Professional Documents
Culture Documents
=
Numerical aperture
27
http://www.microscopy.fsu.edu/primer/java/imageformation/rayleighdisks/index.html
Spectrum of Visible Light
Color Wavelength
violet 380450 nm violet 380450 nm
blue 450495 nm
green 495570 nm
yellow 570590 nm
orange 590620 nm
red 620750 nm
28
http://en.wikipedia.org/wiki/Visible_spectrum
http://science.hq.nasa.gov/kids/imagers/ems/visible.html
Electron Microscopy
The same principles of optics
applies to light microscopy and
electron microscopy.
Wavelength of an electron
beam: ~0.002-0.004nm
Resolution:
TEM: ~0.1-1nm or
SEM: ~10nm
Lower resolution due to the
small NA achievable
29
Different Electron Microscopy Modalities
Two major EM modalities
- Transmission electron microscopy
- Scanning electron microscopy
Transmission electron Transmission electron
microscopy
- High resolution: up to ~1
Samples must be fixed - Samples must be fixed
- Samples must be sectioned
into thin slices (~50-100 nm)
Specific molecules can be
identified using antibody
j t d ld ti l
TEM reveals internal structure.
30
conjugated gold particles.
Different Electron Microscopy Modalities
Scanning electron microscopy
- Lower resolution than TEM; typically 10nm
Oft d t i l l - Often used to image large samples
- Surface scan
- 3D visualization
31
SEM reveals surface structure.
Cryo-EM; 3D Reconstruction
Preservation of samples by rapidly freezing the sample
to a very low temperature to a very low temperature.
Electron-tomography: g p y
- Images from different angles are integrated for 3D reconstruction
in a tomography fashion.
- The crystal structure, if available, can often facilitate this
process.
32
Hepatitis B virus
Comments: Methods in Cell Biology
Cell biology is experimental science and is strongly
dependent on technological advancement. dependent on technological advancement.
Importance of protocols.
- It is common to see different or even conflicting results It is common to see different or even conflicting results
in different studies on the same subject.
Importance of control experiments Importance of control experiments.
Cultural differences from engineering
33
Outline
Basics of cell biology literature reading
Imaging (I): light microscopy
Imaging (II): electron microscopy Imaging (II): electron microscopy
Basics of the diffusion theory
34
References
Howard Berg, Random Walks in Biology,
Princeton University Press 1993 Princeton University Press, 1993.
Jonathon Howard, Mechanics of Motor Proteins Jonathon Howard, Mechanics of Motor Proteins
and the Cytoskeleton, Sinauer Associated, 2001.
35
Introduction
Cell lar molec les are s bject to thermal force d e Cellular molecules are subject to thermal force due
to collisions with water and other molecules.
The resulting motion and energy are called thermal
motion and thermal energy.
36
Movement of a Free Molecule (I)
The average kinetic energy of a particle of mass m
and velocity v is
2
1
2 2
x
kT
mv =
Boltzmann constant=1.38110
-23
J/K
t
K
= t
C
+ 273.15
where k is Boltzmann's constant and T is absolute
temperature (Einstein 1905)
2 2
temperature (Einstein 1905).
Principle of equipartition of energy
2
1 3
2 2
kT
mv
=
37
Movement of a Free Molecule (II)
Molecular mass of GFP is 27 kDa. One atomic mass unit (Da) is
1.660610
-24
g. So the mass of one GFP molecule is 4.4810
-20
g.
At 27 degree C, kT is 4.14 10
-14
gcm
2
/sec
2
.
2
961 3 cm/sec
x
kT
v .
m
= =
38
1D Random Walk in Solution (I)
Assumptions:
(1) A particle i has equal probabilities to walk to the left and to the right.
(2) Particle movement at consecutive time points are independent.
(3) Movement of different particles are independent.
(4) Each particle moves at a average step size of =v
x
( ) ( )
1 o
( ) ( )
1
i i
x n x n o =
( ) ( ) ( )
( ) ( )
1 1
1 1
1
1
1 1
N N
i i
i i
N
i
x n x n x n
N N
x n x n
o
= =
= = (
= =
39
walk remains at the origin.
1D Random Walk in Solution (II)
Property 2: The mean square displacement of a particle
undergoing random walk increases linearly w r t time undergoing random walk increases linearly w.r.t. time.
