Flat-field illumination for quantitative fluorescence imaging

Opt Express. 2018 Jun 11;26(12):15276-15288. doi: 10.1364/OE.26.015276.


The uneven illumination of a Gaussian profile makes quantitative analysis highly challenging in laser-based wide-field fluorescence microscopy. Here we present flat-field illumination (FFI) where the Gaussian beam is reshaped into a uniform flat-top profile using a high-precision refractive optical component. The long working distance and high spatial coherence of FFI allows us to accomplish uniform epi and TIRF illumination for multi-color single-molecule imaging. In addition, high-throughput borderless imaging is demonstrated with minimal image overlap.