FRET (sometimes called Förster Resonance Energy Transfer), is an increasingly popular microscopy technique used to measure the proximity of two fluorophores.Resonance energy transfer occurs only over very short distances, typically within 10nm, and involves the direct transfer of excited state energy from the donor fluorophore to an acceptor fluorophore as an alternative to fluorescence emissive decay from the donor. Upon transfer of energy, the acceptor molecule enters an excited state from which it decays emissively (always of a longer wavelength than that of the acceptor emission). Thus, by exciting the donor and then monitoring the relative donor and acceptor emissions, either sequentially or simultaneously, one can determine when FRET has occurred and at what efficiency.Since fluorophores can be employed to specifically label biomolecules and the distance condition for FRET is of the order of the diameter of most biomolecules, FRET is often used to determine when and where two or more biomolecules, often proteins, interact within their physiological surroundings. Since energy transfer occurs over distances of 1-10nm, a FRET signal corresponding to a particular location within a microscope image provides an additional distance accuracy surpassing the optical resolution (~0.25 mm) of the light microscope. Aside from spatial proximity, for efficient FRET to take place the FRET dye pair must also exhibit significant overlap of the donor's excitation spectrum with the acceptor's absorption spectrum. Herein though lies one of the experimental paradoxes of FRET. The spectral profiles of the FRET pair cannot be so separated that we have poor overlap, yet one wants to avoid "cross-talk" between the two imaging channels, i.e. ideally the donor emission filter set must collect only the light from the donor and none from the acceptor, and vice versa. In practice, this can be somewhat realized by employing short bandpass filters that collect light from only the shorter wavelength side of the donor emission and the longer wavelength side of the acceptor emission. This can limit somewhat the photon flux from both donor and acceptor during a typical exposure, especially when we bear in mind that these measurements are best performed under conditions of reduced excitation power, such that we do not accelerate the rates of bleaching.
FRET Pairs include:
Absorption and emission spectral profiles of the CFP-YFP FRET pair.
Andor's EMCCD cameras, whether as a key component in our Revolution confocal live cell imaging system, or as an EMCCD + iQ imaging software solution, is a well-established technique for FRET imaging. EMCCD enables high resolution, high signal-to-noise (S/N) determination of FRET interactions throughout the imaged area or volume of the cell and help counter the photon throughput sacrifice involved when using narrow-band filters This combined with careful choice of filter sets ensures high integrity of FRET data.Since EMCCDs overcome the noise floor detection limit at any readout speed, molecular interactions can be followed dynamically with high accuracy. Furthermore, through reducing the excitation power, phototoxic and photo-bleach effects are minimized, enabling molecular interactions to be followed for much longer periods.
The iXonEM+ DV887 back-illuminated houses a 512 x 512 back-illuminated sensor offering single photon sensitivity, > 90% Quantum Efficiency (QE) max., 16 x 16µm pixel size, RealGain™ gain control and –100°C Thermoelectric (TE) cooling. Single photon sensitivity @ >34 full frames/sec, ensuring photo-bleaching/phototoxicity is significantly minimized. Lowest dark event specification of any EMCCD on the market. EMCCD and conventional readout options offers complete user flexibility.
The iXonEM+ DV885 houses a 1Mpixel virtual phase sensor offering single photon sensitivity, > 60% QE, 8 x 8μm pixel size, RealGain™ gain control and –100°C TE cooling. Single photon sensitivity @ 30 full frames/sec, ensuring photo-bleaching/phototoxicity is significantly minimized.
LucaEM DL-685M: Andor's newest EMCCD LucaEM camera offering, bringing EMCCD into the mainstream! The highly cost-effective LucaEM provides single photon detection sensitivity and ~ 52% QE at 30 full frames/sec, in a TE cooled USB 2.0 platform.
Quantum Efficiency and Fluorescent Dyes relevant to Fluorescence Resonance Energy Transfer
NewtonEM
Andor's pioneering NewtonEM is a revolutionary range of high-end spectroscopy EMCCD/CCD cameras that provide single photon detection sensitivity, back illuminated QE, and -100°C cooling at rapid frame rates. USB 2.0 connectivity provides plug and play operation. Spectrally overlapped FRET signal can be collected with extremely high spectral resolution and S/N, for subsequent unmixing.
Either iXonEM+, LucaEM or NewtonEM cameras can be used to deliver enhanced EMCCD performance at high frame rate, the choice depending on a number of factors such as:
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iQ Screenshot
The Revolution XD, equipped with emission output splitter, is ideal for dynamic confocal imaging of FRET Pairs, enabling elucidation of quantified molecular dynamics, such as protein-protein interactions, protein-DNA interactions, and protein conformational changes. High S/N multi-dimensional movies can be built through combination of CSU, iXonEM+, iQ software and Andor Laser Combiner with matched solid-state laser lines and AOTF for laser balancing/rapid switching.An Optosplit II or Dual View emission wavelength splitter is placed between the CSU and camera for simultaneous quantitative imaging of donor and acceptor. One can also opt to perform rapid optical sectioning with a Piezo Z100, for 5D imaging - up to 100 sections sec-1. With Revolution XD, image and quantify molecular interactions in intact cells, tissues, and whole organisms, with greater sensitivity and efficiency than any other FRET solution on the market.
Image courtesy of Zhuang Lab, Department of Chemistry and Chemical Biology at Harvard University in Cambridge, Massachusetts.
FRET false-color image captured with the iXon DU-897 camera of single RNA molecules labeled with FRET. A FRET donor and acceptor are attached to either end of an intron that requires magnesium and a protein cofactor (CBP2) to splice together the exon sequences on either side of it. On the left is a false-color image (donor emitting greenish light, acceptor emitting red light)showing low FRET transfer to the acceptor dye due to the dyes being relatively distant, and the intron being in an inactive, unfolded form. On the right is an image taken when the intron folds into its active form, bringing the two exons (and their attached FRET dyes) close together for the splicing reaction, causing high energy transfer to the red dye. Thus, the structural change from an inactive to the active form in the RNA causes the colors emitted by the single molecules to change from green to red due to FRET.
iQ is a state-of-the-art multi-dimensional imaging software package, ideal for even the most complex imaging protocols.This flexible and intuitive platform allows tight synchronization between devices such as camera, z-steppers or fast switchable light sources, enabling unmatched frame rates through multiple dimensions with minimal light loss and minimal photodamage to the cell.iQ is ideal for simultaneous capture and analysis of multiple FRET signals.
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