High Sensitivity NDD Flim and Pattern Matching Based Fluorophore Identification

Abstract

Confocal microscopes with multiphoton excitation and non-descanned detection (NDD) can penetrate deep into tissue. The combination with Fluorescence Lifetime Imaging (FLIM) extends the capability of such an instrument for concentration measurements of specific molecules, superior discrimination against autofluorescence as well as time resolved FRET (FLIM FRET).For optimal detection efficiency the fluorescence light is collected directly above the objective and guided to an Hybrid PMA detector combining GaAsP cathode sensitivity with excellent timing performance. The NDD based FLIM adapter replaces the standard objective holder and is easy to mount. A modular detection assembly allows to perform both confocal and NDD FLIM measurements.The fluorescence lifetime of fluorophores is strongly dependent on their photophysical properties and local environment. Together with the emission profile detected in different spectral channels the fluorescence decay can act as a fingerprint for a dye in a specific environment. We present a pattern-matching analysis technique that allows to identify selected patterns consisting of fluorescence decays in different spectral excitation and detection channels. The technique is easy to apply and allows for an excellent separation of different fluorophores, changes of fluorescence parameters due to differences in the environment as well as their discrimination from autofluorescence in biological samples.