Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes: Energy transfer to nonfluorescent dimers

Chen, Raymond F.; Knutson, Jay R.

doi:10.1016/0003-2697(88)90412-5

Cited by 380 publications

(253 citation statements)

References 44 publications

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“…Atto647N is already established in STED imaging and has previously been used for bleaching experiments (4,11,16,17). The bleaching dynamics of fluorescent dyes strongly depends on their molecular environment, including the dye density (18,19) and oxygen concentration (20,21). To achieve high reproducibility and control over the conditions in the sample, we prepared single-molecule samples of Atto647N-labeled double-stranded DNA (dsDNA) in PBS as described in refs.…”

Section: Resultsmentioning

confidence: 99%

Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent

Göttfert

Pleiner

Heine

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

100

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Photobleaching remains a limiting factor in superresolution fluorescence microscopy. This is particularly true for stimulated emission depletion (STED) and reversible saturable/switchable optical fluorescence transitions (RESOLFT) microscopy, where adjacent fluorescent molecules are distinguished by sequentially turning them off (or on) using a pattern of light formed as a doughnut or a standing wave. In sample regions where the pattern intensity reaches or exceeds a certain threshold, the molecules are essentially off (or on), whereas in areas where the intensity is lower, that is, around the intensity minima, the molecules remain in the initial state. Unfortunately, the creation of on/off state differences on subdiffraction scales requires the maxima of the intensity pattern to exceed the threshold intensity by a large factor that scales with the resolution. Hence, when recording an image by scanning the pattern across the sample, each molecule in the sample is repeatedly exposed to the maxima, which exacerbates bleaching. Here, we introduce MINFIELD, a strategy for fundamentally reducing bleaching in STED/RESOLFT nanoscopy through restricting the scanning to subdiffraction-sized regions. By safeguarding the molecules from the intensity of the maxima and exposing them only to the lower intensities (around the minima) needed for the off-switching (on-switching), MINFIELD largely avoids detrimental transitions to higher molecular states. A bleaching reduction by up to 100-fold is demonstrated. Recording nanobody-labeled nuclear pore complexes in Xenopus laevis cells showed that MINFIELD-STED microscopy resolved details separated by <25 nm where conventional scanning failed to acquire sufficient signal.fluorescence nanoscopy | STED microscopy | photobleaching | superresolution

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Section: Resultsmentioning

confidence: 99%

Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent

Göttfert

Pleiner

Heine

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

100

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“…From the absorption and emission spectra of intact Lip-Q (Figure 2), some level of residual fluorescence can be detected. This can result from non-quenched dye monomers within the liposomes and also from electrostatic interaction and influence of encapsulated dye by phospholipid polar head groups 33 .…”

Section: Discussionmentioning

confidence: 99%

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for <em>In Vivo</em> Optical Imaging

Tansi

Rüger

Rabenhold

et al. 2015

JoVE

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Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed LipdQ) and the free DY-676-COOH were compared with Lip-Q in animal trials. Video LinkThe video component of this article can be found at

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“…The fluorescent dye carboxyfluorescein was used to measure vesicle leakage. The fluorescence of the dye is self-quenched when it is encapsulated in vesicles (37). Loss of membrane integrity allows the dye to escape, relieving self-quenching and leading to enhanced fluorescence.…”

Section: Toxic and Nontoxic Variants Of Iapp Induce Model Membranementioning

confidence: 99%

Islet amyloid polypeptide toxicity and membrane interactions

Cao

Abedini

Wang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

134

143

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Islet amyloid polypeptide (IAPP) is responsible for amyloid formation in type 2 diabetes and contributes to the failure of islet cell transplants, however the mechanisms of IAPP-induced cytotoxicity are not known. Interactions with model anionic membranes are known to catalyze IAPP amyloid formation in vitro. Human IAPP damages anionic membranes, promoting vesicle leakage, but the features that control IAPP-membrane interactions and the connection with cellular toxicity are not clear. Kinetic studies with wildtype IAPP and IAPP mutants demonstrate that membrane leakage is induced by prefibrillar IAPP species and continues over the course of amyloid formation, correlating additional membrane disruption with fibril growth. Analyses of a set of designed mutants reveal that membrane leakage does not require the formation of β-sheet or α-helical structures. A His-18 to Arg substitution enhances leakage, whereas replacement of all of the aromatic residues via a triple leucine mutant has no effect. Biophysical measurements in conjunction with cytotoxicity studies show that nonamyloidogenic rat IAPP is as effective as human IAPP at disrupting standard anionic model membranes under conditions where rat IAPP does not induce cellular toxicity. Similar results are obtained with more complex model membranes, including ternary systems that contain cholesterol and are capable of forming lipid rafts. A designed point mutant, I26P-IAPP; a designed double mutant, G24P, I26P-IAPP; a double N-methylated variant; and pramlintide, a US Food and Drug Administration-approved IAPP variant all induce membrane leakage, but are not cytotoxic, showing that there is no one-to-one relationship between disruption of model membranes and induction of cellular toxicity.

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Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes: Energy transfer to nonfluorescent dimers

Cited by 380 publications

References 44 publications

Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent

Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for <em>In Vivo</em> Optical Imaging

Islet amyloid polypeptide toxicity and membrane interactions

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