G0G77A: Biomolecular Interactions

I0D43A: Molecular Interactions

- Theories and Methods -

Fluorescence microscopy

Prof. Dr. Hideaki Mizuno

Microscopy
Microscope is an instrument to produce magnified images of small objects.
- Magnified image

- Separate details in an image

- Make details visible to eye/camera/screen

 製造手法を確立し、複式顕微鏡の性能を飛躍的に向上させた。

程度）
Optical Microscopes over the years
を観察して小さな部屋 を発見して、 
グラフィア出版）
程度）を作成して、微生物、細菌、

く）
© Science Source/PPS

アントニ・ファン・レーウェンフック
写真提供：学校法人北里研究所 （1632∼1723）
・生理学賞受賞）
1866- Carl
ロベルト・コッホ Zeiss
（左から
北里柴三郎（右端）
Jena
2 人目）と
（1908 年日本で撮影）
アントニ・ファン・レーウェンフックは織物商を営 © Jaroen R
むアマチュア研究者だったが、世界で初めて微生
コッホ（1843∼1910）
はドイツの医師、細菌学者。地方で医師を 物を観察するなど、多くの業績を残した。彼の使っ レーウェンフックの
務めながら、炭疽菌や結核菌、コレラ菌など多くの病原菌を発見。 た単式顕微鏡は 1 枚のレンズを 2 枚の板で挟んだ単 単式顕微鏡
現代細菌学の基礎を築いた。1905年ノーベル生理学・医学賞を受賞。 純な装置だが、その倍率は 270 倍にも達していた。

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程度）
Optical Microscopes over the years
を観察して小さな部屋 を発見して、 
グラフィア出版）
程度）を作成して、微生物、細菌、

く）
・生理学賞受賞）
1866- Carl Zeiss Jena

） 
 Principles governing rey tracking for a
convolve lens
OBJECT–IMAGE MATH 59

A ray passes through the center of

the lens remains undeviated.
(a)

A ray traveling parallel to the optical

axis and refracted by the lens passes
through the rear focal point.
(b)

A ray passing through the front focal

point is refracted and follows a path
(c)
parallel to the optical axis.

Intersection of these rays → image plane

(d)

4.7
this condition are as follows:

Perception of magnified image

• 2F > a > F. A real magnified image is formed. This arrangement is used for pro-
ducing the first real image in a microscope.
• a = 2F. This is a specialized case. Under this condition, b = 2 F also. A real image
is formed, but there is no magnification and M = 1.
focus
• a > 2F. focus
A real demagnified image is formed and M < 1.
real image
In the case of a microscope objective focused on a specimen, the image is both real
specimen
and magnified, meaning that the object is located at an object distance a between 1F
virtual image
and 2 F (2 F > a > F) (Figs. 4.5 and 4.9). Since the focused objective is very near the
specimen, we deduce that the focal length of the objective must be very short, only a
few millimeters. In the course of using the focusing dials of a microscope, the image
comes into sharp focus when the correct object distance a has been obtained, and we
focus focus
obtain the correct adjustment without even thinking about object and image distances.
In practice, focusing a microscope positions the image (the real intermediate image
In the case you’ll
specimen
take a photo, put (or real intermediate)
a camera here
 Finite optical system vs infinity optical system
finite optical system

specimen
real image

focus focus

infinity optical system tube lens

specimen
focus real image

focus
 Conjugate planes and Köhler illumination
FUNDAMENTALS OF LIGHT MICROSCOPY

Köhler illumination
(image forming light rays)

Fundamentals
Figure 1.5 of light microscopy and electronic imaging
2nd Ed.and
Conjugate Murphy
aperture&planes
Davidson, Wiley-Blackwell
in Koehler 2013
illumination. Arrows mark the conjugate focal
planes. Note the locations of four conjugate field planes (red arrows; left) and four conjugate
aperture planes (blue arrows; right) indicated by the crossover points of rays in the diagrams.
 (a)
Propagation of wavefronts(b)

re 5.10
structions of Huygens’ wavelets are used to describe the propagation of (a) planar and
Constructions of Huygens’ wavelets
92 DIFFRACTION AND INTERFERENCE IN IMAGE FORMATION
spherical wavefronts.

e a moment to study the construction for a diffraction grating with spacing d in

ure 5.11, which emphasizes the concept that the diffraction spots occur at angles
re there is constructive interference. At locations between the diffraction spots,
es vary by a fraction of a wavelength and destructively interfere. Although Huygens’
elet construction accounts for the locations of diffraction spots, it does not account
all aspects of the diffraction process. For example, the sum of all of the energy
ent in the luminous regions(a) of the diffraction pattern is (b) known to equal the energy

dent on theFigure
m the geometrical
planar
grating.
5.10
Constructions of Huygens’ wavelets
spherical
This is inconsistent with ray particle models and predictions
construction of wavelets
are used tothat photons
describe are distributed
the propagation uniformly
of (a) planar and
he diffraction screen, being annihilated where there is destructive interference. We
(b) spherical wavefronts.