( ) ( ) ( ) ( )
( )
2 2 2 2
1 1
2 2
1 1
1 2 1
1
N N
i i i
i i
x n x n x n x n
N N
o o
o
= =
( = = +
( )
2 2
1 x n o = +
( )
2 2 2
2
t
x n n Dt o o
t
= = = ( ) ( ) ( )
2 2 2
4 r n x n y n Dt = + =
P t 3 Th di l t f ti l f ll l
t
( ) ( ) ( ) ( )
2 2 2 2
6 r n x n y n z n Dt = + + =
Property 3: The displacement of a particle follows a normal
distribution.
40
1D Random Walk in Solution (III)
( )
( )
! 1 1
! ! 2 2
k n k
n
p k;n
k n k
=
( )
( )
( )
2
2
2
2
2
1
where and
4 2
2
k
n n
p k e
o
o
to
= = =
( ) ( ) ( )
2 x n k n k k n o o = = (
( ) ( )
2 0 x n k n o = =
4 2
2to
( )
( ) ( )
2 2 2 2 2 2 2 2 2
4 4 2 x n k k n n n n n n n o o o = + = + + =
( )
2
2
4
1
where 2
4
x
Dt
p x e n Dt
Dt
o
t
= =
41
Application of the Microscopic Theory (I)
Object Distance diffused
1 m 100 m 1 mm 1 m 1 m 100 m 1 mm 1 m
K
+
0.25ms 2.5s 2.510
4
s
(7 hrs)
2.510
8
s
(8 yrs) (7 hrs) (8 yrs)
Protein 5ms 50s 5.010
5
s
(6 days)
5.010
9
s
(150 yrs)
Organelle 1s 10
4
s 10
8
s 10
12
s g
(3 hrs) (3 yrs) (31710 yers)
K+: Radius = 0 1nm viscosity = 1mPas
-1
; T = 25C; D=2000 m
2
/sec K+: Radius = 0.1nm, viscosity = 1mPa s ; T = 25 C; D=2000 m /sec
Protein: Radius = 3nm, viscosity = 0.6915mPas
-1
; T = 37; D = 100 m
2
/sec
Organelle: Radis = 500nm, viscosity = 0.8904mPas
-1
; T = 25C; D = 0.5 m
2
/sec
42
Application of the Microscopic Theory (II)
Pure diffusion
x
2
>
Diffusion with external flow
Pure diffusion
a
c
e
m
e
n
t
<
u
a
r
e
d
i
s
p
l
a
M
e
a
n
s
q
u
Diffusion in a cage
H. Qian, M. P. Sheetz, E. L. Elson, Single particle tracking:
analysis of diffusion and flow in two-dimensional systems,
Biophysical Journal 60(4):910 921 1991
t
43
Biophysical Journal, 60(4):910-921, 1991.
Questions ?
44
Macroscopic Theory of Diffusion (I)
Fick's first equation
( ) ( )
1
2
N x N x o + (
( ) ( )
1
(
( ) ( )
( ) ( )
0
2
0
1
2
1
2
x
F N x N x / A
N x N x
A A
lim
lim
o
o
o t
o
o
t o o o
= + (
+ (
=
(
( ) ( )
0
1
D C x C x
C
D
x
lim
o
o
o
= + (
c
=
c
45
Macroscopic Theory of Diffusion (II)
Fick's second equation
( ) ( ) ( ) ( )
1
x x
C t C t F x F x A
A
t o t
o
+ = + ( (
( ) ( ) ( ) ( )
( ) ( )
1 1 1
1
x x
C t C t F x F x A
A
F x F x
t o t
t t o
o
+ = + ( (
= + (
( ) ( )
x x
F x F x o
o
+ (
2
x
F C C
D
c c c
2
x
D
t x x
= =
c c c
46
Calculation of Diffusion Coefficient
2
2
2m
o
Einstein-Smoluchowski Relation
1 1
2 2
x
d
F
v a
m
t t = =
2
2
2
2
2
x x
d
m
F mv m kT
f
v D D
t
o t
t
= = = = =
kT
D
f
=
f: viscous drag coefficient
Stokes' relation: the viscous drag coefficient of a
sphere moving in an unbounded fluid
6 f r tq =
q: viscousity
r: radius
47
An example of D calculation
Calculation of diffusion coefficient
6
kT
D
r tq
=
k=1.38110
-23
J/k=1.381 10
-17
Nm/k
q
T = 273.15 + 25
q=0.8904mPas=0.8904 10
-3
10
-12
Nm
-2
s
r= 500nm=0 5m r 500nm 0.5m
D=0.5 m
2
/s
48
An example of direct measurement of D An example of direct measurement of D
M. B. Elowitz et al, Protein mobility in the cytoplasm of E. coli,
J B t i l 181 197 203 1999 J. Bacteriology, 181:197-203, 1999
49