We demonstrated flat-field illumination (FFI) for multi-color wide-field fluorescence
microscopy using a refraction-based 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.
