Microscopy

Prepared and Complied by

Uma Surendran
From Hans &
Zacherias Janssen

• Reproduction of first compound microscope

made by Hans and Zacharias Janssen, circa
1590. From the National Museum of Health
and Medicine, Washington, D.C. (Image
credit: Public domain.)
• The device laid critical groundwork for future
breakthroughs, but only magnified by
between 3x and 9x

This Photo by Unknown author is licensed under CC BY-SA.

to Dr Brad Amos

• http://www.strathclydemesolab.com/
1665- Micrographia
1676- Antony van Leeuwenhoek
 Magnification & Resolution

• https://micro.magnet.fsu.edu/primer/java/electronmicroscopy/magnify1/index.html

Uma Surendran, Assistant Prof, Dept of Zoology, Baselius College,

Kottayam
Magnification & Resolution
• Magnification – the ability to produce an
enlarged image
• Resolution / resolving power- capacity to
distinguish between two closely located
object
• Limit of resolution- the minimum distance
between two points that allows for
their discrimination as 2 points
• Limit of resolution d= 0.61
 • The numerical aperture of a
microscope objective is the measure
Numerical of its ability to gather light and to
resolve fine specimen detail while

Aperture working at a fixed object (or

specimen) distance.
• https://youtu.be/pFSvM8KD5BY

Uma Surendran, Assistant Prof, Dept of Zoology, Baselius College,

Kottayam
Oil immersion

• In light microscopy, oil immersion is a

technique used to increase the resolving
power of a microscope. This is achieved by
immersing both the objective lens and the
specimen in a transparent oil of high
refractive index, thereby increasing
the numerical aperture of the objective
lens.
Light Microscope

Visible light is used for illuminating the object

 • Types of Light
Microscopes

Fluorescent vs confocal
Bright Field
Microscope
• Brightfield microscopy is the most elementary
form of microscope illumination techniques and is
generally used with compound microscopes.
• The name "brightfield" is derived from the fact
that the specimen is dark and contrasted by the
surrounding bright viewing field. Simple light
microscopes are sometimes referred to as
brightfield microscopes.
Simple vs Compound Microscope
Parts of Light
Microscope

OPTICAL MECHANICAL
PART- SERIES
PART
OF LENS
Optical parts
• Objective lens
• Condenser lens
• Diaphragm or Iris: Many microscopes have a rotating disk
under the stage. This diaphragm has different sized holes
and is used to vary the intensity and size of the cone of light
that is projected upward into the slide.

• Eye piece
Mechanical parts
• Base
• Arm
• Stage
• Mirror
• Body tube
• Nose piece
• Draw tube
Image formation
Real image vs virtual image
Application
• Brightfield Microscope is used in several fields, from basic biology to understanding
cell structures in cell Biology, Microbiology, Bacteriology to visualizing parasitic
organisms in Parasitology.
• Most of the specimens to viewed are stained using special staining to enable
visualization. Some of the staining techniques used include Negative staining and
Gram staining.
• Some of its applications include:
• Used to visualize and study the animal cells
• Used to visualize and study plant cells.
• Used to visualize and study the morphologies of bacterial cells
• Used to identify parasitic protozoans such as Paramecium.
Advantages

• It is simple to use with few adjustments involved while viewing the

image.
• It can be used to view both stained and unstained.
• The optics of the microscope do not alter the color of the
specimen.
Disadvantage
• It can not be used to view live specimens such as bacterial cells. Only fixed
specimens can be viewed under the brightfield microscope.
• It has low contrast hence most specimens must be stained for them to be visualized.
• Use of oil immersion may distort the image
• The use of coverslip may damage the specimen
• Staining may introduce extraneously unwanted details into the specimen or
contaminate the specimen.
• The microscope needs a strong light source for magnification and sometimes the
light source may produce a lot of heat which may damage or kill the specimen.
Videos

• https://youtu.be/1ziBoKT_WUc?t=259 - real vs virtual image

• https://youtu.be/tVcEEw6qbBQ?t=65 amoeba sisters
Dark field microscope
• Dark-field microscopy is a technique that can be
used for the observation of living, unstained
cells and microorganisms. In this microscopy,
the specimen is brightly illuminated while the
background is dark. It is one type of light
microscope, others being bright-field, phase-
contrast, differential interface contrast, and
fluorescence.

This Photo by Unknown author is licensed under CC BY-SA.

