Chapter 1 Microscopy with
Light and Electrons
1.1. Methods of Image Formation
1. Projection Image Screen
M=Ii/Io=v/u Magnification
Ii
Io
Base Line
Source Object Image
u
v
Fig. 1.1. Projection type of image formation
Projection Image formation in FEM
Fig. Field emission microscopy
(FEM) has been used to
investigate the field emission of
electrons from carbon nanotubes
(CNTs) and related fibers, viz.,
multi-wall nanotubes (MWNTs),
single-wall nanotubes (SWNTs),
nanofibers (named
“nanografibers”) grown in
hydrogen gas, and vapor grown
carbon fibers (VGCFs). Recent
observations of pentagonal rings
located on the capped tip of a
clean MWNT, and adsorption and
desorption phenomena of residual
gas molecules on the tip, are
presented. Developments of CNT-
based field emission displays are
briefly reviewed.
[Link]
Methods of Image Formation Used in Microscopy
1. Projection
2. Optical
3. Scanning
Optical image is formed by the system of
conventional lenses
Lenses can
• produce magnified or demagnified image of the object;
• focus parallel beam of light to a point (focus).
In our further consideration we will deal with convex and thin lenses only
Back focal plane
Main focus
Light
F F F F
Optic axis
Lens
A: Beams of the light parallel to the optical B: Beams, traveled non parallel to the optical axis
axis convent on the main focus Intersects in the point on the focal plane, but outside
of the optic axis
Fig. 1.2. Schematics showing geometry of the main focus and focal plane in optical system
Optical image: Ray diagram
Two fundamental rules:
1) Beam traveling parallel to the optical axis convents on the main focus;
2) Beam passing through the center of the lens is not affected by the lens.
Object field Image field
#2 u v
Ii v
Ao M = =
#1 C Io u
Io F2 Oi
Oo F1 O
Ii
D
Ai
f f
1 1 1 f v− f
= + Thin lens equitation (Newton) M = =
f u v u− f f
Principle of reciprocity (reversibility), Helmholtz: Rays trace the same path
through all optical systems in either direction
Fig 1.3 (a) The image is magnified, real and inverted if f < u< 2 f
Optical image: Ray diagram (continued)
#2
v
Image field
Ii
#1
Io F
Object field f f
Fig 1.3 (b) The image is magnified, up right but virtual if u < f
Optical image: Ray diagram (continued)
f v− f
M = =
u− f f
u v
#2
F Ii
#1
Io
F
Object field Image field
f f
Fig 1.3 (c) The image is demagnified, real and inverted if u > 2 f
Don’t’ forget that the lenses can be used not only to magnify the object but also to
perform an opposite function, demagnification. Different magnification and
demagnification can be performed with the same lens; position of the object plane will
control the lens magnification and its operation: magnification/demagnification.
Scanning Image: Image Formation
Scanning image is formed serially (each pixel is formed separately)
T1: X1, Y1, I1
l T2: X2, Y2, I2
T3: X3, Y3, I3
Specimen
Ti: Xi, Yi, Ii
L
Tn: Xn, Yn, In
L
M= We don’t need the lens to
magnify the object!
l
Cathode Ray Tube (CRT), TV
1.2. Two Lenses Light Optical System
Magnification of the object without severe distortion is very problematic with only one lens. The
higher magnification causes higher distortion. For higher magnification combination of lenses
is used.
F1 – 2cm
F2 – 1.3 cm
u1 v1 u2 v2 OF1 1.8 cm
L1L2 6.5 cm
f1 f2
F1 F2
O F1 F2 B
A
L1 O – object;
L2
A – intermediate image;
M = M1 M 2 =
(v1 − f1 ) (v2 − f 2 ) B - image
f1 f 2
Fig 1.4 Ray diagram of two-stage magnification optical system
Two Types of Optical Microscope
A. Transmission
B. Reflected
illumination
M=MoMp –total magnification
Condenser system don’t
magnify the object it only
controls: Fig. 1.5. The optical system for the two common types of
microscope.
- brightness of the image;
- aperture angle.
