Lecture 7: Fluorescence: Polarization and FRET Bioc 5085 March 31, 2014.

Slides:



Advertisements
Similar presentations
Big Question: We can see rafts in Model Membranes (GUVs or Supported Lipid Bilayers, LM), but how to study in cells? Do rafts really exist in cells? Are.
Advertisements

Three common mechanisms for bimolecular quenching
METO 621 Lesson 6. Absorption by gaseous species Particles in the atmosphere are absorbers of radiation. Absorption is inherently a quantum process. A.
Some structures Dansyl chloride 1,5-I-AEDANS Fluorescein isothiocyante ANS Ethidium bromide 5-[2-[(2-iodoacetyl)amino]ethylamino] naphthalene-1-sulfonic.
Today’s Topic: Lec 3 Prep for Labs 1 & 2 3-D imaging—how to get a nice 2D Image when your samples are 3D. (Deconvolution, with point scanning or with Wide-field.
Special Applications in Fluorescence Spectroscopy Miklós Nyitrai; 2007 March 14.
Lecture 3 Kinetics of electronically excited states
Today’s Topic (02/03/14) How did 1 st week of labs go? Questions? Comments? Polarization assays—important for  2 and for binding assays (Lab 2)
Oligonucleotides – Primers and Probes by … as quality counts! Competence and Service in Molecular Biology metabion´s history.
Methods: Fluorescence Biochemistry 4000 Dr. Ute Kothe.
Oligonucleotides – Primers and Probes by … as quality counts! Competence and Service in Molecular Biology metabion´s history.
125:583 Biointerfacial Characterization Oct. 2 and 5, 2006 Fluorescence Spectroscopy Prof. Ed Castner Chemistry Chemical Biology Prof. Prabhas Moghe Chemical.
Lecture 3 The Debye theory. Gases and polar molecules in non-polar solvent. The reaction field of a non-polarizable point dipole The internal and the direction.
Fluorescence Resonance Energy Transfer (FRET) Donor Fluorescence Acceptor Absorption INTENSITY WAVELENGTH (nm)
Measurement of Fluorescent Lifetimes: Time-Domain Time-Correlated Single Photon Counting (TCSPC) vs. Stroboscopic (Boxcar) Techniques.
Molecular Luminescence
Study of Protein Association by Fluorescence-based Methods Kristin Michalski UWM RET Intern In association with Professor Vali Raicu.
Lecture 4: Fluorescence UV/Visible and CD are Absorption techniques: The electron absorbs energy from the photon, gets to an excited state. But what happens.
Are You FRETting? Find Out for Sure With FLIM Frequency Domain FLIM for Your Scope Intelligent Imaging Innovations.
FRET(Fluorescent Resonance Energy Transfer)
(D) Crosslinking Interacting proteins can be identified by crosslinking. A labeled crosslinker is added to protein X in vitro and the cell lysate is added.
Fluorescence Techniques
On the orientation of substituents in N-methyl- and N, N-dimethyl- anilines in the excited state Narasimha H Ayachit
CSB Techniques Workshop May 2008 Fluorescence Methods Jeremy Moore.
Fluorescence Absorption of light occurs within ~ seconds, leaving a molecule in an excited state What happens next? –If no photon is re-emitted,
Lecture 5 Intermolecular electronic energy transfer
Fluorescence: Quenching and Lifetimes
Chapter 15 Molecular Luminescence Spectrometry Three types of Luminescence methods are: (i) molecular fluorescence (ii) phosphorescence (iii) chemiluminescence.
Fluorescence Depolarization Martin Cole, Faraz Khan Physics 200 Professor Newman
Two-Focus Fluorescence Correlation  Spectroscopy: A New Tool for Accurate and Absolute Diffusion Measurements Jörg Enderlein et al., ChemPhysChem, 8, 433–443.
