Improving quantitative fluorescence imaging with flat field illumination

摘要

We demonstrated flat-field illumination (FFI) for multi-color wide-field fluorescence microscopy using a refractionbasedbeam shaping system. The non-homogeneous illumination of a Gaussian intensity profile makes quantitativeanalysis in laser-assisted wide-field fluorescence microscopy very difficult. As contrasted with other approaches, ourmethod 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 raysto the intended flattop beam profile in combination with a sophisticated material choice, which decreases the sensitivityto input beam diameter. The homogenous illumination profile of FFI will enable quantitative single-molecule analysisbased on intensity information. This has powerful implications when combined with a pull-down assay, which can probethe oligomerization state of endogenous proteins. When combined with one-to-one fluorophore labeling, thestoichiometry of proteins related to neurodegenerative diseases could be readily determined by intensity distributionanalysis, which is critical to not only diagnosing but also understanding the pathogenesis of these complex disorders thatare particularly difficult to analyze.An additional application of FFI is high quality super-resolution imaging with a uniform spatial resolution over a largeFOV, where the full power of the excitation beam could be utilized. A new optical design approach based on refractivefreeform surfaces generating a square-shaped beam instead of a round one will be presented, which would yield greaterillumination efficiency.