Fast remote focussing for three-dimensional microscopy

共焦显微镜是一种功能强大的成像工具，广泛应用于生物科学领域。它的优势在于它能够以高分辨率对样品进行三维成像，而不是像传统的光学显微镜那样以二维成像。具体地说，三维图像堆叠由在不同焦点设置下拍摄的一系列二维图像构成，其中每个设置对应于样品中的不同深度。然后，可以使用计算机算法以多种不同的方式呈现结果信息，以揭示关于对象结构的丰富信息。近年来，高速采集这些三维图像堆栈以观察生物标本的动态行为已成为人们感兴趣的问题。因此，已经取得了提高图像获取速度的技术进步，并且现在可以高速地从样品中的单个平面获取图像。不幸的是，这些系统可实现的重新对焦速度仍然有限，并成为三维图像获取的真正瓶颈。由于这些系统的光学设计所强加的基本物理原因，必须通过物理改变物镜和样品之间的距离来进行重新聚焦。这是有问题的，原因有二。首先，这一过程通常是缓慢的，其次，它可能导致不受欢迎的样品搅动。为了缓解这些限制，我们建议在我们开发的新的Focus系统架构的基础上构建两个新的显微镜。这些系统将允许以远高于当前技术所允许的速度进行重新聚焦。此外，由于我们方法的特殊性，重新聚焦将在远离标本的地方进行，标本将保留在其自然环境中。第一台显微镜将获得标本的高分辨率、视频率的立体图像，并将这些图像呈现在立体监视器上，以便用户可以实时直接看到三维结构。第二个显微镜将是一个高速双光子显微镜，允许许多不同的成像方式。具体地说，可以快速连续地在三个维度中选择多个任意选择的点进行询问。通过仔细选择这些点，将有可能沿着弯曲的轨迹甚至表面进行扫描。我们将与生物学家合作，开发这些新显微镜的技术并研究其应用。首先，我们将集中于发育中的小鼠胚胎的成像，在这些成像中，提高聚焦速度、非侵入性成像以及增加物镜工作距离的可能性都是可取的。我们还将寻找在神经科学中的应用，其中有必要能够快速和准确地在随机位置定位焦点，以便研究复杂三维结构的功能成像。

The confocal microscope is a powerful imaging tool that is widely used across the biological sciences. Its strength lies in its ability to image specimens at high resolution in three-dimensions, rather than two-dimensions as in conventional optical microscopy. Specifically, a three-dimensional image stack is constructed from a series of two dimensional images taken at different focal settings, where each setting corresponds to a different depth in the specimen. Computer algorithms can then be used to present the resulting information in a number of different ways to reveal a wealth of information about the object structure. Recently, it has become of interest to collect these three dimensional image stacks at high speed in order to observe the dynamic behaviour of biological specimens. As a result, technological advances have been made to improve the image acquisition speed and images from a single plane in the specimen can now be acquired at high speed. Unfortunately, the refocusing speeds that are achieveble with these systems are still limited and provide the real bottleneck in three dimensional image acquisition. For fundamental physical reasons imposed by the optical design of these systems, refocusing must be carried out by physically changing the distance between the objective lens and specimen. This is problematic for two reasons. First, this process is generally slow and second it can lead to undesireable specimen agitation. In order to alleviate these restrictions, we propose to build two new microscopes based on a new focus system architecture that we have developed. These systems will permit refocusing to be carried out at far superior speeds than current technology will allow. Furthermore, due to the specific nature of our approach, the refocusing will be carried out remotely from the specimen which will remain in its natural environment. The first microscope will obtain high resolution, video-rate stereo images of the specimen and present these on a stereo monitor so that the user can see the three-dimensional structure directly in real-time. The second microscope will be a high-speed two-photon microscope permitting a number of different imaging modalities. Specifically, it will be possible to select a number of arbitrarily chosen points in three dimensions to interrogate in quick succession. By careful choice of these points it will be possible to scan along curved trajectories or even surfaces We will work with biologists in order to develop the techniques and investigate applications for these new microscopes. In the first instance, we will concentrate on the imaging of developing mouse embryos where the advantages of increased focusing speed, non-invasive imaging and the possibility to increase the working distance of the objective are all desirable. We will also look for applications in neuroscience where it is necessary to be able to locate the focal spot at random locations with high speed and accuracy so as to study the functional imaging of complex three-dimensional structures.