The current advances in fluorescence microscopy coupled with the development of new fluorescent
probes such as visible fluorescent proteins (VFP) allow Förster (fluorescence) resonance energy transfer
(FRET) to be used to study protein-protein interactions in living specimens. For FRET to occur, the donor
emission spectrum should overlap the acceptor absorption at least about 30%. This spectral overlap generates
donor and acceptor spectral bleedthrough (DSBT and ASBT) in the FRET channel (or acceptor channel) while
exciting the double labeled cell with donor excitation wavelength. The spectral imaging microscopy helps to
avoid the DSBT into FRET channel by linear unmixing. But the ASBT is the same spectral region of the FRET
signal which cannot be removed by linear unmixing. To obtain a quantitative estimation of the C/EBPα protein
dimerization in GHFT1-5 living cell nucleus, we need to remove the ASBT from the FRET channel. So, we
developed an algorithm that removes the ASBT signal from the spectral FRET (sFRET) image. The E%
estimated using the processed spectral FRET (psFRET) algorithm provides excellent comparison with other
techniques such as confocal and lifetime FRET microscopy. This psFRET algorithm was also characterized
with FRET standards, based on constructs with known-length amino acid linkers.
The current advances in fluorescence microscopy coupled with the development of new fluorescent probes and detectors provide the tools to study protein associations in living specimens using FRET microscopy. Upon energy transfer, donor fluorescence is quenched and acceptor fluorescence is increased (sensitized), resulting in a decrease in donor excitation intensity or lifetime. The fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal acquired using one- and two-photon excitation FRET microscopy. There are a number of methods to avoid, minimize or correct the spectral bleedthrough (SBT) contamination in intensity-based FRET, each having specific limitations depending on the level of sensitivity desired. We have developed an algorithm to correct the contamination in the FRET image to estimate the energy transfer efficiency (E%) and the distance (r) between donor and acceptor molecule. In this presentation we explain the influence of back-bleedthrough signal in quenched donor channel, acceptor excitation wavelength exciting donor component of the double labeled specimen (we call them additional SBT in this paper) and its influence in the calculation of energy transfer efficiency and the distance between donor and acceptor molecules. Considerable amount of additional SBT signals were observed in the intensity based multiphoton FRET microscopy compared to the one-photon FRET microscopy.
Remarkable advances have been made in studying the dynamic events of protein molecules in living cells and tissues using advanced light microscopy imaging techniques and green fluorescent proteins (GFPs). Identification of the interacting protein partners is critical in understanding its function and place in the biochemical pathway, thereby establishing its role in important disease processes. FLIM-FRET microscopy technique, allow the study of proteins in multiple ways including what proteins are expressed, where they are expressed- and where they move over time. It has been observed that the eCFP-eYFP FRET pair may not be that suitable to localize the association of protein molecules since the eCFP has two-components lifetime. The new Cerulean green fluorescent protein appears to have only one-component lifetime. We describe the extensive investigation of eCFP and Cerulean to study the dimerization of the transcription factor CCAAT/enhancer binding protein alpha in GHFT1-5 living cell nucleus
using the time-correlated single photon counting (TCSPC) FLIM-FRET microscopy.
Intensity based FRET visualizes protein molecules in living cells and tissues. Lifetime techniques, however, will demonstrate the dynamic functional activity of the protein molecules, because its signal does not depend on changes in fluorophore concentration or excitation intensity. If a laser pulse excites a large number of similar molecules with a similar local environment, and as long as no energy is transferred to another molecule, the lifetime is the “natural fluorescence lifetime”. If energy is transferred, however, the actual fluorescence lifetime is less than the natural lifetime, because an additional path for de-excitation is present. With the occurrence of FRET, strong energy transfer results in extreme quenching of the donor fluorescence and a decrease in the fluorescence lifetime. In this paper we will explain the development of the two-photon FLIM-FRET microscopy with our existing multiphoton microscopy and demonstrate the change in donor lifetime by photobleaching the acceptor molecules in living cells.
Wide-field fluorescence microscopy was used to monitor the co-localization of the homeodomain (HD) transcription factor Pit-1 and the basic-leucine zipper protein CCAAT/enhancer binding protein alpha (C/EBPa), each labeled with fluorescent proteins (FP) in the living cell nucleus. Fluorescence resonance energy transfer (FRET) microscopy was used to resolve the angstrom-scale spatial relationships of these expressed proteins, and the effect of a Pit-1 point mutation on the interaction with C/EBPa was characterized. Two-photon excitation fluorescence lifetime imaging microscopy (2p-FLIM) was then used as an independent method to detect these protein interactions. The excited-state lifetime for the cyan FP (CFP) labeling C/EBPa was determined, and the measurements were repeated in cells co-expressing yellow FP (YFP) labeled-proteins. The CFP lifetime was decreased in the presence of the YFP acceptor, which is consistent with donor quenching by FRET. This was verified by acceptor photobleaching, which caused a shift in the donor lifetime to that similar to the donor alone. However, a significant limitation of this technique was demonstrated by the observation that high-energy 2p-excitation resulted in CFP photobleaching and a parallel decrease in its excited-state lifetime. The key question is whether the sensitivity of this imaging approach will be sufficient to acquire significant data from living cells expressing physiological levels of the labeled proteins.