Experimental methods in Biophysics
Lecture 1: Techniques for observing single molecules
Outline• Lecture 1: Techniques for optical detection of
individual biomolecules
Localizing single molecules
Tracking single molecules in concentrated solutions
Measuring dynamics in single molecules
• Lecture 2: Techniques for imaging single molecules in crowded environments
• Lecture 3: Techniques for manipulating single biomolecules
Distribution-useful if population is
heterogeneous.
time
Time trajectory-useful if the dynamics is
not synchronizable
Ultimate Sensitivity
Why study single molecules
• At most one molecule per detection volume
• Signal > Background
Laser
wavelength
S1
S0
T1
laser
fluorescence
kR
ISC
kISC
1-10 nsec
<1%
How to optically detect single molecules?
Life history of an excited state electron in a
luminescent probe
Localizing Single molecules with high resolution
Image of a point source
Will a geometrically perfect lens generate a mathematical point
focus image from a point source?
What is the size of the blur spot or Point Spread Function (PSF)?
D = 0.61λ/NA
D = center to zero distance
Numerical aperture
Numerical Aperture and PSF
D = 0.61λ/NA
Accuracy and Resolution
Accuracy: minimum distance or volume that one can locate a
particle’s position within a certain time period. Current state of
the art = 1 nm accuracy in 1–500 ms.
Resolution: minimum distance or volume that can be measured
between two (identical) particles in a given period of time. For
visible fluorescence in the far-field, it is λ/2 or 200–300 nm.
However, with modern super-resolution methods, the optical
resolution is 8–25 nm.
Fluorescence Imaging with One Nanometer Accuracy
(1.5 nm, 1-500 msec)
Fluorescence from a single dye
Width = λ/2NA
0
40
80
120
160
200
240
280
05
1015
2025
510
1520
25
Photo
ns
X DataY axis
Prism-type TIR 0.2 sec integration
Z-Data from Columns 1-21
center
width
Enough photons (signal to noise)…Center determined to 1 nm.
Center ≈ width/√N
Accuracy depends on how well you can locate the center of the
fluorescence distribution.
Important Parameters in determining accuracy
1. Number of photons (Signal) must be high. Center of PSF can be
located to within width/√N.
2. Size of detection pixel. If pixel is too large, you cannot resolve the
PSF. If pixel is too small, the signal-to-noise ratio diminishes and the
PSF may become anisotropic.
3. Background noise from the detector or from the sample must be low.
The former can be essentially eliminated with electron multiplying
charge coupled devices (EMCCDs). Autofluorescence can be greatly
minimized using Total Internal Reflection Microscopy
4. Photobleaching time of the fluorophore must be large
Total Internal Reflection microscope
Wide-field
Objective TIR
Laser
Objective
Filter
Dichroic
Sample
CCD
Detector
Lens
Wide-field, Prism-type,
Total Internal Reflection
Microscope
Sample
Laser
Objective
Filter
CCD
Detector
Lens
Calculating Super-Accuracy
Goal is to determine the center, or mean value of the distribution, μ=(x0, y0), and its
uncertainty, standard error of the mean (σμ). σμ tells you how well you can localize the
fluorophore.
Relation between σμ and the number of collected photons (N), the pixel size of the
imaging detector (a), the standard deviation of the background (b), and the width of the
distribution (standard deviation, si, in direction i) in two dimensions is given by
22
2222 812
Na
bs
N
a
N
s ii
i
where the index i refers to the x or y direction. The first term is the photon noise, the
second term is the effect of finite pixel size of the detector, and the last term is the effect
of background
Accuracy from a single dye
0
40
80
120
160
200
240
280
05
1015
2025
510
1520
25
Photo
ns
X DataY axis
Prism-type TIR 0.2 sec integration
Z-Data from Columns 1-21
center
width
PSF of an individual Cy3 dye with an integration time of 0.5 s. N=14,200 photons, a=86
nm, b=11, sy=122 nm, sx=125 nm. The expected σμ is 1.24 nm in each direction.
Photon noise only leads to σμ=1.02 nm, pixelation increases σμ to 1.04 nm, and
background noise increases σμ to 1.24 nm.
22
2222 812
Na
bs
N
a
N
s ii
i
Myosin V (Kinesin): Hand-over-hand or Inchworm?
