Fast remote focussing for three-dimensional microscopy

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.

A common requirement in high-resolution optical microscopy is to obtain a three-dimensional representation of the object under investigation. This is typically achieved via an intermediate step using an optical sectioning technique, such as confocal or two photon microscopy to obtain a through focus series of images from which a three dimensional rendering may be achieved. Although each optical section may be recorded quickly the process of refocusing to successive image planes is usually slow and involves mechanical movement of the objective lens and/or the specimen. This work addresses this problem and presents a fast, all optical method of remote focusing. It is fundamental that one cannot satisfy the optical requirements for the perfect imaging of points lying perpendicular to the optic axis (the sine condition) and those lying along the axis (the Herschel condition) simultaneously together with the need for significant magnification. It is, however, possible to obtain a perfect image if one is prepared to settle for the modest magnification of unity, in the case of dry objectives. This suggests the kernel of the idea, which is to produce a perfect image of the specimen at an intermediate image plane and then to image this 'replica' specimen rather than the actual specimen. In this way refocusing speeds considerably in excess of those currently available may be achieved without introducing spherical aberration and therefore maintaining the quality of the focal spot. The technology will be demonstrated through imaging of biological specimens provided by our collaborators. We will concentrate initially on high-resolution stereo imaging and two-photon imaging of mouse embryos. We will also identify other potential application areas with a view to submitting a full funding proposal before the end of this pilot project.