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We demonstrated flat-field illumination (FFI) for multi-color wide-field fluorescence microscopy using a refractionbased beam shaping system. The non-homogeneous illumination of a Gaussian intensity profile makes quantitative analysis in laser-assisted wide-field fluorescence microscopy very difficult. As contrasted with other approaches, our method is applicable to TIRF illumination, which effectively rejects background fluorescence. Our beam shaping device is extremely tolerant to variations in size of the incoming laser beam by accepting ± 10% variation, while being achromatic as well. This behavior originates from the well-balanced mapping of the incoming rays to the intended flattop beam profile in combination with a sophisticated material choice, which decreases the sensitivity to input beam diameter. The homogenous illumination profile of FFI will enable quantitative single-molecule analysis based on intensity information. This has powerful implications when combined with a pull-down assay, which can probe the oligomerization state of endogenous proteins. When combined with one-to-one fluorophore labeling, the stoichiometry of proteins related to neurodegenerative diseases could be readily determined by intensity distribution analysis, which is critical to not only diagnosing but also understanding the pathogenesis of these complex disorders that are particularly difficult to analyze. An additional application of FFI is high quality super-resolution imaging with a uniform spatial resolution over a large FOV, where the full power of the excitation beam could be utilized. A new optical design approach based on refractive freeform surfaces generating a square-shaped beam instead of a round one will be presented, which would yield greater illumination efficiency.
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