Today’s Announcements 1.Today: finish FRET 2.Diffusion (Why moving in a cell is like swimming in concrete.) 2.Your written Paper, due Nov. 21 st. Today’s.
principle  measures the extent to which light is bent (i.e. refracted) when it moves from air into a sample and is typically used to determine the index.
Scanning excitation and emission spectra I Wavelength (nm) )Scan excitation with emission set at 380 nm -λ ex,max = 280 nm 2) Scan emission.
Electronic excitation energy transfer A Förster energy transfer demonstration experiment 4.
Today’s take-home lessons (i.e. what you should be able to answer at end of lecture) FRET – why it’s useful, R -6 dependence; R 0 (3-7 nm), very convenient.
5. Anisotropy decay/data analysis Anisotropy decay Energy-transfer distance distributions Time resolved spectra Excited-state reactions.
1.Individual Projects due today. 2.Outline of rest of course a.April 9: FRET b.April 11: Diffusion c.April 16: Diffusion II d.April 18: Ion Channels, Nerves.
23.7 Kinetics of photochemical reactions
Firohman Current is a flux quantity and is defined as: Current density, J, measured in Amps/m 2, yields current in Amps when it is integrated.
Chapter 5: Conductors and Dielectrics. Current and Current Density Current is a flux quantity and is defined as: Current density, J, measured in Amps/m.
Announcements Assignment due on Wednesday. I strongly suggest you come and talk to me. (but don’t leave it to the last minute!) Pick: 1) an article, 2)
Förster Resonance Energy Transfer (Chemistry/Biology Interface) Michelle, Pauline, Brad, Thane, Hill, Ming Lee, Huiwang Facilitator: Nancy.
Today’s Announcements 1.Next Tuesday: Diffusion (Why moving in a cell is like swimming in concrete.) 2. Homework assigned today Last graded Homework:
Förster Resonance Energy Transfer (FRET)
FRET 발표자 최예림.
Today’s Topic (02/02/15) How did 1st week of labs go?
Ch 10 Pages ; Lecture 24 – Introduction to Spectroscopy.
1.1 What’s electromagnetic radiation
Electronic Spectroscopy – Emission ( ) Fluorescence is the emission of light by a molecule in the excited state Fluorescence – Decay occurs between.
IPC Friedrich-Schiller-Universität Jena 1 Radiationless excitation energy transfer requires interaction between donor and acceptor  Emission spectrum.
Electrostatic field in dielectric media When a material has no free charge carriers or very few charge carriers, it is known as dielectric. For example.
Fluorescence Spectroscopy
Date of download: 7/7/2016 Copyright © 2016 SPIE. All rights reserved. The spectral overlap of Cerulean or mTFP with Venus is compared. The excitation.
Spectroscopy.
Midterm 2 (53 students wrote the exam)
Measuring Fluorescence Resonance Energy Transfer in vivo
Emission Spectroscopy and Molecular Motions in Biomolecular Systems.
Three common mechanisms for bimolecular quenching
Biophysical Tools '04 - Fluorescence
Fluorescence Lifetimes
Today’s take-home lessons: FRET (i. e
Today’s take-home lessons: FRET (i. e
Gil Rahamim, Dan Amir, Elisha Haas  Biophysical Journal 
Förster Resonance Energy Transfer (FRET)
Three common mechanisms for bimolecular quenching
Vassili Ivanov, Min Li, Kiyoshi Mizuuchi  Biophysical Journal 
Volume 107, Issue 3, Pages (August 2014)
The Effect of Dye-Dye Interactions on the Spatial Resolution of Single-Molecule FRET Measurements in Nucleic Acids  Nicolas Di Fiori, Amit Meller  Biophysical.
Single-Molecule Three-Color FRET
Fluorescence Polarization
Presentation transcript:

