Multiphoton fluorescence lifetime imaging: Enlightening cellular and deep tissue dynamics and mechanics.

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Microscopy gives us an opportunity to observe, record and study a dimension beyond the limits of our eyesight. The latest advances in microscopy means that you can watch life occurring within bacteria, plants and entire organisms such as mice the way one would watch a movie. Often this is done in biology using the principle of fluorescence. Fluorescence occurs when light, in the form of photons, are absorbed by natural occurring or synthetic chemical compounds in the and re-emitted with light of a longer wavelength. The most common usage of fluorescence in imaging biological samples occurs when the absorbed radiation is in the ultraviolet region of the spectrum, and thus invisible to the human eye, while the emitted light is in the visible region, which gives the fluorescent substance a distinct colour and can be captured using a camera. So, to image biological processes live, we often attach fluorescent molecules attached to proteins or bits of DNA that are of interest which is then excited by a high energy laser. In live microscopy, as with our hand-held cameras and phone cameras, the important part is to get the best quality images with the maximum speed. This also often means that we want to have the ability to remove out-of-focus areas to get the best picture and this remains true for imaging biological samples. Limitations exist with many fluorescence microscopy techniques when imaging thick biological samples,because it is hard to focus a laser beam on a single point deep in a tissue and collect all the emitted light thus providing a good signal-to-noise ratio. New microscopy techniques are now available which can both image very deep into the tissue and restrict the imaging to a single point of focus, which means we can get sub-cellular resolution without having stray light making the image blurry. Two-photon microscopy is one such technique which can allow imaging in very dense and thick samples where the images are generated by using two photons to excite fluorescent molecules, rather than a single photon resulting in high signal-to-noise ratio. We propose to use this technique to study process such as cell movement, cell-cell communication and sub cellular processes such as transport of cargo within the cell and physical responses of cells and tissues such as changes to their stiffness and tension. Thus, this technology will support exciting research which fits into several strategic priority areas for BBSRC and contribute to existing BBSRC projects, and planned BBSRC submissions.