1.3. Magnification of the Optical Microscope
Total magnification of the optical microscope is the product of the magnification of the objective lens,
Mo, and projector lens, Mp:
M = Mo M p
( f o ) : 5,10,20,100 70
( f ): 5,7.5,10,12.5
p SEM
How big is real size of the object, l, appeared in the
microscope as 1cm section? We need to know
magnification of the objective and projector lenses we use. 300
Let’s assume Mo=100 and Mp=10
1cm 10 −2 m
l= = = 10 −5 m = 10m
M 100 10
1400
Magnification of regular optical microscope is usually never
higher than 1000X because resolution of human eye is
limited to ~0.2mm and resolution of the optical system
which uses light is ~200 nm. So useful magnification of the Light
light optical system can be calculated: optical
rh.e. 0.2 10 −3 2800
M useful = = 10 3
rl 200 10 −9
Fig 1.6. A series of light and SEM micrographs of the high temperature superconductor barium yttrium copper oxide
at increasing magnification. Original magnifications (a) 70; (b) and (d) 300; (c) and (e) 1400 and (f) 2800
1.4. Resolution of the Optical Image
Resolution, r, is the closest spacing between two points, which can be seen
through the microscope separated completely
screen
d
D
A. Diffraction. When the light passes through
the aperture it transformed into series of cones. C. The intensity of Airy’s rings from two
neighboring pinholes
screen Intensity
D. Reyleigh criterion: when Imax of the first ring
coincide with the first min of another ring two
pinholes will be resolved. So the resolution limit is
d d/2. From the diffraction theory resolution, r, can
be found as:
Where:
0.61 is the wave length of the light;
r= is refractive index of the medium:
sin
1.0003 (in air);
B. Airy’s ring will be formed on the screen. Central is an aperture angle (angle of
collection of the magnifying lens).
spot with diameter d has about 84% of whole intensity.
d~1/D. The effect of diffraction is stronger when the
size of the aperture, D, is smaller!
High resolution means that the value of r is small!
1.4. Resolution of the Optical Image (continued)
Fig. Airy diffraction patterns generated
by light from two points passing
through a circular aperture, such as the
pupil of the eye. Points far apart (top)
or meeting the Rayleigh criterion
(middle) can be distinguished. Points
closer than the Rayleigh criterion
(bottom) are difficult to distinguish.
[Link]
solution
1.5. Depth of Field of Optical Image
The range of the positions for the object, h, for which our eye can detect
no change in sharpness of the image is known as depth of field.
α
0.61
aperture
r
h= =
tan sin tan
h plane of
optimum focus
B. Conditions, which are maximizing h
simultaneously making resolution worse!
r
C. For the light microscope, where α ~ 45
the value of the depth of field is close to the
A. Since the diffraction effect will limit
resolution value: h r.
resolution at the specimen to r it will not
make difference in the sharpness of the
image if the object is anywhere within the
range of h shown on the figure
1.6. Resolution of Scanning Image
Image is formed serially: one pixel (picture element) at the time. Due to the high
frequency of the scanning beam (50 frames per second for the TV signal)
image appears to eye entirely. It is serial type of image.
l L
B. Resolution of scanning image is not better
than the size of the beam!
specimen
beam
L
M =
l specimen 1 2 1 2 1 2
CRT Pixels #1 and #2 are not resolved in the left
L 100m
M = = image. Generally size of the beam must be
l p smaller then the size of the pixel (shown in
the middle) on the sample to distinguish the
Where: contrast between two neighboring pixels.
100 m- size of the pixel on the CRT,
p – is the size of the pixel on the sample
A. In scanning image we don’t need lenses to magnify the object!
1.7. Depth of Field of Scanning Image
p = h is very small, usually less
than 2 deg., so tan
A
p = 100 / M A
sample =
WD 2 WD
plane of the
h optimum focus
0.2 WD
h= mm
p A M
Where:
p -size of the pixel on the sample;
Fig. 1.12. The depth of field in - aperture angle (usually a few degree);
scanning system of image A - aperture diameter (up to few hundreds of microns) ;
formation. WD - working distance (between few mm and few tens of mm);
M - magnification
The depth of field in the SEM can be easily up to several tens of microns at
magnification 1000X times. In general, it is at least two orders of magnitude better than
in light optical microscope.
Depth of Field: Optical and Scanning Image
A. Light optical microscope image B. SEM Image
of the diatom
1.8. Depth of Focus
Depth of focus is the range of the positions of the image when it will be viewed
without appearing out of focus, for a fixed position of the object.
2 ro
tan o = = o
ho
2 ri 2 ro M
tan i = = i
a hi hi
ro o i
ri a = v tan i = v i
a = u tan o = u o
ho v a o o
a M= = =
hi u i a i
2 ro M 2 ro M M
hi = = = ho M 2
u v i o
It is not so critical to know the value of the depth of focus to operate the microscope at
optimal settings, since this value is usually big
Fig. 1.15. The depth of field, ho, and the depth of focus, hi, of an optical system
Optical Image: Spherical Aberrations
Spherical aberration is the most important type of monochromatic aberration (affecting
single wave length). This defect is caused by the lens field acting non homogeneously on
the off-axis rays. The further off axis the beam of light is, the more strongly it is bent back
toward the axis. As a result, a point object is imaged as a disk of finite size, dS (disk of least
confusion), which limits our ability to magnify detail because the detail is degraded by
imaging process.
