At present no effective technique exists for accurately and reliably quantifying cellular interaction forces. An understanding of these forces is crucial to the development of effective vaccinations, skin grafts, tissue regeneration and provides a greater understanding of auto-immune diseases. We propose to develop novel techniques that combine optical trapping with optical sectioning microscopy in order to fully understand and probe cellular interactions. The optical trapping system will be designed specifically for this purpose and will allow the user to directly capture and manipulate the cells of interest without the need to introduce foreign bodies or 'handles' to the sample. The cells will be trapped with a laser beam whose profile has been designed in order to minimize cell roll and hence improve the accuracy of the technique. The position of the cell will be monitored on a nanometer scale and from here the optical force applied by the laser beam will be known and any additional external force, for example from another cell, will be quantified. This specialized optical trapping system will be combined with structured light illumination - a form of optical sectioning microscopy. An optical sectioning microscope provides high quality images with greatly improved axial resolution over conventional widefield microscopy. This enables the user to build up a three-dimensional image, with sub-cellular level resolution, of their sample of interest. As part of this proposal an optical sectioning microscope will be combined with and optical trapping system to gain as much information as possible about the cell properties and configuration whilst quantifying the cellular interaction forces. The type of optical sectioning microscope used will be structured light illumination which can operate in fluorescence and has a similar axial resolution as confocal microscopy. The main benefit of structured light illumination is that it provides rapid imaging of the sample of interest allowing the cellular interactions and information from the image to be determined and compared in real-time. In a confocal microscope, for example, a laser beam would need to be scanned over every point in the image and the signal intensity record, where as in structured light illumination a minimum of three images can be taken and then processed to produce an optically sectioned image. With the technical challenges tackled and the system perfected in order to accurately and reliably quantify cellular interaction forces, it will be employed to answer some fundamental life science questions. There are several scientists currently supporting this proposal and they are interested in the level of interactions between immune cells and how this interaction can be modified and the adhesion properties of a new hydrogel matrix for three-dimensional cell culture.
"This grant developed an optical trapping method for quantifying the cellular interaction force between single immune cell pairs. The technique proved to be a success and we were able to identify a difference in interaction force associated with the absence and presence of specific antigen and also in the case of therapeutic intervention.
In addition we explored techniques to improve the current optical trapping technology and ensure a constant trapping force across a range of trap depths. This we achieved using an aberration correction technique called adaptive optics."