Resonance Energy Transfer Non-Radiative Energy Transfer driven by dipole-dipole coupling • Fluorescence Resonance Energy Transfer • Surface Energy Transfer
Multiplicity : A property of a system due to the spin, or angular momentum, of its component particles ( e.g., electrons) Multiplicity is the quantification of the amount of unpaired electron spin - Hund's rule : favors the single filling of degenerate (same energy) • Number of states with a given angular momentum : 2 S + 1, S= total spin if all electrons are paired, S=0: multiplicity =1; singlet if one unpaired electron, S = 1/2 ; doublet if two unpaired electrons, S =1 ; triplet
Energy scheme used to explain the difference between fluorescence and phosphorescence Singlet state Triplet state • Phosphorescence is a process in which energy absorbed by a substance is released relatively slowly in the form of light
Fluorescence One of a class of luminescence phenomena in which certain molecules may emit light with a longer wavelength than the light with which were excited k f D + hv E D* D + hv F k i D
Quantum yield Q The ratio of the number of photons emitted to the number of photons absorbed • Lifetime The average time spent in the excited state before returning to the ground state
Förster (or Fluorescence)ResonanceEnergy Transfer (FRET) • Non-radiative energy transfer from an energy donor to an energy acceptor • Dipole – dipole coupling • Energy transfer efficiency : • -Degree of spectral overlap between donor fluorescence emission and • acceptor absorption • - Inversely proportional to 6th power of the distance between fluorophores • -~ 10 nm
Use of FRET measurements Calculation of the distance between fluorophores • Detection of target analytes • Analysis of biomolecular interactions • Single molecule analysis • - Protein folding/unfolding • - Protein dynamics
Förster distance The relationship between the transfer efficiency and the distance between the two probe (R) Ro : the Förster distance at which the energy transfer is (on average) 50%
Ro can be calculated using Qd : the quantum yield of the donor, n : the refractive index of the medium (generally assumed to be 1.4 for proteins) Nav : Avogadro's number (Nav= 6.02 x 10 per mole) Kappa squared : the orientation factor J : the overlap integral
Kappa squared Orientation factor , kappa squared
The overlap intergral J The degree of overlap between the donor fluorescence spectrum and the acceptor absorption spectrum λ : the wavelength of the light ε(λ) : the molar extinction coefficient of the acceptor at that wavelength f : the fluorescence spectrum of the donor normalized on the wavelength scale
Surface Energy Transfer • Energy transfer from a dipole to a metallic surface • Interaction of the electromagnetic field of the donor dipole with the nearly free conduction electrons of the accepting metal • Surface energy transfer efficiency : KSET = (1/τD) ( do/d)4 Yun et al., JACS, 2005, 127, 3115-3119
Schematic representation of the system, which consists of a fluorescein moiety (FAM) appended to ds-DNA of length R (varying from 15 to 60bp) with a Au nanoparticle (d = 1.4 nm) appended to the other end. - The flexible C6 linker produces a cone of uncertainty ( R) for both moieties. • Addition of EcoRI (methyltransferase) bends the ds-DNA at the GAATTC site by 128o , producing a new effective distance R'. 15 bp ; 62 A 20 bp ; 96.4 A 30 bp ; 130.4 A 60 bp ; 232.4 A 10 bases per turn 3.4 A per base
Efficiency vs distance -Energy transfer efficiency plotted versus separation distance between FAM and Au(NM). -Filled circles (·) represent DNA lengths of 15bp, 20bp, 30bp, and 60bp. The measured efficiencies of these strands with the addition of M.EcoRI are represented by the open circles. - The dashed line is the theoretical FRET efficiency, while the solid line the theoretical SET efficiency
Conditions -Overlapping of Donar emission and Acceptor Excitation spectrum. -FRET : Donor/Acceptor; <10nm. -SET : Donor/Metal : <20 nm -Spectrally distinct • Applications - Biomolecular interaction study in vivo/vitro - Tracking biomolecualr conformational change -In vivo imaging/co-localization study - Drug discovery - Bio-sensing • Pairs (http://probes.invitrogen.com/resources/; //microscopy.biorad.com) - Organic dye -ALEA-488/RHOD-2; FITC/RHOD-2; FITC/TRITC; GFP/RHOD-2 - Fluorescent protein -BFP/GFP; BFP/YFP; BFP/RFP; CFP/YFP - Nanocrystal -QD/QD; QD/gold
Examples of available fluorescent dye and quencher families. • Tetramethylrhodamine (TMR), carboxytetramethylrhodamine (TAMRA), and carboxy-X-rhodamine (ROX) are all rhodamine-based dyes. • The most common D/A dye combinations: coumarin/fluorescein, fluorescein/rhodamine, and Cy3.5/Cy5. • Popular dye/quencher combinations: rhodamine/Dabcyl and Cy3/QSY9. • Major suppliers: -Molecular Probes (fluorescein, rhodamine, AlexaFluor, BODIPY Oregon Green, Texas Red, and QSY quenchers), -Amersham Biosciences (Cy dyes and Cy5Q/Cy7Q quenchers) - AnaSpec (HiLyteFluors, QXL quenchers) - ATTO-TEC (ATTO dyes and quenchers - Biosearch Technologies (Black Hole). • FITC=fluorescein isothiocyanate.
