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SE-MPM is the gold standard for imaging multiple fluorophores with two-photon excitation.
From the above analysis, we conclude that multiple fluorophores with lifetime contrast cannot be efficiently separated with minimal crosstalk if a single gate of the TPSF is used.
First, once the excitation simulation is computed, it can be used to rapidly calculate the weight functions for multiple fluorophores with different lifetimes.
Combining multiple time gates in one inverse problem allows the simultaneous reconstruction of multiple fluorophores with increased resolution and minimal crosstalk using the proposed formulation.
By recording time dependent fluorescence signals, it is possible to distinguish and estimate the fractional contribution of multiple fluorophores with a monochromatic data set, allowing for fast acquisition protocols.
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Although several methods have been developed based on simultaneous fitting with multiple fluorophores to deal with relatively dense fluorescent data (DAOSTORM, 2011; Huang et al., 2011; Quan et al., 2011), the localization accuracy of fluorophores dramatically decreases as the density of emitters increases.
In summary our novel wide-field multi-parameter FLIM method, based on interleaved synchronized laser excitation of multiple fluorophores in combination with TCSPC by a space sensitive photomultiplier provides several advantages compared to current time-domain FLIM systems.
These probes consist of four linear oligonucleotides labeled with multiple fluorophores, bound together by the biotin-streptavidin linkage.
Future work will involve measuring the fluorescence dynamics with multiple fluorophores, expanding to cell sorting systems, and using the approach to alleviate intensity-related problems such as spectral overlap, autofluorescence noise, and quantification of FRET events.
To eliminate the possibility of 'cross-over' signal, sequential excitation and capture was performed with tissue containing multiple fluorophores analysed by laser confocal microscopy.
Clustering patterns of multiple fluorophores, changes in fluorophore locations over time and with genetic mutations, and recovery patterns of photobleached fluorophores can all be quantitatively analyzed using the model-convolution method as described above.
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