Super-resolution fluorescence microscopy is one of the most important breakthroughs in optical microscopy in the 21st century, envidencing by the awarded Nobel Prize in Chemistry in 2014. Super-resolution localization microscopy (SRLM) is a major super-resolution fluorescence microscopy technique. Based on total internal fluorescence microscopy (TIRF), SRLM achieves ultra-high spatial resolution of 20-30 nm via the combination of single molecule imaging and high-precision molecule localization algorithms. However, limited by the low-light detector --- EMCCD camera, the field-of-view (FOV) of conventional SRLM is only30 μm´30 μm. And, because Gaussian beam illumination is typically used for single molecule fluorescence excitation, conventional SRLM suffers from field-dependent spatial resolution: the edge has always lower resolution than the center. Therefore, in the recent years, there are intensive interests on how to maximize FOV without reducing spatial resolution.
agroup led by Prof. Zhen-li Huang in the Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, developed a high-power homogeneous illumination method for SRLM with large FOV. This method provides field-independent spatial resolution (~40 nm) with an FOV of 221 μm´221 μm, which is 50 times higher than that in conventional SRLM, and is 5 times as the highest reported FOV in SRLM. This method not only provides sufficient illumination intensity for SRLM with large FOV, but also outputs a square-shape illumination beam to match the square-shape sensors in low-light cameras. The results have been recently published in Optics Express ( vol. 25, no. 12, pp. 13382-13395, 2017).
Fig. 1. Characterizing the illumination homogeneity across a FOV of 221 μm × 221 μm. Scale bar: 50 μm
