Optical microscope 1
The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light
and a system of lenses to magnify images of small samples. Optical microscopes are the oldest and simplest of the
microscopes. Digital microscopes are now available which use a CCD camera to examine a sample, and the image is
shown directly on a computer screen without the need for optics such as eye-pieces. Other microscopic methods
which do not use visible light include scanning electron microscopy and transmission electron microscopy.
There are two basic configurations of the conventional optical microscope in use, the simple (one lens) and
compound (many lenses). Digital microscopes are based on an entirely different system of collecting the reflected
light from a sample.
A simple microscope is a microscope that uses only one lens for magnification, and is the original light microscope.
Van Leeuwenhoek's microscopes consisted of a small, single converging lens mounted on a brass plate, with a screw
mechanism to hold the sample or specimen to be examined. Demonstrations  by British microscopist have images
from such basic instruments. Though now considered primitive, the use of a single, convex lens for viewing is still
found in simple magnification devices, such as the magnifying glass, and the loupe. Light microscopes are able to
view specimens in color, an important advantage when compared with electron microscopes, especially for forensic
analysis, where blood traces may be important, for example.
The oldest published image known to have been
made with a microscope: bees by Francesco
It is difficult to say who invented the compound microscope. Dutch
spectacle-makers Hans Janssen and his son Zacharias Janssen are often
said to have invented the first compound microscope in 1590, but this
was a declaration made by Zacharias Janssen himself during the mid
1600s. The date is unlikely, as it has been shown that Zacharias
Janssen actually was born around 1590. Another favorite for the title of
'inventor of the microscope' was Galileo Galilei. He developed an
occhiolino or compound microscope with a convex and a concave lens
in 1609. Galileo's microscope was celebrated in the Accademia dei
Lincei in 1624 and was the first such device to be given the name
"microscope" a year later by fellow Lincean Giovanni Faber. Faber
coined the name from the Greek words μικρόν (micron) meaning
"small", and σκοπεῖν (skopein) meaning "to look at", a name meant to
be analogous with "telescope", another word coined by the Linceans.
Christiaan Huygens, another Dutchman, developed a simple 2-lens
ocular system in the late 1600s that was achromatically corrected, and
therefore a huge step forward in microscope development. The
Huygens ocular is still being produced to this day, but suffers from a
small field size, and other minor problems.
Optical microscope 2
Anton van Leeuwenhoek (1632-1723) is credited with bringing the microscope to the attention of biologists, even
though simple magnifying lenses were already being produced in the 1500s. Van Leeuwenhoek's home-made
microscopes were very small simple instruments, with a single, yet strong lens. They were awkward in use, but
enabled van Leeuwenhoek to see detailed images. It took about 150 years of optical development before the
compound microscope was able to provide the same quality image as van Leeuwenhoek's simple microscopes, due to
timely difficulties of configuring multiple lenses. Still, despite widespread claims, van Leeuwenhoek is not the
inventor of the microscope.
Basic optical transmission microscope elements(1990's)
1. ocular lens, or eyepiece
2. objective turret
3. objective lenses
4. coarse adjustment knob
5. fine adjustment knob
6. object holder or stage
7. mirror or light (illuminator)
8. diaphragm and condenser
All optical microscopes share the same basic
• The eyepiece - A cylinder containing two or more
lenses to bring the image to focus for the eye. The
eyepiece is inserted into the top end of the body tube.
Eyepieces are interchangeable and many different
eyepieces can be inserted with different degrees of
magnification. Typical magnification values for
eyepieces include 5x, 10x and 2x. In some high
performance microscopes, the optical configuration of
the objective lens and eyepiece are matched to give
the best possible optical performance. This occurs
most commonly with apochromatic objectives.
• The objective lens - a cylinder containing one or more
lenses, typically made of glass, to collect light from
the sample. At the lower end of the microscope tube
one or more objective lenses are screwed into a circular nose piece which may be rotated to select the required
objective lens. Typical magnification values of objective lenses are 4x, 5x, 10x, 20x, 40x, 50x and 100x. Some
high performance objective lenses may require matched eyepieces to deliver the best optical performance.
• The stage - a platform below the objective which supports the specimen being viewed. In the center of the stage is
a hole through which light passes to illuminate the specimen. The stage usually has arms to hold slides
(rectangular glass plates with typical dimensions of 25 mm by 75 mm, on which the specimen is mounted).