Dark Field Microscope
• Principle- In dark field microscopy, no direct light
from the condenser enters the objective lens.
• Only light that is reflected, refracted or diffracted by
the specimen enters the objective. The dark field
condenser produces a circle of light.
• The light is at an extremely oblique angle to the
surface of the slide. This oblique light comes to a focus
on the specimen. It then diverges so strongly that no
direct light enters the objective. This type
of illumination is a hollow cone of light
• In order to achieve this the numerical aperture of the
objective should be less than the numerical aperture of
the condensor.
• Reflection involves a change in direction of waves when they
bounce off a barrier; r
• efraction of waves involves a change in the direction of waves as
they pass from one medium to another;
• diffraction involves a change in direction of waves as they pass
through an opening or around a barrier in their path.
Dark Field Microscope
• Dark-field microscopy uses a light microscope
with an extra opaque disc underneath the
condenser lens, or a special condenser having a
central blacked-out area, due to which the light
coming from the source cannot directly enter
into the objective.
• Everything is visible regardless of colour, usually
bright white against a dark background. Pigmented
objects are often seen in "false colours," that is, the
reflected light is of a colour different than the colour
of the object
Uses and Advantages of
Dark Field Microscope
• Uses and advantages
• Live unstained cells/ organisms
• Wet mound.
• Hanging drop preparation- slows down the drying-out
process, so the organisms can be observed for longer
periods.
• It is also useful for the demonstration of the motility of flagellated
bacteria and protozoa.
• It is more useful in examining external details, such as outlines,
edges, grain boundaries and surface defects than internal structure
Disadvantages

• The main limitation of dark-field microscopy is the low light levels

seen in the final image.
• The sample must be very strongly illuminated, which can cause
damage to the sample.
• Low resolution as the numerical aperture of the objective is less
Phase contrast
microscope
• Phase contrast is used to enhance the contrast of
light microscopy images of transparent and
colourless specimens. It enables visualisation of
cells and cell components that would be difficult to
see using an ordinary light microscope.
• Phase contrast does not require cells to be killed,
fixed or stained. The technique enables living
cells, usually in culture, to be visualised in their
natural state. This means biological processes can
be seen and recorded at high contrast and
specimen detail can be observed. Fluorescence
staining can be used in combination with phase
contrast to further improve the visualisation of
samples.
Phase contrast
microscope
• Object is viewed through ordinary microscope due
to the difference in colour intensities of the
specimen.
• This is created by staining the specimens with a
suitable dye. When light passes through a
stained specimen in the ordinary microscope ,
different stains absorb different intensities /
amplitude of light.
• This differential absorption produces and change in
the intensity of transmitted light waves.
• Human eyes can change in amplitude of light waves
and its observed as contrast.
 Human eyes can detect
The human eye can detect the visible
spectrum of the electromagnetic
— a range
spectrum Click to addoftext
wavelengths
between 390 to 700 nanometers.
 What happens when light passes through
transparent objects??
• Image of the effect on light as it passes through different
regions of a sample. The light ray is in phase prior to
reaching the sample. When light passes through the
coverslip and through the medium (A), no diffraction occurs.
As the light passes through different components, such as
the cytoplasm (B), or the cell nucleus (C), the light is
diffracted (phase-shifted) by a certain amount, dependent
upon the RI of the medium it passes through. As such,
waves that pass through thicker parts of the cell are
diffracted to a greater degree (as shown by the red box).
The result is that light that passes through the sample (B or
C), is phase-shifted compared to the light that did not pass
through the sample (A)
What is Phase change?

• Two waves are said to be in phase if any two corresponding points

on those two waves occupy/ arrive at the same point at the same
time.
What happens when
light waves in phase
interact?
Human eye cannot identify this phase change...
• If two waves which are in phase arrive at the
same point at the same time, then their effect
doubles
• If two completely out of phase waves reaches at
a same point at the seem time, the they will
cancel out each other
 • Light waves can interfere with each other if they have the same
frequency and well-ordered phases, with the result dependant

How do we see light

upon the phase difference between waves. For example, waves
that have the same frequency and are perfectly out of
phase (180° phase difference) would align peak to trough,

wave interference?
leading to destructive interference and resulting in 0
amplitude, the two waves perfectly canceling each other out.
Likewise, waves that are perfectly in-phase align peak to
peak (0° phase difference) and thus would constructively
interfere, combining the intensities into a wave with double the
amplitude.
 Phase contrast
microscope
• Principle –
• Phase contrast microscope works by
converting the phase shifts or changes in the
light rays that are induced by differences in the
thickness and refractive index of the different
parts of an object into difference in
https://www.rug.nl/fse/nobelprize-
zernike/fasecontrastmicroscoop brightness or light intensity.
• Phase-contrast microscopy was first described
by Dutch physicist Frits Zernike in the 1930s,
for which he later was awarded the Nobel Prize
in Physics (1953).
Working principle of phase contrast microscope

