Ingaramo Maria, York Andrew G, Andrade Eric J, Rainey Kristin, Patterson George H
National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA.
Nat Commun. 2015 Sep 3;6:8184. doi: 10.1038/ncomms9184.
We describe two-step fluorescence microscopy, a new approach to non-linear imaging based on positive reversible photoswitchable fluorescent probes. The protein Padron approximates ideal two-step fluorescent behaviour: it equilibrates to an inactive state, converts to an active state under blue light, and blue light also excites this active state to fluoresce. Both activation and excitation are linear processes, but the total fluorescent signal is quadratic, proportional to the square of the illumination dose. Here, we use Padron's quadratic non-linearity to demonstrate the principle of two-step microscopy, similar in principle to two-photon microscopy but with orders-of-magnitude better cross-section. As with two-photon, quadratic non-linearity from two-step fluorescence improves resolution and reduces unwanted out-of-focus excitation, and is compatible with structured illumination microscopy. We also show two-step and two-photon imaging can be combined to give quartic non-linearity, further improving imaging in challenging samples. With further improvements, two-step fluorophores could replace conventional fluorophores for many imaging applications.
我们描述了两步荧光显微镜，这是一种基于正可逆光开关荧光探针的非线性成像新方法。蛋白质Padron近似于理想的两步荧光行为：它平衡到非活性状态，在蓝光下转变为活性状态，并且蓝光也激发这种活性状态发出荧光。激活和激发都是线性过程，但总荧光信号是二次方的，与照明剂量的平方成正比。在这里，我们利用Padron的二次非线性来演示两步显微镜的原理，其原理与双光子显微镜相似，但具有几个数量级更好的截面。与双光子一样，两步荧光的二次非线性提高了分辨率并减少了不需要的离焦激发，并且与结构照明显微镜兼容。我们还表明，两步成像和双光子成像可以结合起来产生四次非线性，进一步改善在具有挑战性的样品中的成像。随着进一步改进，两步荧光团可以在许多成像应用中取代传统荧光团。
