A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy

Conibear, Paul B.; Bagshaw, Clive R.

doi:10.1046/j.1365-2818.2000.00774.x

Cited by 25 publications

(31 citation statements)

References 25 publications

(54 reference statements)

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“…Autocorrelation analysis of the fluctuations was performed in an Excel spreadsheet using the Fast Fourier Transform routine (21). Photoactivation was induced with a Xe XF-10 flash lamp, with a 280 to 380 nm band-pass interference filter as described previously (18). Figure 1a shows the pH dependence of the YFP absorption spectra in a buffer comprising 40 mM NaCl, 2 mM MgCl 2 and 20 mM MES/ TES buffer at 20°C.…”

Section: Methodsmentioning

confidence: 99%

Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

et al. 2005

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Yellow fluorescent protein (YFP 10C) is widely used as a probe in biology, but its complex photochemistry gives rise to unusual behavior that requires fuller definition. Here we characterize the kinetics of protonation and reversible bleaching over time scales of picoseconds to hours. Stopped-flow and pressure-jump techniques showed that protonation of the fluorescent YFP -anion state is two-step with a slow transition that accounts for blinking of 527 nm emission at the single molecule level on the seconds time scale. Femtosecond spectroscopy revealed that the protonated excited-state (YFPH*) decayed predominantly by a radiationless mechanism, but emission at 460 nm was detected within the first picosecond. Limited excited-state proton transfer leads to 527 nm emission characteristic of the YFP -* anion. Prolonged continuous wave illumination at the peak of YFP -absorbance (514 nm) yields, irreversibly, a weakly fluorescent product that absorbs at 390 nm. This "photobleaching" process also gives a different species (YFPHrb) that absorbs at 350/430 nm and spontaneously regenerates YFP -in the dark on the time scale of hours but can be photoactivated by UV light to regenerate YFP -within seconds, via a ground-state protonated intermediate. Using a pulsed laser for photobleaching resulted in decarboxylation of YFP as indicated by the mass spectrum. These observations are accounted for in a unifying kinetic scheme.Green fluorescent protein (GFP) 1 and its color variants have found wide use in biology to probe protein dynamics in vitro and within cells (1). They have also attracted the attention of spectroscopists owing to their complex photochemistry (2-5). In particular, those in the YFP class (i.e. containing a T203Y or T203F mutation to shift the emission to 527 nm) have been shown to undergo fluctuations of emission intensity over a wide range of times scales and reversible photobleaching (6-8). Fluctuations in emission were observed by wide field microscopy at the single molecule level by immobilizing individual YFP molecules in acrylamide or agarose gels and using TIRF excitation (6, 9). Molecules were found to blink on the seconds time scale at low laser powers (<500 W cm -2 ). Increasing laser power reduced the lifetime of the emitting state, but not in direct proportion to the laser power. Initial studies discussed the possibility that the fluctuations were related to the protonation of the phenolate moiety of the fluorophore derived from Y66 (6), but later studies found little pH dependence of the onand off-times when monitored at a laser power of 5000 W cm -2 (9).Members of the YFP class also showed a photochromic switching behavior. Molecules that were bleached by 488 nm irradiation, could be induced to fluoresce by illumination with near UV or blue light (6-8). This reversible photobleaching reaction of YFP was observed in FRET measurements (10). Illumination of YFP at 514 nm resulted in bleaching of the YFP acceptor and dequenching of a donor CFP molecule. Once the YFP was bleached, illumina...

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Section: Methodsmentioning

confidence: 99%

Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

et al. 2005

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“…In prism-based TIRF, the evanescent wave is typically generated at the surface of a microscope slide, or possibly at the surface of a cover slip or directly on the prism [5,16]. The function of the prism is to efficiently couple the laser beam (Fig.…”

Section: Prism Tirfmentioning

confidence: 99%

“…Various prism arrangements have been used [5,16], although the most common arrangement is a triangular or cubic prism on the optical axis, as shown in Fig. 6.…”

Section: Prism Objectivementioning

confidence: 99%

Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)

Knight

2014

Pure and Applied Chemistry

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Total internal reflection fluorescence (TIRF) is a popular illumination technique in microscopy, with many applications in cell and molecular biology and biophysics. The chief advantage of the technique is the high contrast that can be achieved by restricting fluorescent excitation to a thin layer. We summarise the optical theory needed to understand the technique and various aspects required for a practical implementation of it, including the merits of different TIRF geometries. Finally, we discuss a variety of applications including super-resolution microscopy and high-throughput DNA sequencing technologies.

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“…Preparation of the sample for single molecule detection To detect the fluorescence signal from the sample we used a flow chamber built between a glass cover slip (0.1 mm) and quartz slide (1 mm) separated by a spacer (0.1 mm) as described (Conibear and Bagshaw 2000). The quartz surface was modified by PEG/PEG-biotin treatment as described (Ha et al 2002).…”

Section: Preparation Of Dna/rna Duplex Oligonucleotidesmentioning

confidence: 99%

“…Single molecule experiments were performed using a custom-built prism-based total internal fluorescence microscope (Conibear and Bagshaw 2000), using a 532 nm laser (Suwtech 50 mW DPSS, sp3plus, Tunbridge Wells, Kent UK) for excitation with an incident power (at the prism) of *100 W/cm 2 (corresponding to *3 9 10 16 photons/s or *2.5 9 10 4 photons/Å 2 s, illumination area is *10 4 lm 2 ) throughout the experiments. Fluorescence emission was collected by a 639 1.2 NA Zeiss C-apochromat water immersion lens, split by a dichroic mirror (645 DRLP, Omega) and projected onto two halves of the detector chip (iXon DV887 emCCD camera, Andor Technology, UK) through emission filters specific for Cy3 and Cy5 chromophores (580 DF30 and 670 DF40, Omega, respectively) using a home-built beam splitter.…”

Section: Detection Of Fluorescence Signalsmentioning

confidence: 99%

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

2008

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Single molecule fluorescent microscopy is a method for the analysis of the dynamics of biological macromolecules by detecting the fluorescence signal produced by fluorophores associated with the macromolecule. Two fluorophores located in a close proximity may result in Förster resonance energy transfer (FRET), which can be detected at the single molecule level and the efficiency of energy transfer calculated. In most cases, the experimentally observed distribution of FRET efficiency exhibits a significant width corresponding to 0.07-0.2 (on a scale of 0-1).Here, we present a general approach describing the analysis of experimental data for a DNA/RNA duplex. We have found that for a 15 bp duplex with Cy3 and Cy5 fluorophores attached to the opposite ends of the helix, the width of the energy transfer distribution is mainly determined by the photon shot noise and the orientation factor, whereas the variation of inter-dye distances plays a minor role.

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A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy

Cited by 25 publications

References 25 publications

Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

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