| 1. |
- Fridberger, Anders, 1966-, et al.
(author)
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Measuring hearing organ vibration patterns with confocal microscopy and optical flow
- 2004
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In: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 86:1, s. 535-543
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Journal article (peer-reviewed)abstract
- A new method for visualizing vibrating structures is described. The system provides a means to capture very fast repeating events by relatively minor modi. cations to a standard confocal microscope. An acousto-optic modulator was inserted in the beam path, generating brief pulses of laser light. Images were formed by summing consecutive frames until every pixel of the resulting image had been exposed to a laser pulse. Images were analyzed using a new method for optical flow computation; it was validated through introducing artificial displacements in confocal images. Displacements in the range of 0.8 to 4 pixels were measured with 5% error or better. The lower limit for reliable motion detection was 20% of the pixel size. These methods were used for investigating the motion pattern of the vibrating hearing organ. In contrast to standard theory, we show that the organ of Corti possesses several degrees of freedom during sound-evoked vibration. Outer hair cells showed motion indicative of deformation. After acoustic overstimulation, supporting cells contracted. This slowly developing structural change was visualized during simultaneous intense sound stimulation and its speed measured with the optical flow technique.
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| 2. |
- Margittai, M., et al.
(author)
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Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1
- 2003
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 100:26, s. 15516-15521
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Journal article (peer-reviewed)abstract
- Protein conformational transitions form the molecular basis of many cellular processes, such as signal transduction and membrane traffic. However, in many cases, little is known about their structural dynamics. Here we have used dynamic single-molecule fluorescence to study at high time resolution, conformational transitions of syntaxin 1, a soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein essential for exocytotic membrane fusion. Sets of syntaxin double mutants were randomly labeled with a mix of donor and acceptor dye and their fluorescence resonance energy transfer was measured. For each set, all fluorescence information was recorded simultaneously with high time resolution, providing detailed information on distances and dynamics that were used to create structural models. We found that free syntaxin switches between an inactive closed and an active open configuration with a relaxation time of 0.8 ms, explaining why regulatory proteins are needed to arrest the protein in one conformational state.
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| 3. |
- Pramanik, Aladdin, et al.
(author)
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Fluorescence Correlation Spectroscopy (FCS)
- 2004
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In: Encyclopedia of molecular cell biology and molecular medicine. - : Wiley-VCH Verlagsgesellschaft. ; , s. 461-500
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Book chapter (other academic/artistic)
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| 4. |
- van den Berg, P. A. W., et al.
(author)
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Fluorescence correlation spectroscopy of flavins and flavoenzymes : photochemical and photophysical aspects
- 2001
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In: Spectrochimica Acta Part A - Molecular and Biomolecular Spectroscopy. - 1386-1425 .- 1873-3557. ; 57:11, s. 2135-2144
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Journal article (peer-reviewed)abstract
- Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity ( < 10 kW/cm(2)), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations ( > 50 mM of I-) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state., thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the enzyme. As the time constant of the process induced at higher light intensities resembles the diffusion time constant of free flavin, photodestruction with the concomitant release of the cofactor offers a reasonable explanation.
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| 5. |
- Widengren, Jerker, et al.
(author)
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Characterization of photoinduced isomerization and back-isomerization of the cyanine dye Cy5 by fluorescence correlation spectroscopy
- 2000
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In: Journal of Physical Chemistry A. - : American Chemical Society (ACS). - 1089-5639 .- 1520-5215. ; 104:27, s. 6416-6428
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Journal article (peer-reviewed)abstract
- Cy5 is one of a few commercially available dyes in the near-infrared wavelength range. In this study, the fluorescence fluctuations of Cy5 have been investigated under steady-state excitation conditions by fluorescence correlation spectroscopy (FCS). The fluctuations in fluorescence are compatible with and can be used to characterize the photoinduced isomerization and back-isomerization, as well as the transitions between the singlet and triplet states of the dye. By employing a simple kinetic model, the rate constants of these processes can be determined. The model was used over a broad range of experimental conditions, where the influence on the isomerization properties of solvent viscosity, polarity, and temperature, excitation intensity and wavelength, and the presence of different side groups was investigated. We propose FCS as a useful and simple complementary approach to study isomerization processes of cyanine dyes yielding information about the rates of both the photoinduced isomerization and the back-isomerization, as well as of the kinetic properties of the triplet states. Our data show that for most excitation conditions relevant for ultrasensitive fluorescence spectroscopy a photostationary equilibrium is established between the isomeric forms, where approximately 50% of the Cy5 dye molecules can be expected to be in their weakly fluorescent cis states. The fluorophores therefore lose about half of their fluorescence capacity. This is of relevance for the performance of the dye in all applications of fluorescence spectroscopy where a high sensitivity or a fast readout is required, such as in single-molecule detection experiments and in many applications of confocal laser scanning microscopy.
