Técnicas avanzadas de espectroscopia confocal FRET, FLIM, FRAP, FCS, RICS, … CIO- León Gto. Octubre 2015 Adán Guerrero [email protected] Av Universidad 2001 Universidad Autónoma del Estado de Morelos, 62210 Cuernavaca, Morelos Fluorescence Resonance Energy Transfer Motivación El límite de difracción limita a la microscopía óptica para resolver estructuras nanoscópicas • FRET permite detectar la interacción entre moléculas que coinciden en el espacio. D D A A Volumen de detección: 10-15 litros Volumen de una proteína 81 kDa: 10-12 litros [5]. R0 was calculated using n = 1.4 andy κ2 = 2/3. bDansyl-labeled phosphatidylethanolamine. ENERGY TRANSFER cEosin-labeled phosphatidylethanolamine. dThe factor of 1028 between J(λ) in M–1 cm3 and M–1 cm3 (nm)4 arises from 1 nm = 10–7 cm, raised to the fourth power. aFrom 44 Fluorescence Resonance Energy Transfer ure the distance from a tryptophan residue to a ligand bindinginsite ligand as the acceptor. fold change the when Förster the distance. Thisserves is because of the 13.2.1. Orientation Factor κ2 sixth-root dependence eq. 13.5. It should also be proteins, noted In the incase of multi-domain RET has been A final factor in the analysis of the energy transfer that the visual impression of overlap is somewhat misleadused to measure conformational changescies that move the factor κ2 which is given by is the orientation ing because the value of J(λ) depends on λ4 (eq. 13.3). domains closer or further apart. Energy transfer can also be en el FRET ocurre una molécula donadora (D) Comparison of the entre spectral overlap for 2,5-DPE and 1,5κ 2 ! and ( cos θ T " 3 cos θ D cos θ A ) 2 DPE suggests Förster the distance for 1,5-DPE, whereusedatolarger measure distance between a site on a protein estado un aceptor (A) en protein el estado estacionario as the excitado calculated value y issurface, smaller. The larger Förster disa membrane association between subunits, tance for 2,5-DPE is due to its larger quantum yield. κ 2 ! ( sin θ D sin θ A cos φ " 2 cos θ D cos θ A ) 2 and lateral association of membrane-bound proteins. In the Because of the complexity in calculating overlap integrals case of macromolecular reactions lessθ is the angle between the e and Förster distances it is convenient to association have several examIn one these relies equations T ples. Values of the overlap integral the on determination of a corresponding precise D–Ato distance, and more onthe donor and the transition ab transition dipole of spectra in Figure 13.3 are summarized in Table 13.1. dipole of the acceptor, the simple fact that energy transfer occurs whenever the θD and θA are the angles these dipoles and the vector joining the donor donors acceptors in 8–9 close proximity comparable to Briefand History of Theodorare Förster acceptor, and φ is the angle between the planes the Förster distance. The theory for resonance energy transfer was developed by Professor Theodor Förster (Figure 13.4). as He a proximity indicator The use of energy transfer was born in Frankfurt, Germany in 1910. He received illustrates an important characteristic of energy transfer. a PhD in 1933 for studies of the polarization of reflected Energy electrons. He then became Research Assistant in transfer canabe reliably assumed to occur whenever Leipzig, Germany, where he studied light absorption the donors and acceptors are within the characteristic of organic compounds until 1942. In this phase of his Figure 13.1. Fluorescence resonance energy transfer (FRET) for a Förster distance, and whenever suitable spectral overlap work he applied the principles of quantum mechanics protein with a single donor (D) and acceptor (A). to chemistry. to 1945 he held occurs. From The 1942 value of R cana Professorbe reliably predicted from the 0 ship in Poznan, Poland. In 1945 he joined the Maxspectral of theindonors Planck Instituteproperties for Physical Chemistry Göttingen,and acceptors. Energy . The rate of energy transfer from a donor to an acceptor where he wroteishisaclassic book Fluoreszenz Organistransfer through-space interaction that is mostly indecher Verbindungen, which has been described as a is given by T(r)La transferencia de energía resulta de "house interacciones dipolo–dipolo pendent ofthethe intervening bible" for German community solvent of spectro-and/or macromolecule. scopists. By 1946 Professor Förster had of written In principle, the orientation the his donors and acceptors can entre D y A. first paper on energy transfer, and pointed out the 1 R0 6 prevent energy transfer between a closely spaced D–A pair, (13.1) importance of energy transfer in photosynthesis syskT (r ) ! tems. wasisalso among first scien- nonexistent in biomolbutProfessor such aFörster result rare andthepossibly τD r to observe excited-state proton transfer, which is ecules. Hence one can assume that RET will occur if the NO involucra la emisión de un fotóntists (no radiativa), la hedistancia now described by the Förster cycle. In 1954 discovproperties are Förster suitable and here τD is the decay time of the donor in the absence of eredspectral excimer formation. Professor died of a the D–A distance is r tiene que ser inferior a la longitud de onda de la luz. attack in his car on the way to work in 1974. For Figure 13.4.interacProfessor Theodor Förster. 15 May 1910–20 Ma comparable to R . A wide variety of biochemical cceptor, R0 is the Förster distance, and r is the donor-to- heart additional information see0 [8] and the introduction Reprinted with permission from [8]. Copyright © 1974, S tions result in inchanges in distance and are thus measurable cceptor distance. Hence, the rate of transfer is equal to the about Theodor Förster [9]. Verlag. using RET. ecay rate of the donor (1/τD)Förster when theT.D-to-A distance (r) Zwischenmolekulare Energiewanderung und Fluoreszenz, Ann. Physik 1948, 437, 55 Donador ( ) Aceptor D emite a longitudes de onda que traslapan con el espectro de excitación de A Revisado en: Molecules 2012, 17,4047-40132 protein withcalculated a single donor (D) and acceptor (A). fer can be easily using 444 photons absorbed by the donor which are t acceptor. This fraction is6 given by Fluorescence Resonance Energy Transfer ENERGY TRANSFER ure the distance from a tryptophan