How can this be

achieved??
Additional
components seen
in Phase contrast
microscope.
• Phase-contrast microscopy is
basically a specially designed light
microscope with all the basic parts
in addition to which an annular
phase plate and annular diaphragm
are fitted.
 Annular
diaphragm
• The annular diaphragm
• It is situated below the condenser.
• It is made up of a circular disc having a
circular annular groove.
• The light rays are allowed to pass
through the annular groove.
• The specimen is illuminated by a
hollow cone of light coming through a
phase annulus in the condenser.
Phase plate
• A phase plate is also called
Phase retardation plate.
• It is located at the back focal plane
of objective lens
• It is a transparent disc with a channel .
• The channel is coated with a material that
absorb light but cannot retard it- used
to reduce the intensity of un diffracted light
• The other portions of the phase plate
is coated with light retarding materials.-
Working
Working
Types

• Positive phase contrast reveals medium to dark gray images on a

lighter grey background; these images often have a bright halo
along the edge of the sample.

• Negative phase contrast is the opposite. The specimen appears

lighter with a dark background; they also have a dark halo
outlining the image.
Working

• https://youtu.be/vr4tYUnaHNQ?t=49
Advantages
• The capacity to observe living cells and, as such, the ability to
examine cells in a natural state
• Observing a living organism in its natural state and/or environment
can provide far more information than specimens that need to be
killed, fixed or stain to view under a microscope
• Ideal for studying and interpreting thin specimens
• Ability to combine with other means of observation, such as
fluorescence
Disadvantages

• This method of observation is not ideal for thick

organisms or particles
• Thick specimens can appear distorted
• Shade-off and halo effect, referred to a phase
artifacts
• Shade-off occurs with larger particles, results in a
steady reduction of contrast moving from the
center of the object toward its edges
• Halo effect, where images are often surrounded
by bright areas, which obscure details along the
perimeter of the specimen
Fluorescence microscopy

• https://youtu.be/AhzhOzgYoqw
Filters- allow the passage of light of a particular
wavelength but act as a barrier for the passage of
other wavelength.
filter
Dichroic mirror-
Video microscopy

• Video camera or fast camera is attached to the

microscope
• To provide with a time lapse video
Polarising Microscope.

• It is a technique where polarized light is used in microscope to

evaluate composition and three-dimensional structure of
anisotropic specimen.
What is a polarised
light??
• We all know that light is an electromagnetic
wave.
• Light that we see coming from an ordinary light
source is composed of zillions of these light
waves at all possible vibration directions . This is
called natural or unpolarized light.
• Light that vibrates in only one vibration
direction or azimuth that is at right angles to the
direction of propagation, is called plane
polarized light
 Isotropic specimens- whose
properties do not change with
directions.- example glass and
What is an metals

anisotropic
specimen? Anisotropic specimens-
whose properties change with
direction- example- wood.
Spindle fibres, crystals, quartz....
Refraction
• Refraction, in physics, the change in direction of a
wave passing from one medium to another caused
by its change in speed
Anisotropic materials
are usually birefringent
• Birefringence is the optical property of a material
having a refractive index ( refractive index of a
material describes how fast light travels through it)
that depends on the polarization and propagation
direction of light
• Birefringence is the phenomenon exhibited by
certain materials in which an incident ray of light is
split into plane polarised rays, called an ordinary ray
and an extraordinary ray
Working of polarising microscope.

• Polarized light microscopes work by converting unpolarized light

to polarized light. Using this polarized light structure and
properties of transparent anisotropic birefringent objects can be
studied.
• Additional components needed
• A polarizer
• An analyzer
Components

• The polarizer is located below the

specimen stage and can be rotated
through 360°.Polarizer produce
plane polarised light from natural
light
Components

