Summary of research projects
Confocal, multiphoton and non-linear microscopy
Our research into laser-scanning microscopes encompasses confocal fluorescence, multiphoton fluorescence and harmonic generation microscopes. In each of these microscopes, a laser spot is scanned through the specimen in three dimensions as the image signal is acquired. Volumetric images are constructed from this signal in a point-by-point manner. In the confocal microscope, a pinhole is used to exclude out-of-focus light – this leads to the effect of optical sectioning, whereby high resolution 3D images can be obtained. Multiphoton fluorescence and harmonic generation microscopes use non-linear effects in focussed short-pulsed lasers to produce a similar optical sectioning effect. These microscopes are widely used for imaging in the life sciences.
Widefield sectioning microscopy
The conventional optical microscope becomes an even more powerful imaging tool when it is combined with three-dimensional imaging properties of the kind displayed in the confocal microscope. In order to introduce a three-dimensional imaging capability into the conventional microscope we have discarded the traditional confocal principle and have developed a fringe projection (structured illumination) technique based on spatial heterodyning. This approach, which requires the minimum modification to the illumination path of a conventional microscope, has been implemented in a number of different ways to provide high quality real-time optically sectioned imaging.
The imaging quality of a confocal microscope is severely compromised when aberrations are present in the optical system. These aberrations lead to reduced signal level and degraded lateral and, more significantly, axial resolution. Often aberrations can be introduced not only by misalignment of optical components in the microscope but also by the specimen itself. We are developing adaptive optics systems to overcome these limitations. We have implemented adaptive optics in a range of microscopes and have demonstrated the benefits of aberration correction. The methods have been further applied to optical tweezers, data storage and optical fabrication techniques.
Rapid remote focusing
Most microscopes are designed to acquire images from a lateral (x-y) plane. Three dimensional representations of volume objects require the acquisition of a series of such images from a range of focusing depths. In most systems, refocusing is performed by mechanical movement of the specimen relative to the objective lens. This has two major drawbacks. First, the axial scan speeds are slow. Second, the scanning movements can disturb the specimen during the imaging process. An alternative focusing method that does not require mechanical movement of the specimen is clearly preferable. We have developed scanning systems based upon remote focusing that avoid specimen agitation and permit axial scan speeds higher than those in existing microscopes.
Active optics for photonic engineering
Laser-induced optical processes are widely used for fabrication. Taking advantage of non-linear optical processes, one can create structures with sub-micron 3D resolution as the fabrication effects are confined to the laser focus. There are however limitations that obstruct the further development of such techniques into a widely used fabrication process. One issue is the loss of resolution and effectiveness of fabrication as one focuses deeper into a 3D material. Another limitation is speed – existing processes are often slow. We are developing adaptive optical techniques, based around deformable mirror and liquid crystal devices, to overcome these limitations. These techniques are used for applications including waveguides, photonic crystals and metamaterials.
Spectral confocal microscopy
Photonic crystal fibre-based supercontinuum light sources have opened up a range of possibilities in confocal microscopy. Confocal microscopes have relied upon the use of lasers to produce a bright, point-like source of light. However, lasers that might be practically used for microscopy have only been available in a limited number of discrete wavelengths. For this reason, commercial confocal microscopes have only typically incorporated two or three laser wavelengths. Supercontinuum sources act as laser-like point sources but include a wide range of illumination wavelengths. Combined with spectral detection, we have used these sources to implement new reflection and fluorescence spectral microscopes for three-dimensional imaging.