r here a murky area where the wave and particle natures of light are difficult to
Constructive interference d sin θ = mλ
Geometrical determination of
ncile.
Take a moment to study the construction for a diffraction grating with spacing d in
Figure 5.11, which emphasizes the concept that the diffraction spots occur at angles
scattering angles in a diffraction grating
where there is constructive interference. At locations between the diffraction spots,
waves vary by a fraction of a wavelength and destructively interfere. Although Huygens’
wavelet construction accounts for the locations of diffraction spots, it does not account
for all aspects of the diffraction process. For example, the sum of all of the energy
present in the luminous regions of the diffraction pattern is known to equal the energy
incident on the grating. This is inconsistent with ray particle models and predictions
from the geometrical construction of wavelets that photons are distributed uniformly
on the diffraction screen, being annihilated where there is destructive interference. We
enter here a murky area where the wave and particle natures of light are difficult to Destructive interference
reconcile.
⎛ 1⎞
d sin θ = ⎜ m + ⎟ λ
⎝ 2⎠

re 5.11
metrical determination of scattering angles in a diffraction grating using the construction
 “double slit” experiment

https://web2.ph.utexas.edu/~coker2/index.files/diff.htm
https://qiita.com/onhrs/items/9bf83c61fcfcff43011f
 (a)
Abbe’s theory for image formation(b)

re 5.10 d sin θ = mλ
structions of Huygens’ wavelets are used to describe the propagation of (a) planar and
spherical wavefronts.
grating
Interference between 0th and
1st order
e a moment to study the construction for a diffraction grating with spacing d in higher order diffracted rays in
ure 5.11, which θ
d emphasizes the concept that the diffraction spots occur at angles
re there is constructive interference. At locations between the diffraction spots,
θ ofλa wavelength and destructively0th order the image plane generates
es vary by a fraction interfere. Although Huygens’
elet construction accounts for the locations of diffraction
all aspects of the diffraction process. For example,
spots, it does not account
1st order
the sum of all of the energy
image contrast and
f
ent in the luminous regions of the diffraction pattern is known to equal the energy
dent on the grating. This is inconsistent with ray particle models and predictions determines the limit of spatial
m the geometrical construction of wavelets that photons are distributed uniformly
he diffraction screen, being annihilated wherediffraction
specimen
plane (rear focalnatures
plane interference.
there is destructive
planeofoflightlens)
imageWe
planeto
resolution that can be
r here a murky area where the wave and particle are difficult
ncile. provided by an objective.

re 5.11
metrical determination of scattering angles in a diffraction grating using the construction
higher numerical aperture (NA) is required to
achieve higher resolution
d sin θ = mλ

NA = nsin θ
refractive index
of media = n
θ
SPATIAL R

High frequency component

(small d) gives larger fringe f
pattern (large θ), and 1st order
can be outside of the lens.

A lens with larger NA covers

higher frequency component,
which means higher resolution.
Figure 6.2
Due to closer reflective
Effect of immersion index of oilthe to
oil on increasing coverslip,
angular extent over which diffracted
accepted by an objective. For dry objectives, NA is limited, because rays subte
refraction at thegreater
coverslip-air
than about 39° (angleinterface
formed at dotted lineiswitheliminated.
the normal) are lost by
reflection and never enter the lens (downward deflected red arrows). This com
- avoid total internal
an oil lens torefraction
acceptance angle of 67° in the case of an oil immersion lens and accounts fo
collect much larger angles of diffracted light. Note, however, that
maximum NA of 0.95 for the dry lens is actually calculated from the acceptance
- increase effective halfarrow).
(double-headed angleThe larger acceptance angle is due to the refraction
air : coverslip interface. Snell’s law, the critical angle for total internal reflection, a
tive indices of the glass, oil, and air are all that are required to calculate these
The newest TIRF objectives designed for use with special immersion oil have
up to 1.49.

Diffraction pattern of a point source of light
(point spread function)
TION AND INTERFERENCE IN IMAGE FORMATION
observed to consist of a central spot or diffraction disk surrounded by a se
fraction rings. In the nomenclature of diffraction, the bright central spot is
0th order diffraction spot, and the rings are called the 1st, 2nd, 3rd, . . . . ord
tion rings (see Fig. 5.4). When the objective is focused properly, the intens
the center circle is
“Airy disc”

(a )

0.61λ 1.12 λ
NA NA
(a) (b)

7
angle determines the size of the diffraction spot. Point source P and its conjugate 1.12 λ 0.61λ
n the image plane. Point P″ is moved laterally in the focal plane away from P′ until − −
Destructive
e interference Constructive
at a certain distance determines the location of the first diffraction
NA NA
and thusinterference interference
the radius of the diffraction spot. (a) Points A and B in the wavefront with (b )
perture. (b) Points A and B in the wavefront with reduced aperture angle caused
y stopping down the condenser iris diaphragm.
Resolution of Optical Microscopy
Rayleigh criterion
The limit at which two Airy disks can be resolved into separate entities is often called the
Rayleigh criterion.

λ: around 500 nm
Resolution max.NA: around 1.4
R = 0.61λ / NA
λ : wavelength
NA : numerical aperture
R ≈ 200 nm
Resolution of Optical Microscopy
NA and resolution
R = 0.61λ / NA

NA = 0.10 NA = 0.18 NA = 0.36

High NA : better resolution

Resolution of Optical Microscopy
Wavelength and resolution
R = 0.61λ / NA

wavelength = 400 nm wavelength = 550 nm wavelength = 700 nm

Short wavelength : better resolution

Colocalization = interaction?

CFP

YFP
 epifluorescence microscopes

upright inverted

Figure 1.1 Figure 1.2

The research light microscope with upright stand. Two lamps provide transmitted and reflected
The research light microscope with inverted stand. As in upright designs, two lamps pro
light illumination. Note the locations of the knobs for the specimen and condenser lens focus
transmitted and reflected light illumination. Note the locations of the knobs for the specim
adjustments. Also note the positions of two variable iris diaphragms: the field diaphragm near
the illuminator, and the condenser diaphragm at the front aperture of the condenser. Each and condenser lens focus adjustments, which are often in different locations on inverted mi
has an optimum setting in a properly adjusted microscope. Above: Nikon Eclipse 80i uprightscopes. Also note the positions of two variable iris diaphragms: the field diaphragm near
microscope; below: Olympus BX71 upright microscope. illuminator, and the condenser diaphragm at the front aperture of the condenser. Each has
Fluorescence Microscopy
Epi fluorescence microscopy

Advantage
• The objective, first serving as a well corrected
condenser and then as the image-forming light
gatherer, is always in correct alignment relative to each
of these functions.