Fluorephore materials used in bioanalytical FRET • Organic materials -Available in reactive form from commercial sources : activated with N- hydroxysuccinimide (NHS) ester, maleimide, hydrazide, amine functionality Ex) Fluorescein dyes : very popular because of their high quantum yield, solubility, ease of bioconjugation. Excitation with a standard argon-ion laser (488 nm) High rate of photo-bleaching, pH sensitive, self-quenching - Alternatives : AlexaFluore, Cy family, BODIPY • Inorganic materials - Metal chelates, semiconductor nanocrystals • Biological origins - Fluorescent proteins
Structures of common UV/Vis fluorescent dyes. Typical substituents at the R position include CO2-, SO3-, OH, OCH3, CH3, and NO2; Rx marks the typical position of the bioconjugation linker.
Experimental methods Conventional filter FRET Apply filter/emission band configurations for donor, acceptor and FRET (donor excitation and acceptor emission) to acquire single images or time series. If the donor signal decreases, acceptor and FRET signal increases. Acceptor photobleaching Apply donor/ acceptor configurations to acquire single images or time series. After some control images, acceptor (with 514 nm) is bleached. Donor signal increases after acceptor bleach
Analysis of FRET Fluorescence lifetime imaging microscopy (FLIM) Information about the interactions between, and the structural states of, signaling molecules needs to be obtained as a function of space and time in a living cell. By using FLIM, the nanosecond decay kinetics of the electronic excited-state of fluorophores can be mapped spatially. Fluorescence lifetime The average amount of time that a molecule spends in the excited state upon absorption of a photon of light. Fluorescence lifetime is independent of fluorophore concentration and light-path length.
Fluorescent proteins • Green Fluorescence Protein (GFP) from jellyfish • Widespread use by their expression in other organisms • Key internal residues are modified during maturation to form the p-hydroxybenzylideneimidazolinon chromophore, located in the central helix and surrounded by 11 ß-strands (ß-can structure) • In-vivo labeling of cells ; Localization and tracing of target protein • GFP variants : BFP, CFP, YFP • Red fluorescent protein (DS Red) from coral reef : tetrameric, slow maturation • Monomeric RFP by protein engineering • Quantum yield : 0.17 (BFP) ~ 0.79 (GFP) • BFP/CFP ; CFP/YFP( high change in the FRET signal ratio) : fused to N- or C terminus of proteins by gene manipulation
GFP (Green Fluorescent Protein) • Jellyfish Aequorea victoria • A tightly packed -can (11 -sheets) enclosing an -helix containing the chromophore • 238 amino acids • Chromophore • Cyclic tripeptide derived from Ser-Tyr-Gly • The wt GFP absorbs UV and blue light (395nm and 470nm) and emits green light (maximally at 509nm)
a)Normalized absorption and b) fluorescence profiles of representative fluorescent proteins: cyan fluorescent protein (cyan), GFP, Zs Green, yellow fluorescent protein (YFP), and three variants of red fluorescent protein (DS Red2, AS Red2, HC Red). From Clontech.
Analysis of biomolecular interactions using FP Inter-molecular FRET
FRET-based Sensors Intra-molecular FRET
Calmodulin • Calcium ions : crucial for the metabolism and physiology of eukaryotes • Regulate many cellular processes, ranging from transcription control and cell survival to neurotransmitter release and muscle function. • Calmodulin (CaM, 148 aa); a ubiquitous, calcium-binding protein ( typically binds 0, 2 or 4 Ca+2) Regulate a multitude of different protein targets, affecting many different cellular functions. • CaM : mediates processes such as inflammation, metabolism, apoptosis, muscle contraction, intracellular movement, short-term and long-term memory, nerve growth and the immune response. • In the absence of Ca+2, the two main helical domains have hydrophobic cores. On the binding of a calcium ion, conformational changes exposes hydrophobic regions which have the potential to act as docking regions for target proteins ( over 100 proteins including kinases, phosphatases etc.) In the absence of Ca+2 In the presence of Ca+2
- Modified MBP fluorescent indicator. ECFP as donor was fused to the N terminus of MBP, and YFP as a FRET acceptor was fused to the C terminus. - H indicates the portion of protein functioning as a hinge between the two lobes of the MBP. - The central binding pocket of the MBP is located between the two lobes. - In the absence of maltose, the two FPs are at their maximum distance from each other and FRET is minimal. Upon binding maltose, the MBP undergoes a conformation change that brings the two FPs into close proximity and increases FRET, which can be monitored by the change in ratio of the YFP and CFP emission
a) Confocal image of a maltose-FP sensor expressed in yeast. Fluorescence is detected in the cytosol but not in the vacuole. Scale bar=1 um. b) Changes of the maltose concentration in the cytosol of yeast that expresses a maltose sensor with a Kd value of 25 nM. The graph indicates emission ratio as a function of maltose uptake for a single yeast cell.
