Alexa Dyes, a Series of New Fluorescent Dyes that Yield Exceptionally Bright, Photostable Conjugates

Panchuk-Voloshina, Nataliya; Haugland, Rosaria P.; Bishop-Stewart, Janell; Bhalgat, Mahesh K.; Millard, Paul J.; Mao, Fei; Leung, Wai‐Yee; Haugland, Richard P.

doi:10.1177/002215549904700910

Cited by 778 publications

(613 citation statements)

References 18 publications

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“…Since autofluorescence is less pronounced toward the red spectral region, the preferred donor acceptor dye pair should be red-shifted as well, such as the AlexaFluor546 -AlexaFluor647 pair with 543 nm and 633 nm laser excitations, respectively. In addition to their spectral advantages, these dyes are more photostable than their commonly used spectral analogues (18). For flow cytometric FRET, this dye pair was found to be the optimal choice (19) and it can be extrapolated that this FRET pair is also favorable in image cytometry.…”

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confidence: 96%

Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

et al. 2009

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The intensity-based ratiometric FRET (fluorescence resonance energy transfer) method is a powerful technique for following molecular interactions in living cells. Since it is not based on irreversibly destroying the donor or the acceptor fluorophores, the time course of changes in FRET efficiency values can be monitored by this method. ImageJ, a sophisticated software tool for many types of image processing allows users to extend it with programs for various purposes. Implementing intensity-based ratiometric FRET with ImageJ vastly enhances the applicability of the FRET method. We developed an efficient ImageJ plugin, RiFRET, which calculates FRET efficiency on a pixel-by-pixel basis from ratiometric FRET images. It allows the user to correct for channel cross-talk (bleed-through) and to calculate FRET from image stacks, i.e., from 3D data sets. Semiautomatic processing for larger datasets is also included in the program. Furthermore, several options for calibrating FRET efficiency calculations were tested and their applicability to various expression systems is discussed. Although the ratiometric FRET method is widely applied, our plugin is the first freely available software for evaluating such FRET data. The program is user friendly and provides reliable, standardized results. ' 2009 International Society for Advancement of Cytometry Key terms fluorescence resonance energy transfer; ratiometric FRET; intensity-based FRET; FRET calibration; confocal laser scanning microscopy; ImageJ; 3D image processing IN biological systems, fluorescence resonance energy transfer (FRET) is a widely used method for studying molecular interactions and processes (1). FRET is a nonradiative transfer of energy from an excited fluorophore, the donor, to another fluorophore, the acceptor, that are at a distance of 1-10 nm (2). The efficiency of FRET (E) depends on the inverse sixth power of the distance between the donor and the acceptor, so monitoring E can serve as a spectroscopic ruler (3). Several spectroscopic parameters (e.g., intensity, lifetime, anisotropy) can be used to determine E (1,4-8). In flow cytometry, the intensity-based ratiometric FCET (flow cytometric FRET) method provides statistics for large cell populations. On the other hand, when subcellular details are needed, various microscopic FRET methods can be used (9-16). The ratiometric method (10), which was adapted from flow cytometry to microscopy (17), has the advantage of preserving the donor and acceptor dyes, which is necessary for following the time course of molecular interactions in living cells. Although the ratiometric approach is similar to the 3-cube FRET method (14), it uses less complicated calculations.The achievable signal to noise ratio is usually limited by the autofluorescence of the cells. Since autofluorescence is less pronounced toward the red spectral region, the preferred donor acceptor dye pair should be red-shifted as well, such as the AlexaFluor546 -AlexaFluor647 pair with 543 nm and 633 nm laser excitations, respectively. In a...

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confidence: 96%

Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

et al. 2009

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“…Of note, the fluorophore used in this work (AlexaFluor 680) is insensitive to pH over a wide pH window (Fig. S1) 40 . Here we designed programmable DNA-based triplex pHtriggered nanoswitches that form an intramolecular triplex structure through the formation of a Watson-Crick (dashed) pH-insensitive hairpin and a second Hoogsteen (dots) pH-sensitive hairpin.…”

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Programmable pH-Triggered DNA Nanoswitches

