Fluorescence microscopy provides a non-invasive means for visualising dynamic protein interactions. As well as
allowing the calculation of kinetic processes via the use of time-resolved fluorescence, localisation of the protein within
cells or model systems can be monitored. These fluorescence lifetime images (FLIM) have become the preferred
technique for elucidating protein dynamics due to the fact that the fluorescence lifetime is an absolute measure, in the
main independent of fluorophore concentration and intensity fluctuations caused by factors such as photobleaching. In
this work we demonstrate the use of a time-resolved fluorescence microscopy, employing a high repetition rate laser
excitation source applied to study the influence of a metal surface on fluorescence tagged protein and to elucidate
viscosity using the fluorescence lifetime probe DASPMI. These were studied in a cellular environment (yeast) and in a
model system based on a sol-gel derived material, in which silver nanostructures were formed in situ using irradiation
from a semiconductor laser in CW mode incorporated on a compact time-resolved fluorescence microscope (HORIBA
Scientific DeltaDiode and DynaMyc).
We report the first read-out module for use with single- photon timing array detectors such as multi-anode MCP-PMTs. The IBH Model 5000MXR interfaces to the time-correlated single-photon counting (TCSPC) technique using a single time-to-amplitude converter. In addition to performing established multiplexing tasks, such as simultaneous acquisition of fluorescence and excitation and anisotropy, the new module enables spectral and spatial imaging of kinetic parameters such as fluorescence lifetimes and amplitudes. The system retains the inherent advantages of TCSPC with respect to picosecond time resolution and wide dynamic range, while featuring parallel data acquisition and enhanced data acquisition rates. Unlike early TTL implementations of multiplexing which were limited to four channels, our system uses an application specific integrated circuit (ASIC) which can read out the data from up to sixteen detection channels with higher reliability and less time-dispersion. The Model 5000MXR can be packaged as a NIM standard module, packaged to serve more channels or be close coupled to detector arrays for specific applications such as microscopy and lifetime based sensors. The theory, design and performance of ASIC data read-out will be described. Other applications include photon migration in tissue, time- of-flight reflectometry/mass spectrometry and nucleonics.
The fluorescence properties of tryptophan derivatives in dioctyl sodium sulfosuccinate (AOT)/iso-octane/buffer reverse micelles were studied using the intrinsic fluorescence of the indole group. The fluorescence decay is more complex for both N-acetyl- 1-tryptophan-amide (NATA) and Human Serum Albumin (HSA) in AOT reverse micelles than in aqueous solution. Time-resolved anisotropy and fluorescence quenching studies using carbon tetrachloride suggest one species of NATA lies on the internal micellar interface and another lies embedded in the interfacial region. The fluorescence decay of HSA in AOT reverse micelles has three unchanging exponential components over the (omega) 0 (the ratio of the concentration of water to AOT) range 9 to 51, indicating the environment of the tryptophan residue does not change in this range of waterpool size. Fluorescence quenching experiments of HSA in reverse micelles using acrylamide and carbon tetrachloride show that, like NATA, HSA also lies in the interfacial region. There is a minimum in the static component of quenching by CCL4 of HSA in reverse micelles at (omega) - 0)$AP21. This may be due to conformational stability around this waterpool size, and providing this is not an HSA-specific effect, may correlate with the enhancement of enzyme activity often observed in reverse micelles at a particular waterpool size.
A picosecond single-photon fluorescence lifetime instrument has been constructed for sensor development using liquid light guide coupling. The performance and application of the instrument are demonstrated with respect to examples of a proposed new type of energy transfer sensor for the detection of metal ions in solution. Novel instrumental features include the capability for performing multiplexed measurements using a distributed sensor network.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.