Lecture 7: Fluorescence: Polarization and FRET Bioc 5085 March 31, 2014

Flourescence Resonance Energy Transfer (FRET) 1 = Absorbance 2 = Emission 3 = Absorbance 4 = Emission D = Donor A = Acceptor FRET occurs and can be readily measured when: 1. Donor (D) has a high quantum yield (  f ) 2. Donor emission and acceptor (A) absorption spectra overlap (designed by J DA ) 3. Donor-acceptor distances are < 1.5 R o (R o is known as the Förster distance, see below)

Förster Theory and Förster Distances (R o ) Förster Distances = Distance between the donor and acceptor at which energy transfer is (on average) 50% efficient. According to Förster theory: 8.79 x is a combination of several physical constants  2 is the Förster orientation factor  is the refractive index of the medium (typically 1.4 for proteins)  d is the donor fluorescence quantum yield J DA is the Förster overlap integral All of the above can be determined experimentally, except for     is indeterminate since it depends not only on the orientation of the donor and acceptor dipoles, but also on the dynamics of these relative to one another   is theoretically 2/3 when the donor and acceptor fluorophores reorient isotropically relative to one another; this has been found to realized in most cases for probes that are not greatly restricted

FRET transfer efficiencies can be used to measure distances Importance of FRET to biochemistry is that the transfer efficiency, E, is a function of the separation of the fluors. Together with the known R o, E can be used to measure molecular distances:

Measurement of Transfer Efficiencies Measure Donor Emission Intensity ( 2 ) in the Presence and Absence of the Acceptor

FRET Donor-Acceptor Pairs: Trp as a Donor Wu & Brand, Anal. Biochem., 218, 1-13 (1994)

FRET Donor-Acceptor Pairs: “Attached” Donors and Acceptors Wu & Brand, Anal. Biochem., 218, 1-13 (1994)

Applications of FRET Wu & Brand, Anal. Biochem., 218, 1-13 (1994)

Fluorescence Polarization (FP) Polarizers transmit light that is either plane polarized along y or z (can usually be adjusted back-and-forth between these two positions).

Fluorescence Polarization (FP) Excitation Absorption Dipole I // II P //000  01 FP is based on selectively exciting molecules with their absorption transition moments (or equivalently absorption dipole) aligned parallel to the electric vector of polarized light (known as photoselection) Source “parallel” polarizer Absorption Transition Moment Angle Between Excitation & Emission Dipoles  0°  //101  000 0° // 0° Detect

Limiting Values of FP (+1 and -1) will never be obtained experimentally: 1)Assumed all dipoles in sample are identically aligned (not going to be true for liquid samples). 2) Assumed all molecules are fixed (not going to be true for liquid samples). 3)Assumed that the absorption and emission dipoles within fluorophore are collinear (not generally true). Factors that determine the extent of FP Limiting polarization (P o ) for molecules tumbling (isotropically) in solution is given by the Perrin-Weber Equation (addresses assumptions 1 and 3):  = Angle between absorption and emission dipoles

Extent to which molecule reorients relative to its fluorescence lifetime determines the extent of polarization (addresses second assumption)  = excited state lifetime  = rotational correlation time (i.e. rotational diffusion constant)  = solvent viscosity, V = volume of the fluorophore R = gas constant, T = temperature P  1/  hence, decreasing  will yield increased P P   hence, increasing  will yield increased P (   V and V  MW (by Stokes Law), hence P  MW)

Factors that effect extent of reorientation (and hence, extent of polarization) Figure 2. Simulation of the relationship between molecular weight (MW) and fluorescence polarization (P). Simulations are shown for dyes with various fluorescence lifetimes (  ): 1 ns (cyanine dyes) in purple, 4 ns (fluorescein and Alexa Fluor 488 dyes) in red, 6 ns (some BODIPY dyes) in green and 20 ns (dansyl dyes) in blue. At MW = 1000, P = for  = 1 ns, P = for  = 4 ns, P = for  = 6 ns and P = for  = 20 ns. Simulations assume P o (the fundamental polarization) = 0.5 and rigid attachment of dyes to spherical carriers. -Polarization increases as the MW increases (or as the solvent viscosity increases) -Polarization decreases as the excited state lifetime (  ) increases

FP Applications

Green Fluorescent Protein