Single Molecule Fluorescence

fluorescence cuvette,fluorophore ~10-6M

1015 molecules

Bulk experiment Single macromolecule,several fluorophores

Single fluorophore

Advantages of Single-Molecule Fluorescence

Distribution

# m

ole

cule

s

EFRET, r, If

Bulk experiment gives the same result in the 3 cases: the average value

DynamicsBiochemical reactions can be monitored by temporal changes in ensemble FRET only if the reaction can be prepared in one state before initiating the reaction by an external trigger.

Acceptor Intensity

Donor Intensity

660

601dR

R

I

IE

D

DA

DA

The study of single molecules can also detect rare transitions that are difficult to detect using bulk techniques.

time

Me

asu

red

pro

per

ty

molecule #1

molecule #2

molecule #3

• Fluorescence intensity and/or lifetime:

Molecular-scale motion that brings a quencher in and out of close range of the fluorophore can be detected as a temporal fluctuation of the fluorescence intensity of lifetime.

Observables A single dye molecule can report on the host molecule to which it is attached to in a number of ways.

time

Flu

ores

cenc

eIn

tens

ity

quencher

• Energy transfer:

A small change in the distance between two sites of a biological molecule where the donor and acceptor are attached can result in a sizeable change in the efficiency of transfer. Structural changes of biological molecules or relative motion and interaction between two different molecules can be detected by changes in FRET.

• Fluorescence polarization:

The temporal variation in dipole orientation of a rigidly attached probe can provide information on the angular motion of the macromolecule.

Dye attachment via a flexible linker can be used to provide information on changes in its mobility.

fluorophore is fixed with respect to the macromolecule fluorophore rotation in ns-timescale

)(2)(

)()()(

||

||

tItI

tItItr

I//

I

Fluorescence Anisotropy

excitationpolarizer

emissionpolarizer

)()(

)()()(

||

||

tItI

tItItP

Polarization:

r

rP

2

3

absorption transition dipole moment

emission transition dipole moment

II

IIr

2||

||

Fluorescence anisotropy

I//I r

r

I

I

1

12//

Fluorescence Anisotropy

r = 1

Single molecules fixed during the measurement

I//I

-0.5 < r < 1

I//I

r = -0.5

(or single molecule experiment where the molecule rotates freely in the time-scale of the measurement (>1ms) )

Bulk experiment

Probability of absorption and emision is proportional to cos2

r = 0.4 I /I=3

Fluorescence anisotropy

II

IIr

2||

||

Rotational diffusion depolarizes the emission

0<r < 0.4=r0 1<I /I<3

Depends on viscosity, temperature, specific interactions with the environment, shape and

volume of fluorophore.

Effect of rotational diffusion

I//I

ns-timescale

Bulk experiment Single molecule experiment where the molecule rotates freely in the time-scale of the measurement (>1ms)

assuming that the absorption and emission dipole moments are parallel

Detection of Single Molecules

In order to be able to detect fluorescence from single molecules, the detected signal must exceed the signal from impurities in the solvent, glass coverslips, and optical components, as well as the signal associated with the dark current of the detector.

Therefore, it is necessary to use a small excitation volume to reduce the background, high-efficiency collection optics, and detectors with high quantum efficiency and low dark noise.

Confocal scanning optical microscope

Point detection using APDs (avalanche photodiodes).

Good time resolution and sensitivity.

Observation of only one molecule at the time.

Wide field microscopy

two-dimensional detectors such as CCD cameras.

Several single-molecules can be detected simultaneously.

Sensitivity and time-resolution not as good.

Confocal Microscopy

Basement membrane labeled with cy2 (green)Neurons labeled with cy3 (red)

http://www.atto.com/Carv/CarvSkinSection.htm

Evanescent waves are formed when sinusoidal waves are (internally) reflected off an interface at an angle greater than the critical angle so that total internal reflection occurs.The intensity of evanescent waves decays exponentially (rather than sinusoidally) with distance from the interface at which they are formed.

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/tirf/olympusaptirf.html

reflected ray

refracted ray

n1

n2

i = r

t

n1

n2

i = r

t= 90º

c

n1.sin i = n2.sin t n1.sin c = n2

Total internal Reflection

In fluorescence mode, having focused on the beads, the bead fluorescence is very difficult to distinguish because of the obscuring background fluorescence from the cheek cells. In TIRFM imaging mode, the image contrast is dramatically high, and beads can be observed easily.

laser

timecoun

ts p

er

seco

nd

photobleaching

APD 1

APD 2

color beam splitter or polarizing beam splitter

laser

sample mounted on a piezo scanner

CCDConfocal microscopy Total internal reflection

Cy5 (650 nm)

Alexa Fluor 488 (520 nm)

TAMRA (570 nm)

O NH2NH2

COO

+

-

SO3SO3--

2 Li+

N N

SO3SO3

+

--

N N

SO3SO3

+

--

Cy3 (570 nm)

O N(CH3)2(H3C)2N

COO-

+

Ideal dyes for SMF have to possess as many as possible from the following characteristics:

-photostability

- high fluorescence quantum yield

- high extinction coefficient

- small intensity fluctuations

- absorb and emit in the visible

- small size to introduce minimum perturbations to the host molecule.