residue to a ligand binding site when the ligand serves as the acceptor. In the case of multi-domain proteins, RET has been used to measure conformational changes that move the domains closer or further apart. Energy transfer can also be used to measure the distance between a site on a protein and a membrane surface, association between protein subunits, and lateral association of membrane-bound proteins. In the case of macromolecular association reactions one relies less on determination of a precise D–A distance, and more on the simple fact that energy transfer occurs whenever the donors and acceptors are in close proximity comparable to the Förster distance. The use of energy transfer as a proximity indicator illustrates an important characteristic of energy transfer. Energy transfer can be reliably assumed to occur whenever the donors and acceptors are within the characteristic Förster distance, and whenever suitable spectral overlap occurs. The value of R0 can be reliably predicted from the spectral properties of the donors and acceptors. Energy transfer is a through-space interaction that is mostly independent of the intervening solvent and/or macromolecule. In principle, the orientation of the donors and acceptors can prevent energy transfer between a closely spaced D–A pair, but such a result is rare and possibly nonexistent in biomolecules. Hence one can assume that RET will occur if the spectral properties are suitable and the D–A distance is comparable to R0. A wide variety of biochemical interactions result in changes in distance and are thus measurable using RET. It is important to remember that resonance energy transfer is a process that does not involve emission and reabsorption of photons. The theory of energy transfer is based on the concept of a fluorophore as an oscillating dipole, which can exchange energy with another dipole with a similar resonance frequency.4 Hence RET is similar to the behavior of coupled oscillators, like two swings on a common supporting beam. In contrast, radiative energy transfer is due to emission and reabsorption of photons, and ( ) 1 R0 from a donor to an acc Å. The rate of energy transfer (13.10) kT (r ) ! kT(r) is by de transferencia τ D r de energía de D a A La given velocidad esta dada por: kT (r ) E ! 6 "1 If the transfer rate is much faster than decay rate, then R 1 the 0 τ # k (r ) T D kT (r ) If!the transfer rate is slow- ( energy transfer will be efficient. τD er than the decay rate, then little transfer will occur during donde τis el tiempo de of decaimiento de D en ausencia D esthe which ratio the transfer rate to the the excited-state lifetime, and RET will be inefficient. R0 = 3 nm la distancia wheredeτA,D risesthe decayentre timeD yofA.the donor in the absen 1.0 of the donor in the presence of acceptor. Re The efficiency of energy transfer (E) is the fraction of R0 es R la0distancia de Förster (tipicamente en nm). acceptor, is the Förster distance, and r is the don D 6,donor –1(R photons absorbed by the which are transferred to the 0.8 = τ /r) one can easily rearrange eq. 1 A D 0 acceptor distance. Hence, the rate of transfer is equal ( ) La eficiencia deistransferencia de energía (E) es la fracción acceptor. This fraction given by 0.6 decayderate of the donorpor (1/τ the D-to-A fotones absorbidos D que son transferidos a A. distan D) when 0.4 is equal to the Förster distance (R0), and6the transfer R k (r ) 0 donor em T 0.2 ciency is 50%.EAt this distance (r = R ) the 0 (13.11) ! E ! D "1 6 in the 6 absen τ # k (r ) would be decreased to half its intensity 0.0 R0 # r T D 0.0 0.5 1.0 1.5 2.0 acceptors. The rate of RET dependsSpectroscopy. strongly Joseph on distR Principles of Fluorescence A r/R0 Lakowicz. Thirdrate Editio which and is the of the transfer rate 13.1). to the total decay is ratio proportional to r–6 (eq. Figure 13.1. Fluorescence resonance energy transfer (FRET) for a protein with a single donor (D) and acceptor (A). Å. The rate of energy transfer from a donor to an acceptor kT(r) is given by 1 R0 kT (r ) ! τD r E (r) 6 (13.1) where τD is the decay time of the donor in the absence of acceptor, R0 is the Förster distance, and r is the donor-toacceptor distance. Hence, the rate of transfer is equal to the decay rate of the donor (1/τD) when the D-to-A distance (r) is equal to the Förster distance (R0), and the transfer efficiency is 50%. At this distance (r = R0) the donor emission would be decreased to half its intensity in the absence of acceptors. The rate of RET depends strongly on distance, and is proportional to r–6 (eq. 13.1). Förster distances ranging from 20 to 90 Å are convenient for studies of biological macromolecules. These distances are comparable to the size of biomolecules and/or the distance between sites on multi-subunit proteins. Any –1. The term κ2 is a factor des orescence quenching, or cm g, or cm–1. The term κ2 is a factor describing the relative ori her fluorescence phenomtion in space of the transition nomtion in space of thede transition dipoles of the donor Teoría FRET 2 is usually assumed e fluorophore with other acceptor. κ 2 otherR0 depende acceptor. κ is usually assumedyto be equal to 2/3, whi de parámetros de orientación espectroscópicos vent shell. These nearby appropriate for dynamic random earby appropriate for dynamic random averaging of the dono Índicetransfer, de refracción Rendimiento cuántico de Dbelow or energy except acceptor (Section 13.2.1, xcept acceptor (Section 13.2.1, below). In eq. 13.2 the tra of theisdonor andas a function rate is written function of roperties and rate written of r, kTas (r),a to emphasiz ransfer effective over dependence on distance. over isdependence on distance. ent intervening solvent or integralThe overlap integral or The overlap (J(λ)) expresses the (J(λ) degre n the efficiency energybetween spectral overlap between nergy spectralofoverlap the donor emission andthe thedon ac distance. In tor absorption: ce.onInthe D–A tor absorption: basic adia- theory of non-radiaIntegral de traslape Factor de orientación ∞ lications of RET to∞ biobio∞ 4 "0 FD (λ )εA (λ )λ4 dλ 4 cal applications of RET RET J(λ ! FD (λ ) ε (λ ) λ dλ ! (1 J(λ ) ! FD (λ )εA (λ )λ dλ) ! A ∞ F (λ )dλ " D 0 reviews (additional referefer0 