定义，在许多患病的心脏中，情况并非如此——生物学家对此特别感兴趣。我们将通过开发一种极快的体成像方法来克服这个问题。通常情况下，成像速度受限于我们改变显微镜焦点的速度，但波前编码将允许我们在计算机上进行重新聚焦，从而使我们能够更快地获得3D图像。这些技术将为显微镜成像提供新的和改进的方法，以观察活体动物的内部，帮助生物学家更好地了解心脏是如何发育和功能的，最终目的是改善人类心脏病的医学治疗。

Recent advances in light sheet fluorescence microscopy have allowed biomedical researchers to watch and study living animals such as the zebrafish as they grow from a single cell to a fully functioning organism. However obtaining continuous 3D images presents a particular challenge in the heart (since it is constantly beating) and images become clouded and blurred as the animal grows larger and it is necessary to image through increasing amounts of overlying tissue to see the organ of interest.We will acquire microscope images using specific new illumination and imaging techniques we will develop, to allow us to obtain higher quality images than previously possible inside living tissue. In their raw form these images will not resemble conventional images, but with the help of the powerful imaging processing capabilities of modern computers, we will be able to analyze and combine the raw images to recover better images than would have otherwise been possible with conventional microscope imaging.Specifically, we will research and implement three techniques:1. Speckle light sheet imaging. Here instead of illuminating our sample with uniform light we will illuminate it with a random speckle field. Our raw images will therefore appear "dappled" and unclear, but following computer image processing the resultant images will be much sharper and less affected both by shadowing effects and by the overlying tissue that the light has passed through.2. Wavefront coding for focus-invariant synchronized heart imaging. When we take 3D video images of the heart, we have to cope with the fact that the heart is beating faster than we can normally obtain a complete 3D image of it. We overcome this by using image analysis and computer control to synchronize our image acquisition with the heartbeat. However this is particularly difficult because we would usually move the sample around in order to take the 3D image, and this spoils the synchronization. Wavefront coding lets us acquire images that no longer look like a clear image of the heart, but which remain the same as we move the sample, thus allowing us to build a much simpler and cheaper synchronized imaging system.3. Wavefront coding for snapshot volume imaging. Our synchronized imaging technique assumes that the heart is beating regularly, and by definition that will not be the case in many diseased hearts - which biologists are particularly interested in studying. We will overcome this problem by developing a method for extremely fast volume imaging. Normally the imaging speed is limited by how fast we can change the focus of our microscope, but wavefront coding will allow us to do the refocusing on a computer afterwards, thus allowing us to obtain 3D images much faster.These techniques together will offer new and improved methods for microscope imaging to look inside living animals, to help biologists better understand how the heart develops and functions - with the ultimate aim of improving medical treatments for human heart diseases.