Oheim Martin, Salomon Adi, Brunstein Maia
Saints-Pères Paris Institute for the Neurosciences, Université de Paris, CNRS, Paris, France.
Saints-Pères Paris Institute for the Neurosciences, Université de Paris, CNRS, Paris, France; Department of Chemistry, Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan, Israel.
Biophys J. 2020 May 19;118(10):2339-2348. doi: 10.1016/j.bpj.2020.03.029. Epub 2020 Apr 11.
Fluorescence detection, either involving propagating or near-field emission, is widely being used in spectroscopy, sensing, and microscopy. Total internal reflection fluorescence (TIRF) confines fluorescence excitation by an evanescent (near) field, and it is a popular contrast generator for surface-selective fluorescence assays. Its emission equivalent, supercritical angle fluorescence (SAF), is comparably less established, although it achieves a similar optical sectioning as TIRF does. SAF emerges when a fluorescing molecule is located very close to an interface and its near-field emission couples to the higher refractive index medium (n >n) and becomes propagative. Then, most fluorescence is detectable on the side of the higher-index substrate, and a large fraction of this fluorescence is emitted into angles forbidden by Snell's law. SAF, as well as the undercritical angle fluorescence (UAF; far-field emission) components, can be collected with microscope objectives having a high-enough detection aperture (numerical aperture >n) and be separated in the back focal plane by Fourier filtering. The back focal plane image encodes information about the fluorophore radiation pattern, and it can be analyzed to yield precise information about the refractive index in which the emitters are embedded, their nanometric distance from the interface, and their orientation. A SAF microscope can retrieve this near-field information through wide-field optics in a spatially resolved manner, and this functionality can be added to an existing inverted microscope. Here, we describe the potential underpinning of SAF microscopy and spectroscopy, particularly in comparison with TIRF. We review the challenges and opportunities that SAF presents from a biophysical perspective, and we discuss areas in which we see potential.
荧光检测，无论是涉及传播发射还是近场发射，都广泛应用于光谱学、传感和显微镜技术中。全内反射荧光（TIRF）通过倏逝（近）场限制荧光激发，它是用于表面选择性荧光检测的一种常用对比度生成方法。其发射等效方法，即超临界角荧光（SAF），虽然能实现与TIRF类似的光学切片效果，但相对来说应用较少。当荧光分子非常靠近界面时，SAF就会出现，其近场发射耦合到更高折射率的介质（n>n）并变为传播性发射。然后，大部分荧光在高折射率衬底一侧可被检测到，并且其中很大一部分荧光以违反斯涅尔定律的角度发射。SAF以及亚临界角荧光（UAF；远场发射）成分，可以用具有足够高检测孔径（数值孔径>n）的显微镜物镜收集，并通过傅里叶滤波在背焦平面上分离。背焦平面图像编码了有关荧光团辐射模式的信息，可以对其进行分析以获取有关发射体所嵌入的折射率、它们与界面的纳米距离以及它们的取向的精确信息。SAF显微镜可以通过宽场光学以空间分辨的方式检索此近场信息，并且此功能可以添加到现有的倒置显微镜中。在此，我们描述了SAF显微镜和光谱学的潜在基础，特别是与TIRF相比。我们从生物物理学角度回顾了SAF所带来的挑战和机遇，并讨论了我们认为有潜力的领域。