2014

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ABSTRACT:Here we have rationally designed a tunable DNA-based nanoswitch whose closing/opening can be triggered over specific different pH windows. This nanoswitch forms an intramolecular triplex DNA structure through pH-sensitive parallel Hoogsteen interactions. We demonstrate that by simply changing the relative content of TAT/CGC triplets in the switch we can rationally tune its pH-dependence over more than 5 pH units. By using a combination of such nanowitches with different pH sensitivity, we also demonstrate how we can engineer a pH nanosensor that can precisely monitor pH variations over 5.5 units of pH. With their fast response time (<200 msec) and high reversibility, these pH-triggered nanoswitches appear particularly suitable for applications ranging from the real-time monitoring of pH changes in-vivo to the development of pH sensitive smart nanomaterial.Nature often employs finely pH-regulated biomolecules to modulate and tune a number of biological activities 1 ranging from enzyme catalysis 2 to protein folding 3 , membrane function 4 and apoptosis 5 . For these reasons, developing probes, switches or nanomaterials that are able to respond to specific pH changes should prove of utility for several applications in the fields of in-vivo imaging, clinical diagnostics, and drug-delivery [6][7][8] .By taking advantage of the high versatility and designability of DNA chemistry 9-19 several groups have recently developed pH-triggered DNA-based probes or nanomachines [20][21][22][23][24][25][26][27][28][29][30] . Such probes typically exploit DNA secondary structures that display pH-dependence due to the presence of specific protonation sites. These structures include I-motif [21][22][23]26,29,31 , intermolecular triplex DNA 25,28,32 , DNA tweezers 20 and, more recently, the Amotif 33 . Despite the promising and advantageous characteristics of some of these DNA-based nanomachines, which include fast response times and sustained efficiency over several cycles, a drawback inevitably affects their performances: they all respond (with an exception 33a ) over a fixed pH window that typically spans 1.5 to 2 pH units 26,34,33b . These nanomachines, therefore, cannot be adapted to provide a useful output outside these fixed pH-windows.Here we describe a method to rationally design and program pH-triggered DNA-based nanoswitches whose pH-dependence can be finely tuned and modulated over more than 5 units of pH. We created our switches by taking advantage of the wellcharacterized pH sensitivity of the parallel Hoogsteen (T,C)-motif in triplex DNA [34][35][36] . To do so we have designed a DNAbased triplex pH-triggered nanoswitch that consists in a double intramolecular hairpin stabilized with both Watson-Crick (W-C) and parallel Hoogsteen interactions (Fig. 1). More specifically, one hairpin of the triplex nanoswitch is formed by the W-C hybridization of two 10-base complementary portions separated by a 5 base loop. This duplex DNA is then able to form a triplex structure via the formation of a second hairpin through...

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“…1,2 Considerable efforts have been devoted to suppressing photobleaching, which culminates primarily in chemical-based strategies that aim to make the local environment more chemically inert to fluorophores, 3,4 or modifying the structures and energy landscapes of fluorophores to be more resistant to photobleaching. 5,6 Progress has been achieved along these routes, including the discovery of oxygen scavenger systems, 3 photostable green fluorescent proteins, 5 and semiconductor quantum dots. 7 However, the increasing demand for more photons from a single molecule 1,8 requires new strategies beyond these chemical approaches.…”

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confidence: 99%

Giant Suppression of Photobleaching for Single Molecule Detection via the Purcell Effect

et al. 2013

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ABSTRACT:We report giant suppression of photobleaching and a prolonged lifespan of single fluorescent molecules via the Purcell effect in plasmonic nanostructures. The plasmonic structures enhance the spontaneous emission of excited fluorescent molecules, reduce the probability of activating photochemical reactions that destroy the molecules, and hence suppress the bleaching. Experimentally, we observe up to a 1000-fold increase in the total number of photons that we can harvest from a single fluorescent molecule before it bleaches. This approach demonstrates the potential of using the Purcell effect to manipulate photochemical reactions at the subwavelength scale. KEYWORDS: Nano-optics, single-molecule fluorescence spectroscopy, plasmonics P hotobleaching is a photochemical reaction of fluorescent molecules that irreversibly destroys their fluorescence capability. It determines the lifespan of a single fluorescent molecule and imposes a fundamental limit on the total number of photons that can be harvested from the molecule. 1,2 Considerable efforts have been devoted to suppressing photobleaching, which culminates primarily in chemical-based strategies that aim to make the local environment more chemically inert to fluorophores, 3,4 or modifying the structures and energy landscapes of fluorophores to be more resistant to photobleaching. 5,6 Progress has been achieved along these routes, including the discovery of oxygen scavenger systems, 3 photostable green fluorescent proteins, 5 and semiconductor quantum dots. 7 However, the increasing demand for more photons from a single molecule 1,8 requires new strategies beyond these chemical approaches.Recent progress in plasmonics manifests a "physical" approach to address the challenge. Plasmonics 9−11 offer a new way to mediate the interaction between a molecule and its local electromagnetic environment. 12,13 The surface plasmon of metallic nanostructures allows for deep subwavelength confinement of light 14,15 and enhancement of the local density of optical states. 16,17 These structures can substantially enhance the spontaneous emission of fluorophores near the metal surface, that is, the so-called Purcell enhancement. 18−20 In contrast to photonic crystals, 21−24 the plasmonic Purcell enhancement is broadband. The impacts of the plasmonic Purcell enhancement on photostability have been noticed previously. For instance, Hale et al. have observed reduction of photo-oxidation of semiconducting polymer when doped with silica core-gold nanoshells, 25 and Muthu et al. and Malicka et al. have observed decreasing photobleaching of fluoresce dyes on the surface of thin silver films 26 and silver nanoparticles. 27 Recently, the Purcell effect has been further expanded to tune the spectroscopic properties of dye molecules to selectively remove a long-lived triplet state, 28 enhance the fluorescence signal, 29 suppress quantum dot blinking, 30 and manipulate selection rules. 31 A novel core−shell type of plasmonic nanocomposite structures has also been developed as nanoc...

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Alexa Dyes, a Series of New Fluorescent Dyes that Yield Exceptionally Bright, Photostable Conjugates

Cited by 778 publications

References 18 publications

Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

Evaluation of intensity‐based ratiometric FRET in image cytometry—Approaches and a software solution

Programmable pH-Triggered DNA Nanoswitches

Giant Suppression of Photobleaching for Single Molecule Detection via the Purcell Effect

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