• Most of the unwanted or unused excitation light

reaching the specimen travels away from the objective.

• The area being illuminated is restricted to the area

being observed

• The full NA of the objective is utilizable.

• It is possible to combine with transmitted light

observation.
 Two illumination modes for excitation
(infinity corrected optical system)
Khöler illumination Critical illumination
(for wide field microscopy) (for confocal microscopy)

If we focused the If we introduce parallel

excitation beam at the beam perpendicular to
back focal plane, we could the lens, we can
illuminate the specimen illuminate a single point
uniformly on the focal plane.
 Confocal microscopy
Critical illumination

The incident angle of the

excitation beam determine Pinhole position determined the
the focal position of the focal position of the emission
excitation

Adjust the the focuses of the excitation confocal

and emission to the same plane
PSF, wide field vs confocal
Rxy = 0.44λ / NA
Rlat = 0.61λ / NA

Rz = 2nλ / NA2
 Laser scanning to acquire 2D and 3D confocal
269
images
THE OPTICAL PRINCIPLE OF CONFOCAL IMAGING

THE OPTICAL PRINCIPLE OF CONFO

Figure 13.3
Optical pathway in a confocal microscope point-scanning unit. A laser beam is reflected by
the excitation dichromatic mirror (DM) onto the galvanometer scanning mirrors of the scan-
control mechanism, which sweep the beam in a raster pattern back and forth across the
specimen to excite fluorophores. The objective simultaneously collects fluorescent emission,
which is passed back to the scanning unit and descanned by the same galvanometer mirrors,
transmitted through the confocal pinhole, reflected from the appropriate emission
Figure 13.5dichromatic
mirror, and passed through an emission filter to the detection PMT. The The excitation
scan-controllight is
mechanism in a CLSM. The sketch shows the delivery of
colored green and the emission light is colored red (mixed together inlaserthebeam
objective). For
to the specimen by one of two galvanometer-driven mirrors that v
 Optical cross-section image can be acquired
by confocal microscopy
CONFOCAL LASER SCANNING MICROSCOPY
CONFOCAL LASER SCANNING MICROSCOPY

wide field confocal

autofluorescence in
a pollen grain

(a) (b)
(a) (b)

a thick section of rat brain

GFAP - Alexa568
neurofilament - Alexa488
Dapi (nuclei)

(c) (d)
 Cameleon, a genetically-encoded Ca2+ indicator

HeLa cell

10 µM histamine
Fluorescence

3
Ratio

0 500 1000
Time (msec)
PKC interacts with DG and stays on the
plasma membrane upon the stimulation

CHO-K1 cells (Chinese hamster ovary cells)

PKCγ labeled with eGFP
stimulation with 100 µM ATP
10 sec/frame
How large is the confocal observation volume?

Rxy = 0.44λ / NA

Rz = 2nλ / NA2
 How many molecules are there in the
illumination volume?

1M 10 µM 1 µM 0.1 µM 0.01 µM

1l 6 x 1023 6 x 1017 6 x 1016 6 x 1015 6 x 1014

1 fl 6 x 108 6000 600 60 6

0.1 fl 6 x 107 600 60 6 0.6

0.01 fl 6 x 106 60 6 0.6 0.06

Fluorescence correlation spectroscopy (FCS)

Count&Rate&R6G&&(kHz)&
46#

44#

42#

Count&Rate&(kHz)&
40#

38#

36#

34#

32#

30#
0# 50# 100# 150# 200# 250# 300# 350# 400#
/me&(s)&

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1.2$

1.15$

1.1$

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1$ 10$ 100$ 1000$ 10000$ 100000$ 1000000$ 10000000$
Relationship between behavior of molecules
and fluorescence signals
fluorescence

fluorescence
time time
Size of the fluctuation is
Speed of the fluctuation is related to “number” of
related to “size” and molecules
“shape” of molecules
fluorescence

fluorescence
time time
 Autocorrelation analysis
C (τ ) = I ( t ) ⋅ I ( t + τ ) δI(t) δI(tn)
δI(tn+τ)

fl intensity (I(t))
average fluorescence intensity δI(t+τ)
δI(t+τ)
difference in fluorescent intensity
〈I〉
from 〈I〉 at time “t”

t t+τ tn tn+τ
Since 〈δI(t)〉 and 〈δI(t+τ)〉 are average of time (t)
fluctuation, these becomes 0.