0 Principles of Fluorescence Spectroscopy. Joseph R. Lakowicz. Third Edition nd areoflisted near the end of ! ! D emite a longitudes de onda que traslapan con el espectro de excitación de A Revisado en: Molecules 2012, 17,4047-40132 Parejas FRET populares Revisado en: Molecules 2012, 17,4047-40132 Parejas FRET populares Tres tipos de sensores FRET Revisado en: Molecules 2012, 17,4047-40132 Configuraciones posibles de la apolipoproteínaENERGY A1 TRANSF 454 454 ENERGY TRANSFER Figure 13.13. Emission spectra of labeled apoA-I in HDL. Revise from [36]. Figure 13.13. Emission spectra of labeled apoA-I in HDL. Revised from [36]. tra of labeled apoA-I in discoidal HDL. The spectrum of t D–A apoA-I pair shows a decrease intensity tra of labeled in discoidal HDL. in Thedonor spectrum of the and increase D–A pair showsinaacceptor decreaseintensity, in donorconsistent intensity with and about an 40 transfer. The presence of RET apoA-I increaseenergy in acceptor intensity, consistent withproves aboutthat 40% in the belt conformation 13.12) because energy transfer. The presence of (Figure RET proves that apoA-IRET is wou notconformation occur for the picket-fence conformation where t in the belt (Figure 13.12) because RET would Figure 13.12. Possible conformations for apolipoprotein apoA-I donor are 104D apart. Otherwhere groupsthe agree w not occur forand theacceptor picket-fence conformation when bound to lipids. Reprintedforwith permission from [36]. Figure 13.12. Possible conformations apolipoprotein apoA-I theacceptor belt Joseph structure, butapart. believe thegroups peptides are with in a hairp donor and are 104D Other Principles of Fluorescence Spectroscopy. R. Lakowicz. Thirdagree Edition Figure courtesy of Dr. Mary with G. Sorci-Thomas from Wake Forest when bound to lipids. Reprinted permission from [36].the Figure 37 Estudio FRET sobre el plegamiento intracelular de proteínas PRINCIPLES OF FLUORESCENCE SPECTROSCOPY ORESCENCE SPECTROSCOPY 455 Figure 13.14. Schematic neuronales, of labeled Apo E3 and Apo E4, and donor Células Neuro-2a (CFP) and RET images in Neuro-2a cells. Reprinted with permission from [38]. Principles of Fluorescence Spectroscopy. Joseph R. Lakowicz. Third Edition Indicador FRET para estrógeno ENERGY TRANSFE gure 13.21. RET indicator for estrogens using the ligand-binding domain of estrogen receptor. The color scale shows the intensity at 480 nm dividby the intensity at 535 nm. Revised and reprinted with permission from [45]. Copyright © 2004, American Chemical Society. Principles of Fluorescence Spectroscopy. Joseph R. Lakowicz. Third Edition Visualización de la activación de Rac en células vivas Donador: GFP-Rac Aceptor: Alexa546-Cinasa Principles of Fluorescence Spectroscopy. Joseph R. Lakowicz. Third Edition La activación de Rab13 ocurre en el extremo de migración de las células ublished February 23, 2015 Down J Cell Biol 2015 208:629-648 yversely, of non-RET donor quenching can addressed around the an donor in 1979. RET toformultiple acc wavelengths. Hence thebe intensity measured atthat the transfer efficiency quickly thei racceptor will be larger thanwith the the true value. In suchHence by it the fact analytical expression the donor complete labeling acceptor. is essential to obtain complete labeling wi and if rthe = transfer 0.5R0 then the efficiency is contribuarison of efficiencies observed from one,energy two, and threeindimensions is described in m F acceptor wavelength typically contains some DA if r is greater than R . Because E depends nsfer efficiency determined from enhanced sity for transfer two dimensions only appear 0 acceptor, pletely labeled with the acceptorexp[-σS(t)] or to knowdescribes the extent of portion acceptor labeling. In these equations that of the 57 (13.13) E ! 1 " ractical to use RET to measure distances nching and acceptor sensitization. See Problem in Chapter 15. Several of these results are presen 69 from the donor. sion is thought to be the correct value. The 1964, and was extended to allow an excluded vo stance, measurements of the distance (r) F would be larger than the true value, D 13 donor decay due to RET, σ is the surface density of the illustrate the general form of the expected data. of r = 0.5R to r = 2R . 59 0 acceptor 0 can be addressed non-RET donor quenching around the donor in 1979. RET to multiple accepto The use of intensities isR further complicated when r is within a factor of 2 of . If r is nce too large. We are less concerned 0 acceptor, and rc is the distance of closest approach between Aand general description of described energy transfer o efficiency is typically measured using the nthe of need the transfer efficiencies observed from one, two, three dimensions is in more to(r account for directly excitedeffiacceptor emisGr distance = 2R0the ) can then the transfer he donor because protein moleransfer efficiency also be calculated from the life2 the donor and acceptors. The energy-transfer efficiency can dimensional surface has been given by Fung an 57in κ 13.3.3. Effect of on the Possible Range of nce intensity of the donor, the absence ng and acceptor sensitization. See Problem in Chapter 15. Several of these results are present he ERGY TRANSFER IN MEMBRANES 13 n,and which is almost always present. In the case of and if these r do = 0.5R then the efficiency is Assuming no homotransfer between the donors, an in donors not to the be calculated by an equation analogous to eqs. 13.13 and 0contribute under respective conditions (τ and τ ): Distances Advanced T illustrate the general form of the expected data. La eficiencia de FRET se mide usando la intensidad relativa del donador (D), en la ausencia DA D (FDA ) of acceptor: eled apoA-I (Figure 13.13) the directly excited acceptor of amples oftoresonance energy transfer described fusion during the donor of excited-state lifetime, the ractical use RET to measure distances 13.14, except that the intensities or lifetimes are calculated ng the extent of donor labeling is the A general description energy transfer on a (FD) y presencia del aceptor (FDA) ssion accounted for about halffrom the totaldonor acceptor emisre was a single acceptor attached to each decay of intensity the donor is been givengiven by by Fung and bra integrals of the donor decay: of r = 0.5R to r = 2R . e and donor–acceptor pair. dimensional surface has St 0 0 In distance measurements using RET there is often co GY TRANSFER MEMBRANES n. acceptor isFbecomes almost always directly to some . The situationIN more τexcited complex forAssuming DA DA for FRET en membranas no homotransfer between the donors, and n efficiency is typically measured using the elingEwith donor (f ) is known then 2 aE (13.13) ! 