• The illumination source - below the stage, light is provided and controlled in a variety of ways. At its simplest,
daylight is directed via a mirror. Most microscopes, however, have their own controllable light source that is
focused through an optical device called a condenser, with diaphragms and filters available to manage the quality
and intensity of the light.
The whole of the optical assembly is attached to a rigid arm which in turn is attached to a robust U shaped foot to
provide the necessary rigidity. The arm is usually able to pivot on its joint with the foot to allow the viewing angle to
be adjusted. Mounted on the arm are controls for focusing, typically a large knurled wheel to adjust coarse focus,
Optical microscope 3
together with a smaller knurled wheel to control fine focus.
Updated microscopes may have many more features, including reflected light (incident) illumination, fluorescence
microscopy, phase contrast microscopy and differential interference contrast microscopy, spectroscopy, automation,
and digital imaging.
On a typical compound optical microscope, there are three objective lenses: a scanning lens (4×), low power lens
(10×)and high power lens (ranging from 20 to 100×). Some microscopes have a fourth objective lens, called an oil
immersion lens. To use this lens, a drop of immersion oil is placed on top of the cover slip, and the lens is very
carefully lowered until the front objective element is immersed in the oil film. Such immersion lenses are designed
so that the refractive index of the oil and of the cover slip are closely matched so that the light is transmitted from the
specimen to the outer face of the objective lens with minimal refraction. An oil immersion lens usually has a
magnification of 50 to 100×.
The actual power or magnification of an optical microscope is the product of the powers of the ocular (eyepiece),
usually about 10×, and the objective lens being used.
Compound optical microscopes can produce a magnified image of a specimen up to 1000× and, at high
magnifications, are used to study thin specimens as they have a very limited depth of field.
Optical path in a typical microscope
The optical components of a modern
microscope are very complex and for a
microscope to work well, the whole
optical path has to be very accurately
set up and controlled. Despite this, the
basic operating principles of a
microscope are quite simple.
The objective lens is, at its simplest, a
very high powered magnifying glass
i.e. a lens with a very short focal
length. This is brought very close to
the specimen being examined so that the light from the specimen comes to a focus about 160 mm inside the
microscope tube. This creates an enlarged image of the subject. This image is inverted and can be seen by removing
the eyepiece and placing a piece of tracing paper over the end of the tube. By carefully focusing a brightly lit
specimen, a highly enlarged image can be seen. It is this real image that is viewed by the eyepiece lens that provides
In most microscopes, the eyepiece is a compound lens, with one component lens near the front and one near the back
of the eyepiece tube. This forms an air-separated couplet. In many designs, the virtual image comes to a focus
between the two lenses of the eyepiece, the first lens bringing the real image to a focus and the second lens enabling
the eye to focus on the virtual image.
In all microscopes the image is viewed with the eyes focused at infinity (mind that the position of the eye in the
above figure is determined by the eye's focus). Headaches and tired eyes after using a microscope are usually signs
that the eye is being forced to focus at a close distance rather than at infinity.
The essential principle of the microscope is that an objective lens with very short focal length (often a few mm) is
used to form a highly magnified real image of the object. Here, the quantity of interest is linear magnification, and
this number is generally inscribed on the objective lens casing. In practice, today, this magnification is carried out by
means of two lenses: the objective lens which creates an image at infinity, and a second weak tube lens which then
forms a real image in its focal plane.
Optical microscope 4
Optical microscopy is used extensively in microelectronics, nanophysics, biotechnology, pharmaceutic research and
Optical microscopy is used for medical diagnosis, the field being termed histopathology when dealing with tissues,
or in smear tests on free cells or tissue fragments.
Modern stereomicroscope optical design.
A - Objective B - Galilean telescopes (rotating
objectives) C - Zoom control D - Internal
objective E - Prism F - Relay lens G - Reticle H -
The stereo or dissecting microscope is designed differently from the
diagrams above, and serves a different purpose. It uses two separate
optical paths with two objectives and two eyepieces to provide slightly
different viewing angles to the left and right eyes. In this way it
produces a three-dimensional visualization of the sample being
The stereo microscope is often used to study the surfaces of solid
specimens or to carry out close work such as sorting, dissection,
microsurgery, watch-making, small circuit board manufacture or
inspection, and the like.
