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.
"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 , Mercaptoethanol – BME, or recently TCEP ) remaining the most widely used –. 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. "
[Show abstract][Hide abstract] 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
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: A D-π-A type phenothiazine derivative 4-((E)-2-(10-ethyl-10H-phenothiazin-3-yl)-vinyl)-benzaldehyde (abbreviated as L) was designed, synthesized and characterized by single crystal X-ray diffraction analysis. Dispersion-corrected theoretical calculation revealed that L molecules tended to grow along 1-diamentional (1-D) orientation. Further, L was used to prepare nanohybrid consisting of silver nanoparticles (NPs) dispersed on L nanoribbons. L molecules coupled with Ag NPs through S atoms, which brought about significant blue-shift of the fluorescence (FL) and an obvious increase of FL lifetime. Through fluorescence microscopy, the spectral change was used as a wavelength-based biodetection tool to study the possible mechanism for enhanced inhibitory effect of L/Ag nanohybrid on both gram-negative bacterium Escherichia coli and gram-positive bacterium Staphylococcus aureus compared to either pure Ag NPs or pure L.
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