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Single Molecule Fluorescence
Bulk experiment
fluorescence cuvette,
fluorophore ~10-6M
1015 molecules
Single macromolecule,
several fluorophores
Single fluorophore
Advantages of Single-Molecule Fluorescence
# molecules
Distribution
EFRET, r, If
Bulk experiment gives the same result in the 3 cases: the average value
Dynamics
Biochemical 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.
D
A
Donor Intensity
R06
I DA
E  1
 6
I D R0  d 6
Acceptor Intensity
The study of single molecules can also detect rare transitions that are difficult to detect
using bulk techniques.
Measured property
molecule #1
molecule #2
molecule #3
time
Observables
A single dye molecule can report on the host molecule to
which it is attached to in a number of ways.
quencher
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.
• 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
Intensity
• Fluorescence intensity and/or lifetime:
time
• 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
Fluorescence Anisotropy
absorption transition dipole moment
emission transition dipole moment
I//
excitation
polarizer
emission
polarizer
r (t ) 
I
Polarization:
P(t ) 
I|| (t )  I  (t )
I|| (t )  I  (t )
P
3r
2r
I|| (t )  I  (t )
I|| (t )  2I  (t )
Fluorescence Anisotropy
I//
I
Fluorescence anisotropy
r
I||  I 
I||  2 I 
I // 2r  1

I 1 r
r=1
Single molecules fixed during the measurement
I
I//
Bulk experiment
-0.5 < r < 1
(or single molecule experiment where the
molecule rotates freely in the time-scale of the
measurement (>1ms) )
I//
I
Probability of absorption and
emision is proportional to cos2
r = 0.4
r = -0.5
I /I=3
Effect of rotational diffusion
I
I//
Fluorescence anisotropy
r
ns-timescale
I||  I 
I||  2 I 
Depends on viscosity,
temperature, specific interactions
with the environment, shape and
volume of fluorophore.
Rotational diffusion depolarizes
the emission
0<r < 0.4=r0
1<I /I<3
assuming that the absorption and
emission dipole moments are parallel
 Bulk experiment
 Single molecule experiment where the molecule rotates freely in
the time-scale of the measurement (>1ms)
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, highefficiency collection optics, and detectors with high quantum efficiency and low dark noise.
Confocal scanning optical microscope
Wide field microscopy
Point detection using APDs (avalanche
photodiodes).
two-dimensional detectors such as CCD
cameras.
Good time resolution and sensitivity.
Several single-molecules can be detected
simultaneously.
Observation of only one molecule at the
time.
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
c
Total internal Reflection
reflected ray
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.
i = r
i = r
n1
n1
n2
n2
t
t= 90º
refracted ray
n1.sin i = n2.sin t
n1.sin c = n2
http://micro.magnet.fsu.edu/primer/techniques/fluorescence/tirf/olympusaptirf.html
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.
Confocal microscopy
Total internal reflection
APD 1
color beam splitter or
polarizing beam splitter
CCD
APD 2
laser
counts per second
sample mounted on a
piezo scanner
photobleaching
time
laser
Fluorophores and biological constructs
SO3
SO3
N
Ideal dyes for SMF have to possess as many as possible
from the following characteristics:
+N
-photostability
Cy3 (570 nm)
- high fluorescence quantum yield
-
SO3
SO3
- high extinction coefficient
N
+N
- small intensity fluctuations
Cy5 (650 nm)
SO3
NH 2
- absorb and emit in the visible
SO3
+
NH 2
O
COO
2 Li+
TAMRA (570 nm)
(H3C) 2N
- small size to introduce minimum
perturbations to the host molecule.
O2 scavengers reduce photobleaching:
Glucose
glucose oxidase
gluconic acid
+
O
N(CH 3)2
O2
H2 O 2
-
COO
H2 O 2
Alexa Fluor 488 (520 nm)
catalase
H2O + 1/2 O2
Immobilization of macromolecules to glass surfaces
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.
biotin-streptavidin
biotinylated macromolecule
streptavidin
glass surface
biotinylated BSA
> Ni-NTA (Nickel-nitrilotriacetic acid)- 6xHis tagged proteins
> antidigoxigenin- digoxigenin
Recent applications of SMF to biophysical research
Fluorescence from single diffusing molecules
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.
R6G dissolved in Tris buffer and in a
50/50 mixture of buffer and glycerol
J. Phys. Chem. B, 104 (6), 1382 -1390, 2000
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.
Fluorescence from single immobilized molecules
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
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
F1FO ATP Synthase:
F1FO-ATPase:
ATP
ADP + Pi
ATP
ADP + Pi
V
H

time
Stepping rotation of F1-ATPase visualized through angle-resolved single-fluorophore imaging.
Proc Natl Acad Sci U S A 2000 Jun 20;97(13):7243-7
Adachi K, Yasuda R, Noji H, Itoh H, Harada Y, Yoshida M, Kinosita K Jr.
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
(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°.
Recommended reviews
Single-Molecule Fluorescence Resonance Energy Transfer
Methods: A Companion to Methods in Enzymology vol. 25, No. 1, September 2001 pp. 78-86.
Single-molecule fluorescence methods for the study of nucleic acids
Taekjip Ha
CURR OPIN STRUC BIOL 11 (3): 287-292 JUN 2001
Measuring conformational dynamics of biomolecules by single molecule
fluorescence spectroscopy
Shimon Weiss
Nature Structural Biology Vol.7 Number 9- September 2000.
Fluorescence Spectroscopy of Single Biomolecules
Shimon Weiss
Science March 12 1999 pp.1676-1683