• The analyzer is a second

polarizing filter is placed above
the objective and may be
rotatable in some cases. It
combines the different rays
emerging from the specimen to
generate the final image.
• Both are placed are right angles
to each other.In this configuration,
the polarizer and analyzer are said
to be crossed, with no light
passing through the system and a
dark viewfield present in the
eyepieces.
Working
• If a birefringent specimen is placed on the microscope and
rotated, it will be seen to reach a maximum intensity on a black
background at some particular angles. The intensity will gradually
decrease as the specimen is rotated until it completely disappears.
The specimen will also show polarization colors (Newton’s colors)
• This common setup of a polarizing microscope can be used to
determine if polarizing materials are present in a biological
specimen such as amyloid, uric acid crystals and different collagen
types
Videos

• https://youtu.be/W6v7JdK4sps - working

• https://youtu.be/fzfnLHi09gA apple-green birefringence

Electron microscope
• https://youtu.be/eSKTFXv5rdI
• Electrons are such small particles that, like photons in light, they act as waves.
• An electron microscope uses an 'electron beam' to produce the image of
the object and magnification is obtained by 'electromagnetic fields'.
• The wavelength of electrons is much smaller than that of light, hence the
resolution obtained in electron microscope pictures are much higher that light
microscope. https://youtu.be/ljTEG-B-kGc
• Source of illumination- beam of electrons
• Lens- electromagnetic coils
Magnification & Resolution
• Magnification – the ability to produce an
enlarged image
• Resolution / resolving power- capacity to
distinguish between two closely located
object
• Limit of resolution- the minimum distance
between two points that allows for
their discrimination as 2 points
• Limit of resolution d= 0.61
 • The numerical aperture of a

Numerical microscope objective is the measure

of its ability to gather light and to

Aperture resolve fine specimen detail while

working at a fixed object (or
specimen) distance.
• https://youtu.be/pFSvM8KD5BY

Uma Surendran, Assistant Prof, Dept of Zoology, Baselius College,

Kottayam
Resolution and wavelength

• Wave length of light was always a limiting factor in providing high

resolution.
• But as electrons behave like light waves in vacuum and their
wavelength is much smaller (10 lakh times)- if they are used it
could solve the limitation issues.
• Since normal lens could not focus electrons, the discovery that
electric and magnetic fields can influence electron waves just like
mirrors and lens led to the invention of electromagnetic lens .
Invention

• Ernst Ruska at the University of Berlin, along with

Max Knoll, combined these characteristics and
built the first transmission electron
microscope (TEM) in 1931, for which Ruska was
awarded the Nobel Prize for Physics in 1986.
Electron microscope.
• The object is placed between the condenser and
objective.
• The magnified image of the object is formed on the
fluorescent screen or on photographic film rather
than being observed through eye piece.
• Since the image produced by electrons does not
have the colour, the electron micrograph always has
shades of black, grey and white.
• It gives information about the topography,
morphology, composition of any object
Types of Electron Microscope

TEM
• Transmission Electron Microscope

SEM
• Scanning Electron Microscope
What is voltage and current?

• Voltage is the difference in charge between two points.

• Current is the rate at which charge is flowing
 TEM
• The transmission electron microscope is used to view
thin specimens (tissue sections, molecules, etc)
through which electrons can pass generating a
projection image.
• The TEM is analogous in many ways to the
conventional (compound) light microscope.
• TEM is used, among other things, to image the
interior of cells (in thin sections), the structure of
protein molecules (contrasted by metal shadowing),
the organization of molecules in viruses and
cytoskeletal filaments (prepared by the negative
staining technique), and the arrangement of protein
molecules in cell membranes (by freeze-fracture).
Components
• There are four main components to a transmission
electron microscope:
• an electron optical column- all the components
are placed here
• a vacuum system-otherwise the electrons would
collide with air molecules and be absorbed
• the necessary electronics -lens supplies for focusing
and deflecting the beam and the high voltage
generator for the electron source
• Software https://www.youtube.com/watch?v=PmfjYk
KeVEA
Components • EM is in the form of a tall vacuum column which is
vertically mounted. It has the following components:
• Electron gun
• The electron gun is a heated tungsten filament, which
generates electrons.
• Electromagnetic lenses
• Condenser lens focuses the electron beam on the
specimen. A second condenser lens forms the
electrons into a thin tight beam.
• The electron beam coming out of the specimen passes
down the second of magnetic coils called
the objective lens, which has high power and forms
the intermediate magnified image.
• The third set of magnetic lenses called projector
(ocular) lenses produce the final further magnified
image.
• Each of these lenses acts as an image magnifier all the
while maintaining an incredible level of detail and
resolution.
Components