Unlike compound microscopes, illumination in a stereo microscope
most often uses reflected (episcopic) illumination rather than
transmitted (diascopic) illumination, that is, light reflected from the
surface of an object rather than light transmitted through an object. Use
of reflected light from the object allows examination of specimens that
would be too thick or otherwise opaque for compound microscopy.
However, stereo microscopes are also capable of transmitted light
illumination as well, typically by having a bulb or mirror beneath a
transparent stage underneath the object, though unlike a compound
microscope, transmitted illumination is not focused through a
condenser in most systems. Stereoscopes with specially-equipped
illuminators can be used for dark field microscopy, using either
reflected or transmitted light.
Optical microscope 5
Scientist using a stereo microscope outfitted with
a digital imaging pick-up
Great working distance and depth of field here are important qualities
for this type of microscope. Both qualities are inversely correlated with
resolution: the higher the resolution (i.e. the shorter the distance at
which two adjacent points can be distinguished as separate), the
smaller the depth of field and working distance. A stereo microscope
has a useful magnification up to 100×. The resolution is maximally in
the order of an average 10× objective in a compound microscope, and
often much lower.
There are two major types of magnification systems in stereo
microscopes. One is fixed magnification in which primary
magnification is achieved by a paired set of objective lenses with a set
degree of magnification. The other is zoom or pancratic magnification,
which are capable of a continuously variable degree of magnification
across a set range. Zoom systems can achieve further magnification
through the use of auxiliary objectives that increase total magnification
by a set factor. Also, total magnification in both fixed and zoom
systems can be varied by changing eyepieces.
Intermediate between fixed magnification and zoom magnification systems is a system attributed to Galileo as the
"Galilean optical system" ; here an arrangement of fixed-focus convex lenses is used to provide a fixed
magnification, but with the crucial distinction that the same optical components in the same spacing will, if
physically inverted, result in a different, though still fixed, magnification. This allows one set of lenses to provide
two different magnifications ; two sets of lenses to provide four magnifications on one turret ; three sets of lenses
provide six magnifications and will still fit into one turret. Practical experience shows that such Galilean optics
systems are as useful as a considerably more expensive zoom system, with the advantage of knowing the
magnification in use as a set value without having to read analogue scales. (In remote locations, the robustness of the
systems is also a non-trivial advantage.)
The stereo microscope should not be confused with a compound microscope equipped with double eyepieces and a
binoviewer. In such a microscope both eyes see the same image, but the binocular eyepieces provide greater viewing
comfort. However, the image in such a microscope is no different from that obtained with a single monocular
Optical microscope 6
Digital display with stereo microscopes
Recently various video dual CCD camera pickups have been fitted to stereo microscopes, allowing the images to be
displayed on a high resolution LCD monitor. Software converts the two images to an integrated anaglyph 3D image,
for viewing with plastic red/cyan glasses, or to the cross converged process for clear glasses and somewhat better
color accuracy. The results are viewable by a group wearing the glasses.
A miniature digital microscope.
Low power microscopy is also possible with
digital microscopes, with a camera attached
directly to the USB port of a computer, so
that the images are shown directly on the
monitor. Often called "USB" microscopes,
they offer high magnifications (up to about
200×) without the need to use eyepieces,
and at very low cost. The precise
magnification is determined by the working
distance between the camera and the object,
and good supports are needed to control the
image. The images can be recorded and
stored in the normal way on the computer.
The camera is usually fitted with a light
source, although extra sources (such as a
fiber-optic light) can be used to highlight
features of interest in the object. They also offer a large depth of field, a great advantage at high magnifications.
They are most useful when examining flat objects such as coins, printed circuit boards, or documents such as
banknotes. However, they can be used for examining any object which can be studied in a standard
stereo-microscope. Such microscopes offer the great advantage of being much less bulky than a conventional
microscope, so can be used in the field, attached to a laptop computer. Although convenient, the magnifying abilities
of these instruments are often overstated; typically offering 200x magnification, this claim is based usually on 25x to
30x actual magnification PLUS the expansion of the image facilitated by the size of the available screen- so for
genuine 200x magnification a ten-foot screen would be required.
