Stemmer Andreas, Beck Markus, Fiolka Reto
Nanotechnology Group, Department of Mechanical and Process Engineering, ETH Zurich, Tannenstrasse 3, 8092, Zurich, Switzerland.
Histochem Cell Biol. 2008 Nov;130(5):807-17. doi: 10.1007/s00418-008-0506-8. Epub 2008 Sep 23.
Widefield fluorescence microscopy is seeing dramatic improvements in resolution, reaching today 100 nm in all three dimensions. This gain in resolution is achieved by dispensing with uniform Köhler illumination. Instead, non-uniform excitation light patterns with sinusoidal intensity variations in one, two, or three dimensions are applied combined with powerful image reconstruction techniques. Taking advantage of non-linear fluorophore response to the excitation field, the resolution can be further improved down to several 10 nm. In this review article, we describe the image formation in the microscope and computational reconstruction of the high-resolution dataset when exciting the specimen with a harmonic light pattern conveniently generated by interfering laser beams forming standing waves. We will also discuss extensions to total internal reflection microscopy, non-linear microscopy, and three-dimensional imaging.
宽场荧光显微镜在分辨率方面正取得显著进步，如今在所有三个维度上都能达到100纳米的分辨率。这种分辨率的提升是通过摒弃均匀的柯勒照明实现的。取而代之的是，应用在一个、两个或三个维度上具有正弦强度变化的非均匀激发光模式，并结合强大的图像重建技术。利用荧光团对激发场的非线性响应，分辨率可进一步提高到几十纳米。在这篇综述文章中，我们描述了在用由形成驻波的干涉激光束方便地产生的谐波光模式激发样品时，显微镜中的图像形成以及高分辨率数据集的计算重建。我们还将讨论全内反射显微镜、非线性显微镜和三维成像的扩展。