C (τ ) = δ I ( t ) ⋅ δ I ( t + τ ) + I
2

C (τ ) δ I (t ) ⋅ δ I (t + τ ) C(τ) 〈I〉2
G (τ ) = 2 = 1+ 2
I I
log τ
 What G(0) value means?
δ I (t ) ⋅ δ I (t + τ ) Poisson distribution
G (τ ) = 1+ 2 0.5
P(X = k) =
k −λ
λ ⋅e
I 0.4
k! λ=4

λ=3

Fluorescence intensity is proportional λ=2

P(X=k)
0.3 λ=1
to the number of fluorescent molecules
0.2

I = kN 0.1

δ N (t )
2

G ( 0 ) = 1+
0
0 1 2 3 4 5 6 7 8 9
2 k
N
expected value (λ)= average = variance
Since each molecule is
independent, and moving
randomly, number of molecules
shows Poisson distribution. From
the property of Poisson
G(τ)

distribution, the expected value

and also to its variance
N = δ N (t )
2

1
G ( 0 ) = 1+ Time (ms)
N
 Determination of diffusion coefficient from the
autocorrelation curve
δ I (t ) ⋅ δ I (t + τ )
G (τ ) = 1+ 2
I
Assuming the illumination area as a cylinder along the light
axis, with the radius of w and the half of the height of z.
The structure parameter s is determined to s= z/w.
For fluorescence fluctuation caused by translational
diffusion motion of molecules,
Rigler et al. introduced the following equation.
1

1⎛ 1 ⎞⎛ ⎞ 2
1
G (τ ) = 1+ ⎜ ⎜ ⎟
N ⎝ 1+ τ τ D ⎟⎠ ⎝ 1+ (1 s )2 (τ τ D ) ⎠

τ D = w 4D
2
D: translational diffusion coefficient
1
1⎛ 1 ⎞⎛ 1 ⎞ 2
G (τ ) = 1+ ⎜
N ⎝ 1+ 4Dτ w 2 ⎟⎠ ⎜⎝ 1+ 4Dτ z 2 ⎟⎠
Effect of number of molecules and size of
molecules to correlation function 1
1⎛ 1 ⎞⎛ 1 ⎞ 2
G (τ ) = 1+ ⎜
N ⎝ 1+ 4Dτ w 2 ⎟⎠ ⎜⎝ 1+ 4Dτ z 2 ⎟⎠
molecular number
G(τ)

molecular size

Time (ms)
② contains two components
Confocal set-up for FCCS
 Interacting molecules move together
Interacting molecules move together

Autocorrelation

Cross correlation
large overlap of
signals
Autocorrelation

Non-interacting molecules move separately

Autocorrelation

Cross correlation
small overlap of
signals
Autocorrelation
 Cross correlation analysis

cross correlation
δ I g (t ) ⋅ δ I r (t + τ )
Ggr (τ ) = 1+
Ig ⋅ Ir

Fluorescence

G(τ)
Time Time
Green fl

Fluorescence

G(τ)
Time

Time Time
Red fl

Time
Fluorescence cross correlation spectroscopy
(FCCS)
interacting molecules
Autocorrelation

Cross correlation
large overlap of
signals large correlation
Autocorrelation

non-interacting molecules
Autocorrelation

Cross correlation
small overlap of
signals
small correlation
Autocorrelation
 EGFR dimerization upon EGF binding
EGF EGF

EGFR EGFR

DG PIP2
RAS
IP3

PKC SOS
CHO cells expressing
Ca 2+ RAF
IP3R PLCγ Grb2
EGFR-GFP + EGFR-mRFP
MEK SW-FCCS Protein Dimerization Measurement
CaM blue: cross correlation
CaMK ERK

F
ti
c
m
ti
a
Liu, P. et al., Biophys J 93, 684–698 (2007)
was almost unchanged. Thus, although focusing m
o
re
Total internal reflection Microscopy: TIRF
providing so-called TIRF objectives with very large NAs. working-distance lenses for physiology experiments; there is greater freedo
ulating the is
angle of incidence of the laser beam to obtain TIR; and th
Modes of TIRF illumination
An alternative method that can be used to advantage on inverted microscopes
often improved, because the incident and return laser beams do not scatte
“prism-TIRF.” In this method, a small glass prism (triangle, trapezoid) with polished
objective.
optical surfaces is placed in contact with the coverslip with immersion oil, and a
laser beam is directed at the prism to give TIR at the coverslip surface (Fig. 12.12).
prism TIRF through the lensTIRF TIRF Objectives

TIRF illumination is commonly obtained by focusing a laser beam at on

rear aperture of a high NA objective. The reflected beam enters the othe
lens and exits back out the rear aperture (Fig. 12.13). This method of ill
called “through-the-lens-TIRF.” As described above, the beam must be del

(a) (b)

Figure 12.12
Modes of TIRF illumination. (a) Prism TIRF, and (b) through-the-lens TIRF.

Figure 12.13
Adjustment of the critical angle for TIRF illumination. The laser beam is focused
 TIRF MICROSCOPY: EXCITATION BY AN EVANESCENT WAVE 253

(b)

(a) (c)

Figure 12.10 RFP-paxillin

Principles of TIRF microscopy. (a) Cartoon of a cell on the surface of a coverslip labeled with
 PLCγ is recruited to EGFR in the plasma
membrane upon the stimulation
EGFR

pY783
P
P pY992 ?
2+
P
P
P pY1148
P pY1173
Ca
PLCγ PKC

under TIRF illumination

PLCγ labeled with eGFP
stimulation with EGF
5 sec/frame
 Single molecule imaging under TIRF illumination
405 nm

6
Single molecule detection
Susana Rocha et al.

. 7 Fitting of the PSF with a 2D Gaussian function. (a, c) PSF of a fluorescent bead. (a) Intensity plot. (c)
face plot. (b, d) The fitting result. (b) The fitted 2D Gaussian function is shown in light blue. The right and
tom are the y and x profiles of the fitting (data points are shown in black); the fitting is shown in red. (d)
face plot of the fitted 2D Gaussian
Fitting to 2D Gaussian
4. Drift correction.
function gives coordinates
The fluorescent beads are used as fiducial of
markers to compen-
sate movement of the sample during the data acquisition (see
the molecule Note 26).with
Drifting ofprecision in is corrected in
the positions in each frame
such a way that the points corresponding to the fiducial
tens of nanometer
markers are within a 10–20 nm precision (Fig. 8).
5. Image rendering.
Once all the positions have been calculated, the superresolution HeLa cells expressing EGFR-mEOS2
 Analysis of molecular dynamics of EGF
receptor by single particle tracking

labeled EGF binding to EGFR

De Keersmaecker, Rocha et al, Biophys Rev and Lett 8 p 229 (2014)

