Aberration free extended depth of field microscopy

In recent years, the confocal and two photon microscopes have become ubiquitous tools in life science laboratories. The reason for this is that both these systems can acquire three dimensional image data from biological specimens. Specifically, this is done by acquiring a series of two-dimensional images from a set of equally spaced planes within the specimen. The resulting image stack can be manipulated and displayed on a computer to reveal a wealth of information. These systems can also be used in time lapse studies to monitor the dynamical behaviour of specimens by recording a number of image stacks at a sequence of time points. The time resolution in this situation is, however, limited by the maximum speed at which each constituent image stack can be acquired. Various techniques have emerged to speed up image acquisition and in most practical implementations a single, in-focus, image can be acquired very quickly. However, the real bottleneck in three dimensional imaging is the process of refocusing the system to image different planes. This is commonly done by physically changing the distance between the specimen and imaging lens, which is a relatively slow process. It is clear with the ever-increasing need to image biologically relevant specimens quickly that the speed limitation imposed by the refocusing process must be overcome. This thesis concerns the acquisition of data from a range of specimen depths without requiring the specimen to be moved. A new technique is demonstrated for two photon microscopy that enables data from a whole range of specimen depths to be acquired simultaneously so that a single two dimensional scan records extended depth of field image data directly. This circumvents the need to acquire a full three dimensional image stack and hence leads to a significant improvement in the temporal resolution for acquiring such data by more than an order of magnitude. In the remainder of this thesis, a new microscope architecture is presented that enables scanning to be carried out in three dimensions at high speed without moving the objective lens or specimen. Aberrations introduced by the objective lens are compensated by the introduction of an equal and opposite aberration with a second lens within the system enabling diffraction limited performance over a large range of specimen depths. Focusing is achieved by moving a very small mirror, allowing axial scan rates of several kHz; an improvement of some two orders of magnitude. This approach is extremely general and can be applied to any form of optical microscope with the very great advantage that the specimen is not disturbed. This technique is developed theoretically and experimental results are shown that demonstrate its potential application to a broad range of sectioning methods in microscopy.This thesis is not currently available in OR