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Agenda• Jen Lee: Introduction to FRET

• Marla Feller: Using FRET sensors to look at time resolved measurements

• Becky Lamason: Using FRET to determine if a bacterial protein manipulates cell-cell junctional tension

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What is FRET?Förster Resonance Energy Transfer

1946-1948, Theodor Förster

Defined as: non-radiative, dipole-dipole resonance energy transfer

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What is FRET?Förster Resonance Energy Transfer

Defined as: non-radiative, dipole-dipole resonance energy transfer

i.e., no emission of a photon

http://nikon2.magnet.fsu.edu/articles/fluorescence/fret/fretintro.html

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What is FRET?Förster Resonance Energy Transfer

Defined as: non-radiative, dipole-dipole resonance energy transfer

Ishikawa-Ankerhold, et. al., Molecules 2012, 17(4)

Open MIC @ Berke

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What is FRET?Förster Resonance Energy Transfer

Defined as: non-radiative, dipole-dipole resonance energy transfer

http://ascensionglossary.com/index.php/Law_of_Resonance

Open MIC @ Berke

ley

What is FRET?Förster Resonance Energy Transfer

Defined as: non-radiative, dipole-dipole resonance energy transfer

http://mlilm.iqfr.csic.es/materiales_laser_ing/index_ing.htmlOpen MIC @

Berkeley

Why FRET?• “To detect interaction by spatial coincidence of

molecules” - Philippe Bastiaens (iBiology)

• Molecular interactions within 10 nm range, better resolution than traditional colocalization experiments

• Allows for detection of dynamic events in vivo (biosensors)

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FRET Biosensors

http://zeiss-campus.magnet.fsu.edu/tutorials/spectralimaging/fretbiosensors/indexflash.html

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Overlapping Spectra is Required for FRET, But Causes Bleedthrough

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http://zeiss-campus.magnet.fsu.edu/articles/spectralimaging/spectralfret.html

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FRET Efficiencydistance between

donor & acceptor (nm)

Förster Radius

k2 = orientation of transition dipoles J(l) = overlap integral of emission spectra n = refractive index of medium QD = quantum yield of donor

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FRET Efficiency

Ishikawa-Ankerhold, et. al., Molecules 2012, 17(4)

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Quantifying FRET = “Apparent Efficiency” (Eapp)

Eapp = E⍺FRET efficiency

geometric conformation distances & angles global parameter

Donor-Acceptor Reaction [DA]/[Dtotal]

How many molecules are in a complex?

biologically relevant local parameterOpen MIC @

Berkeley

How to Measure FRET• Sensitized Emission (Ratiometry)

• Acceptor Photobleaching (Donor Dequenching)

• Fluorescence Lifetime Imaging Microscopy (FLIM)

• Spectral Imaging

• Fluorescence Polarization Imaging (Anisotropy)Open MIC @ Berke

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Measuring FRET: Sensitized Emission (Ratiometric Imaging)

• Excite donor, then measure donor emission (DD) and acceptor emission (DA)

• Take the ratio of [DA/DD] • For more quantitative measurements, one can correct for

bleed through

DD DA Eapp= DADDOpen MIC @

Berkeley

Measuring FRET: Sensitized Emission (Ratiometric Imaging)

Wang, et. al., Molecular Imaging 12(2), 2013

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Measuring FRET: Sensitized Emission (Ratiometric Imaging)

• Pros: • Fast, good for live imaging • Easily implemented on standard scopes

• Cons: • Very sensitive to noise • May require a lot of image processing (shade/flat-

field correction, bleedthrough correction, background subtraction, image alignment, photobleaching correction)Open MIC @

Berkeley

Measuring FRET: Acceptor Photobleaching (Donor Dequenching)

• Excite donor, look at donor emission (DD). If FRET is occurring, then donor should be quenched.

• Ask: what is the donor intensity in the absence of the acceptor? • Bleach acceptor • Excite donor, then measure donor emission post-bleach

(DDpb). • Donor should unquench, resulting in higher intensity

from no FRET. • Eapp = 1 - (DD/DDpb)Open MIC @

Berkeley

Measuring FRET: Acceptor Photobleaching (Donor Dequenching)

Majoul, et. al., J Biotechnol. 2002;82(3).

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Measuring FRET: Acceptor Photobleaching (Donor Dequenching)

• Pros: • Easily implemented on standard scopes • No external calibration • Robust, reliable, and semi-quantitative

• Cons: • Fixed samples or one time point only

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Measuring FRET: Fluorescence Lifetime Imaging Microscopy (FLIM)

• Every fluorophore has an exponential decay curve, a.k.a. a fluorescence lifetime. When FRET occurs, the fluorescence lifetime of the donor decreases.

• Process: excite donor, then measure donor lifetime (DD). If FRET, then there will be faster donor decay.

http://nikon2.magnet.fsu.edu/articles/fluorescence/fret/fretintro.html

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Measuring FRET: Fluorescence Lifetime Imaging Microscopy (FLIM)

• Pros: • Direct measure of FRET efficiency • Independent of concentration • Acceptor doesn’t need to be imaged

• Cons: • Specialized equipment required (we have one

instrument at the MIC!)

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Measuring FRET: Spectral Imaging

• Every fluorophore has a unique emission spectra. • Spectral imaging uses an array of highly sensitive

detectors to acquire intensities at specified wavelengths to plot out an emission spectra. Then, reference spectra are used to identify the fluorophore.

• Similar approach to sensitized emission, but detection is more specific.

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Measuring FRET: Spectral Imaging

http://zeiss-campus.magnet.fsu.edu/articles/spectralimaging/spectralfret.html

Open MIC @ Berke

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Measuring FRET: Spectral Imaging

• Pros: • Direct measure of FRET efficiency • Can correct for bleed through • Does not discard any signal (vs. sensitized emission)

• Cons: • Specialized equipment required (we have many

instruments at the MIC!) • Requires reference spectra, controls

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Measuring FRET: Polarization Anisotropy

https://www.microscopyu.com/articles/fluorescence/fret/fretintro.html

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Measuring FRET: Polarization Anisotropy

• Pros: • Independent of concentration • Relatively easy, inexpensive, and fast • Able to detect homo-FRET

• Cons: • Not quantitative • Some common optical components destroy

polarization (e.g., high NA objectives)

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Summary

• FRET is a very powerful method of detecting molecular interactions within 10 nm range.

• No FRET approach is perfect. Goals, equipment, and experimental system must all be considered.

• FRET pairs should be chosen with care.

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For more info… • iBiology FRET lecture by Philippe Bastiaens

• Nikon, Zeiss, & Olympus education pages (all written by Mike Davidson, Florida State University)

• Cold Spring Harbor Quantitative Imaging Course

★ Links & slides will be available on the blog! openmicberkeley.wordpress.com

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