Evaluation of fluorescent proteins for compatibility with STED microscopy systems using two-color spectroscopies

Stimulated emission depletion (STED) microscopy is a super-resolution fluorescence imaging technique that achieves high spatial and temporal resolution by exploiting stimulated emission to induce fluorescence depletion (FD) and is expected to have substantial utility for imaging applications using fluorescent proteins. However, the compatibility of fluorescent proteins with STED microscopy systems has been understood primarily through empirical observations, and there is no established methodology for the rational selection of fluorescent proteins for STED microscopy. In this study, we systematically evaluated the compatibility of commonly used fluorescent proteins with STED microscopy systems by measuring FD properties using transient absorption spectroscopy and fluorescence dip spectroscopy, both of which are classified as two-color spectroscopy (TCS). Fluorescent proteins identified as compatible with the STED microscopy system based on the TCS measurements were employed for three-dimensional STED imaging of cellular samples expressing each protein. In all samples, three-dimensional spatial resolution was improved relative to confocal laser microscopy, with particularly marked improvements in z-axis resolution. These findings demonstrate that measurements of FD properties via TCS provide a robust approach for evaluating the compatibility of fluorescent proteins with the STED microscopy system and for selecting suitable fluorescent proteins for STED imaging.