 0.03
05 0.03
A interactions between resident proteins.
R0R ITCIinteraction
T0.2
yAprotein C
L EL0.6SE 0.8
0.4 S 1.0kinet- 0 0.2 0.4
tors in
0.6 0.8 1.0
the absence ofprior work
the ligand describing
(QD-VHHs) ‘actina more
showed than19,25
corrals’ four,

Interacting molecule diffuse together

03 Such microdomains are consistent with
developed
0 0.2 0.4 0.6a0.8 mathematical times faster off-rate Supplementary 34 or ‘protein
Methodsislands’ 35.
and Supplementary
‘actin (see ‘lipid rafts’
1.0 19,25
prior work describing corrals’ ,
el (HMM) approach 33. This Video 2). We conclude
otion (Fig. 2h). This Our
‘lipid rafts’ two-color
34 or ‘protein islands’35.that
tracking andpreformed
three-state dimers
HMM areapproach
highlyFigure
transient,
resulted con- in
od a
Our
a
estimate
two-colorof b
the
b
trackingkinetic
and sistent
three-state with
HMM c
the
c lack
approach of d d
detectable
resulted in correlated motion
Figure
(Fig.
2
2g).
2 Direct
Direct
Dimers
visualization
visualization of of erbB1
erbB1
1 do form, as previ- improved QD-EGF quantification of erbB1 dimer off-rates (Fig. 3c). A summary of by two-color single-
QD-VHH
QD-VHH QD-EGF
QD-EGF dimerization is captured by two-color single-
QD-EGF dimerization is captured
improved
ates. Forquantification
a set of observa- of erbB1 dimer off-ratesof
composed (Fig.
one 3c). A summary of
ligand-bound erbB1 and
is one unoccupied receptor were
particle tracking. (a)(a) Sample time series
kinetic parameters derived from these data reported in Supplementary
particle
showing
tracking.
dimer
Sample
formation
time
(white)
series
between
okinetic parameters
receptor derived from
trajectories, the these data
also is reported
relatively in Supplementary
unstable, withTable
a two2. times fasterthe
off-rate showing
than dimercom-
dimers formation (white) between
Methods and Supplementary Methods
Table 2. and
Notably, Supplementary
the k values for ligand- Notably, k off values for
QD585-EGF–erbB1ligand-
QD585-EGF–erbB1 (green)
(green) andand QD655-
QD655-
hat reflect the underlying posed of two EGF-bound erbB1 molecules. Pretreatment
off of cells
EGF–erbB1 with(b)(b)Cartoon
(pink). Cartoon of of tracking
ncy
bound 0 shomodimers are50similar, bound homodimers
regardless of whether 3 s are
the similar,
dimers regardless
were of whether the dimers
EGF–erbB1
condition were
(pink).
(top), 3D trajectories
tracking
(middle) and
urately represent 50
0s
the data, latrunculin B (LatB; disrupts50actin) or nystatin (Nys; sequesters
3 s 50
condition (top),choles-3D trajectories (middle) and
cate
treatedthe presence
with the kinase 40or
40 treated
inhibitor. withthat
Dimers theformed
kinase inhibitor.
between recep- 40Dimers that formed between distance
distance recep-
between
between receptors
receptors (bottom)
(bottom) asas
a a
with free, co-confined and terol) did not markedly alter 40
dimer stability. Our experiments
function in HeLa
tors
in in the absencekinet-
interaction of30the
30 ligand (QD-VHHs) showed a more than four
tors in the absence of the ligand (QD-VHHs) showed a more of of
than time areare
four shown forfor
thethe indicated
Time (s)

Time (s)
30 function time shown indicated
Time (s)

Time (s)
30
on offaster
times a state representing
off-rate cells showed consistent trends,20with similar off-rates for
20 Supplementary Methods and Supplementary
(see 20 ligand-bound
receptors
receptors a.
in a. (c) Sample time series forfor
in (c) Sample time series
ped
n was a required
mathematical in10order timesdimers
to faster in off-rate
the (see Supplementary
absence
20
and presence Methods and
of PD153035. Thus, Supplementary
QD655-EGF–erbB1
EGF binding toandandQD585-VHH–erbB1
QD655-EGF–erbB1 QD585-VHH–erbB1
Video 4.52).
s We conclude
33. This that
10
preformed dimers are highly transient, con-
10
10
M)
ary4.5approach
Methods,
s
‘A two-state
0
sistent with the lack of115detectable
0
Video 2).
both We conclude
receptors
115 116 117 correlated
18 s
18 s
is that
required
motion (Fig. 2g). Dimers preformed
for the 0 most dimers
stable are highly
dimer transient,
show
show
formation.interactions con-
interactions for a 1:2 EGF–erbB1
for a 1:2 EGF–erbB1 dimer.
dimer.
(d)(d) Cartoon of of tracking condition (top),
3D3D
78 79 0 93 122 124 Cartoon tracking condition (top),
mate
ved erbB1
composed of the kinetic
behaviors’
of one sistent
116 X117
1.5,X anderbB1 and
ligand-bound
78 79
77with
77
the lack of detectable
Y one unoccupied receptor were
Y
93 94 94 95
correlated
1.5 X
95
X 120 motion
122 124 (Fig.
120
Y 2g). Dimers
trajectories
trajectories (middle)
(middle) andand distance
distance between
between

