Burghardt Thomas P, Hipp Andrew D, Ajtai Katalin
Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, Minnesota 55905, USA.
Appl Opt. 2009 Nov 10;48(32):6120-31. doi: 10.1364/AO.48.006120.
Total internal reflection fluorescence (TIRF) microscopy uses the evanescent field on the aqueous side of a glass/aqueous interface to selectively illuminate fluorophores within approximately 100 nm of the interface. Applications of the method include epi-illumination TIRF, where the exciting light is refracted by the microscope objective to impinge on the interface at incidence angles beyond critical angle, and prism-based TIRF, where exciting light propagates to the interface externally to the microscope optics. The former has higher background autofluorescence from the glass elements of the objective where the exciting beam is focused, and the latter does not collect near-field emission from the fluorescent sample. Around-the-objective TIRF, developed here, creates the evanescent field by conditioning the exciting laser beam to propagate through the submillimeter gap created by the oil immersion high numerical aperture objective and the glass coverslip. The approach eliminates background light due to the admission of the laser excitation to the microscopic optics while collecting near-field emission from the dipoles excited by the approximately 50 nm deep evanescent field.
全内反射荧光（TIRF）显微镜利用玻璃/水界面水侧的倏逝场来选择性地照亮界面约100纳米范围内的荧光团。该方法的应用包括落射照明TIRF，其中激发光由显微镜物镜折射，以超过临界角的入射角照射到界面上；以及基于棱镜的TIRF，其中激发光在显微镜光学系统外部传播到界面。前者来自物镜玻璃元件的背景自发荧光较高，激发光束聚焦于此；而后者不收集来自荧光样品的近场发射。本文开发的物镜周围TIRF通过调节激发激光束，使其通过油浸高数值孔径物镜和玻璃盖玻片形成的亚毫米间隙来产生倏逝场。该方法消除了由于激光激发进入显微镜光学系统而产生的背景光，同时收集由约50纳米深的倏逝场激发的偶极子的近场发射。