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| 6. |
- Widengren, Jerker, et al.
(author)
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Manipulation and characterization of photo-induced transient states of Merocyanine 540 by fluorescence correlation spectroscopy
- 2000
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In: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 2:15, s. 3435-3441
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Journal article (peer-reviewed)abstract
- In this study, fluorescence correlation spectroscopy (FCS) is used to investigate the photo-induced transient states of Merocyanine 540 (MC540), a fluorescent agent for photodynamic therapy. Two relaxation processes are observed in the FCS measurements, which can be attributed to trans-cis isomerization and triplet state formation. Under the photostationary conditions present in the detection volume of the FCS measurements, the steady state populations of the photo-isomer and the triplet state, as well as their relaxation rates, can be determined. The population of the triplet states was noticeably reduced by light-induced deactivation at 515 nm excitation, and by simultaneous excitation at 647 nm the triplet state build-up could be almost eliminated with a concomitant increase in fluorescence intensity. By applying a simplified kinetic model for the measured fluorescence fluctuations it is possible to determine the rates for intersystem crossing, triplet state decay, as well as photo-induced isomerization and back-isomerization. In relation to other present techniques, FCS offers a relatively simple way to monitor photo-induced trans-cis isomerization, and cis-trans back-isomerization. For MC540, it is desirable to increase the triplet state formation at the expense of trans-cis isomerization in order to optimize the photodynamic action. FCS is well suited to monitor these processes on a microscopic scale, and thus to follow the local potency of MC540 as a photodynamic agent, at its site of action in target cells.
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| 7. |
- Widengren, Jerker, et al.
(author)
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Two new concepts to measure fluorescence resonance energy transfer via fluorescence correlation spectroscopy : Theory and experimental realizations
- 2001
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In: Journal of Physical Chemistry A. - : American Chemical Society (ACS). - 1089-5639 .- 1520-5215. ; 105:28, s. 6851-6866
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Journal article (peer-reviewed)abstract
- In this study, we demonstrate two new concepts, using fluorescence correlation spectroscopy (FCS), to characterize fluorescence resonance energy transfer (FRET). The two approaches were tested experimentally by measuring a series of double-stranded DNA molecules, with different numbers of base-pairs separating the donor (Alexa488) and acceptor (Cy5) fluorophores. In the first approach, FRET efficiencies are determined from the detected acceptor fluorescence rate per molecule. Here, the unique possibility with FCS to determine the mean number of molecules within the detection volume is exploited, making a concentration calibration superfluous. The second approach takes advantage of FRET-dependent fluorescence fluctuations of photophysical origin, in particular fluctuations generated by trans-cis isomerization of the acceptor dye. The rate of interchange,between the trans and cis states is proportional to the excitation rate and can be conveniently measured by FCS. Under FRET-mediated excitation, this rate can be used as a direct measure of the FRET efficiency. The measured isomerization rate depends only on the fluctuations in the acceptor fluorescence, and is not affected by donor, fluorescence cross-talk, background, dye labeling efficiencies, or by the concentration of molecules under study. The measured FRET efficiencies are well in agreement with a structural model of DNA. Furthermore, additional structural information is obtained from simulations of the measured fraction of acceptor dyes being in a nonfluorescent cis conformation, from which differences in the position and orientation of the trans and cis form of the acceptor dye can be predicted.
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