Separation distance (µm)

Y
Separation distance (µm)

1.5 1.5
or arelatively
set are
of unstable,
observa- composed of one ligand-bound erbB1 and one unoccupied receptor wereas a function of time

Separation distance (µm)

Separation distance (µm)

also with ato two times faster off-rate than dimers com- receptors (bottom) as a function of time
areare
domains considered receptors (bottom)
eposed
tor
and35 of twoprovide
EGF-bound
trajectories,
that the
a 1.0 erbB1
barrier also a
molecules.
relatively Interaction
Pretreatment
unstable, distance
of cells
with a with
two c
1.0 times faster off-rate thanfor
1.0
shown
shown forfor
dimers
a and
thethe
com-
receptors
receptors c. c.
in in Scale
Scale bars,
bars, 0.5m m
0.5
c. c. (e–h) Ensemble correlated motion
1.0
atrunculins
B (LatB; disrupts posed actin) orofnystatin
34 s
(Nys; sequesters choles- for a and (e–h)
34 s Ensemble correlated motion A431 HeLa
flect
35 s
the underlying two EGF-bound erbB1 molecules. Pretreatment plots ofsummarize
plots cells with
summarize allall two-color
two-color data
data forfor EGF,
EGF,
terol) did not markedly 0.5 alter dimer stability. Our experiments in HeLa
0.5 0.5 1.6 EGF+PD and VHH conditions. A decrease in in
ycells
represent
showed
the
by aconsistent
data, latrunculin B (LatB; disrupts
trends, with similar off-rates for ligand-bound
LA QD655 LAactin)
0.5
QD585 or nystatin (Nys; sequesters
EGF+PD choles-
and
uncorrelated
VHH
jump
conditions.
distance
A decrease
(blue)
cterized three-state uncorrelated jump distance (blue) at at short
short
e, co-confined
dimers
tes. (a)inDefinition
the absence and
of 0and 0 terol)
0presence
a dimer. 10 20 of30
did not
PD153035.
40 50
markedly alter dimer 0 stability. Our experiments in
separations HeLa
indicates that receptors
Thus, EGF binding0 to 0 10 1.220 30 40 50 separations indicates that receptors are moving are moving
together. A concurrent drop in jump magnitude
aboth
state
44 s representing each forcells
0
showed
b(s)stable consistent
10 20 30 40 50
trends, with 0
similar
Time (s)off-rates for ligand-bound
10 20 30 40 50

Off rate (s–1)

44 sreceptors
(LA) isfor
required the
Timemost dimer39 s formation.
ccuracies together. A concurrent drop in jump magnitude
ligand bound 2:2 short-lived 1:1
Time 39 s Time (s)
(s) (red) demonstrates decreased diffusion.
ents
e e
required
the areainEGForder
within
EGF which af
to f dimers in the absence
EGF+PD
EGF+PD
g and
g 3 - free presence
VHH
VHH
h
hof PD153035.
0.8
EGF&VHH
(red) demonstrates decreased diffusion.
EGF&VHH Thus, EGF binding to
State
ID) 0.09
ethods,is 0.09
a dimer
defined by the crystal
‘A two-state 0.09both receptors is
0.09
c required for the most
0.09
0.09 stable dimer formation.
0.09
erbB1 homodimer erbB1 homodimer
Displacement (µm)

0.09
Interaction distance
Displacement (µm)

und and the diameters

bB1 behaviors’
ee states
0.07
0.07
are defined , and as free,
0.07
0.07
0.07
A431
0.07 HeLa 0.07 0.4
0.07 to to free
free diffusion,
diffusion, causing
causing receptors
receptors toto deflect
deflect
ns are considered
d separation. Six kinetic to 0.050.05 Jump 1.6
Jump mag 1.24 offoffthetheboundaries,
boundaries,facilitating
facilitatingrepeated
repeated
(s–1) for a Uncorrelated c 0 A R T I Cinteractions
0.05 0.05 0.05
mag interactionsbetween
betweenresidentresidentproteins.
proteins.
hat
0.05 LA QD655
provide
states. a barrier
(c) Off-rates
LA QD585
jump
Interaction0.05
Uncorrelated
jump distance
distance
distance 0.05
L E S
Such microdomains are consistent with
0.8 1.0 Such microdomains are consistent with
0.03 0.03 0.03 0.03

H
F+GF
VHD
ys
F+GF
VH V D

F+at B
EG+L HH
D
1.2 0.6 0.8 1.0 0 0.2 0.4 0.6 A431 HeLa EG H HH
e is 0.03
defined as 0.4
the0.6 sum of
0.03 0.03 0.03

P
P

EG F& +P
0 0.2 0.8 1.0 0 0.2 0.4 0 0.2 0.4 0.6 0.8 1.0 19,25

N
prior work describing ‘actincorrals’
corrals’
19,25, ,
Off rate (s )

