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

显微镜使我们有机会观察、记录和研究超出我们视力极限的维度。显微镜技术的最新进展意味着你可以像看电影一样观察细菌、植物和整个生物体（如老鼠）内发生的生命。这通常是在生物学中利用荧光原理来完成的。当光以光子的形式被自然界中天然存在的或合成的化合物吸收，并以较长波长的光重新发射时，就会产生荧光。荧光在生物样品成像中最常见的用途是，当吸收的辐射在光谱的紫外区域，因此人眼不可见，而发射的光在可见光区域，这使荧光物质具有独特的颜色，可以用相机捕获。因此，为了对生物过程进行实时成像，我们经常将荧光分子附着在感兴趣的蛋白质或DNA片段上，然后用高能激光激发这些荧光分子。在实时显微镜中，就像我们的手持相机和手机相机一样，最重要的部分是用最快的速度获得最好质量的图像。这也通常意味着我们希望能够去除失焦区域以获得最佳图像，这对于成像生物样本来说仍然是正确的。许多荧光显微镜技术在对厚的生物样本成像时存在局限性，因为很难将激光束聚焦在组织深处的一个点上并收集所有发射的光，从而提供良好的信噪比。现在有了新的显微镜技术，既可以对组织的深处进行成像，又可以将成像限制在单个焦点上，这意味着我们可以获得亚细胞分辨率，而不会有杂散光使图像模糊。双光子显微镜就是这样一种技术，它可以在非常密集和厚的样品中成像，其中图像是通过使用两个光子来激发荧光分子而不是单个光子产生高信噪比而产生的。我们建议使用这种技术来研究细胞运动、细胞间通讯和亚细胞过程（如细胞内货物运输）以及细胞和组织的物理反应（如刚度和张力的变化）。因此，这项技术将支持适合BBSRC几个战略优先领域的令人兴奋的研究，并为现有的BBSRC项目和计划提交的BBSRC做出贡献。

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.