Ruckstuhl Thomas, Verdes Dorinel, Winterflood Christian M, Seeger Stefan
Institute of Physical Chemistry, University of Zurich, Zurich, Switzerland.
Opt Express. 2011 Mar 28;19(7):6836-44. doi: 10.1364/OE.19.006836.
A new microscope objective is presented for the parallel fluorescence detection below and above the critical angle of total internal reflection with single molecule sensitivity. The collection of supercritical angle fluorescence (SAF) leads to a strongly surface confined detection volume whereas the collection of undercritical angle fluorescence (UAF) allows for the observation of deeper axial sections of the specimen. By simultaneous detection of the near-field-mediated SAF and the far-field UAF emission modes the z-position of emitters can be obtained on the nanometer scale. We investigate the point spread function of the optics and demonstrate nanoscopic z-localization of single molecules. The oil immersion objective, developed for use on common microscope bodies, opens up new possibilities for the study of topographies and dynamics at surfaces and on membranes.
本文提出了一种新型显微镜物镜，用于在全内反射临界角上下进行具有单分子灵敏度的平行荧光检测。超临界角荧光（SAF）的收集导致检测体积强烈地局限于表面，而亚临界角荧光（UAF）的收集则允许观察标本更深的轴向截面。通过同时检测近场介导的SAF和远场UAF发射模式，可以在纳米尺度上获得发射体的z位置。我们研究了该光学系统的点扩散函数，并展示了单分子的纳米级z定位。这种为普通显微镜机身开发的油浸物镜，为研究表面和膜上的形貌及动力学开辟了新的可能性。
