Fluctuation‐based Fluorescence Labeling and Biosensing for Multi‐color Super‐resolution Imaging

Abstract

Multi-color super-resolution microscopy has become a desirable method for exploring the dynamic interaction and compartmentalization of biomolecules in living cells. Among numerous super-resolution microscopy concepts, photochromic stochastic optical fluctuation imaging (pcSOFI) has been widely recognized and applied in the life sciences due to advantages such as use of off-the-shelf equipment and high temporal resolution. Furthermore, with reversibly switchable fluorescent proteins (RSFP), pcSOFI could be used to localize biomolecules in high resolution in living systems. Previously, we discovered a phenomenon called fluorescence fluctuation increase by contact (FLINC), in which the RSFP Dronpa can specifically enhance the ability of fluorescence fluctuations of non-RSFP TagRFP-T when they are spatially close to each other. We developed FLINC-based A kinase-activity reporter (AKAR) and achieved super-resolution PKA activity imaging by pcSOFI in living cells. However, FLINC-based sensors contain two FPs, presenting challenges in multi-color super-resolution imaging. To overcome this issue, we identified mutations that disrupt fluorescence of Dronpa but maintain FLINC. We then introduced a Dronpa-removed FLINC (DrFLINC) as an enhanced red label, in combination with pcSOFI, to localize biomolecules in super-resolution in different compartments of living cells. Applying this new red fluorescent label in dual-color pcSOFI, we revealed that type II PKA regulatory subunit-α (RIIα) mainly located on microtubule in MIN6 beta cells. Furthermore, we developed DrFLINC-AKAR and demonstrated the multiplexed super-resolution imaging to reveal the regulatory context of PKA activity microdomains in living cells.