Acid selective pro-dye for cellular compartments (original) (raw)
Related papers
Selective Imaging of Late Endosomes with a pH-Sensitive Diazaoxatriangulene Fluorescent Probe
Journal of the American Chemical Society, 2016
Late endosomes are a major trafficking hub in the cell at the crossroads between endocytosis, autophagy, and degradation in lysosomes. Herein is disclosed the first small molecule allowing their selective imaging and monitoring in the form of a diazaoxatriangulene fluorophore, 1a (hexadecyl side chain). The compound is prepared in three steps from a simple carbenium precursor. In nanospheres, this pH-sensitive (pK a = 7.3), photochemically stable dye fluoresces in the red part of visible light (601 and 578 nm, acid and basic forms, respectively) with a quantum yield between 14 and 16% and an excited-state lifetime of 7.7−7.8 ns. Importantly, the protonated form 1a•H + provokes a specific staining of late endosome compartments (pH 5.0−5.5) after 5 h of incubation with HeLa cells. Not surprisingly, this late endosome marking depends on the intra-organelle pH, and changing the nature of the lipophilic chain provokes a loss of selectivity. Interestingly, fixation of the fluorophore is readily achieved with paraformaldehyde, giving the possibility to image both live and fixed cells.
The Histochemical Journal, 1991
Cultured rat fibroblasts were exposed to 50 fluorescent probes of varied physicochemical characteristics. Probe concentrations, fluorochrome excitation wavelength and period of illumination, and cell-probe contact time were varied. Structureactivity relationships defining a number of classes of fluorescent probes for lysosomes and related processes and compartments were demonstrated. Numerical specifications are now available for several familiar classes of probes: (a) acidotropic weak bases, used as markers for low pH compartments; (b) markers of adsorptive pinocytosis, involving non-specific protein binding; (c) markers for fluid phase pinocytosis; and (d) viability stains involving intralysosomal enzymic activity. Two novel classes of probes have also been specified numerically: (a) acid-precipitated weak acids, as markers for low pH compartments; and (b) lipid-binding markers of adsorptive pinocytosis. Overall, these structure-activity models provide a tool for predicting whether or not compounds enter cells; and whether they accumulate in lysosomes and related compartments. Pathways of entry are also predicted. This tool should permit design and selection of improved probes, and provide a better understanding of existing reagents. Moreover these models are expected to be applicable to interactions between any non-polymeric xenobiotic with lysosomes and related compartments.
Scientific reports, 2015
Intracellular pH plays an important role in the response to cancer invasion. We have designed and synthesized a series of new fluorescent probes (Superior LysoProbes) with the capacity to label acidic organelles and monitor lysosomal pH. Unlike commercially available fluorescent dyes, Superior LysoProbes are lysosome-specific and are highly stable. The use of Superior LysoProbes facilitates the direct visualization of the lysosomal response to lobaplatin elicited in human chloangiocarcinoma (CCA) RBE cells, using confocal laser scanning microscopy. Additionally, we have characterized the role of lysosomes in autophagy, the correlation between lysosome function and microtubule strength, and the alteration of lysosomal morphology during apoptosis. Our findings indicate that Superior LysoProbes offer numerous advantages over previous reagents to examine the intracellular activities of lysosomes.
Cell Biochemistry and Biophysics, 2013
Two novel precursors of fluorescent dyes (PFD813 and PFD814) have been studied for their ability to photo-activation, transfer across the biomembrane and cells staining. The fluorescent dyes Rho813 and Rho814 formed by photo-activation of their precursors PFD813 and PFD814 inside cells were used for the optical detection of particular features in vitro (HaCat cells, human epithelial carcinoma A431, epidermoid carcinoma of the cervix HeLa and chinese hamster ovary CHO cells). One of the possibilities to visualize and track the pathways of macromolecules or organelles in a "living" cell is to monitor them after staining with these PFDs during the real time measurements. A bright fluorescent signal from the photoactivated dye molecules inside the small spot in the cell can be monitored during their movement into the cell dark region (where the dye was not activated and did not fluoresce). The obtained data are important for further application of these precursors of the fluorescent dyes ("caged" dyes) for microscopic probing of biological objects.
Highly Stable and Sensitive Fluorescent Probes (LysoProbes) for Lysosomal Labeling and Tracking
Scientific reports, 2015
We report the design, synthesis and application of several new fluorescent probes (LysoProbes I-VI) that facilitate lysosomal pH monitoring and characterization of lysosome-dependent apoptosis. LysoProbes are superior to commercially available lysosome markers since the fluorescent signals are both stable and highly selective, and they will aid in characterization of lysosome morphology and trafficking. We predict that labeling of cancer cells and solid tumor tissues with LysoProbes will provide an important new tool for monitoring the role of lysosome trafficking in cancer invasion and metastasis.
Photochemistry and Photobiology, 1996
Neutral red is a lysosomal probe and a biological pH indicator. In aqueous solutions, the protonated (NRW) and neutral (NR) forms of monomeric neutral red exhibit distinct absorption maxima (535 and 450 nm, respectively) but have the same fluorescence with a maximum at 637 nm and a quantum yield of 0.02. The similarity of the fluorescence spectra at acidic and basic pH suggests deprotonation of cationic species in the first singlet excited state. The NR fluorescence strongly depends on the solvent polarity as shown by addition of increasing amounts of water to pure dioxane, which gradually shifts the fluorescence maximum from 540 nm in pure dioxane to 637 nm in water. The fluorescence quantum yield increases from 0.17 in dioxane to 0.3 upon addition of 7% water and then decreases, reaching 0.02 in pure water. Immediately after incubation of human skin fibroblasts with neutral red, excitation with 435 nm light produces a fluorescence whose maximum is recorded at 575 nm. This fluorescence is located in the perinuclear region and originates from large fluorescent intracytoplasmic spots, suggesting staining of the endoplasmic reticulum-Golgi complex. At longer times, this fluorescence is shifted to 606 nm, suggesting slow diffusion of the lysosomotropic dye toward the more hydrated and acidic interior of lysosomes. Addition of a lysosomotropic detergent to cells previously incubated with neutral red shifts the fluorescence to the blue. Thus, in complex biological systems, this probe cannot be a good pH indicator but is a very sensitive probe of lysosomal rnicroenvironrnents.