1 " ! 1 " (13.14) thewaveeffects of the0 orientation factor κ . At pr 5 In this case the bulk about donors and acceptors. conent because the acceptor absorbs at the excitation Fenergy es be ofintensity resonance transfer fusion duringIDthe excited-state lifetime, the (t ) donor ! I(tD)exp("t/τ ) # inte ete τdescribed D donor, I D ) exp""σS(t nce the in the an used to of calculate the transfer D absence 1 D 2 there isEno! way to measuredtκ , except by determinati ofaused acceptors is important because thedonor acceptor gth to acceptor excite the donor, resulting in acceptor emis1 " (13.28) as single attached to each decay of the donor is given by 13 no 0 q.(F13.14 becomes etion ) of acceptor: τ I DA D determines the D–A proximity. Also, onex-raywhere the crystal structure, D or NMR structure, in which nimportant without RET. eency situation becomes more complex for can also be calculatedthe from the life- donde and to remember assumptions involved in Efectos de marcaje incompleto onsider the presence of more than a single accepthe distance would be known and thus there would 5 Inthe rsrespective and acceptors. this case the bulk conCalculation of transfer efficiency from the 0 conditions (τDA and 13.13 τDUse ): RET (1 ID (t )only ! is ID moderately exp("t/τ D ) complex exp""σS(tand )# reg of eqs. 13.26–13.28 Equations and 13.14 are dacceptors each In spite of the complexity, has 1these # fAdonor. )equations. F F 1 requires reason to use energy∞ transfer. However, it is possible DADAbecause is important the acceptor ancedEacceptor emission careful consideration (13.17) !donor-acceptor 1# (13.13) ! 1for " ide requires use of numerical integration. However, the ble potential studies of lateral organization in 2 cable to pairs that are separated by a 6 determines the D–A proximity. Also, one limits on donor κ that setexp""(t/τ limits on the range of po FDFintensities. S(t ) in ! turn {1 " A D fA all the interrelated Assuming that the D ) (R0 /r ) # } 2πr dr where es. For example,ofτconsider a membrane thatisconapproach quite general, and can be applied to a wide varibo DA der the presence more than a single accep- Recapitulando y mensajes para llevar a casa sobre la implementación de FRET ! ( ) ! D–A distances. These limits are determined from athesituation frequently encountered for r s distance, not Eemit acceptor wavelength, the efficiency of ! 1at" (13.14) etyRET of circumstances by using different expressions for S(t) h donor. In spite of the complexity, has τ anisotropies of the donor and acceptor, which reflec ∞ T donor quenching, (FDA /F << 1),the Efectos deby sangrado espectral iency also be Dcalculated from lifeed proteins. However, a Dsingle fixed donor–acceptor sfer iscan given that correspond to different geometric conditions. Figure gra otential for studies of lateral organization in 6 the dynamic (excitación directa del aceptor) extentand of S(t orientational averaging toward unlabeled acceptor can result in a respective conditions (τmixture and τ ): (1 ) ! { 1 " exp""(t/τ ) (R /r ) # } 2πrin dr nce is not found for a of donors acceptors D 0 DA D 13.28 shows the calculated transfer efficiencies for a case FRET en solución…. lip orremember example, consider a membraneinvolved that con- in the assumptions 2 = 2/3. age of κ r ex em ution, nor for donors and acceptors dispersed randomwhich the donor to acceptors are constrained to the tra ε (λ ) F (λ ) 1 A AD D A ions.EEquations 13.13 and 13.14 are only The(13.25) 2 has been discussed in detail by problem of κ ! " 1 interface region of a bilayer. Several features of lab exτ DA em lipid–water f ε (λ ) F (λ ) Dand or-acceptor by a coworkers10–12 and summarized by Cheung.13 The E ! 1D pairs "D thatA areA separated (13.14) these predicted data are worthy of mention. The efficiency com τ D a situation frequently encountered for is thatwith the Rdonor andefficiency acceptor of move freely wit of transferidea increases and the energy aci c [ ]( ) ex ex ! c 0 Fluorescence Lifetime Imaging Microscopy NCIPLES OF FLUORESCENCE SPECTROSCOPY e-domain and frequency-domain measurements are in espread use. 1. Meaning of the Lifetime or Decay Time or to further discussion of lifetime measurements, it is ortant to have an understanding of the meaning of the ime τ. Suppose a sample containing the fluorophore is ted with an infinitely sharp (δ-function) pulse of light. s results in an initial population (n0) of fluorophores in excited state. The excited-state population decays with te Γ + knr according to dn(t ) ! (Γ " knr ) n(t ) dt The denominator is equal to τ parts, one finds the numerator single exponential decay the a remains in the excited state is e <t> ! It is important to note that complex decay laws, such as decays. Using an assumed dec can always be calculated using age lifetime can be a complex describing the actual intensity n(t)(4.1) = n0 exp(–t/τD). this reason, caution is necessary lifetime. the instrument to a zero lifetime sample. This curve typmethods. In time-domain or pulse fluorometry, the is sample that sources. The TAC has to be reset and set to zero before each collected using of a light dilute(Figure scattering such isically excited with a pulse 4.1).solution The width of as the start pulse, which takes a finite amount of time. The TAC es be colloidal silica (Ludox) and no emission filter. This decay pulse is made as short as possible, and is preferably much can be constantly in reset mode if the start signals arrive too of th represents the shortest time profile that can be measured by shorter than the decayTIME-DOMAIN time τ of the sample. The timerapidly. The emission signals occur about 1 per 100 excitaa mi 4 LIFETIME MEASUREM dependent intensity is measured following the excitation tion pulses, and thus much less frequently than the excitathe