• Specimen Holder
• The specimen holder is an extremely thin film of carbon or
collodion held by a metal grid.
• Image viewing and Recording System.
• The final image is projected on a fluorescent screen.
• Below the fluorescent screen is a camera for recording the image.
Thermionic Gun
• The tungsten filament is hairpin-shaped and heated
to about 2700 degree Celsius. By applying a very high
positive potential difference between the filament and
the anode, electrons are extracted from the electron
cloud round the filament and accelerated towards the
anode. The anode has a hole in it so that an electron
beam in which the electrons are travelling at several
hundred thousand kilometres per second emerges at
the other side
Working of TEM
• The beam emerging from the gun is condensed beam
at the specimen by the condenser lenses
• after passing through the specimen, projected as a
magnified image of the specimen onto the fluorescent
screen at the bottom of the column.
• If the specimen were not thin, the electrons would
simply be stopped and no image would be formed
• Specimens for the TEM are usually 0.5 micrometres or
less thick.
• The Specimen should be prepared in such a way that it
can withstand vacuum and electron wave, is coated
with heavy metals.
• Imaging methods in TEM use the information
contained in the electron waves exiting from the
sample to form an image.
 Differences between Light Microscope and Electron Microscope

Light Microscope Electron Microscope

Illuminating source is the Light. Illuminating source is the beam of electrons.

Specimen preparation takes usually few minutes to Specimen preparation takes usually takes few
hours. days.

Live or Dead specimen may be seen. Only Dead or Dried specimens are seen.

Condenser, Objective and eye piece lenses are

All lenses are electromagnetic.
made up of glasses.

It has high resolving power (0.001µm), about 250

It has low resolving power (0.25µm to 0.3µm).
times higher than light microscope.

It has a magnification of of 500X to 1500X. It has a magnification of 100,000X to 300,000X.

The object is 5µm or thicker. The object is 0.1µm or thinner.

Image is Colored. Image is Black and White.

 Differences between Light Microscope and Electron Microscope

Light Microscope Electron Microscope

Vacuum is not required. Vacuum is essential for its operation.

High voltage electric current is required (50,000 Volts and

There is no need of high voltage electricity.
above).

It has a cooling system to take out heat generated by high

There is no cooling system.
electric current.

Filament is not used. Tungsten filament is used to produce electrons.

Radiation risk is absent. There is risk of radiation leakage.

Specimen is coated with heavy metals in order to reflect

Specimen is stained by colored dyes.
electrons.

Image is received in Zinc Sulphate Fluorescent Screen or

mage is seen by eyes through ocular lens.
Photographic Plate.

It is used in the study of external surface, ultra structure of

t is used for the study of detailed gross internal structure.
cell and very small organisms.
SEM
• The SEM produces images of the specimen with a focused electron
beam that is scanned across a rectangular area of the specimen.
• Just like the way you read something written on the wall of a dark room
with a torch light.
• observe the surface phenomena of the materials.
• The sample is exposed in SEM to the high-energy electron beam.
• The electrons interact with atoms in the sample producing various
signals that gives information about topography, morphology,
composition, chemistry, orientation of grains, crystallographic
information
 SEM

• Backscattered electrons are reflected back after

elastic interactions between the beam and the
sample. Secondary electrons, however, originate
from the atoms of the sample.
• The scanning electron microscope (SEM)
produces images by scanning the sample with a
high-energy beam of electrons. As the electrons
interact with the sample, they produce secondary
electrons, backscattered electrons, and
characteristic X-rays. These signals are collected
by one or more detectors to form images which
are then displayed on the computer screen. .
Components