Other types of optical microscope include:
• the inverted microscope for studying samples from below; useful for cell cultures in liquid;
• the student microscope designed for low cost, durability, and ease of use;
• the petrographic microscope whose design usually includes a polarizing filter, rotating stage and gypsum plate to
facilitate the study of minerals or other crystalline materials whose optical properties can vary with orientation.
• the polarizing microscope
• the fluorescence microscope
• the phase contrast microscope
Optical microscope 7
At very high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded by diffraction
rings. These are called Airy disks. The resolving power of a microscope is taken as the ability to distinguish between
two closely spaced Airy disks (or, in other words the ability of the microscope to reveal adjacent structural detail as
distinct and separate). It is these impacts of diffraction that limit the ability to resolve fine details. The extent of and
magnitude of the diffraction patterns are affected by both by the wavelength of light ( ), the refractive materials
used to manufacture the objective lens and the numerical aperture (NA) of the objective lens. There is therefore a
finite limit beyond which it is impossible to resolve separate points in the objective field, known as the diffraction
limit. Assuming that optical aberrations in the whole optical set-up are negligible, the resolution d, is given by:
Usually, a λ of 550 nm is assumed, corresponding to green light. With air as medium, the highest practical NA is
0.95, and with oil, up to 1.5. In practice the lowest value of d obtainable is about 200 nm.
A modern microscope with a mercury bulb for
fluorescence microscopy. The microscope has a
digital camera, and is attached to a computer.
Other optical microscope designs can offer an improved resolution.
These include ultraviolet microscopes, which use shorter wavelengths
of light so the diffraction limit is lower, Vertico SMI, near field
scanning optical microscopy which uses evanescent waves, and
Stimulated Emission Depletion (STED) microscopy which is used for
observing self-luminous particles. In the latter, non-self-luminous
particle is illuminated by an external source, and thus this microscope
is not diffraction limited by the Abbe's theory. Stefan Hell of the Max
Planck Institute for Biophysical Chemistry was awarded the 10th
German Future Prize in 2006 for his development of the Stimulated
Emission Depletion (STED) microscope.
Several other optical microscopes have been able to see beyond the
theoretical Abbe limit of 200 nm. In 2005, a microscope capable of detecting a single molecule was described as a
teaching tool. A holographic microscope described by Courjon and Bulabois in 1979 is also capable of breaking
this magnification limit, although resolution was restricted in their experimental analysis.
As a sensitivity improvement, a sarfus method was developed, which uses contrast-enhancing substrates and thereby
allows to directly visualize films as thin as 0.3 nanometers.
In order to overcome the limitations set by the diffraction limit of visible light other microscopes have been designed
which use other waves.
• Atomic Force Microscope (AFM)
• Scanning Electron Microscope (SEM)
• Scanning Ion-Conductance Microscope (SICM)
• Scanning Tunneling Microscope (STM)
• Transmission Electron Microscope (TEM)
• X-ray microscope
The use of electrons and x-rays in place of light allows much higher resolution - the wavelength of the radiation is
shorter so the diffraction limit is lower. To make the short-wavelength probe non-destructive, the atomic beam
imaging system (atomic nanoscope) has been proposed and widely discussed in the literature, but it is not yet
competitive with conventional imaging systems.
Optical microscope 8
STM and AFM are scanning probe techniques using a small probe which is scanned over the sample surface.
Resolution in these cases is limited by the size of the probe; micromachining techniques can produce probes with tip
radii of 5-10 nm.
Additionally, methods such as electron or X-ray microscopy use a vacuum or partial vacuum, which limits their use
for live and biological samples (with the exception of ESEM). The specimen chambers needed for all such
instruments also limits sample size, and sample manipulation is more difficult. Color cannot be seen in images made
by these methods, so some information is lost. They are however, essential when investigating molecular or atomic
effects, such as age hardening in aluminium alloys, or the microstructure of polymers.
• Digital microscope
• Köhler illumination
• Microscope slide
• "Metallographic and Materialographic Specimen Preparation, Light Microscopy, Image Analysis and Hardness
Testing", Kay Geels in collaboration with Struers A/S, ASTM International 2006.