Glucose gluconic acid

H2O2 H2O + 1/2 O2

O2 H2O2

glucose oxidase

catalase

Fluorophores and biological constructs

O2 scavengers reduce photobleaching:

Immobilization of macromolecules to glass surfaces

glass surface

biotinylated BSA

streptavidin

biotinylated macromolecule

> Ni-NTA (Nickel-nitrilotriacetic acid)- 6xHis tagged proteins> antidigoxigenin- digoxigenin

biotin-streptavidin

The most exciting promise of single-molecule fluorescence studies is the observation of conformational dynamics of biological molecules. This requires a long observation time and hence some form of immobilization of the molecules.If performed improperly, immobilization can perturb the integrity of the molecule.

Detection of fluorescence from fluorescein-labeled ss-DNA diffusing through a focused laser beam: (a) Tris buffer solution; (b) 1.6 × 10-10 M; (c) 1.6 × 10-9 M. J. Phys. Chem. B, 104 (6), 1382 -1390, 2000

R6G dissolved in Tris buffer and in a 50/50 mixture of buffer and glycerol

Fluorescence from single diffusing molecules

Recent applications of SMF to biophysical research

Ratiometric single-molecule studies of freely diffusing biomolecules Ashok A Deniz, Ted A Laurence, Maxime Dahan, Daniel S Chemla, Peter G Schultz, and Shimon Weiss . Annu. Rev. Phys. Chem. 2001. 52:233-253.

Protein-induced conformational changes of single RNA molecules measured using FRET. (a) An RNA three-helix junction folds upon the specific binding of ribosomal protein S15. Donor (D) and acceptor (A) dyes attached to two arms of the junction move closer to each other when the protein binds and FRET increases. The RNA junction was attached to a surface in a specific way using a well-known `molecular glue', biotin¯streptavidin binding.

(b) Fluorescence images of donor and acceptor dyes from dozens of single RNA molecules were obtained simultaneously. The donor image was colored green and the acceptor image was colored red, and their overlay image is shown. Green spots are due to protein-free RNA molecules and red spots, with high FRET, represent protein-bound, folded RNA

Fluorescence from single immobilized molecules

Real-time observation of single RNA molecule conformational changes on buffer exchange. Time traces (integration time, 5 ms) of donor (solid line) and acceptor signal (dotted line) on buffer exchange. [Mg2+] was alternated between 0 and 1 mM every 200 ms (starting from 0). Significant donor signal reduction is seen every time Mg2+ buffer is present. Vertical grids denote buffer exchange periods (400 ms). Three-point averaging was applied to reduce noise. Donor photobleaching is marked by an arrow.

Ligand-induced conformational changes observed in single RNA molecules Taekjip Ha, Xiaowei Zhuang, Harold D. Kim, Jeffrey W. Orr , James R. Williamson , and Steven Chu,

PNAS Vol. 96, Issue 16, 9077-9082, August 3, 1999

time

V

H

Stepping rotation of F1-ATPase visualized through angle-resolved single-fluorophore imaging.Proc Natl Acad Sci U S A 2000 Jun 20;97(13):7243-7Adachi K, Yasuda R, Noji H, Itoh H, Harada Y, Yoshida M, Kinosita K Jr.

F1FO ATP Synthase: ADP + Pi ATP

F1FO-ATPase: ATP ADP + Pi

(A) Sequential fluorescence images, at 167-ms intervals, of a single Cy3-F1 molecule. V, vertically polarized

fluorescence; H, horizontally polarized fluorescence. (B) Time courses of spot intensities for V and H in A. (C) Time courses of the polarization, P = (V   H)/(V + H), and total intensity, I = V + H, calculated from B. The fluorophore photobleached at 55 s. Dashed lines (a, b, and c) are calculated P for the three orientations in D: P = 0.4 × [sin2( + 18°)   cos2(  + 18°)], where  = 0°, 120°, and 240°.

The fluorophore Cy3 attached to the

subunit of F1-ATPase revealed that the

subunit rotates in the molecule in discrete 120° steps and that each step is driven by

the hydrolysis of one ATP molecule

Single-Molecule Fluorescence Resonance Energy TransferMethods: A Companion to Methods in Enzymology vol. 25, No. 1, September 2001 pp. 78-86.

Single-molecule fluorescence methods for the study of nucleic acidsTaekjip HaCURR OPIN STRUC BIOL 11 (3): 287-292 JUN 2001

Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopyShimon WeissNature Structural Biology Vol.7 Number 9- September 2000.

Fluorescence Spectroscopy of Single BiomoleculesShimon WeissScience March 12 1999 pp.1676-1683

Recommended reviews