Fluorescence. I. Fluorescence. Figure 1: Fluorometer

Transcription

1 luorescence I. luorescence h igure 1: luorometer The luorescence, = 2.3 I 0 Φ (θ)g(λ)εlc where I 0 is the intensity o the incient light; (θ) is the luorescence that reaches the etector; g(λ) is the eiciency o the etector; ε is the molar extinction coeicient; l is the pathlength.; c is the molar concentration; Φ is the quantum yiel Quantum yiel = where 0 The quantum yiel is essentially the emission eiciency o a given luorochrome. Once the luorophore, M, has absorbe a photon an goes to the excite state M*, it can ecay in a number o ways. The rate constants or the ierent processes are: t : thermal inactivation p : photochemistry (photobleaching) : luorescence Q : quenching Lecture 18 Photochemical reaction: luorescence 1 o 6

2 M + h M* N p An as a result, M* M + h t M = [M*] = + t + p + [Q]Q = [M*] + t [M*] + p [M*] + Q [M*][Q] M* + Quencher M + Q* Q* Q + Heat [ M*] [ M*] 0 Slope = - τ = 1/ ln [M*] = - t + ln [ ] [M*] Ln = - t + constant Two amino aci sie chains have substantial luorescence: Time tryptophan an tyrosine. A polar environment next to a luorescent group results in Φ an a non-polar environment results in Φ. Thereore the luorescence o the sie chains give inormation about the environment. Conormational changes that change the environment aroun a luorescent group can be etecte as a change in luorescence. Amino aci sie chains with reactive groups such as SH,, or COOH can be chemically labele with a luorophore an use to etect the changes in conormation, bining to ligans, etc.. Some co-actors such as NADH are naturally luorescent. NADH Lecture 18 Photochemical reaction: luorescence 2 o 6

3 Oxiation to NAD+ results in loss o luorescence. Thus one can ollow this changes in intact cells by measuring the luorescence. II. Examples o the use o luorescence: Lateral Motion an Vesicle usion The motion o proteins, lipis, etc. insie a cell or in a cell membrane can be etecte using luorescent molecules. The protein or lipi is tagge with a luorophore either chemically or with a luorescently labele antiboy. Processes such as enocytosis can be monitore as a unction o time. Lateral iusion in a membrane can be measure by photobleaching an area an measuring the rate o recovery o the luorescence. igure 2: Photobleaching experiment where a component in the plasma membrane is tagge with a luorescent ye. Then a small area is bleache with a laser beam o intensity strong enough to estroy the luorescent ye by wea enough to not estroy the cellular structures. The luorescence o the bleache patch recovers as tagge molecules iuse into the patch rom the rest o the membrane. The larger molecules move slowly than the smaller ones (Picture courtesy o NIH) Another experiment that can be use to monitor vesicle usion. Three ierent experimental protocols are illustrate below. (a) the outer lealets o the vesicles are ierentially labele with two ierent luorophores. (b) same but the inner lealets are labele (c) the vesicle contents are lebele. The mixing o the two ierent luorescent molecules is use to show mixing o the ierent compartments. Alternatively, 1 luorescent molecule an quencher can be use. Mixing woul iminish the luorescence. Below is the membrane usion rom Kason et al. (PNAS P P.11921, 2006) that utilize two ierent luorescent molecules. Lecture 18 Photochemical reaction: luorescence 3 o 6

4 III. Examples o the use o luorescence: Membrane Potential The potential ierence between insie an outsie o the cells can be measure by using a luorescent molecule that is amphipathic an has either a ipole or an asymmetric net charge allowing it to respon to the electric iel. Thus the electric iel avors the spanne state. in this state the environment aroun the luorophore changes resulting in changes in luorescence. Which orm woul you expect to be more luorescent? _ + igure3: The membrane potential can cause the charge luorophore to change location. Lecture 18 Photochemical reaction: luorescence 4 o 6

5 SP As psi Base on igure 3 an graph 1, Asorbe ( ) unerstoo quantitatively: Span ( ). This process can be ( G B E) RT [ Spanne] e where is ipole moment an is the electric iel. [ Asorbe] IV. Examples o the use o luorescence: Vesicle Permeation + Q Q E Sel-quenching ( Lecture 18 Photochemical reaction: luorescence 5 o 6

6 To be able to see, sel-quenching is use. X Detergent 100% % Time (X) Vesicles are ille either with (a) a high concentration o luorophore so that there is strong selquenching or (b) with a combination o luorophore an a quenching agent. Thus release o the vesicle contents will increase the luorescence either by (a) elimination o sel-quenching by ilution or (b) elimination o quenching by the quenching agent by ilution. V. Quantitation o Quenching 0 This is the quantum yiel without the quencher. Here inclues all the rate constants EXCEPT Q Q [Q] 0 Q[ Q] 1 An thereore Stern Volmer equation can be erive as: [ Q] Q 0 is the luorescence without the quencher. τ 0 is the time constant o the luorescence ecay without the quencher. A plot o 0 / vs [Q] yiels the Q. With this constant one can now unerstan/preict how the luorescence will change as [Q] changes. Lecture 18 Photochemical reaction: luorescence 6 o 6