• Electron gun consisting of cathode and anode

• The condenser lens controls the amount of electrons travelling down
the column.
• The objective lens focuses the beam into a spot on the sample.
• Deflection coil helps to deflect the electron beam.
• https://myscope-explore.org.au/2_7_deflector.html
• Secondary electron detector attracts the secondary electrons.
• Additional sensors detect backscattered electrons and x-rays.
SEM
• Resolution of SEM is lower that TEM.
• https://www.youtube.com/watch?v=GY9lfO-tVfE
• The specimen does not need special treatment for visualization under
the SEM, even air-dried samples can be examined directly. However,
microbial specimens need fixation, dehydration, and drying in order to
maintain the structural features of the cells and to prevent collapsing of
the cells when exposed to the high vacuum of the microscope.
• The samples are mounted and coated with thin layer heavy metal
elements to allow spatial scattering of electric charges on the surface of
the specimen allowing better image production, with high clarity
• Advantages of SEM
• It gives detailed 3d and topographical imaging and the versatile information.
• This works very fast.
• Modern SEMs allow for the generation data ni digital form.
• Most SEM samples require minimal preparation actions.
• Disadvantages
• SEMs are expensive and large.
• Special training is required to operate an SEM.
• SEMs are limited to solid samples.
• SEMs carry a small risk of radiation exposure associated with the electrons that scatter from beneath the
sample surface.
• Advantages of SEM
• It gives detailed 3d and topographical imaging and the versatile information.
• This works very fast.
• Modern SEMs allow for the generation data ni digital form.
• Most SEM samples require minimal preparation actions.
• Disadvantages
• SEMs are expensive and large.
• Special training is required to operate an SEM.
• SEMs are limited to solid samples.
• SEMs carry a small risk of radiation exposure associated with the electrons that scatter from beneath the
sample surface.
Properties Scanning Electron Microscopy (SEM) Transmission Electron Microscopy (TEM)
SEM is based on scattered electrons, i.e. electrons Electrons are used as “light source”. TEM is based
Light Source emitted from the surface of a specimen. It is the EM on transmitted electrons and operates on the same
analog of a stereo light microscope. basic principles as the light microscope.

Transmission electron microscope is used to view

SEM provides detailed images of the surfaces of
thin specimens (tissue sections, molecules, etc). TEM
cells. SEM focuses on the sample’s surface and its
Purpose can show many characteristics of the sample, such as
composition, so SEM shows only the morphology of
internal composition, morphology, crystallization,
samples.
etc.
The sample in TEM has to be cut thinner (70-90 nm)
Sample is coated with a thin layer of heavy metal
Sample Preparation because electrons cannot penetrate very far into
such as gold or palladium.
materials.
TEM has a much higher resolution than SEM. It can
Resolution SEM can resolve objects as close as 20 nm. resolve objects as close as 1 nm i.e. down to near-
atomic levels.
Magnification The magnifying power of SEM is up to 50,000X. The magnifying power of TEM is up to 2 million times.
SEM allows for a large amount of sample to be With TEM only a small amount of samples can be
Processing of sample (s)
analyzed at a time analyzed at a time.
Transmitted electrons hit a fluorescent screen giving
Secondary or backscattered electrons arising from rise to a “shadow image” of the specimen with its
the interaction of electron beam and metal-coated different parts displayed in varied darkness
Image formation
specimen are collected and the resulting image is according to their density. The image can be studied
displayed on a computer screen. directly by the operator or photographed with a
camera.
3D picture SEM provides a 3-dimensional image TEM provides a 2-dimensional picture.

To image the interior of cells (in thin sections), the

structure of protein molecules (contrasted by metal
To study topography and atomic composition of
shadowing), the organization of molecules in viruses
Current Applications specimens, process control and also, for example,
and cytoskeletal filaments (prepared by the negative
the surface distribution of immuno-labels
staining technique), and the arrangement of protein
molecules in cell membranes (by freeze-fracture).
Scanning Transmission Electron Microscope

• Scanning transmission electron microscopy (STEM) combines the principles

of transmission electron microscopy and scanning electron microscopy and can be
performed on either type of instrument. Like TEM, STEM requires very thin samples
and looks primarily at beam electrons transmitted by the sample. One of its principal
advantages over TEM is in enabling the use of other of signals that cannot be
spatially correlated in TEM, including secondary electrons, scattered beam electrons,
characteristic X-rays, and electron energy loss.

• Like SEM, the STEM technique scans a very finely focused beam of electrons across
the sample in a raster pattern.
Camera Lucida
• Is an optical instrument patented in 1806 by William Hyde
Wollaston to facilitate accurate sketching of objects.
• The camera lucida performs an optical superimposition of
the subject being viewed and the surface on which the
observer is drawing .

• The person sees both the image of the specimen and

drawing surface simultaneously
• Two types
• -Prism Type
• Mirror Type
Prism Type
• It consists of a four-sided prism mounted on a small
stand above a sheet of paper.
• By placing the eye close to the upper edge of the
prism so that half the pupil of the eye is over the
prism, the observer is able to see a reflected image
of an object situated in front of the prism,
apparently lying on the paper.
• He can then trace the image with a pencil. In its
original form the camera lucida was extremely
difficult to focus properly, and a weak spectacle lens
was added between the prism and the paper.
Mirror type

https://youtu.be/uxx8sJBMFi8
https://youtu.be/Opb3meFarm4 -
as a drawing tool
https://youtu.be/y_mB7-
oKJFM app