d tion pulses. These emission pulses are used to start the TAC, pulse, andthat the decay is calculatedwhen from the slopefluoropho of effec form wouldtime beτ observed many and the next laser pulse is used to stop the TAC. a plot of log I(t) versus t, or from the time at which the late excited and numerous photons are observed. Howev There are many subtleties in TCSPC that are not obviintensity decreases to 1/e of the intensity at t = 0. The intenthe TCSPC the conditions are adjusted that less tha ous at first examination. Why is the photon counting rate sity decays are often measured through a polarizersooriented limited to 1 photon per 100 laser pulses? Present electronphoton is detected per laser pulse. In fact, the detecti ics for TCSPC only allow detection of the first arriving phois typically 1 photon per 100 excitation pulses. The ton. The dead times range from 10 microseconds in older measured between the excitation pulse and the ob systems to about 120 ns with modern TCSPC electronics. photon and stored in a histogram. The x-axis is the tim These times are much longer than the fluorescence decay. The dead time in the electronics prevents ference and the y-axis the number of photons detec detection of another photon resulting from the same excithis time difference. When much less than 1 pho tation pulse. Recall that emission is a random event. Foldetected per excitation pulse, the histogram represe lowing the excitation pulse, more photons are emitted at early times than at late times. If all these photons could be waveform of the decay. If the count rate is higher t measured, then the histogram of arrival times would repretogram is biased to shorter times. This is becaus sent the intensity decay. However, if many arrive, and only TCSPC only the first photon can be observed. At p the first is counted, then the intensity decay is distorted the electronics are not fast enough to measure multip to shorter times. This effect is described in more detail in Section 4.5.6. tons per pulse when the lifetimes are in the nano Another important feature of TCSPC is the use of the Fig range. Multiple photons per pulse can be measur rising edge of the photoelectron pulse for timing. This me Figure 4.1. Pulse or time-domain lifetime measurements. decay times near a microsecond or longer. Specialize sio allows phototubes with ns pulse widths to provide subnanosecond resolution. This is possible because the rising tronics are used for measuring the time delay betwe edge of the single-photon pulses in is the usually steeper than one Figure 4.7. Principle of TCSPC. The pulses middle panel repexcitation and emission (Figure 4.8). The experimen Figure 4.9. TCSPC data for 2,5-diphenyl-1,3,4-oxadiazole (PPD) in would expect from thefraction time response of the PMT. Also, the esent the output from a constant discriminator (see Figure TCSPC data for 2,5-diphenyl-1,3,4-oxadiazole (PPD) incavity ethanol. The with the excitation pulse that excites the samples and ethanol. The light source was an R6G dye laser, dumped at 1 of a[11]. constant fraction discriminator provides improved 4.22). Reviseduse from light source was anMHz. laser, dumped at 1 MHz. The dye detector was ancavity R2809 MCP PMT (Hamamatsu). TheThe left time resolution by removing the variability due to the asideR6G signal to the electronics. This signal is passed thr of the residuals (lower panel) show some minor systematic error. detector was an R2809 MCP PMT (Hamamatsu). amplitude of each pulse. From [15]. constant function discriminator (CFD), which acc The lifetime is the average amount of time a fluorophore remains in the excited state following excitation. Principles of Fluorescence Spectroscopy. Joseph R. Lakowicz. Third Edition of is called demodulation, also beinused to calcuChapter 5, but it is valuable to understand the basi usualeffect expression for a singleand candetail he late the decay time. FD measurements typicallyrelating use both lifetimes to phase and modulation. Th equations nthe phase and modulation information. At present, both modulation of the excitation is given by b/a, where a is th ed Phase-modulation or frequency-domain lifetime measurements. average intensity and b is the peak-to-peak height (4.2) exp (#t / τ ) time 0. The lifetime τ is the e, τ = (Γ + knr)–1. In general, the sum of the rates which depopfluorescence lifetime can be a plot of log I(t) versus t (Figy by fitting the data to assumed of th incident light (Figure 4.2). The modulation of the emissio The modulation of the excitation is defined similarly, B/A, except using the intensities of th is given by b/a, where a is the emission (Figure 4.2). The modulation of the emission average intensity and b is the measured relative to the excitation, height m = (B/A)/(b/a). Whil peak-to-peak of the m is actually a demodulation incidentfactor, light. it is usually called th modulation. The other experimental observable is the phas The modulation of the emission delay, called the phase is angle (φ),similarly, which is B/A, usually measure defined except from the zero-crossing using timesthe of the modulated intensities of thecomponent modulation of the The phase angle (φ) emission. and theThe modulation (m) can b emission is measured relative to employed to calculate the lifetime using erage amount of time a fluoted state following excitation. the excitation, m = (B/A)/(b/a). ating the average time in the is obtained t over Figureby 4.2. averaging Phase-modulation or frequency-domain lifetime measure#1 (4.5 tan φ ! ωτ , τ ! ω tan φ φ φ ments. The ratios B/A (φ) and b/a represent the modulation of the emisThe phase angle and the orophore: sion and excitation, respectively. modulation (m) can be employed to calculate the lifetime using ∞ !0 t exp (#t / τ ) dt 1 1 1 1/2 (4.3) ! ∞ m! (4.6 , τ ! # 1 m 2 ω m !0 exp (#t / τ )dt √1 " ω2τ 2m [ ] Principles of Fluorescence Spectroscopy. Joseph R. Lakowicz. Third Edition Comparison of time-domain (left) and frequency-domain (right) decay time measurements of N-acetyl-L-tryptophanamide (NATA). TIME-DOMAIN LIFETIME MEASUR 3. Comparison of time-domain (left) andoffrequency-domain decay time measurements N-acetyl-L-tryptophanamide Principles Fluorescence(right) Spectroscopy. Joseph