By measuring head (foot)-step size, can differentiate models
Hand-over-hand: Head (foot) takes 74 (16.6) nm steps
16 nm
Adapted from Hua, Chung, Gelles, Science, 20028 nm 8 nm
Inchworm: Head (foot) takes 37 (8.3) nm steps
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
0
5
10
15
20
25
30
35
40
duration between adjacent steps (sec)
num
ber
of
step
s
0 5 10 15 20 25
200
300
400
500
600
700
800
900
1000
MyoV steps
Time (Sec)
Positio
n (
nm
)
72 74 76 780
1
2
no. of
ste
ps
step size (nm)
nm
nm
Myosin V Steps
Steps: 75,0,75,0...
0 10 20 30 40 50 60 70 80 90 100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
60 65 70 75 80 85 900
10
20
30
40 = 74.08 nm
= 5.25 nm
R2 = 0.99346
2 = 1.6742
Nmol
= 31
Nstep
= 227
histogram of 74-0 nm steps
step size (nm)
num
ber of
ste
ps
75.4 nm
82.4 nm
65.1 nm
83.5 nm
69.8 nm
64.2 nm
78.8 nm
70.0 nm
70.9 nm
79.1 nm
67.4 nm
68.8 nm
73.7 nm
70.7 nm
70.1 nm
68.9 nm
71.3 nm
72.0 nm
time (sec)
Pos
itio
n (n
m)
74 nm +/- 5nm
Strongly supports
hand-over-hand
Tracking single molecules in concentrated solutions
Detecting Single Molecules in reduced volume
Laurence, et. al. Science (2003), 299, 667 - 668
Conventional observation of single molecules require 10-12 to 10-9 M concentrations of
fluorophore in order to isolate individual molecules in solution. However, many enzymes
work at much higher ligand concentrations. Working at biologically relevant, micromolar
concentrations requires reduced observation volume. In addition, temporal resolution of
conventional approaches is often limited by the time it takes for molecules to diffuse out
of the observation volume, usually on the order of several hundred microseconds.
Confocal Microscopes TIR Microscopes Near Field Scanning Optical Microscopes
Solution: Zero Mode Waveguides (ZMW)
Zero Mode waveguides (ZMW
•Circular apertures fabricated on a thin layer of aluminum on a glass coverslip.
•ZMW exhibit a cut-off wavelength of λc=1.7d (where d is ZMW diameter), above which
no propagating guided modes exist in the metallic system.
• Light of wavelengths longer than λc decay exponentially along the length of the
aperture
•For smallest ZMW, the depth of decay is 10nm to 20nm. Effective volume is < 10-20 to
10-21 L
Zero-mode waveguides (ZMW)
Levene, et. al. Science (2003), 299, 682 - 686
How to measure dynamics of single molecules?
Fluorescence Resonance Energy Transfer
(FRET)
Fluorescence Resonance Energy Transfer (FRET):
conformational changes of single biomolecules
Distance dependent interactions between green and red light
bulbs can be used to deduce the shape of the scissors during
the function.
Energy
Transfer
DONOR ACCEPTOR
Energy transfer efficiency
6
0 )/(1
1
RRE
R0=50% transfer efficiency distance
3nm~7nm
“Spectroscopic Ruler”
AD
R0AD
D A 0 25 50 75 100
0.0
0.2
0.4
0.6
0.8
1.0
E
R (Å)
R0=50 Å
Fluorescence Resonance Energy Transfer
Calculating R0
Single Molecule Techniques; Paul Selvin and Taekjip Ha, CSHL Press
Choosing fluorophore pairs for FRET experiments
1. Photostable: Should emit millions of photons before photobleaching
2. Bright: high extinction coefficient and quantum yield
3. Show little to no intensity fluctuation
4. Excitable and emitting in visible wavelengths
5. Relatively Small
6. Commercially available in a form that can be conjugated to
biomolecules
Single RNA Folding via FRET
0 20 40 60 80 100 120 140 160 180 200
0
500
1000
1500
Fluo
resc
ence
Time (sec)
0 50 100 150 200
0.0
0.2
0.4
0.6
0.8
1.0
1.2
EFR
ET=I
Acc
epto
r/(ID
onor+I
acce
ptor)
Time (sec)
FR
ET
Effic
iency
Folding Unfolding
Analyzing conformational dynamics with single
molecule FRET
Single Molecule Techniques; Paul Selvin and Taekjip Ha, CSHL Press