Fluorescence lifetime imaging microscopy (FLIM) and spectral imaging are two broadly applied methods for increasing dimensionality in microscopy. However, their combination is typically inefficient and slow in terms of acquisition and processing. By integrating technological and computational advances, we developed a robust and unbiased spectral FLIM (S-FLIM) system. Our method, Phasor S-FLIM, combines true parallel multichannel digital frequency domain electronics with a multidimensional phasor approach to extract detailed and precise information about the photophysics of fluorescent specimens at optical resolution. To show the flexibility of the Phasor S-FLIM technology and its applications to the biological and biomedical field, we address four common, yet challenging, problems: the blind unmixing of spectral and lifetime signatures from multiple unknown species, the unbiased bleedthrough- and background-free Förster resonance energy transfer analysis of biosensors, the photophysical characterization of environment-sensitive probes in living cells and parallel four-color FLIM imaging in tumor spheroids.

Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K⁺ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.

Bioluminescence is a powerful method for imaging in vivo, but applications at the microscale are far from routine. This is due, in part, to a lack of versatile tools for visualizing dynamic events. To address this void, we developed a new platform-Bioluminescence Resonance Energy mAKe over with a Fluorescence-Activating absorption-Shifting Tag (BREAKFAST). BREAKFAST features a bright luciferase combined with a chemogenetic tag (pFAST) for rapid color switching. In the presence of luciferin and a discrete fluorogenic ligand, signal is observed via resonance energy transfer. We evaluated spectral outputs with various fluorogens and established the utility of BREAKFAST for combined fluorescence and bioluminescence imaging. Dynamic, four-color visualization was achieved with sequential ligand addition and spectral phasor analysis. We further showed selective signal quenching with a dark fluorogen. Collectively, this work establishes a new method for bioluminescence imaging at the cellular scale and sets the stage for continued probe development.