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– Light Microscopy-Introduction, Geometrical optics, Image
formation, Magnification and Resolution, Lens aberrations,
Distortion of image and curvature of field, Types of
microscopes- Compound, Comparison, Fluorescence,
– Their basic principles, working and Forensic Applications.
• Electron Microscopy-
– Introduction, Historical review, Scanning electron microscopy
(SEM), Transmission electron microscopy (TEM),
– Theory and basic principles , Instrumentation, Forensic
• The applications of microscopy in forensic science
– The ability of microscopes to locate/detect, to recover,
to resolve, to compare and to image the smallest
items of evidence, often without alteration or
Laws governing the image formation
– law of reflection determines the imaging
properties of mirrors,
– Snell’s law of refraction determines the imaging
properties of lenses.
Light rays undergoing reflection (a) and refraction (b) at plane surfaces
• Lenses are at the heart of many optical devices (cameras,
microscopes, binoculars, and telescopes).
• Lenses are essentially light-controlling elements, used
primarily for image formation with visible light.
• A lens is made up of a transparent refracting medium,
generally of some type of glass, with spherically shaped
surfaces on the front and back.
• A ray incident on the lens refracts at the front surface
(according to Snell’s law) propagates through the lens, and
refracts again at the rear surface.
Types of Lens
• Positive lens converges parallel incident rays and forms
a real image; such a lens is thicker in the middle than at
the periphery and has at least one convex surface.
– Positive lenses magnify when held in front of the eye.
• Negative lens causes parallel incident rays to diverge;
negative lenses are thinner in the middle than at the
periphery, and have at least one concave surface.
– Negative lenses do not form a real image, and when held
in front of the eye, they reduce or demagnify.
• The focal length is shown as the distance ‘f’ from the principal
plane of the lens to its focal point F, the front and rear focal lengths
having the same value.
• The optic axis is shown by a horizontal line passing through the
centre of the lens and perpendicular to its principal plane.
• The object distance ‘a’; distance from the object to the principal
plane of the lens.
• The Image distance ‘b’; distance from the image to the principal
plane of the lens.
• Front face & Rear face
•Geometrical optics of a simple lens.
The focal length f, focal point F, object-lens distance a, and lens-image
distance b are indicated.
The well-known lens equation describes the relationship between focal
length ‘f’ and object and image distances, ‘a’ and ‘b’:
The magnification factor ‘M’ of an image is
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Image formation by convex lens
• An optical aberration is a departure of the
performance of an optical system from the
predictions of paraxial optics.
• Aberration leads to blurring of the
image produced by an image-forming optical
• Makers of optical instruments need to correct
optical systems to compensate for aberration.
Types of Aberrations
• Monochromatic aberrations are caused by the
geometry of the lens or mirror and occur both when
light is reflected and when it is refracted.
– They appear even when using monochromatic light.
• Chromatic aberrations are caused by dispersion, the
variation of a lens's refractive index with wavelength.
– They do not appear when monochromatic light is used.
• Chromatic Aberrations - This type of optical defect is a
result of the fact that white light is composed of
• When white light passes through a convex lens, the
component wavelengths are refracted according to
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• Blue light is refracted to the
greatest extent followed by
green and red light, a
referred to as dispersion.
• The inability of the lens to
bring all of the colours into a
common focus results in a
slightly different image size
and focal point for each
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Chromatic Aberration in an image of a bird.
• Dollond, Lister reduce longitudinal chromatic aberration:
– By combining crown glass and flint glass, where each lens has a
different refractive index and dispersive properties.
– They succeeded in bringing the blue rays and the red rays to a
common focus, near the green rays.
– This combination is termed a lens doublet or achromatic
doublet (a – without & chroma - colour) which means the lens
that makes image without colours.
• Different lens materials (crystal fluorite) may also be used
to minimise chromatic aberration.
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• Spherical aberration causes beams
parallel to, but distant from, the lens
axis to be focused in a slightly
different place than beams close to
• Variation of focus
• This manifests itself as a blurring of
• Rays striking the surface at a greater
distance (marginal rays) are focused
closer to the vertex ‘V’ than are the
paraxial rays and creates spherical
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•Marginal rays are bent too much and focused in front of paraxial rays.