R.ofLakowicz. Third Edition (NA detector phase angles can be fit to determine the gle and modulation of the emission. Since the nsitive intensities were measured using an imaging the data can be used to create an image when the or color is based on phase angle, modulation or 4 lifetime. The data in Figure 22.5 can be used to create a calibration curve for calcium (Figure 22.6). These curves can be used to determine the concentration of calcium from the phase or modulation of the emission. It is important to rec- Fluorescence Lifetime Imaging Microscopy FLUORESCENCE-LIFETIME IMAGING MICROSCOPY FETIME IMAGING OF CALCIUM QUIN-2 Determination of Calcium Concentration etime imaging depends on the use of a probe that lifetime in response to a change in conditions. of calcium concentrations requires a probe that calcium-dependent lifetimes. Figure 22.5 shows ging frequency-domain data for the calcium probe with various concentrations of calcium.6 The freesponse shifts dramatically to lower frequencies at Figure 22.3. Schematic of lifetime imaging using phase-sensitive images. 2.1.1. FLIM Using Known Fluorophores Figure 22.5. Frequency-domain intensity decays for Quin-2 with varying concentrations of calcium. The data were fit globally with two lifetimes τ1 = 1.38 ns and τ2 = 11.58 ns. Figure 22.4. Phase-sensitive intensities of standard fluorophores at various detector phase angles. The decay times from left to right are 0.04 ("), 1.10 (∆), 3.75 (!), and 9.90 ns (!). The modulation frequency was 49.53 MHz. DMSS, 4-dimethylamino-ω-methylsulfonyltrans-styrene; 9-CA, 9-cyanoanthracene; POPOP, p-bis[2-(5-phenyloxazazolyl)]benzene. θ1 is the arbitrary phase angle of the incident light. Principles of Fluorescence Spectroscopy. Joseph R. Lakowicz. Third Edition Fluorescence Lifetime Imaging Microscopy PRINCIPLES OF FLUORESCENCE SPECTROSCOPY e and modulation of Quinom [7]. ons can be determined ulation frequency withnential decay. This is ase angle and modulathe complexity of the ration curve would be frequencies. Figure 22.6. Dependence of the phase angle and modulation of Quin2 on the calcium concentration. Revised from [7]. Cells calcium concentrations can be determined theognize phase that and the modulausing measurements calcium concentrationsat a single modulation frequency withof the multi-exponential decay. This is gesout for resolution Quin-2 in three because there is always a single phase angle and modulaThe intensity of Quin-2 tion of the emission irrespective of the complexity of the s of the cells (top). The decay. Of course, a different calibration curve would be e if the intensity differobtained using different modulation frequencies. n-2 concentrations or to Phase-sensitive images 22.2.2. Lifetime Images of Cos Cells hown in Figure 22.2 and Principles of Fluorescence Spectroscopy. Joseph R. Lakowicz. Third Edition 7 Protein Kinase C Activation Wide-field frequency-domain FLIM can be used to study the activation of intracellular proteins. One example is the central cell decreases because of energy tran 3.5-labeled antibody. These images are not co difficult to know if phosphorylated PKC appe throughout the cell or is localized near the m Activation of lipid/calcium-dependent protein kinase C (PKC) in Cos7 cells GFP-tagged PKC. GFP-lifetime images the central cell was injected with Cy3.5IgG specific for the phosphorylated epitope of PKC. All the cells were treated with phorbol myristoyl acetate (PMA). Figure 22.8. Activation of lipid/calcium-dependent protein kinase C (PKC) in Cos7 cells. The top panels show the intensity images Principles Fluorescence Spectroscopy. Joseph R.show Lakowicz. Third Edition PKC. All the cells were of treated with phorbol myristoyl acetate (PMA). The lower panels the GFP-lifetime images the central c Figure 22.17. pH-dependent phase and modulation values of BCECF for 820 nm excitation with a Ti:sapphire laser, 80 MHz. Revised from [57]. image shows that BCECF is present in the interstitial spaces. The modulation is higher is lower in the interstitial spaces. These data be imaged within and around the keratinoc shows the ability of FLIM to provide quan lar imaging using minimal perturbation of Registros de pH en células epiteliales de cornea a una profundidad def 6.8 micras usando BCECF PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 751 except that the phase and modulation of the emission is measured rather than using TCSPC. An example of such measurements is lifetime imaging of the stratum corneum, which is the outermost layer of the skin. The lifetime probe was BCECF,57 which displays a pH-dependent change in lifetime. This change was used to obtain a calibration curve of phase and modulation versus pH (Figure 22.17). The stratum corneum was imaged using two-photon excitation of BCECF at 820 nm. It was possible to obtain images at various depths in the stratum corneum because the long wavelength used for two-photon excitation can penetrate tissues and two-photon excitation is intrinsically confocal due to localized excitation at the focal point of the laser. The images of BCECF at a depth of 6.8 microns in the stratum corneum are shown in Figure 22.18. The intensity image shows that BCECF is present in the cells and in the interstitial spaces. The modulation is higher and the lifetime is lower in the interstitial spaces. These data allow the pH to Figure 22.18.ofpH lifetime imaging of the skin stratum corneum at a depth of 6.8 microns using BCECF. See Figure 22.17. η is refr Figure 22.17. pH-dependent phase and modulation values BCECF environment. withimaged permission from [57]. Images courtesy of the Dr. Kerry M. Hanson from the University of within and around keratinocytes. This result for 820 nm excitation with a Ti:sapphire laser, 80surrounding MHz. Revised from Reprintedbe Champaign. [57]. shows the ability of FLIM to provide quantitative molecular imaging using minimal perturbationThird of tissues. Principles of Fluorescence Spectroscopy. Joseph R. Lakowicz. Edition Medidas FLIM-FRET multifotónicas en células HeLa co-expresando EGFP y mCherry. Revisado en: Molecules 2012, 17,4047-40132 Fenómeno de transporte que ocurre en la naturaleza Generalización del concepto de difusión Traslacional Rotacional Anómala (fluorescence recovery eaching) Métodos que permiten cuantificar la difusión uctuations nd dynamic ICS ❖ Basados en una perturbación ❖ FRAP tal difference between Basados en fluctuaciones although they are FCS, ICS,RICS sical phenomena they ❖ ❖ http://www.lfd.uci.edu/workshop/2013/ Schematic representation of a FRAP and iFRAP experiment. (A) A region of interest (ROI) is selected, bleached with an intense laser beam, and the fluorescence recovery in the ROI is measured over time. (B) In iFRAP, the reverse is done and a ROI is selected to remain intact, while the rest of the cell is bleached. This is particularly useful when studying dynamic movement in organelles such as the nucleus. Revisado en: Molecules 2012, 17,4047-40132 Example of a FRAP experiment to show that monomeric GFP can pass the nuclear membrane. A) Myoblast cell line (myo3) homogenously expressing GFP-Myosin III before bleaching. (B) A region of interest (ROI) is bleached with high intensity laser light. Directly after bleaching, the cell shows a dark area in which the fluorochromes were permanently damaged and thus no longer emit light (C). The fluorescence in the photobleached region recovers via replacement with intact fluorochrome molecules from the surrounding area (D). Note that the total amount of fluorescence has decreased during the experiment, because a substantial amount of fluorochromes were irreversibly damage. Revisado en: Molecules 2012, 17,4047-40132 Anatomy of a typical FRAP curve (A) From the initial (pre-bleach) fluorescence intensity (Ii), the signal drops to a particular low value (I0) as the high intensity laser beam bleaches fluorochromes in the ROI. Over time the signal recovers from the post-bleach intensity (I0) to a maximal plateau value (I∞). From this plot and equations 11–12, the mobile fraction (Mf), immobile fraction (IMf), I½ and corresponding time (τ½ – the time for the exchange of half the mobile fraction between bleached and unbleached areas) can be calculated (Light blue line: reference photobleaching curve to correct for fluorescence loss during data acquisition). The information from the recovery curve (from I0 to I∞) can be used to determine the diffusion constant and the binding dynamics of fluorescently labeled proteins. Revisado en: Molecules 2012, 17,4047-40132 Anatomy of a typical FRAP curve Based on different recovery profiles, the protein mobility can be classified as (B) highly mobile with virtually no immobile fraction, (C) intermediate mobile with an immobile fraction, or (D) immobile. Revisado en: Molecules 2012, 17,4047-40132 Simultaneous FRAP and FRET measurements to separately determine the mobility of interacting and noninteracting CFP- and YFP-tagged proteins in a single cell nucleus. (A) Schematic representation of the method. A 100 ms high-intensity bleach pulse at 514 nm is applied to irreversibly photobleach YFPs in a narrow strip spanning the nucleus. Redistribution of YFP and CFP fluorescence is recorded at 100 ms intervals at 458 nm. Donor (CFP) emission (increased because of unquenching as a result of acceptor [YFP] bleaching) represents the mobility of interacting molecules only (donor-FRAP). Acceptor emission represents the total pool of YFP-tagged molecules irrespective of interaction (acceptor-FRAP). Revisado en: Molecules 2012, 17,4047-40132 Simultaneous FRAP and FRET measurements to separately determine the mobility of interacting and noninteracting CFP- and YFP-tagged proteins in a single cell nucleus. (B) Graph showing CFP and YFP fluorescence intensities in the bleached strip plotted against time. Experiments were performed in Hep3B cells expressing CFP-YFP fusions (red line indicates CFP fluorescence [donor-FRAP], and blue line indicates YFP fluorescence [acceptor-FRAP]), or in Hep3B cells expressing separate CFPs and YFPs (yellow line indicates CFP, and green line indicates YFP; n = 30). (C) Inverted donor-FRAP (red line) and acceptor-FRAP (blue line) plotted against time, showing similar kinetics. The curves were normalized by calculating Inorm = (Iraw − I0)/(Ifinal − I0), where I0 and Ifinal are the fluorescence intensities immediately after the bleach and after complete recovery, Revisado en: Molecules 2012, 17,4047-40132 respectively. (fluorescence recovery eaching) Métodos que permiten cuantificar la difusión uctuations nd dynamic ICS ❖ Basados en una perturbación ❖ FRAP tal difference between Basados en fluctuaciones although they are FCS, ICS,RICS sical phenomena they ❖ ❖ http://www.lfd.uci.edu/workshop/2013/ Fluctuaciones coaccionadas por el movimiento de las partículas Se requiere un volumen de observación reducido ❖ Parámetros registrados: ❖ Velocidad de movimento. ❖ Concentración de partículas. ❖ Cambios en la intensidad de la fluorescencia durante el proceso re observación, por ejemplo cambios de forma. Note que el espectro de las fluctuaciones creadas por el movimiento de las partículas es “plano”mientras que el espectro fluctuaciones en la fluorescencia no lo es. http://www.lfd.uci.edu/workshop/2013/ Análisis espectroscópico de moléculas fluorescentes https://www.dkfz.de/Macromol/research/fcs.html Factores que determinan la intensidad de la señal de fluorescencia What determines the intensity of the fluorescence signal?? This is the fundamental equation in FCS F t kQ = quantum yield and detector sensitivity (how bright is our probe). This term could contain the fluctuation of the fluorescence intensity due to internal processes Q d W C W(r) describes the profile of illumination t C(r,t) is a function of the fluorophore concentration over time. This is the term that contains the “physics” of the diffusion processes The value of F(t) depends on the profile of illumination! http://www.lfd.uci.edu/workshop/2013/ La definición de la función de autocorrelación The definition of the Autocorrelation Function F t F t G Photon Counts F t F t F t F t time Average Fluorescence t t+ http://www.lfd.uci.edu/workshop/2013/ La función deTheauto correlación Autocorrelation Function G(0) 1/N As time (tau) approaches 0 Diffusion In the simplest case, two parameters define the autocorrelation funct http://www.lfd.uci.edu/workshop/2013/ El efectoThe de la concentración de las moléculas en la curva de Effects of Particle