| 1. |
- Blom, Hans, et al.
(författare)
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Triplet-State Investigations of Fluorescent Dyes at Dielectric Interfaces Using Total Internal Reflection Fluorescence Correlation Spectroscopy
- 2009
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Ingår i: Journal of Physical Chemistry A. - Washington, DC : American Chemical Society. - 1089-5639 .- 1520-5215. ; 113:19, s. 5554-5566
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Tidskriftsartikel (refereegranskat)abstract
- The triplet-state kinetics of several fluorescent dyes used in ultrasensitive fluorescence microscopy are investigated using total internal reflection fluorescence correlation spectroscopy (TIR-FCS). A theoretical outline of the correlation analysis and the physical aspects of evanescent excitation and fluorescence emission at dielectric interfaces are given. From this analysis, the rates of intersystem crossing and triplet decay are deduced for fluorescein, ATTO 488, rhodamine 110, rhodamine 123, and rhodamine 6G in aqueous buffer solutions. All investigated dyes show slightly higher triplet rates at the dielectric interface compared to bulk solution measurements. We attribute this enhancement to possible modifications of the dyes’ photophysical properties near a dielectric interface. In the case of rhodamine 6G, the impact of changes in the dye concentration, ionic strength of the solvent, and potassium iodide concentration are also investigated. This leads to a better understanding of the influences of dye−dye, dye−solvent, and dye−surface interactions on the increased triplet intersystem crossing and triplet decay rates. The study shows that analysis of triplet-state kinetics by TIR-FCS not only results in a better understanding of how the photophysical properties of the dyes are affected by the presence of an interface, but also provides a means for probing the microenvironment near dielectric interfaces.
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| 2. |
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| 3. |
- Chmyrov, Andriy, 1979-, et al.
(författare)
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Characterization of new fluorescent labels for ultra-high resolution microscopy
- 2008
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Ingår i: Photochemical and Photobiological Sciences. - : Springer Science and Business Media LLC. - 1474-905X .- 1474-9092. ; 7:11, s. 1378-1385
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Tidskriftsartikel (refereegranskat)abstract
- Photo-induced switching of dyes into dark, long-lived states, such as a triplet state, has recently gained increasing interest, as a means to achieve ultra-high optical resolution. Additionally, these long lived states are often highly environment-sensitive and their photodynamics can thus offer additional independent fluorescence-based information. However, although providing a useful mechanism for photo-induced switching, the triplet state often appears as a precursor state for photobleaching, which potentially can limit its usefulness. In this work, a set of rhodamine and pyronin dyes, modified by substitution of heavy atoms and nitrogen within or close to the central xanthene unit of the dyes, were investigated with respect to their triplet state dynamics and photostabilities, under conditions relevant for ultra-high resolution microscopy. Out of the dyes investigated, in particular the rhodamine and pyronin dyes with a sulfur atom replacing the central oxygen atom in the xanthene unit were found to meet the requirements for ultra-high resolution microscopy, combining a prominent triplet state yield with reasonable photostability.
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| 4. |
- Chmyrov, Andriy, et al.
(författare)
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Characterization of new fluorescent labels for ultrahigh resolution microscopy
- 2009
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Ingår i: Novel Techniques in Microscopy (NTM) 2009. - : Optical Society of America. - 9781557528711
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Konferensbidrag (refereegranskat)abstract
- A set of modified dyes was investigated, of which several candidates combine prominent triplet state yield with reasonable photostability. They can be used to achieve ultrahigh optical resolution by photo-induced switching into dark (triplet) states.
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| 5. |
- Eggeling, C., et al.
(författare)
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Analysis of photobleaching in single-molecule multicolor excitation and forster resonance energy transfer measurement
- 2006
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Ingår i: Journal of Physical Chemistry A. - : American Chemical Society (ACS). - 1089-5639 .- 1520-5215. ; 110:9, s. 2979-2995
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Tidskriftsartikel (refereegranskat)abstract
- we investigated the influence of photobleaching in fluorescence experiments applying multicolor laser as well as Forster resonance energy transfer (FRET) mediated excitation using several red-emitting dyes frequently used in multicolor experiments or as FRET acceptors. The chosen dyes (cyanine 5 (Cy5), MR121, Alexa660, Alexa680, Atto647N, Atto655) have chemically distinct chromophore systems and can be excited at 650 nm. Several fluorescence analysis techniques have been applied to detect photobleaching and to disclose the underlying photophysics, all of which are based on single-molecule detection: (1) fluorescence correlation spectroscopy (FCS) of bulk solutions, (2) fluorescence cross-correlation of single-molecule trajectories, and (3) multiparameter fluorescence detection (MFD) of single-molecule events. The maximum achievable fluorescence signals as well as the survival times of the red dyes were markedly reduced under additional laser irradiation in the range of 500 nm. Particularly at excitation levels at or close to saturation, the 500 nm irradiation effectively induced transitions to higher excited electronic states on already excited dye molecules, leading to a pronounced bleaching reactivity. A theoretical model for the observed laser irradiance dependence of the fluorescence brightness of a Cy5 FRET acceptor dye has been developed introducing the full description of the underlying photophysics. The model takes into account acceptor as well as donor photobleaching from higher excited electronic states, population of triplet states, and energy transfer to both the ground and excited states of the acceptor dye. Also, photoinduced reverse intersystem crossing via higher excited triplet states is included, which was found to be very efficient for Cy5 attached to DNA. Comparing continuous wave (cw) and pulsed donor excitation, a strong enhancement of acceptor photobleaching by a factor of 5 was observed for the latter. Thus, in the case of fluorescence experiments utilizing multicolor pulsed laser excitation, the application of the appropriate timing of synchronized green and red laser pulses in an alternating excitation mode can circumvent excessive photobleaching. Moreover, important new single-molecule analysis diagnosis tools are presented: (1) For the case of excessive acceptor photobleaching, cross-correlation analysis of single-molecule trajectories of the fluorescence signal detected in the donor and acceptor detection channels and vice versa shows an anticorrelated exponential decay and growth, respectively. (2) The time difference, T-g - T-r of the mean observation times of all photons detected for the donor and acceptor detection channels within a single-molecule fluorescence burst allows one to identify and exclude molecules with an event of acceptor photobleaching. The presented single-molecule analysis methods can be constrained to, for example, FRET-active subpopulations, reducing bias from FRET-inactive molecules. The observations made are of strong relevance for and demand a careful choice of laser action in multicolor and FRET experiments, in particular when performed at or close to saturation.