Enzyme-generated Bioluminescence • BRET ( Bioluminescence RET): - Donor : Luciferase ; Acceptor : GFP - No excitation light source to excite the donor, which avoids problems such as light scattering, high background noise, and direct acceptor excitation • In-vivo monitoring of protein-protein interactions such as circadian clock proteins, insulin receptor activity, real-time monitoring of intracellular ubiquitination • The firefly luciferase/luciferin system : the best candidate for a BRET-based donor ; high quantum yield ( 0.88)
Bioluminescent substrates and enzymatic reactions of several common luciferases: a) the aliphatic aldehyde substrate of bacterial luciferase; b) structure and reaction of luciferin, the substrate of firefly luciferase; c) colenterazine, the substrate for Renilla luciferase and also part of apoaequorin.
Enzyme-generated Chemiluminescence :Luminophore : synthetic substrate that is excited through an enzymatically catalyzed reactions Chemiluminescent substrates and the enzymatic reactions of horseradish peroxidase (HRP) and alkaline phosphatase. a) Luminol; b) Acridan (also available as an ester); c) Adamantyl-1,2-dioxetane (substrate for alkaline phosphatase and other enzymes).
Self-illuminating quantum dot conjugates for in-vivo imaging • Unique optical property of Qd: - High quantum yields, large molar extinction coefficients, size-dependent tunable emission and high photostability Fluorescent probes for biological imaging • Challenging issues - Requirement for external illumination strong background auto-fluorescence from ubiquitous endogenous chromophores such as collagen, porphyrins and flavins little light is available for quantum dot excitation at non-superficial locations due to absorption and scattering of optical photons in tissues • Ideal quantum for in-vivo imaging - Light emission with no requirement for external excitation Quantum dot conjugates based on the principle of BRET So et al., Nature Biotech., 24, 339-343 (2006)
Construction of self-illuminating QDs conjugates • Use of luciferase from Renilla reniformis • Luc8 : Eight mutation variant : more stable in serum and higher catalytic activity - emit blue light with a peak at 480 nm upon addition of its substrate coelenterazine • Conjugation of Luc8 to polymer-coated CdSe/ZnS core-shell QD 655 through coupling of the amino groups on LUC8 to carboxylated on the QD - The hydrodynamic diameter of QD655-Luc8 : ~ 2 nm - The conjugate contains six copies of Luc8 on average
Gold nanoparticles • Exceptional quenching ability • Plasmon resonances in the visible range with large extinction coefficient (105 /cm/M) • Stable • Unfluctuating signal intensities • Resistant to photo-bleaching
Gold Nano Particles (AuNPs) • Core Materials for NPs - Au, Ag, Pt : Electron transporter, Catalysis, NPs coating for electrode - Mg, Co, Fe : Magnetic behavior, Sample purification, MRI signal enhancing - CdSe, ZnS, InP : Semiconductor QDs • Stabilization by surfactants in synthesis of AuNPs - Reduction of HAuCl4in the presence of surfactant - Citrate, tannic acid, white phosphorus : > 3 nm - Alkanethiol : Monolayer protected cluster (MPC), 2 ~ 3 nm - Dendrimer : Dendrimer encapsulated nanocluster (DEN). < 2 nm • Characteristics of AuNPs - Surface Plasmon Resonance Band . Absorbance band near 520 nm in 5 ~ several tens nm of AuNPs . SPB shift responding to surface modification and environmental condition - Photoluminescence as Gold QDs . <2nm of AuNPs : smaller Bohr radius than semiconductor . Size dependent excitation/emission spectrum
Reduction NaBH4 DSN Surfactant NP MPC Gold Nanoparticles Gold Quantum Dot before reduction Citric Acid DEN (G4) DEN MPC AuCl4- DSN (G2-NH2/OH) Synthesis of AuNPs • NP : Nanoparticle capped with surfactant (ex) sodium citrate • MPC : Monolayer-Protected Clusters with alkanethiol (ex) 1-OT / 11-MUA • DEN / DSN : Dendrimer-Encapsulated (or Stabilized) Nanoclusters
FRET-based probe : Sensitive and no separation step Real time analysis of amplicons by PCR Sequence specific multiplex analysis Molecular Beacon (MB) Single-stranded oligonucleotide molecular probe Target strand • Donor : FAM (Ex 494 nm, Em 520 nm) • Quencher : Dacyl ( Abs 380 ~600 nm) Quenching by FRET