Live-cell fluorescence nanoscopy is a powerful tool to study cellular biology on a molecular scale, yet its use is held back by the paucity of suitable fluorescent probes. Fluorescent probes based on ...regular fluorophores usually suffer from a low cell permeability and an unspecific background signal. Here we report a general strategy to transform regular fluorophores into fluorogenic probes with an excellent cell permeability and a low unspecific background signal. Conversion of a carboxyl group found in rhodamines and related fluorophores into an electron-deficient amide does not affect the spectroscopic properties of the fluorophore, but allows us to rationally tune the dynamic equilibrium between two different forms: a fluorescent zwitterion and a non-fluorescent, cell-permeable spirolactam. Furthermore, the equilibrium generally shifts towards the fluorescent form when the probe binds to its cellular targets. The resulting increase in fluorescence can be up to 1,000-fold. Using this simple design principle, we created fluorogenic probes in various colours for different cellular targets for wash-free, multicolour, live-cell nanoscopy.
Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with ...either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.
The quality and application of super-resolution fluorescence imaging greatly lie in the dyes' properties, including photostability, brightness, and Stokes shift. Here we report a synergistic strategy ...to simultaneously improve such properties of regular fluorophores. Introduction of quinoxaline motif with fine-tuned electron density to conventional rhodamines generates new dyes with vibration structure and inhibited twisted-intramolecular-charge-transfer (TICT) formation synchronously, thus increasing the brightness and photostability while enlarging Stokes shift. The new fluorophore YL578 exhibits around twofold greater brightness and Stokes shift than its parental fluorophore, Rhodamine B. Importantly, in Stimulated Emission Depletion (STED) microscopy, YL578 derived probe possesses a superior photostability and thus renders threefold more frames than carbopyronine based probes (CPY-Halo and 580CP-Halo), known as photostable fluorophores for STED imaging. Furthermore, the strategy is well generalized to offer a new class of bright and photostable fluorescent probes with long Stokes shift (up to 136 nm) for bioimaging and biosensing.
Monitoring metabolites at the point of care could improve the diagnosis and management of numerous diseases. Yet for most metabolites, such assays are not available. We introduce semisynthetic, ...light-emitting sensor proteins for use in paper-based metabolic assays. The metabolite is oxidized by nicotinamide adenine dinucleotide phosphate, and the sensor changes color in the presence of the reduced cofactor, enabling metabolite quantification with the use of a digital camera. The approach makes any metabolite that can be oxidized by the cofactor a candidate for quantitative point-of-care assays, as shown for phenylalanine, glucose, and glutamate. Phenylalanine blood levels of phenylketonuria patients were analyzed at the point of care within minutes with only 0.5 microliters of blood. Results were within 15% of those obtained with standard testing methods.
We introduce a new class of semisynthetic fluorescent biosensors for the quantification of free nicotinamide adenine dinucleotide (NAD
) and ratios of reduced to oxidized nicotinamide adenine ...dinucleotide phosphate (NADPH/NADP
) in live cells. Sensing is based on controlling the spatial proximity of two synthetic fluorophores by binding of NAD(P) to the protein component of the sensor. The sensors possess a large dynamic range, can be excited at long wavelengths, are pH-insensitive, have tunable response range and can be localized in different organelles. Ratios of free NADPH/NADP
are found to be higher in mitochondria compared to those found in the nucleus and the cytosol. By recording free NADPH/NADP
ratios in response to changes in environmental conditions, we observe how cells can react to such changes by adapting metabolic fluxes. Finally, we demonstrate how a comparison of the effect of drugs on cellular NAD(P) levels can be used to probe mechanisms of action.
We introduce color‐shifting fluorophores that reversibly switch between a green and red fluorescent form through intramolecular spirocyclization. The equilibrium of the spirocyclization is ...environmentally sensitive and can be directly measured by determining the ratio of red to green fluorescence, thereby enabling the generation of ratiometric fluorescent probes and biosensors. Specifically, we developed a ratiometric biosensor for imaging calcium ions (Ca2+) in living cells, ratiometric probes for different proteins, and a bioassay for the quantification of nicotinamide adenine dinucleotide phosphate.
Environmentally sensitive color‐shifting fluorophores that reversibly switch between a green and red fluorescent form were developed. The equilibrium of the spirocyclization is environmentally sensitive, thereby enabling the generation of ratiometric fluorescent probes and biosensors, including probes for imaging calcium ions (Ca2+) in living cells and a bioassay for the quantification of NADPH.
The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection ...or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.
We introduce here a new class of BODIPY-based Ca2+ indicators which can be derivatized with biological ligands that permit the localization of the indicators in living cells. The underivatized ...BODIPY-based Ca2+ indicator (BOCA-1) shows a 250-fold increase in fluorescence intensity upon Ca2+ binding. We also prepared its O 6-benzylguanine (BG) derivative, BOCA-1-BG, which can be covalently and specifically linked to SNAP-tag fusion proteins in living cells. The indicator retains its properties as a highly sensitive Ca2+ indicator after conjugation to proteins, displaying a 180-fold increase in fluorescence intensity upon Ca2+ binding. We further demonstrated that BOCA-1-BG through reaction with localized SNAP-tag fusion proteins can be used to sense changes in Ca2+ concentrations in the nuclei and in the cytosol of live CHO-K1 cells. The high sensitivity of the indicator together with the possibility to selectively couple it to proteins of interest makes it a powerful tool for measuring local changes in Ca2+ concentrations in living cells.
Acetylcholine is a key neurotransmitter, and anticholinesterase agents are essential compounds used as medical drugs, pesticides, and chemical warfare agents. A semisynthetic fluorescence‐based probe ...for the direct, real‐time detection of acetylcholine and anticholinesterase compounds is introduced. The probe possesses good sensitivity, tunable detection range, and can be selectively targeted to cell surfaces, thereby making it an attractive tool for applications in analytical chemistry and quantitative biology.
A semisynthetic fluorescence‐based probe (ACh‐SNIFIT) for the direct, real‐time detection of acetylcholine and anticholinesterase compounds such as drugs and nerve agents is introduced. The probe possesses good sensitivity, tunable detection range, and can be selectively targeted to cell surfaces, thereby making it an attractive tool for applications in analytical chemistry and quantitative biology.
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