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Polarity-sensitive fluorogenic dyes raised considerable attention because they can turn on their fluorescence after binding to biological targets, allowing background-free imaging. However, their brightness is limited, and they do not operate in the far-red region. Here, we present a new concept of fluorogenic dye based on a squaraine dimer that unfolds on changing environment from aqueous to organic and thus turns on its fluorescence. In aqueous media, all three newly synthesized dimers displayed a short wavelength band characteristic of an H-aggregate that was practically nonfluorescent, whereas in organic media, they displayed a strong fluorescence similar to that of the squaraine monomer. For the best dimer, which contained a pegylated squaraine core, we obtained a very high turn-on response (organic vs aqueous) up to 82-fold. Time-resolved studies confirmed the presence of nonfluorescent intramolecular H-aggregates that increased with the water content. To apply these fluorogenic dimers for targeted imaging, we grafted them to carbetocin, a ligand of the oxytocin G protein-coupled receptor. A strong receptor-specific signal was observed for all three conjugates at nanomolar concentrations. The probe derived from the core-pegylated squaraine showed the highest specificity to the target receptor together with minimal nonspecific binding to serum and lipid membranes. The obtained dimers can be considered as the brightest polarity-sensitive fluorogenic molecules reported to date, having ~660,000 M-1 cm-1 extinction coefficient and up to 40% quantum yield, whereas far-red operation region enables both in vitro and in vivo applications. The proposed concept can be extended to other dye families and other membrane receptors, opening the route to new ultrabright fluorogenic dyes. [ABSTRACT FROM AUTHOR]

We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca2+) indicators as new tools for biological Ca2+ imaging. The specificity of this Ca2+-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca2+-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 μM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca2+ uncaging or optogenetic stimulation with Ca2+ imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca2+ transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca2+-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca2+ chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca2+-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca2+ concentrations are feasible. [ABSTRACT FROM AUTHOR]