Phosphine Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy

Journal of the American Chemical Society (Impact Factor: 12.11). 01/2013; 135(4). DOI: 10.1021/ja3105279
Source: PubMed


We report that the cyanine dye Cy5 and several of its structural relatives are reversibly quenched by the phosphine TCEP (tris(2-carboxyethyl)phosphine). Using Cy5 as a model, we show that the quenching reaction occurs by 1,4-addition of the phosphine to the polymethine bridge of Cy5 to form a covalent adduct. Illumination with ultraviolet light dissociates the adduct and returns the dye to the fluorescent state. We demonstrate that TCEP quenching can be used for superresolution imaging as well as for other applications, such as differentiating between molecules inside and outside the cell.

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    • "was released and analyzed by HPLC with fluorescence detection, which facilitated separation from TCEP, which is reported to affect the fluorescence of various fluorophores (Vaughan et al. 2013). For accurate quantification of dansylcadaverine released from the fluorophore-modified Fe 3 O 4 @SiO 2 MNPs, the calibration standard, 3-MPA-Dansyl, was synthesized (Fig. S3 Supporting Information) and characterized by fluorescence spectroscopy (Fig. S4), mass spectrometry , and NMR (Supporting Information). "
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    ABSTRACT: The N-hydroxysuccinimide (NHS) ester moiety is one of the most widely used amine reactive groups for covalent conjugation of proteins/peptides to other functional targets. In this study, a cleave-analyze approach was developed to quantify NHS ester groups conjugated to silica-coated iron oxide magnetic nanoparticles (Fe3O4@SiO2 MNPs). The fluorophore dansylcadaverine was attached to Fe3O4@SiO2 magnetic nanoparticles (MNPs) via reaction with NHS ester groups, and then released from the MNPs by cleavage of the disulfide bond in the linker between the fluorophore and the MNPs moiety. The fluorophore released from Fe3O4@SiO2 MNPs was fluorometrically measured, and the amount of fluorophore should be equivalent to the quantity of the NHS ester groups on the surface of Fe3O4@SiO2 MNPs that participated in the fluorophore conjugation reaction. Another sensitive and semiquantitative fluorescence microscopic test was also developed to confirm the presence of NHS ester groups on the surface of Fe3O4@SiO2 MNPs. Surface-conjugated NHS ester group measurements were primarily performed on Fe3O4@SiO2 MNPs of 100–150 nm in diameter and also on 20-nm nanoparticles of the same type but prepared by a different method. The efficiency of labeling native proteins by NHS ester-coated Fe3O4@SiO2 MNPs was explored in terms of maximizing the number of MNPs conjugated per BSA molecule or maximizing the number of BSA molecules conjugated per each nanoparticle. Maintaining the amount of fresh NHS ester moieties in the labeling reaction system was essential especially when maximizing the number of MNPs conjugated per protein molecule. The methodology demonstrated in this study can serve as a guide in labeling the exposed portions of proteins by bulky multivalent labeling reagents.
    Journal of Nanoparticle Research 09/2015; 17(9). DOI:10.1007/s11051-015-3133-z · 2.18 Impact Factor
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    • "To date, the composition of STORM-buffers has scarcely evolved since the first demonstrations of single dye molecule controlled switching, with a combination of an enzymatic oxygen-scavenging system and a reducing agent (usually a thiol: Mercaptoethilamine –MEA [10], Mercaptoethanol – BME, or recently TCEP [11]) remaining the most widely used [10]–[16]. Here, we show that STORM-buffer optimization using the polyunsaturated hydrocarbon cyclooctatetraene (COT) can provide significantly increased photon yields and therefore localization precision for the dye Alexa-647. "
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    ABSTRACT: Super-resolution imaging methods have revolutionized fluorescence microscopy by revealing the nanoscale organization of labeled proteins. In particular, single-molecule methods such as Stochastic Optical Reconstruction Microscopy (STORM) provide resolutions down to a few tens of nanometers by exploiting the cycling of dyes between fluorescent and non-fluorescent states to obtain a sparse population of emitters and precisely localizing them individually. This cycling of dyes is commonly induced by adding different chemicals, which are combined to create a STORM buffer. Despite their importance, the composition of these buffers has scarcely evolved since they were first introduced, fundamentally limiting what can be resolved with STORM. By identifying a new chemical suitable for STORM and optimizing the buffer composition for Alexa-647, we significantly increased the number of photons emitted per cycle by each dye, providing a simple means to enhance the resolution of STORM independently of the optical setup used. Using this buffer to perform 3D-STORM on biological samples, we obtained images with better than 10 nanometer lateral and 30 nanometer axial resolution.
    PLoS ONE 07/2013; 8(7):e69004. DOI:10.1371/journal.pone.0069004 · 3.23 Impact Factor
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    ABSTRACT: Advances in far-field fluorescence microscopy over the past decade have led to the development of super-resolution imaging techniques that provide more than an order of magnitude improvement in spatial resolution compared to conventional light microscopy. One such approach, called Stochastic Optical Reconstruction Microscopy (STORM) uses the sequential, nanometer-scale localization of individual fluorophores to reconstruct a high-resolution image of a structure of interest. This is an attractive method for biological investigation at the nanoscale due to its relative simplicity, both conceptually and practically in the laboratory. Like most research tools, however, the devil is in the details. The aim of this chapter is to serve as a guide for applying STORM to the study of biological samples. This chapter will discuss considerations for choosing a photoswitchable fluorescent probe, preparing a sample, selecting hardware for data acquisition, and collecting and analyzing data for image reconstruction.
    Methods in cell biology 08/2013; 114:561-92. DOI:10.1016/B978-0-12-407761-4.00024-5 · 1.42 Impact Factor
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