Multiplexed In Vivo Imaging with Fluorescence Lifetime-Modulating Tags.

Fluorescence lifetime imaging microscopy (FLIM) opens new dimensions for highly multiplexed imaging in live cells and organisms using differences in fluorescence lifetime to distinguish spectrally identical fluorescent probes. Here, a set of fluorescence-activating and absorption-shifting tags (FASTs) capable of modulating the fluorescence lifetime of embedded fluorogenic 4-hydroxybenzylidene rhodanine (HBR) derivatives is described. It is shown that changes in the FAST protein sequence can vary the local environment of the chromophore and lead to significant changes in fluorescence lifetime. These fluorescence lifetime-modulating tags enable multiplexed imaging of up to three targets in one spectral channel using a single HBR derivative in live cells and live zebrafish larvae. The combination of fluorescence lifetime multiplexing with spectral multiplexing allows to successfully image six targets in live cells, opening great prospects for multicolor fluorescence lifetime multiplexing.

Genetic Targeting of Solvatochromic Dyes for Probing Nanoscale Environments of Proteins in Organelles.

A variety of protein tags are available for genetically encoded protein labeling, which allow their precise localization and tracking inside the cells. A new dimension in protein imaging can be offered by combining protein tags with polarity-sensitive fluorescent probes, which provide information about local nanoscale environments of target proteins within the subcellular compartments (organelles). Here, we designed three fluorescent probes based on solvatochromic nile red dye, conjugated to a HaloTag reactive targeting group through polyethylene glycol linkers of varying lengths. The probe with medium linker length, NR12-Halo, was found to label specifically a large variety of proteins localized in defined cell compartments, such as plasma membranes (outer and inner leaflets), endoplasmic reticulum, Golgi apparatus, cytosol, microtubules, actin, and chromatin. Owing to its polarity-sensitive fluorophore, the probe clearly distinguished the proteins localized within apolar lipid membranes from other proteins. Moreover, it revealed dramatic changes in the environment during the life cycle of proteins from biosynthesis to their expected localization and, finally, to recycling inside lysosomes. Heterogeneity in the local polarity of some membrane proteins also suggested a formation of low-polar protein aggregates, for example, within cell-cell contacts. The approach also showed that mechanical stress (cell shrinking by osmotic shock) induced a general polarity decrease in membrane proteins, probably due to the condensation of biomolecules. Finally, the nanoenvironment of some membrane proteins was affected by a polyunsaturated fatty acid diet, which provided the bridge between organization of lipids and proteins. The developed solvatochromic HaloTag probe constitutes a promising tool for probing nanoscale environments of proteins and their interactions within subcellular structures.

Isolating and Engineering Fluorescence-Activating Proteins Using Yeast Surface Display.

This protocol describes the workflow to isolate and engineer fluorescence-activating proteins by yeast surface display. Fluorescence-activating proteins are an emerging class of fluorescent chemogenetic reporters for monitoring gene expression and protein localization in living cells and organisms. They become fluorescent upon binding exogenously applied fluorogenic organic dyes. Efficient fluorescence-activating proteins can be selected from yeast-displayed libraries by iterative rounds of fluorescence-activated cell sorting. The overall strategy is described, as well as a strategy for characterizing the affinity and spectroscopic properties of the selected clones.

An expanded palette of fluorogenic HaloTag probes with enhanced contrast for targeted cellular imaging.

We report the development of HaloTag fluorogens based on dipolar flexible molecular rotor structures. By modulating the electron donating and withdrawing groups, we have tuned the absorption and emission wavelengths to design a palette of fluorogens with emissions spanning the green to red range, opening new possibilities for multicolor imaging. The probes were studied in glycerol and in the presence of HaloTag and exhibited good fluorogenic properties thanks to a viscosity-sensitive emission. In live-cell confocal imaging, the fluorogens yielded only a very low non-specific signal that enabled wash-free targeted imaging of intracellular organelles and proteins with good contrast. Combining experimental studies and theoretical investigation of the protein/fluorogen complexes by molecular dynamics, these results offer new insight into the design of molecular rotor-based fluorogenic HaloTag probes in order to improve reaction rates and the imaging contrast.

Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging.

Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we report the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. The ability to tune the fluorescence color and properties through simple molecular modulation provides a broad experimental versatility for imaging proteins in live cells, including neurons, and in multicellular organisms, and opens avenues for optimizing Förster resonance energy transfer (FRET) biosensors in live cells. The ability to tune the spectral properties and fluorescence performance enables furthermore to match the specifications and requirements of advanced super-resolution imaging techniques.