This preview shows pages 1–5. Sign up to view the full content.
1
Biophysical Methods
Slide 1
Phase Contrast and EM
Phase contrast
objects consisting of brighter and darker
spots are amplitude objects.
Image
objective
condenser
bright
spot
dim
spot
low
intensity
high
intensity
Focal plane
=transform plane
object
Biophysical Methods
Slide 2
Phase Contrast and EM
In unstained biological objects light absorption is usually very weak
but a cell has features with different refractive index, different optical
density. Depending on their optical thickness they generate phase
shifts of the light waves.
Image
objective
condenser
phase object
Small
Phase shift
Large
phase shift
focal plane
=transform plane
E
(
z
=
0,
t
)
=
E
0
sin
!
t
,
=
2
"
f
=
2
"
c
#
Incoming wave (black)
1.0
0.5
0.0
0.5
1.0
6
5
4
3
2
1
0
!
E
(
x
,
y
,
z
=
t
)
=
E
0
sin
t
+
"
(
x
,
y
)
[ ]
Phase modulated wave (red)
This preview has intentionally blurred sections. Sign up to view the full version.
View Full Document2
Biophysical Methods
Slide 3
Phase Contrast and EM
Since the phase shifts are small, we can approximate:
1.0
0.5
0.0
0.5
1.0
6
5
4
3
2
1
0
!
The phase modulated wave (red)
E
(
x
,
y
,
z
=
0,
t
)
=
E
0
sin
!
t
+
"
(
x
,
y
)
[ ]
sin(
+
#
)
=
sin
$
cos
+
cos
$
sin
E
0
sin
%
t
+
&
(
x
,
y
)
[ ] =
E
0
sin
t
[ ]
cos
(
x
,
y
)
[ ]+
E
0
cos
t
[ ]
sin
(
x
,
y
)
[ ]
cos
(
x
,
y
)
[ ]
"
1,
sin
(
x
,
y
)
[ ]
"
(
x
,
y
)
E
0
sin
t
+
(
x
,
y
)
[ ]=
E
0
sin
t
E
0
(
x
,
y
)cos
t
[ ]
independent
of object
dependent
on object
Our eyes as well as our cameras only see light intensities.
How to make the phase change visible?
Using trigonometric relations and some tricks
Use the sine sum formula
Biophysical Methods
Slide 4
Phase Contrast and EM
phase object
To get the phase advance of the undisturbed light we insert a phase plate with an indentation
such that the unscattered light gets
π
/2 less phase delay
Image
objective
condenser
Small
Phase shift
Large
phase shift
Focal plane
=transform plane
1.0
0.5
0.0
0.5
1.0
6
5
4
3
2
1
0
!
E
0
sin
t
+
(
x
,
y
)
E
0
sin
t
E
0
(
x
,
y
)cos
t
[ ]
independent
of object
(unscattered)
dependent
on object
(scattered)
The trick is to shift the undisturbed light by a phase
π
/2 or
λ
/4
We give it a phase advance such that the scattered wave is delayed by
π
/2:
3
phase object
Image
objective
condenser
Small
Phase shift
Large
phase shift
Focal plane
=transform plane
1.0
0.5
0.0
0.5
1.0
6
5
4
3
2
1
0
!
E
0
sin
!
t
+
"
(
x
,
y
)
[ ]=
E
0
sin
t
[ ]+
E
0
(
x
,
y
)cos
t
[ ]
independent
of object
(unscattered)
dependent
on object
(scattered)
E
0
sin
t
+
#
(
x
,
y
)
[
] =
E
0
sin
t
[
]+
E
0
(
x
,
y
)cos
t
+
$
2
%
&
’
(
)
*
=
E
0
sin
t
[
E
0
(
x
,
y
) cos
t
+
cos
$
2
,
sin
t
+
sin
$
2
%
&
’
(
)
*
=
E
0
sin
t
[
]
,
E
0
(
x
,
y
)sin
t
[
]
=
E
0
1
,
(
x
,
y
)
[
]
sin
t
[
]
Thus we have converted the phase contrast into amplitude contrast
Biophysical Methods
Slide 6
Phase Contrast and EM
To get brighter images, we use a broad light source
illuminating an annular
diaphragm in the back focal plane of the condenser. The annular diaphragm
may be viewed as a ringshaped light source instead of the point source.
Accordingly the phase plate is also ringshaped
This preview has intentionally blurred sections. Sign up to view the full version.
View Full Document4
Biophysical Methods
Slide 7
Phase Contrast and EM
Phase contrast has been invented by Zernicke and has been awarded the
Nobel Prize in 1953. It is widely used to look at cells under the light
microscope but is also of great importance in electron microscopy.
This is the end of the preview. Sign up
to
access the rest of the document.
This note was uploaded on 03/29/2009 for the course A&EP 470 taught by Professor Lindau during the Fall '08 term at Cornell University (Engineering School).
 Fall '08
 LINDAU

Click to edit the document details