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How Fine can You See with an
Optical Microscope?
Magnification M = 25L/f
o
f
e
If we can make lenses with extremely
short focal length, can we design an
optical microscope for seeing atoms?
Can you tell the difference between
magnification and resolution?
Imagine printing a JPEG file of
resolution 320240 to a A4 size print!!
Empty Magnification
Higher resolution Lower resolution
Diffraction of Light
Sinu=/d
film
1
st
2
nd
3
rd
Light waves interfere constructively
and destructively.
Distribution
http://micro.magnet.fsu.edu/primer/java/diffraction/basicdiffraction/index.html
http://www.youtube.com/watch?v=-mNQW5OShMA
Resolution of an Optical Microscope
Physical Limit
Owing to diffraction, the
image of a point is no
longer a point but an
airy disc after passing
through a lens with
finite aperture!
The disc images
(diffraction patterns) of
two adjacent points may
overlap if the two points
are close together.
The two points can no
longer be distinguished
if the discs overlap too
much
Resolution of Microscope
Rayleigh Criteria
Rayleigh Criteria: Angular separation
of the two points is such that the
central maximum of one image falls
on the first diffraction minimum of
the other
=u
m
~ 1.22/d
Resolution of Microscope
Rayleigh Criteria
Image 1
Image 2
http://micro.magnet.fsu.edu/primer/java/imageformation/rayleighdisks/index.html
Resolution of Microscope in
terms of Linear separation
To express the resolution in
terms of a linear separation r,
have to consider the Abbes
theory
Path difference between the
two beams passing the two
slits is
Assuming that the two beams
are just collected by the
objective, then i = o and
d
min
= /2sino
o = + sin sin d i d
I II
I II
Resolution of Microscope
Numerical Aperture
If the space between the specimen and the
objective is filled with a medium of refractive index
n, then wavelength in medium
n
= /n
The d
min
= /2n sino = /2(N.A.)
For circular aperture
d
min
= 1.22/2(N.A.)=0.61/(N.A.)
where N.A. = n sino is called numerical aperture
Immersion oil n=1.515
http://micro.magnet.fsu.edu/primer/java/imageformation/rayleighdisks/index.html
NA of an objective is a measure of its ability to
gather light and resolve fine specimen detail at
a fixed object distance.
NA = n(sin o)
n: refractive index of the imaging medium between
the front lens of objective and specimen cover glass
Numerical Aperture (NA)
o
Angular aperture
One half of A-A
NA=1 - theoretical
maximum numerical
aperture of a lens
operating with air as
the imaging medium
(s72 degrees)
http://micro.magnet.fsu.edu/primer/java/microscopy/immersion/index.html
Factors Affecting Resolution
Resolution = d
min
= 0.61/(N.A.)
Resolution improves (smaller d
min
) if + or n| or o|
Assuming that sino = 0.95 (o = 71.8)
(The eye is more sensitive to blue than violet)
Wavelength
Red
Yellow
Green
Blue
Violet
Air (n= 1) Oil (n = 1.515)
0.42 m
0.39 m
0.35 m
0.31 m
0.27 m
0.28 m
0.17 m
0.20 m
0.23 m
0.25 m
650 nm
600 nm
550 nm
475 nm
400 nm
The smallest distance between two specimen points
that can still be distinguished as two separate entities
d
min
= 0.61/NA NA=nsin(o)
illumination wavelength (light)
NA numerical aperture
o-one half of the objective angular aperture
n-imaging medium refractive index
d
min
~ 0.3m for a midspectrum of 0.55m
Resolution of a Microscope (lateral)
Optical Aberrations
Spherical (geometrical) aberration related to the
spherical nature of the lens
Chromatic aberration arise from variations in the
refractive indices of the wide range of frequencies in
visible light
Two primary causes of non-ideal lens action:
Astigmatism, field curvature and comatic aberrations
are easily corrected with proper lens fabrication.
Reduce the resolution of microscope
Aberration in optical systems (lenses intended to produce a
sharp image) generally leads to blurring of the image. It
occurs when light from one point of an object after transmission
through the system does not converge into a single point.
http://www.youtube.com/watch?v=XGjg64rayfM Aberration of microscope
Defects in Lens
Spherical Aberration
Peripheral rays and axial
rays have different focal
points (caused by spherical
shape of the lens surfaces).
causes the image to appear
hazy or blurred and slightly
out of focus.
very important in terms of
the resolution of the lens
because it affects the
coincident imaging of points
along the optical axis and
degrade the performance of
the lens.
http://micro.magnet.fsu.edu/primer/java/aberrations/spherical/index.html
Chromatic Aberration
Axial - Blue light is refracted to
the greatest extent followed by
green and red light, a
phenomenon commonly referred
to as dispersion
Lateral - chromatic difference of
magnification: the blue image of a
detail was slightly larger than the
green image or the red image in
white light, thus causing color
ringing of specimen details at the
outer regions of the field of view
Defects in Lens
A converging lens can be combined
with a weaker diverging lens, so that
the chromatic aberrations cancel for
certain wavelengths:
The combination achromatic doublet
weaker diverging lens
http://micro.magnet.fsu.edu/primer/java/aberrations/chromatic/index.html
Astigmatism - The
off-axis image of a
specimen point
appears as a disc or
blurred lines instead
of a point.
Depending on the
angle of the off-axis
rays entering the
lens, the line image
may be oriented
either tangentially
or radially
Defects in Lens
o
A
http://micro.magnet.fsu.edu/primer/java/aberrations/astigmatism/index.html
http://www.youtube.com/watch?NR=1&v=6YxffFmi4Eo&feature=endscreen
Curvature of Field
- When visible light
is focused through a
curved lens, the
image plane
produced by the lens
will be curved
The image appears
sharp and crisp
either in the center
or on the edges of
the viewfield but not
both
Defects in Lens
http://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffield/index.html
Coma - Comatic
aberrations are
similar to spherical
aberrations, but they
are mainly
encountered with off-
axis objects and are
most severe when the
microscope is out of
alignment.
Defects in Lens
Coma causes the image of a non-axial point to be reproduced
as an elongated comet shape, lying in a direction
perpendicular to the optical axis.
http://micro.magnet.fsu.edu/primer/java/aberrations/coma/index.html
Depth of focus (f mm)
The distance above and below
geometric image plane within
which the image is in focus
The axial range through which
an object can be focused without
any appreciable change in image
sharpness
(F m)
M NA f F
M NA f F
Axial resolution Depth of Field
Depth of Field Ranges (F m)
F is determined by NA.
NA f F
0.1 0.13 15.5
0.4 3.8 5.8
.95 80.0 0.19
http://micro.magnet.fsu.edu/primer/anatomy/objectives.html
Optical Microscopy
Introduction
Lens formula, Image formation and
Magnification
Resolution and lens defects
Basic components and their
functions
Common modes of analysis
Specialized Microscopy Techniques
Typical examples of applications
Scanning Electron Microscopy
Do review problems (1-9) on OM
Read dispersion and refraction of
light and lens
http://www.doitpoms.ac.uk/tlplib/optical-microscopy/questions.php