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1 Welcome! openmicberkeley.wordpress.com

2 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

3 What is FRET? Förster Resonance Energy Transfer , Theodor Förster Defined as: non-radiative, dipole-dipole resonance energy transfer

4 What is FRET? Förster Resonance Energy Transfer Defined as: non-radiative, dipole-dipole resonance energy transfer i.e., no emission of a photon

5 What is FRET? Förster Resonance Energy Transfer Defined as: non-radiative, dipole-dipole resonance energy transfer Ishikawa-Ankerhold, et. al., Molecules 2012, 17(4)

6 What is FRET? Förster Resonance Energy Transfer Defined as: non-radiative, dipole-dipole resonance energy transfer

7 What is FRET? Förster Resonance Energy Transfer Defined as: non-radiative, dipole-dipole resonance energy transfer

8 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)

9 FRET Biosensors

10 Overlapping Spectra is Required for FRET, But Causes Bleedthrough k r y e l e p O n e C I e B

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12 FRET Efficiency distance between donor & acceptor (nm) Förster Radius k 2 = orientation of transition dipoles J(l) = overlap integral of emission spectra n = refractive index of medium QD = quantum yield of donor

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

14 Quantifying FRET = Apparent Efficiency (E app ) FRET efficiency Eapp = E geometric conformation distances & angles global parameter Donor-Acceptor Reaction [DA]/[Dtotal] How many molecules are in a complex? biologically relevant local parameter

15 How to Measure FRET Sensitized Emission (Ratiometry) Acceptor Photobleaching (Donor Dequenching) Fluorescence Lifetime Imaging Microscopy (FLIM) Spectral Imaging Fluorescence Polarization Imaging (Anisotropy)

16 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= DA DD

17 Measuring FRET: Sensitized Emission (Ratiometric Imaging) Wang, et. al., Molecular Imaging 12(2), 2013

18 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/flatfield correction, bleedthrough correction, background subtraction, image alignment, photobleaching correction)

19 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 (DD pb ). Donor should unquench, resulting in higher intensity from no FRET. Eapp = 1 - (DD/DD pb )

20 Measuring FRET: Acceptor Photobleaching (Donor Dequenching) Majoul, et. al., J Biotechnol. 2002;82(3).

21 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

22 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.

23 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!)

24 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.

25 Measuring FRET: Spectral Imaging

26 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

27 Measuring FRET: Polarization Anisotropy

28 Measuring FRET: Polarization Pros: Anisotropy 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)

29 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.

30 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