Enzyme-Induced Staining of Biomembranes with Voltage-Sensitive Fluorescent Dyes
The Journal of Physical Chemistry B, 2004
We consider the physicochemical basis for enzyme-induced staining of cell membranes by fluorescent voltagesensitive dyes, a method that may lead to selective labeling of genetically encoded nerve cells in brain for studies of neuronal signal processing. The approach relies on the induction of membrane binding by enzymatic conversion of a water-soluble precursor dye. We synthesized an amphiphilic hemicyanine dye with and without an additional phosphate appendix at its polar headgroup. The fluorescence of these dyes is negligible in water but high when bound to lipid membranes. By fluorescence titration with lipid vesicles it was shown that the phosphate group lowers the partition coefficient from water to membrane by more than an order of magnitude. By isothermal titration calorimetry, we showed that the dye phosphate was a substrate for a watersoluble alkaline phosphatase following Michaelis-Menten kinetics. In a suspension of lipid vesicles, the enzyme reaction led to a fluorescence increase due to enhanced membrane binding of the product dye in accord with the Michaelis-Menten kinetics of the reaction and the partition coefficients of substrate and product. We successfully tested the staining method by fluorescence microscopy with individual giant lipid vesicles and with individual red blood cells. In both systems, the membrane fluorescence due to bound hemicyanine was enhanced by an order of magnitude, proving the feasibility of enzyme-induced staining with voltage-sensitive dyes.
Fluorescence Behavior of the pH-Sensitive Probe Carboxy SNARF-1 in Suspension of Liposomes¶
When exposed to the intracellular environment fluorescent probes sensitive to pH exhibit changes of photophysical characteristics as a result of an interaction of the dye molecule with cell constituents such as proteins, lipids or nucleic acids. This effect is reflected in calibration curves different from those found with the same dye in pure buffer solutions. To study an interaction of the probe 5(and 6)-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7,1(3H)-isobenzofuran]-3one (carboxy SNARF-1) with membrane lipids, we measured its fluorescence in model systems of large unilamellar vesicles (LUV) prepared by extrusion. When the dye was removed from the bulk solution by gel filtration the relative fluorescence intensity of the lipid-bound dye form was enhanced, showing a strong interaction of the dye molecule with LUV membrane lipids. Surprisingly, the dye molecules seem to be bound predominantly to the outer surface of the lipid bilayer. The same situation was found with small unilamellar vesicles prepared by sonication. This effect makes it difficult to use carboxy SNARF-1 for measurements of the internal pH in suspensions of liposomes.
Journal of Biomedical Optics, 2005
Liposomes are known to be taken up by the liver cells after intravenous injection. Among the few techniques available to follow this process in vivo are perturbed angular correlation spectroscopy, nuclear magnetic resonance spectroscopy, and scintigraphy. The study of the intracellular pathways and liposomal localization in the different liver cells requires sacrifice of the animals, cells separation, and electronic microscopy. In the acidic intracellular compartments, the in situ rate of release of liposomes remains poorly understood. We present a new method to follow the in situ and in vivo uptake of liposomes using a fluorescent pH-sensitive probe 5,6carboxyfluorescein (5,6-CF). 5,6-CF is encapsulated in liposomes at high concentration (100 mM) to quench its fluorescence. After laparotomy, liposomes are injected into the penile vein of Wistar rats. Fluorescence images of the liver and the skin are recorded during 90 min and the fluorescence intensity ratio is calculated. Ratio kinetics show different profiles depending on the liposomal formulation. The calculated intracellular liver pH values are, respectively, 4.5 to 5.0 and 6.0 to 6.5 for DSPC/chol and DMPC liposomes. After sacrifice and flush with a cold saline solution, the pH of the intracellular site of the liver (ex vivo) is found to be 4.5 to 5.0. This value can be explained by an uptake of liposomes by the liver cells and subsequent localization into the acidic compartment. An intracellular event such as dye release of a drug carrier (liposomes loaded with a fluorescent dye) can be monitored by pH fluorescence imaging and spectroscopy in vivo and in situ.
Cell Staining by Novel Derivatives of Fluorescent Rhodamine Dyes
Two novel precursors of fluorescent dyes (PFD813 and PFD814) have been studied for their ability to photo-activation, transfer across the biomembrane and cells staining. The fluorescent dyes Rho813 and Rho814 formed by photo-activation of their precursors PFD813 and PFD814 inside cells were used for the optical detection of particular features in vitro (HaCat cells, human epithelial carcinoma A431, epidermoid carcinoma of the cervix HeLa and chinese hamster ovary CHO cells). One of the possibilities to visualize and track the pathways of macromolecules or organelles in a " living " cell is to monitor them after staining with these PFDs during the real time measurements. A bright fluorescent signal from the photoactivated dye molecules inside the small spot in the cell can be monitored during their movement into the cell dark region (where the dye was not activated and did not fluoresce). The obtained data are important for further application of these precursors of the fluorescent dyes (" caged " dyes) for microscopic probing of biological objects.