EG E
EG E

State 1 - 1.0
dimer 0 0.2 0.4 0.6 0.8 1.0
b 0 Separation
0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 1.0
F V
–1

distance ( µm) prior work describing ‘actin

domain–confined
dand
Separation distance

athree-state
aHeLa cells
and(µm)
are shown.
dimer-
0.8
LA QD655
State 2
co-confined
cLA71QD585
- 1.6
‘lipid
‘lipid
Condition
rafts’
rafts’
34
34 oror ‘protein
‘protein islands’
islands’
35
35. . Off rate: dissociation rate of
byunliganded
unliganded
State 3 - free
receptor demonstrated correlated motion
0.8receptor demonstrated correlated motion (Fig. 2h).
3 (Fig. 2h). This Our
This Our two-color
two-color tracking
tracking andand three-state
three-state HMM
HMM approach
approach resulted
resulted inin 0.738
Definition
shows that ofdimers
a dimer.
in inthethe ratio
of of1:21:2 EGF:erbB1 dodo form, as30as improved
previ- improved quantificationof of erbB1 dimer off-rates (Fig. 3c).
AA summary ofof respective molecule = k-1
Percent dimer events > 4s

1.2
shows that dimers ratio EGF:erbB1
0.4 form, previ- 30 quantification erbB1 dimer off-rates (Fig. 3c). summary
Off rate (s )

ously forreported 9. .
9
b
kinetic parameters derived from these data is reported in Supplementary
–1

es (LA) each
events

ously reported kinetic parameters derived from these data is reported in Supplementary
0.271
Separation distance (µm)

area VOLUME
within
0.6 which 18 aNUMBER 11 NOVEMBER 2011 NATURE STRUCTURAL Methods and &Supplementary
MOLECULAR Table BIOLOGY2. Notably, the k values for ligand-
Methods and Supplementary Table 2. Notably, the koffoff values for ligand-
k1
2s 9.5 s 0 42 s 25 20
0.8
Dimer stability is governed by ligand State occupancy
3 - free bound homodimers are similar, regardless of whether the dimers were
EGdimer

efined by the crystal bound homodimers are similar, regardless of whether the dimers were
H
F+GF
VHD
ys
F+GF
V V D

F+at B
EG+L HH
D
EGHH HH

Dimer stability is governed by ligand occupancy

P
P

EG F& +P

Although
1 - dimer correlated motion analysis can indicate 2 the presence or treated with the kinase inhibitor. Dimers that formed between recep-
E
EG E

State
F V

20
er and the diameters
State

Although correlated
0.4 motion
State 2 -analysis can indicate the presence or treated
10 with the kinase inhibitor. Dimers that formed between recep-
E+L EL
Percent of total

es absence
are defined of dimerization, it cannot
co-confined quantify
as free, it cannot quantify proteinCondition protein interaction kinet- tors
0.4 in the absence of the ligand (QD-VHHs) showed a more than four
absence of dimerization, interaction 15kinet- tors in the absence of the ligand (QD-VHHs) showed a more than four
ics. To extract dimerization kinetics, we developed a mathematical times
ation.
ics. To Six
model based
kinetic
extract dimerization23kinetics, s we developed a mathematical
upon a hidden Markov model (HMM) approach 10 33 . This Video
faster off-rate (see Supplementary Methods and Supplementary
times0faster off-rate (see Supplementary Methods and Supplementary
2). We conclude that preformed dimers are highly transient, con-
k-1
model 0.2
based
(c) Off-rates (s–1a) hidden
upon for Markov model (HMM) approach33. This Video 0 2).EGF VHH
We conclude that preformed dimers are highly transient, con-
method generates 2011 a NATUREa maximum likelihood& estimate
STRUCTURAL MOLECULAR of the 5 kinetic sistent
BIOLOGY sistent with the lack of detectable correlated motion (Fig. 2g). Dimers
H
F+GF

NOVEMBER with the lack of detectable correlated motion (Fig. 2g). Dimers
VHD
ys
F+GF
VH V D

F+at B
EG+L HH
D
EG H HH

method
ined as generates
the sum of maximum likelihood estimate of the kinetic
P
P

EG F& +P

rate constants for transitions between states. For1 a set of observa- composed of one ligand-bound erbB1 and one unoccupied receptor were
EG E
EG E

State 1 - dimer
F V

rate constants
–confined 0and fordimer-
transitions between states. For a set ofState observa-
0 2 - the composed of one ligand-bound erbB1 and one unoccupied receptor were
bles, in this case separation between two receptor trajectories, also relatively unstable, with a two times faster off-rate than dimers com-
bles,
LaHMM in this
cells are 0case
shown. separation
10 20between 30 two 40 receptor 50trajectories, 0 the 10also relatively
co-confined 20 unstable,40with a50
30 Condition two times faster off-rate than dimers com-
is used to identify hidden states that reflect the underlying posed of two EGF-bound erbB1 molecules. Pretreatment of cells with
HMM is used to identify hidden Time (s)states that reflect the underlying posed of Low-Nam,
Dimer two EGF-bound
duration (s)
T.erbB1 molecules. Pretreatment 18,of1244–1249
cells with (2011).
behaviors of the proteins. In order to accurately represent the data, latrunculin B (LatB;S. et al.
disrupts NatorStruct
actin) Mol
nystatin Biolsequesters
(Nys; choles-
behaviors of the proteins. In order to accurately represent the data, latrunculin B (LatB; disrupts actin) or nystatin (Nys; sequesters choles-
e z-direction
light path to be(Fig. 2a).with
conjugate Wethedecreased the The
specimen plane. illumination
image of illumination inclination increased the ratio of image to background
ess of a few micrometers derived from tary Fig. 3c). Reduction of the illumination diameter R furth
with
calof the
ditional inportin β interaction to nuclear pore
thestopreduction
the field
optics
illumination
thickness
of specimen
is formed at the
thickness
the illumination
R/tany
at the edge of
of a few
plane so that the
(Fig.
the field-stop
micrometers
(Fig. 1b and Supplementary Fig. 1a,b online).
diameter
image2b).
divergence R, (signal/background) up to 3.1–3.5-fold (Fig. 2e and Supplemen-
is minimizeddecreased the background intensity (Fig. 2d and Supplementar
derived from tary Fig. 3c). Reduction of the illumination diameter R further
f the
maximum of the profile wasis less than Fig. 3e). Consequently, reduction
intensityof(Fig.
the2ddiameter R increase
ullbeam
ow 20complexes observed under HILO illumination
Onegeometrical
width
of the features
mm.
always
optics
of HILO
passesmaximum
at half
thickness
microscopy
through the ofcenter
R/tany
that
theofprofile
the
the specimen
(Supplementary Fig. 1c–f), which means the illumination beam
(Fig. 2b).
illumination
was less plane
decreased the background and Supplementary
than Fig. 3e). Consequently, reduction of the diameter R increased
diameter Rz-directional
ignal/background below 20shift mm.
ratio of imagesplane. Thisin
HILO:
follows the highly inclined
of the specimen and feature is
imaging. ratio of images GFP-importin
valuated
powerful for
fluorescent the signal/background
three-dimensional
microspheres (Fig. 2c–e) in
a a β cc
o laminated
ways, using fluorescent opticalmicrospheres
sheet (Fig. 2c–e)
a 200
Epi
b

