Of all the ways in which complex materials (including many biological systems) can be explored, imaging is perhaps the most powerful because delivering high information content directly. This is particular relevant in aspects of cellular localization where the physical proximity of molecules is crucial in biochemical processes. A great deal of effort in imaging has been spent on enabling chemically selective imaging so that only specific features are revealed. This is almost always achieved by adding fluorescent chemical labels to specific molecules. Under appropriate illumination conditions only the molecules (via their labels) will be visible. The technique is simple and elegant but does suffer from fundamental limitations: (1) the fluorescent labels may fade when illuminated (a phenomenon called photobleaching) thereby constantly decreasing signal contrast over the course of image acquisition. To combat photobleaching one must reduce observation times or apply unfavourably low excitation levels all of which reduce the information content of images; (2) the fluorescent species may be deactivated by various environmental factors (the general term is fluorescence quenching); (3) the presence of fluorescent labels may introduce unexpected complications or may interfere with processes of interest (4) Some molecules of interest cannot be labelled. In these circumstances we require a fundamentally different strategy. One of the most promising alternative is based on a technique called Coherent Anti-Stokes Raman scattering (CARS). CARS is a fundamentally more complex process than is fluorescence and the experimental procedures and optical systems required to deliver high quality CARS images are intricate. However, the rewards are correspondingly very high: CARS probes the chemically distinct vibrations of the constituent molecules in a complex system and is therefore also chemically selective as are fluorescence-based methods. Moreover,the potentially severe problems of fluorescence bleaching and quenching are circumvented and high-resolution three dimensional images can be obtained on completely unlabelled specimens. We review here aspects of CARS and Multiphoton fluorescence techniques to cellular localization and measurement.

作者：A, McVey；J, Crain

来源：Methods (San Diego, Calif.) 2014 年 68卷 2期