•Distance between the intersection of marginal rays and the paraxial focus is
known as the LSA (longitudinal spherical aberration).
•TSA (transverse SA) is the transverse deviation between the marginal and
paraxial rays on a screen placed at F.
•Rays close to optical axis come to focus near the paraxial focus position.
•As height increases, the focus moves farther.
• ‘Coma’ aberration is a result of refraction differences by light rays passing
through the various lens zones as the incident angle increases and derives its
name from comet shaped aberrated images.
• This type of aberrations are only encountered with off-axis objects.
• Variation of magnification.
• Oblique rays incident on a lens with coma, the rays passing through the edge may
be imaged at a different height than those passing through the center.
• It is also one of the easiest aberrations to demonstrate.
– On a bright sunny day, use a magnifying glass to focus an image of the sun on the
sidewalk and slightly tilt the glass with respect to the principal rays from the sun.
– The sun's image, when projected onto the concrete, will then elongate into a comet-
like shape that is characteristic of comatic aberration.
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•The imaging of a point at ‘S’ can
result in a “comet-like” tail, known
as a coma flare and forms a
“comatic” circle on the screen
(positive coma in this case).
•This is often considered the worst
out of all the aberrations, primarily
because of its asymmetric
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Marginal rays give smaller
image negative coma
Marginal rays give larger
image positive coma
• Astigmatism occurs when the tangential and sagittal images do not coincide.
• The image of a point turns into two separate lines.
• An optical system with astigmatism is one where rays that propagate in two perpendicular planes have
• If an optical system with astigmatism is used to form an image of a cross, the vertical and horizontal lines
will be in sharp focus at two different distances.
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Rays along x-axis: sagittal;
along y-axis: tangential
Astigmatism increases when moving
further from the axis.
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• Astigmatism causes difficulties in seeing fine detail.
• The amount of aberration due to astigmatism is proportional to
the square of the angle between the rays from the object and the
optical axis of the system.
• With care, an optical system can be designed to reduce or
eliminate astigmatism. Such systems are called anastigmats.
• Astigmatism can be often corrected by-
1. Glasses with a lens that has different radii of curvature in different
planes (a cylindrical lens),
2. Contact lenses, or
3. Refractive surgery of cornea.
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Petzval Field Curvature
• Petzval field curvature, named for Joseph Petzval, describes
the optical aberration in which a flat object normal to the optical
axis cannot be brought into focus on a flat image plane.
• This makes a planar object looks curved in its image.
• Image points near the optical axis will be in perfect focus, but rays
off axis will come into focus before the image sensor.
• This is less of a problem when the imaging surface is spherical, as
in the human eye.
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Field curvature: the image "plane" (the arc) deviates from a flat surface (the vertical line).
• Distortion is a deviation from rectilinear
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• Distortion can be irregular or follow many
• As we are mainly concerned with the distortion
caused by symmetrical photographic lens, so we
will be discussing the radially symmetric
distortions which falls in two categories :
– Barrel distortions or
– Pincushion distortions.
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Barrel distortion &
1. In barrel distortion, the
image magnification decreases
with distance from the optical
•The apparent effect is that of an
image which has been mapped
around a sphere (or barrel).
2. In pincushion distortion, image
magnification increases with the
distance from the optical axis.
•The visible effect is that lines that
do not go through the centre of the
image are bowed inwards, towards
the centre of the image, like
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Images of a square grid for “pincushion distortion” (left)
and “barrel distortion” (right).
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Aberration Character Correction
Chromatic Aberration White light, on & off axis,
Contact doublet, spaced
doublet, crystal fluorite
Spherical Aberration Monochromatic light, on &
off axis, image blur
COMA Monochromatic light, off
axis only, comet shaped
Spaced doublet, stoppers
Field Curvature Monochromatic light, off
Flat field objectives, spaced
Distortion Monochromatic light, off
axis only, distorted image
Astigmatism Monochromatic light, off
axis, image blur
• Spherical Aberration: limiting the outer edges of the lens from
exposure to light using diaphragms
• Chromatic Aberrations: combining crown glass and flint glass,
• Field Curvature Aberrations: flat-field objectives.
• Comatic aberrations: spherical aberrations or by designing lens
elements of various shapes
• Astigmatism aberrations: design of the objectives to provide precise
spacing of individual lens
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