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| 6. |
- Hassler, Kai, et al.
(författare)
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Dynamic disorder in horseradish peroxidase observed with total internal reflection fluorescence correlation spectroscopy
- 2007
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Ingår i: Optics Express. - 1094-4087. ; 15:9, s. 5366-5375
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Tidskriftsartikel (refereegranskat)abstract
- This paper discusses the application of objective-type total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to the study of the kinetics of immobilized horseradish peroxidase on a single molecule level. Objective-type TIR-FCS combines the advantages of FCS with TIRF microscopy in a way that allows for simultaneous ultra-sensitive spectroscopic measurements using a single-point detector and convenient localization of single molecules on a surface by means of parallel imaging.
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| 7. |
- Leutenegger, Marcel, et al.
(författare)
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Dual-color total internal reflection fluorescence cross-correlation spectroscopy
- 2006
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Ingår i: Journal of Biomedical Optics. - : SPIE-Intl Soc Optical Eng. - 1083-3668 .- 1560-2281. ; 11:4
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Tidskriftsartikel (refereegranskat)abstract
- We present the development and first application of a novel dual-color total internal reflection (TIR) fluorescence system for single-molecule coincidence analysis and fluorescence cross-correlation spectroscopy (FCCS). As a performance analysis, we measured a synthetic DNA-binding assay, demonstrating this dual-color TIR-FCCS approach to be a suitable method for measuring coincidence assays such as biochemical binding, fusion, or signal transduction at solid/liquid interfaces. Due to the very high numerical aperture of the epi-illumination configuration, our setup provides a very high fluorescence collection efficiency resulting in a two- to three- fold increase in molecular brightness compared to conventional confocal FCCS. Further improvements have been achieved through global analysis of the spectroscopic data.
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| 8. |
- Lin, Hongzhen, et al.
(författare)
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Fate of Excitations in Conjugated Polymers : Single-Molecule Spectroscopy Reveals Nonemissive "Dark" Regions in MEH-PPV Individual Chains
- 2009
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Ingår i: Nano letters (Print). - : American Chemical Society (ACS). - 1530-6984 .- 1530-6992. ; 9:12, s. 4456-4461
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Tidskriftsartikel (refereegranskat)abstract
- Single chains of the conjugated polymer MEH-PPV (poly(2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene)) were studied with wide-field fluorescence microscopy (dispersion in inert polymer matrices) and with fluorescence correlation spectroscopy (chloroform solution). The fluorescence yield of individual molecules in matrices was found to be 1-2 orders of magnitude lower than that in solution and it decreased substantially with increasing chain length. It suggests that isolation of MEH-PPV molecules in polymer matrices creates favorable conditions for photogeneration of nonemissive primary excited states.
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| 9. |
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| 10. |
- Persson, Gustav, et al.
(författare)
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Fluorescence cross-correlation spectroscopy of a pH-sensitive ratiometric dye for molecular proton exchange studies
- 2009
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Ingår i: Physical Chemistry, Chemical Physics - PCCP. - Cambridge, UK : RSC Publishing. - 1463-9076 .- 1463-9084. ; 11:21, s. 4410-4418
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Tidskriftsartikel (refereegranskat)abstract
- Fluorescence fluctuation analysis of individual pH-sensitive fluorophores has recently proven to be a useful approach for biomolecular proton exchange studies. In this work, dual-color fluorescence cross-correlation spectroscopy (FCCS) is demonstrated on a ratiometric pH-sensitive dye, for which both the excitation and emission spectra shift as a function of pH. In the FCCS measurements, the fluorescence signal from the predominant emission wavelength range of the protonated form of the dye is cross-correlated with that of the deprotonated form. Two lasers are used alternatingly to excite predominantly the protonated and the deprotonated form of the dye. The alternating excitation modulation scheme is combined with detection gating, and is based on a recently developed concept that allows extraction of correlation data for all correlation times regardless of the chosen modulation period. The scheme can thus be applied without concern for the time-scales of the molecular dynamic processes to be studied. By this combined discrimination based on both excitation and emission, spectral cross-talk is dramatically reduced and a very distinct and unambiguous anticorrelation can be recorded in the correlation curves as a consequence of the proton exchange. The strong discrimination power makes the approach applicable also to ratiometric dyes with less pronounced spectral shifts. It should also be useful in combination with ratiometric dyes sensitive to other ambient conditions and ions, such as the biologically very important calcium ion.
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