Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis

Abstract

Förster resonance energy transfer (FRET) detected via fluorescence lifetime imaging microscopy (FLIM) and global analysis provide a way in which protein-protein interactions may be spatially localized and quantified within biological cells. The FRET efficiency and proportion of interacting molecules have been determined using bi-exponential fitting to time-domain FLIM data from a multiphoton time-correlated single-photon counting microscope system. The analysis has been made more robust to noise and significantly faster using global fitting, allowing higher spatial resolutions and/or lower acquisition times. Data have been simulated, as well as acquired from cell experiments, and the accuracy of a modified Levenberg-Marquardt fitting technique has been explored. Multi-image global analysis has been used to follow the epidermal growth factor-induced activation of Cdc42 in a short-image-interval time-lapse FLIM/FRET experiment. Our implementation offers practical analysis and time-resolved-image manipulation, which have been targeted towards providing fast execution, robustness to low photon counts, quantitative results and amenability to automation and batch processing.

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

“…In the beginning of 2009, the proceedings of the first international Theodor Förster lecture series, held in Cambridge UK, were published offering a wealth of information on state-of-the-art quantitative optical microscopy (Kaminski 2009). Recent reviews on fluorescence lifetime imaging and on FRET-FLIM with many back references have also appeared in current literature (Festy et al 2007;Barber et al 2009). …”

Coordinated RhoA signaling at the leading edge and uropod is required for T cell transendothelial migration

“…An inverted microscope (TE2000E; Nikon) combined with an in-house scanner (Peter et al, 2005) and either a solid-state-pumped (Verdi; Coherent) or self mode-locked Ti:Sapphire laser (Mira or Chameleon; Coherent) for multiphoton excitation was used. All images were acquired at a suitable spatial and time resolution to provide enough photon arrival times for accurate fluorescence decay fitting, while avoiding detector pile up, and analyzed by performing a single-exponential pixel fit in time-resolved image analysis software (TRI2; Peter et al, 2005;Barber et al, 2009;Makrogianneli et al, 2009;Carlin et al, 2010).…”

“…GFP alone lifetimes were measured at 2.25 (fixed) and 2.35 ns (live). Images were analyzed using TRI2 (Peter et al, 2005;Barber et al, 2009;Makrogianneli et al, 2009;Carlin et al, 2010) and ImageJ.…”

Upgrading time domain FLIM using an adaptive Monte Carlo data inflation algorithm

“…The benefits of the AMDI algorithm are not limited to this fitting method and may easily be extended to all the minimization algorithms classically used in time domain FLIM image analysis (such as the Newton trust region regression method, which is more robust to dispersive elements). Moreover, it should be noted that since the AMDI algorithm is an inflation of temporal fluorescence intensity decays, it is also compatible with all existing time domain FLIM image analysis strategies (28,(31)(32)(33)(34)(35).…”