Fast remote focussing for three-dimensional microscopy

共聚焦显微镜是一种强大的成像工具，广泛应用于生物科学。它的优势在于它能够以高分辨率对样品进行三维成像，而不是像传统光学显微镜那样进行二维成像。具体地，三维图像堆栈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本中的不同深度。然后，计算机算法可以用于以许多不同的方式呈现所得到的信息，以揭示关于对象结构的丰富信息。最近，它已成为感兴趣的，以高速收集这些三维图像堆栈，以观察生物标本的动态行为。因此，已经取得了技术进步，以提高图像采集速度，并且现在可以高速采集来自样本中单个平面的图像。不幸的是，这些系统可接受的重新聚焦速度仍然有限，并且在三维图像采集中提供了真实的瓶颈。由于这些系统的光学设计所施加的基本物理原因，必须通过物理地改变物镜透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，这个过程通常是缓慢的，其次，它可能会导致不必要的标本搅拌。为了减轻这些限制，我们建议建立两个新的显微镜的基础上，我们已经开发了一个新的聚焦系统架构。这些系统将允许以比当前技术所允许的高得多的上级速度进行重新聚焦。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，标本将保持在自然环境中。第一台显微镜将获得标本的高分辨率、视频速率立体图像，并将其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，允许一些不同的成像模式。具体地说，它将有可能在三维空间中选择一些任意选择的点来快速连续地询问。通过仔细选择这些点，将有可能沿着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，以开发这些新显微镜的技术和研究应用。在第一种情况下，我们将集中在发展中的小鼠胚胎的成像，提高聚焦速度，非侵入性成像和增加物镜的工作距离的可能性的优点都是可取的。我们还将寻找在神经科学中的应用，其中有必要能够以高速度和准确性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.