Lens C A- marginal focus
A B
CS=0 B- axial focus
C- disk of least
P α dS confusion
Fig. 1.16. Ray diagram illustrating spherical aberration Plane of least Gaussian image
confusion plane
dS=2CS·α3
Cs ~ 2 cm for the pinhole lens and SEM
Where;
Cs is coefficient of spherical aberration
Cs ~ 3 mm for conventional TEM and about
α is the aperture angle 1 mm for HRTEM
Optical Image: Spherical Aberrations (cont.)
Spherical aberration is the most important type of aberrations in EM because of the difficulty of its
correction! It is also very important in light optical systems. Hubble Space telescope (deployed in 1990) is
a good example of its correction. The curvature of 2.4 m mirror was 2 mkm (!) off. In 1994 NASA
announced that the new correcting optics installed on Hubble during servicing mission.
Fig. This comparison image of the core of the galaxy M100 shows the dramatic improvement in the Hubble
Space Telescope's view of the universe after the first servicing mission in December 1993. The original
view, taken a few days before the servicing mission, is on the left. Credits: NASA
[Link]
Optical image: Chromatic aberration
Chromatic aberration causes light to be deviated by an amount depending on its
wavelength. Thus a lens will have different focal point for light with different wavelength.
Plane of least
confusion
dmin
P α
White
Light
Blue light Red light
Lens focus focus
Fig. 1.17. Ray diagram illustrating chromatic aberration.
Light of the shorter wavelength (blue) is brought to a focus nearer to the lens than the longer
wavelength (red) light. The smallest spot size, dC is the plane of least confusion. There is always some
energy spread of electrons, E, in EM (about 0.3-3 eV) causing electrons having slight variation of their
wave length. Chromatic aberration in EM can be calculated: E
1/2·drCc = Cc
E0
Optical Image: Astigmatism
Astigmatism is related to the monochromatic aberration effect. It occurs when the focus
of the rays traveling in the horizontal plane is different from the focus of the rays traveling
in the vertical direction.
vertical
focal plane
horizontal
focal plane
plane of least
confusion
Fig. 1.18. Ray diagram illustrating the formation of astigmatism for a lens.
Astigmatism in SEM
A B Fig. 1.19. Astigmatism
adjustment is usually important
at high magnifications. This
figure shows images of the tin
balls sample taken at 20,000X
before (A,B and C) and after
(D) astigmatism adjustment in
the SEM. Due to astigmatism
operator has problems to adjust
image to its perfect focus. A.
Shows the image of tin balls
sample before the adjustment.
B. After the focus control is
turned slightly left (underfocus).
C D
All features on the image are
elongated in vertical direction.
C. The same conditions, but the
focus control is turned slightly
to the right (overfocus). All
features on the image are
elongated now in horizontal
direction. D. After proper
adjustment of astigmatism
image appears in perfect focus.
Optical Image: Barrel and Pincushion
(a) (b)
Fig. 1.19. The appearance of the square grid in presence of (a) barrel and (b) pincushion distortion.
Electron Versus Light
A. Light and electrons are electromagnetic radiation
B. Both type of description: wave and particles are applicable to light and electrons.
Differences:
Light Electron
Wave length, 400-700 nm 0.001-0.01 nm (1MV – 20kV)
Scattering by the In most cases can be Significant. Vacuum is required, usually
air neglected better than 10-4 Pa
Refractive index, 1.0003 in the air 1 (in the vacuum)
Aperture angles, Usually 20-50 Commonly a few degrees (less than 5 deg)
Aberration Compound lenses, Extremely difficult to correct. Operation with
correction filters small is required!
Depth of field Compatible with Extremely good in SEM
resolution
Charge No charge Negatively charged particles. Electro-
magnetic field can be used to control
(focus) electrons
Generally, electron microscopy offers higher resolution, magnification and greater
depth of field compare to the light microscopy.
Effect of the Aperture Angle on the Resolution of
the Optical System: Light and Electron Optical
r = rd + rs + rc - Real resolution of the optical (light and
electron) systems
E
rd = 0.61
rs = C s 3
rc = Cc (EOM)
E0
Effect of chromatic aberration is much smaller compare to rs and rd, so it can be neglected
in most cases.
Conditions for the aperture angle, , which reduces spherical aberration, makes
diffraction-limited resolution worse!
1 −1
opt = 0.67 Cs 4 4 Optimum aperture angle of the electron-optical
system. (TEM)
3 1 Optimum (real) resolution of the electron optical
ropt = 1.21 Cs 4 4 system, which accounts for effect of diffraction
(property of light/electrons) and spherical
aberration (lens defect).