Protoporphyrin IX (PpIX) occurs naturally as part of the haem pathway. The photophysics of PpIX exhibits potential in photodynamic therapy, hence its study and the ability to rapidly image its localisation is important, especially in the field of fluorescence guided surgery. The use of the fluorescence lifetime imaging (FLIM) is advantageous, but for practical purposes imaging needs to be rapid and preferably in real time. Advances in CMOS based technology has enabled widefield sensor arrays with in-pixel timing to allow for the Fluorescence Lifetime Acquisition by Simultaneous Histogramming (FLASH). Here we use PpIX in a tissue mimic construct imaged using FLASH – FLIM on a commercial widefield TCSPC camera based on a sensor chip with 192 x 128 pixels. The potential use in visualising tumour boundaries in a model system using FLIM is shown.
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).
Fluorescence imaging techniques are powerful tools in the biological and biomedical sciences, because they are
minimally invasive and can be applied to live cells and tissues. The fluorescence emission can be characterized not only
by its intensity and position, by also by its fluorescence lifetime, polarization and wavelength. Fluorescence Lifetime
Imaging (FLIM) in particular has emerged as a key technique to image the environment and interaction of specific
proteins in living cells. Using a time-correlated single photon counting (TCSPC)-based FLIM set-up, we find that the
fluorescence lifetime of GFP-tagged proteins in cells is a function of the refractive index of the medium the cells are
suspended in. In addition, combining Fluorescence Recovery After Photobleaching (FRAP) of fluorescently labeled
proteins of different sizes in sol gels with time-resolved fluorescence anisotropy measurements, we demonstrate that we
can measure their lateral and rotational diffusion. This allows us to infer the size and connectivity of the pores in the sol
gel matrix. Moreover, wide-field photon counting imaging, originally developed for astronomical applications, is a
powerful imaging method because of its high sensitivity and excellent signal-to-noise ratio. It has a distinct advantage
over CCD-based imaging due to the ability to time the arrival of individual photons. The potential of time-resolved wide-field
photon counting imaging with a fast CMOS camera applied to luminescence microscopy is demonstrated.
Both micellar and lipid membrane systems have been used as models to provide further information regarding peptide properties in biological systems. Peptides are basic architectural units in nature and the study of their properties in membranes and other non-homogeneous media is of fundamental importance. We present the results for a time-resolved and steady state fluorescence study of two 4-methoxy-naphthalene labelled twelve amino acid residue model peptides that we have synthesised. One peptide, with a N-tert-Butoxycarbonyl (BOC) modified N-terminal, was incorporated into small unilamellar vesicles of L-(alpha) dipalmitoylphosphatidycholine (DPPC) and the other, with a free amino group, into inverse micelles of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) in 2,2,4-trimethylpentane. Steady state fluorescence and time-resolved fluorescence lifetime and anisotropy measurements show the first peptide to be situated in the lipid bilayer. In the inverse micelle system there is evidence for the peptide being situated at the surfactant waterpool interface.
KEYWORDS: Systems modeling, Luminescence, Proteins, Time resolved spectroscopy, Absorption, Data modeling, Dielectrics, Spectroscopy, Photosynthesis, Bioalcohols
At present there is a great deal of interest in the study of the transference of energy in biological systems. For example, electron transfer is of major importance in many synthetic and biological processes and in nature is mediated by proteins. Information regarding this process is therefore useful in leading to a greater understanding of phenomena such as photosynthesis and respiration. Previous work on protein systems has shown the electron transfer process to be complex to analyze because of the presence of competing pathways. This has led to the use of model systems to simplify the kinetics. We have synthesized novel model systems using peptides containing both a fluorescent methoxy- naphthalene donor and a dicyanoethylene group as a potential electron acceptor and observed fluorescence quenching for both dipeptide and oligopeptide systems. Biexponential fluorescence decay behavior was observed for all donor acceptor systems, with an increase in the amount of the shorter fluorescence decay component on increasing temperature.
The application of our development of 750 nm excitation from an argon-hydrogen filled spark source to time-resolved fluorescence probe studies of lipid membranes and inverse micelles is reported. The laser dye IR-140 was studied using the single-photon counting technique both in a lipid membrane of L-(alpha) dipalmitoylphosphatidylcholine (DPPC) and in sodium sulfosuccinic acid bis (2-ethylhexyl) ester (AOT) in iso-octane. In DPPC a dramatic change in the fluorescence behavior of IR-140 is observed between the gel and liquid crystalline phases. In inverse micelles of AOT an increase in the intensity of the peak fluorescence emission and a decrease in fluorescence lifetime is noticed on increasing the water content.
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