Confocal and multi-photon imaging have become the most important tools in live cell imaging of gene expression, and cell development and behaviour. A new challenge is the specific imaging of molecules in tissues without the need for staining or marker expression. The group is interested in:
Non-linear imaging
Advanced non-linear imaging combines the application of multi-photon imaging with fluorescence lifetime information as well as second harmonic imaging (SHG) for tumour detection in live cells and biopsies.
We are applying new imaging technologies based on nonlinear optics, in which two or more photons (usually infra-red light) interact with molecules, either to excite fluorescence or stimulate scattering at multiples of the incident frequency.
Results from our work show several advantages over current confocal methods. For example, interaction is with the specimen only at the point of focus. And perhaps the greatest advantage is that this method allows the excitation of ultraviolet absorbers (both endo- and exogenous) without the need for penetration of the specimen by UV, whilst simultaneously allowing better penetration depth for IR. There is better penetration into thick specimens, and the absence of a confocal aperture allows more efficient detection of emitted light.
Second harmonic imaging (SHG)
In the case of second harmonic scattering, the signal requires asymmetry in the macromolecular structure and reveals, in addition to specific molecular probes and unstained collagen fibres. This technique is applied to breast cancer biopsies in order to distinguish cancerous tumour from healthy tissue. Current work is directed particularly to assessing the value of non-linear optics in biological and medical microscopy, and to seek methods for improving optical resolution.
Fluorescence life-time (FLIM)
Lifetime measurements can yield information on the molecular micro-environment of a fluorescent molecule. Factors such as ionic strength, hydrophobicity, oxygen concentration, binding to macromolecules and the proximity of molecules that can deplete the excited state by resonance energy transfer, can all modify the lifetime of a fluorophore. Measurements of lifetimes can therefore be used as indicators of these parameters.
Furthermore, these measurements are generally absolute, being independent of the concentration of the fluorophore. Lifetime imaging, in conjunction with spectral imaging should greatly facilitate studies using ion indicator probes and FRET studies of intermolecular distances.
Confocal 'Macroscope'
The project is a novel lens system with an unprecedented ratio of resolving power to magnification. Designed by WB Amos of the MRC Laboratory of Molecular Biology (LMB), the system will allow the observation of whole mouse embryos or large tissue biopsies at optimal confocal image resolution and quality. The system will be highly sensitive, thus allowing the detection of low-intensity fluorescence, e.g. from rare antigens or bio-luminescence.
The system's unique combination of features will make possible work that cannot be achieved with any other system, and will give the Institute's researchers a great advantage over other labs, as they will have access to the prototype system before it becomes commercially available. The project is expected to be completed sometime mid-2006.