Number of molecules per NPC

Specimen HILO 200 20

Number of molecules per NPC

R 20
Coverslip Laser
Specimen 15
15
Oil θ beam
Specimen 150
150
Objective plane dz 10
10
lization of singlebGFP–importin
GFP–importin molecules and nuclearand nuclear
b molecules 55
Back Laser Coverslip
etic analysis
he interactions.
focal beam of the interactions. (a) Fluorescence
(a) Fluorescence image Objective of image of 100
100 00 0.1
plane x 0.1 11 1010
ortin
mediating b molecules mediating
the cargo the cargo
transport transport
atFluorescence
the bottom at the bottom
image
Lower
leus TIR (Supplementary
HILO
φ
Epi Video 2). (b) NPCs on the bottom of a
TIR 50 Lower
tary Video 2). (b) NPCs on the bottom of a
ars, 5 mm. (c,d) The number of molecules bound to a single
50 Higher affinity affinity
affinity
Higher affinity
he cnumber of molecules bound to a single 0
as the ratio of the fluorescence intensities against that of 0.1 1 10 100 1,000
es. fluorescence intensities against thatthe of NPC in 0
(c) Binding of cargo-free importin b with 0.1 10 β (nM) 100
1GFP-importin 1,00
argo-free sourcesbaswith
importin the NPC in importin b GFP-importin β (nM)
Ran
nurces
and energy
the incubation medium.
a function of the
b d
of Inset, an enlarged b graph near the
ng gave a sum of two binding functions (solid line), thus b d
as a function the importin 200

Number of molecules per NPC

20
medium. Inset, an enlarged graph near the
the interaction was composed of two types of binding, namely 200 15
two binding functions (solid line), thus

Number of molecules per NPC

150 20
binding (dotted red line) and lower-affinity binding (broken 10
asbarscomposed of two 155
indicate s.d.; n ¼ types
213, 134,of binding,
273, 286, namely235, 254, 128,
redforline)
Cs and lower-affinity
GFP–importin b ¼ 0.1, 0.3,binding
1, 2, 10,(broken
30, 100, 300 and Permeabilized
150
100
100 0.1 1 MDCK cells
10
5
GFP-importin β
2
;ctively.
n
ishima,¼ (d) Binding
213,
Shizuoka 134,
411-8540, of the286,
273,
Japan. number
Research 235, of for
Center cargo
254, (IBB)-bound
128,
Allergy and Immunology,
ent of Genetics, School of Life Science, The Graduate University for Advanced 10050 0 Lower
thebNPC in theMishima
absence No0.1
higher affinity
in
nstitute ¼Genetics,
of 0.1, 0.3, 2,of10,
1,Shizuoka Ran and 100,
30,
411-8540, energy
Japan. sources.
300
5Cellular
and Note that
Dynamics 1 10 affinity

ng
ressed to M.T.
inding
of the number
Error bars indicate s.d.; n ¼ 139, 239,
(mtoku@lab.nig.ac.jp).
disappeared.
of cargo (IBB)-bound in the 0 absence of Ran
50 0.1 1 10 100 1,000
008; DOI:10.1038/NMETH.1171
327, 207 and 258 NPCs for GFP–importin b ¼ 0.3, 1, 3, 10, Lower
bsence No higher GFP-importin
affinity β (nM)
d 1,000 of nM,Ran and energy sources. Note that
respectively. affinity
ed. Error bars NATURE indicate s.d.;| VOL.5
METHODS n ¼NO.2 139, | FEBRUARY
239, 2008 | 159 0
1 10 100 0.1 1,00
NPCs for GFP–importin b ¼ 0.3, 1, 3, 10,
O.2 | FEBRUARY 2008 | NATURE METHODS
ectively.
NPC GFP-importin β (nM)
Tokunaga et al. Nat Methods 5, 159–161 (2008).
 Summary

• Resolution of optical microscope is limited by diffraction

• Lens with larger NA value gives better resolution
• Achievable resolution is not enough to directly visualize interacting
molecules
• Signal from respective molecules is detectable if the molecular
density is low enough and background is effectively diminished
• If isolated single molecule is detected, its coordinates can be
calculated with certain precision
• Molecular interaction can be detected by making use of a property
that interacting molecule move together
• A fast-moving molecule is invisible due to motion blur, but it can
become visible once the movement is restricted by interacting with
slowly-moving molecule