The combination of time-resolved and spectral resolved techniques as achieved by SLIM (spectrally resolved
fluorescence lifetime imaging) improves the analysis of complex situations, when different fluorophores
have to be distinguished. This could be the case when endogenous fluorophores of living cells and tissues are
observed to identify the redox state and oxidative metabolic changes of the mitochondria. Other examples
are FRET (resonant energy transfer) measurements, when different donor/acceptor pairs are observed
simultaneously. SLIM is working in the time domain employing excitation with short light pulses and
detection of the fluorescence intensity decay in many cases with time-correlated single photon counting
(TCSPC). Spectral resolved detection is achieved by a polychromator in the detection path and a 16-channel
multianode photomultiplier tube with the appropriate routing electronics.
Within this paper special attention will be focused on FRET measurements with respect to protein
interactions in Alzheimers disease. Using global analysis as the phasor plot approach or integration of the
kinetic equations taking into account the multidimensional datasets in every spectral channel we could
demonstrate considerable improvement of our calculations.
In many fields of life science, visualization of spatial proximity, as an indicator of protein interactions in living cells, is of outstanding interest. A method to accomplish this is the measurement of Förster resonant energy transfer (FRET) by means of spectrally resolved fluorescence lifetime imaging microscopy. The fluorescence lifetime is calculated using a multiple-wavelength fitting routine. The donor profile is assumed first to have a monoexponential time-dependent behavior, and the acceptor decay profile is solved analytically. Later, the donor profile is assumed to have a two-exponential time-dependent behavior and the acceptor decay profile is derived analytically. We develop and apply a multispectral fluorescence lifetime imaging microscopy analysis system for FRET global analysis with time-resolved and spectrally resolved techniques, including information from donor and acceptor channels in contrast to using just a limited spectral data set from one detector only and a model accounting only for the donor signal. This analysis is used to demonstrate close vicinity of β-secretase (BACE) and GGA1, two proteins involved in Alzheimer's disease pathology. We attempt to verify if an improvement in calculating the donor lifetimes could be achieved when time-resolved and spectrally resolved techniques are simultaneously incorporated.
The fluorescence lifetime of different molecular species is calculated from the measured fluorescence intensity decrease
following short pulsed laser excitation, by a multi-channel fitting procedure. In a FRET (Förster Resonant Energy
Transfer) experiment the time dependent behaviour of the donor profile is assumed in a first view mono-exponential and
the acceptor decay profile is solved analytically. A global minimization fitting algorithm has increased information
content than a single channel fitting routine. In a normal FRET-FLIM experiment, the efficiency of FRET is calculated
only by considering the kinetics of the donor. However, as will be shown, a considerable improvement could be achieved
when time-resolved and spectral-resolved techniques are simultaneously incorporated.
We propose a cancer diagnostics method using 3D reconstruction of fluorescence based optical imaging data. The
reconstruction of luminescence sources in biological tissue is investigated using data obtained from Monte Carlo
simulations as well as simulated data using the diffusion approximation. The absolute determination of the tumor
locations is dependent on the information gathered by the recorded data. To tackle the forward problem we used the
solution of the diffusion equation for a cube. The inverse problem is solved.
We propose a cancer diagnostics method using 3D reconstruction of fluorescence based optical
imaging data. The system was tested with analytical simulations. Phantom measurements will be
acquired and compared with the simulations.
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