FRET
The Problem:
When and where proteins or other molecules associate with each other in living cells is a key question to learn about their function. To address this question, proteins are labeled with different fluorophores. However, the optical resolution of light microscopes limits the detection of protein proximities to about 0.2 µm. A higher resolution is necessary to study the physical interaction of protein partners.		 Overview LSM

FRET
Emission Fingerprinting and FRET
Quantitative colocalization

appropriate products:
LSM 7 LIVE
LSM 710
LSM 710 NLO
LSM 700
LSM 710 ConfoCor 3

Deutsche Version
The solution:
Fluorescence Resonance Energy Transfer (FRET) experiments with the LSM 710.
What Is FRET?
FRET is the non-radiative transfer of photon energy from an excited fluorophore (the donor) to another fluorophore (the acceptor) when both are located within close proximity (1-10 nm).
Using FRET one can resolve the relative proximity of molecules beyond the optical limit of a light microscope to reveal for example (1) molecular interactions between two protein partners, (2) structural changes within one molecule (e.g. enzymatic activity, DNA/RNA conformation), (3) ion concentrations using special FRET-tools like the CFP-YFP Cameleon.
No FRET Signal
FRET, no signal
  • CFP is excited by light and emits light
  • CFP is more than 10 nm distant from YFP
  • YFP is not excited and does not emit light
FRET Signal
FRET, signal
  • CFP is excited by light but does emit little light
  • CFP is in close proximity (1-10 nm) to YFP
  • YFP is not excited by light but does emit light
The Principle of FRET
An excited fluorophore (the donor) transfers its excited state energy to a light absorbing molecule (the acceptor). This transfer of energy is non-radiative, due primarily to a dipole-dipole interaction between donor and acceptor.
There are only certain pairs of fluorophores suitable for FRET experiments since, besides other prerequisites (e.g. dipole orientation, sufficient fluorescence lifetime), the donor emission spectrum has to overlap the excitation spectrum of the acceptor. Known FRET pairs are CFP/YFP, BFP/GFP, GFP/Rhodamine, FITC/Cy3.
Energy diagram of CFP/YFP FRET:
CFP (donor) is excited but most of its energy is not resulting in cyan emission, instead it is transferred to the YFP (acceptor); thus, the resulting emission is mainly yellow.
FRET Using Fluorescent Proteins
Fluorescent proteins (FPs) like the green-fluorescent-protein (GFP) are very attractive for FRET experiments. They can be genetically fused to proteins of interest and expressed in cells making them an excellent reporter system for gene expression and protein localization in living cells. Several enhanced FP variants with different spectral properties are available.

The cyan-colored CFP as donor and the yellow YFP as acceptor are best suited for FRET experiments in living cells, since the emission spectrum of CFP partially overlaps the excitation spectrum of YFP.
FRET, fluorescent proteins
Current FRET Methods
There are several different methods in use to detect FRET events. All these methods can be carried out with the LSM 700 or LSM 710.
FRET - current methods
Conventional filter FRETApplies filter/emission band configurations for donor, acceptor and FRET (donor excitation and acceptor emission) to acquire single images or time series.
In case of FRET the donor signal decreases, acceptor and FRET signal increases.

FRET:
donor , acceptor , FRET
Acceptor bleachApplies donor, acceptor configurations to acquire single images or time series. After some control images, acceptor (with 514 nm) is bleached.
In case of FRET, donor signal increases after acceptor bleach.

FRET (before acceptor bleach):
donor ,acceptor
FRET (after acceptor bleach):
no acceptor, donor
: signal increase
: signal decrease
With LSM 710 Carl Zeiss introduces a completely new approach that makes FRET experiments easy and reliable.
LSM 710 and FRET
The concept of LSM 710 is ideal for FRET experiments. Emission Fingerprinting with Lambda Stack acquisition for complete emission detection and Linear Unmxing for clear separation of the fluorescent signals, lead to a greater dynamic range and a better signal-to-noise ratio resulting in a higher sensitivity of FRET detection than emission band-based methods.


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