Concentration on the autocorrelación Autocorrelation Curve Observation volume <N> = 2 <N> = 4 http://www.lfd.uci.edu/workshop/2013/ El efecto delThe tamaño las partículas en la curva de Effectsde of Particle Size on the autocorrelación Autocorrelation Curve Diffusion Constants 300 um2/s 90 um2/s 71 um2/s Slow Diffusion Fast Diffusion Stokes Einstein Equation: D k T r and MW Volume r Monomer > Dimer Only a change in D by a factor of 21/3, or 1.26 http://www.lfd.uci.edu/workshop/2013/ Recapitulando http://www.picoquant.com/applications/category/life-science/fluorescence-correlation-spectroscopy-fcs Correlación entre dos canales de fluorescencia Two Channel Cross correlation lfd The cross correlation ONLY if particles are observed in both channels Red filter Each detector observes particles with a particular color The cross correlation signal: Only the green red molecules are observed!! http://www.lfd.uci.edu/workshop/2013/ Sample Green filter La función de correlación cruzada Calculating the Cross correlation Function lfd Detector 1: Fi time t+t t Gij dFi t dF j t Fi t Fj t Detector 2: Fj time http://www.lfd.uci.edu/workshop/2013/ Parámetros celulares que se pueden cuantificar con FCS y FCCS PERSPECTIVE a Size Viscosity, hindered diffusion, membrane phase b Directed transport Membrane binding Stoichiometry G Affinity Oligomerization Bacia K, Kim SA, Schwille Pby (2006) cross-correlation spectroscopy in living cells. Nat Methods 3: 83–89 igure 1 | Parameters assessed FCSFluorescence and FCCS. (a) Applications of fluorescence autocorrelation (top) and c Espectroscopía de correlación de la fluorescencia por barrido espacial Fluctuation analysis: single point and scanning Single point FCS Time 0 Correlation 1 1.0 1.0 2 4 8 0.9 0.6 0.3 RICS Single point FCS Scanning FCS and RICS Shift (pixel) 0 Correlation 1.00 1 1.00 http://www.lfd.uci.edu/workshop/2013/ 2 0.66 4 8 0.14 0.00 Escaneo por entramado (RICS: Raster Image Correlation Spectroscopy) Raster Scanning lfd http://www.lfd.uci.edu/workshop/2013/ En RICS lainformation información temporal mediante el Temporal hiddenesinobtenida the raster-scan espacial image: escaneo the RICS approach lfd http://www.lfd.uci.edu/workshop/2013/ Relación entre el tiempo y el espacio en RICS lfd Prt The RICS approach for diffusion G RICS S Dt r Dt http://www.lfd.uci.edu/workshop/2013/ G http://www.lfd.uci.edu/workshop/2013/ Autocorrelation Adenylate Kinase EGFP Diffusion constants (um2/s) of AK EGFP in the cell (HeLa). At the membrane, a dual diffusion rate is calculated from FCS data. Away from the plasma membrane, single diffusion constants are found. http://www.lfd.uci.edu/workshop/2013/ Mapeo de las constantes de difusión de paxilina-EGFP http://www.lfd.uci.edu/workshop/2013/ Conclusions lfd Techniques FCS Temporal-ICM RICS Line-RICS RICS Time Res. Spatial Res. Correlación temporal sec m (Escaneo por <0.5 línea) Used to Study Protein aggregates Transmembrane proteins Soluble proteins ~2 m Correlación temporal Binding interactions en imágenes (detectores CCD) msec Soluble proteins <0.5 m sec-msec Binding interactions Resolución temporal de cada técnica http://www.lfd.uci.edu/workshop/2013/ ¡Gracias! FRET, FLIM, FRAP, FCS, RICS, … CIO- León Gto. Octubre 2015 Adán Guerrero [email protected] Biot ecnología EN MOVIMIENT REVISTA DE DIVULGACIÓN DEL INSTITUTO DE BIOTECNOLOGÍA DE LA UNAM Biotecnología en Movimiento Revista de Divulgación del Instituto de Biotecnología de la UNAM Ejemplar número: 2, julioagosto-septiembre de 2015 UN MODELO EXPERIMENTAL para estudiar la obesidad EL ÁCIDO FÓLICO y las plantas EMPRENDER con compuestos que pican LA DESAPARICIÓN de las abejas 2da. parte LA ECONOMÍA en el SIGLO XXI LAS NUEVAS TECNOLOGÍAS y los patógenos DEL PULQUE Y LA SEQUÍA en las plantas LOS INICIOS del IBt 2da. parte Unidad de Secuenciación Masiva y Bioinformática Disponible en: www.ibt.unam.mx NUMERO 2 JULIO-AGOSTO-SEPTIEMBRE DE 2015 www.ibt.unam.mx Principles of Fluorescence Spectroscopy Joseph R. Lakowicz Third Edition • Fluorescence Resonance Energy Transfer • • • • Fluorescence Lifetime Imaging Microscopy • • • • • Ch. 13. EnergyTransfer Ch. 14. Time-Resolved Energy Transfer and Conformational Distributions of Biopolymers Ch. 15. Energy Transfer to Multiple Acceptors in One,Two, or Three Dimensions Ch. 4. Time-Domain Lifetime Measurements Ch. 5. Frequency-Domain Lifetime Measurements Ch. 22. Fluorescence-Lifetime Imaging Microscopy Fluorescence Recovery After Photobleaching Fluorescence Correlation Spectroscopy / Raster Image Correlation Spectroscopy • Ch. 24. Fluorescence Correlation Spectroscopy Molecules 2012, 17, 4047-4132; doi:10.3390/molecules17044047 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Review Advanced Fluorescence Microscopy Techniques—FRAP, FLIP, FLAP, FRET and FLIM Hellen C. Ishikawa-Ankerhold 1,†,*, Richard Ankerhold 2 and Gregor P. C. Drummen 3,†,* 1 Ludwig Maximilian University of Munich, Institute of Anatomy and Cell Biology, Schillerstr. 42, Abstract: 80336 München, Germany 2 Carl Zeiss Microimaging GmbH, Kistlerhofstr.recovery 75, 81379after München, Germany (FRAP), the related …The techniques described here are fluorescence photobleaching 3 fluorescence loss in photobleaching localization photobleaching Bionanoscience and Bio-Imaging (FLIP), Program,fluorescence Cellular Stress and Ageingafter Program, Bio&Nano(FLAP), Förster or fluorescence resonance energy transfer (FRET) and the different ways how to Solutions, Helmutstr. 3A, 40472 Düsseldorf, Germany measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and † fluorescence lifetime imagingequally microscopy These authors contributed to this(FLIM). work. First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and * Authors to whom correspondence should be addressed; fluorescence, confocal, and multiphoton microscopy is given. E-Mail: [email protected] (H.C.I.-A.); Subsequently, these advanced microscopy techniques are introduced in more detail, with a practical [email protected] (G.P.C.D.); advantages they can bring to cell biological research.Tel.:+49-89-218075-873 (H.C.I.-A.); Fax: +49-89-218075-004 (H.C.I.-A.); Tel.: +49-211-2297-3648 (G.P.C.D.); Fax: +49-3222-240-7500 (G.P.C.D.).
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