• A collection of early microscopes 
• Historical microscopes , an illustrated collection with more than 3000 photos of scientific microscopes by
European makers (German)
• Molecular Expressions , concepts in optical microscopy
• Online tutorial of practical optical microscopy 
• OpenWetWare 
• Interactive Overview of a Light Microscope 
 http:/ / www. brianjford. com/ wavrbcs. htm
 "The Lying stones of Marrakech", by Stephen Jay Gould, 2000
 Fi.it (http:/ / brunelleschi. imss. fi. it/ esplora/ microscopio/ dswmedia/ risorse/ testi_completi. pdf), "Il microscopio di Galileo"
 Stephen G. Lipson, Ariel Lipson, Henry Lipson, Optical Physics 4th Edition, Cambridge University Press, ISBN 9780521493451
 O1 Optical Microscopy (http:/ / www. fy. chalmers. se/ microscopy/ students/ imagecourse/ O1. pdf) By Katarina Logg. Chalmers Dept.
Applied Physics. 2006-01-20
 "Introduction to Stereomicroscopy" (http:/ / www. microscopyu. com/ articles/ stereomicroscopy/ stereointro. html) by Paul E. Nothnagle,
William Chambers, and Michael W. Davidson, Nikon MicroscopyU.
 "Illumination for Stereomicroscopy: Reflected (Episcopic) Light" (http:/ / www. microscopyu. com/ articles/ stereomicroscopy/
stereoreflected. html) by Paul E. Nothnagle, William Chambers, Thomas J. Fellers, and Michael W. Davidson , Nikon MicroscopyU.
 "Illumination for Stereomicroscopy: Darkfield Illumination" (http:/ / www. microscopyu. com/ articles/ stereomicroscopy/ stereoreflected.
html) by William Chambers, Thomas J. Fellers, and Michael W. Davidson , Nikon MicroscopyU.
 "German Future Prize for crossing Abbe's Limit" (http:/ / www. heise. de/ english/ newsticker/ news/ 81528). . Retrieved Feb 24, 2009.
 "Demonstration of a Low-Cost, Single-Molecule Capable, Multimode Optical Microscope" (http:/ / chemeducator. org/ bibs/ 0010004/
1040269mk. htm). . Retrieved February 25, 2009.
 D. Courjon and J. Bulabois (1979). Real Time Holographic Microscopy Using a Peculiar Holographic Illuminating System and a Rotary
Shearing Interferometer. 10.
 http:/ / www. antique-microscopes. com
 http:/ / www. musoptin. com/ mikro1. html
 http:/ / micro. magnet. fsu. edu/ primer/ anatomy/ anatomy. html
Optical microscope 9
 http:/ / www. doitpoms. ac. uk/ tlplib/ optical-microscopy/ index. php
 http:/ / openwetware. org/ wiki/ Microscopy
 http:/ / smartymaps. com/ map. php?s=lightMicroscope
Article Sources and Contributors 10
Article Sources and Contributors
Optical microscope Source: http://en.wikipedia.org/w/index.php?oldid=358448708 Contributors: 2over0, 3dnatureguy, A Karley, Aaagmnr, Aazn, Ajraddatz, Alansohn, Alipson, Amaltheus,
Andy Nestl, Archer3, Arnero, Axl, Beetstra, Bobo192, Bochica, Boing! said Zebedee, Brian the Editor, Bryan Derksen, Can't sleep, clown will eat me, Cancun771, Captain-tucker, Chizeng, Chris
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Dreadstar, Edward Z. Yang, Egil, Ellergodt, EntertainU, Erkcan, Escape Orbit, EyeTrawl, Favonian, Femto, Fountains of Bryn Mawr, Fritzpoll, G. C. Hood, Gaff, Gaius Cornelius, Galoubet,
Ganjil213, Gerry Ashton, Goodparley, HaeB, Haiqu, HalfShadow, Hao2lian, Haydood23, HeartofaDog, Hema 030, Heron, HexaChord, HiraV, Hu12, Hugh2414, Huntthetroll, Hurricanehink,
Iridescent, J.delanoy, JTN, Jagged 85, Jakehuston, Jasper33, Jeff G., Jmlk17, John Nevard, John254, JohnCD, Julius Sahara, Keyence, Kkmurray, Klapi, Knight1993, Kopeliovich, Kosebamse,
Kyle1278, Larryisgood, LeaveSleaves, Leonard G., Liamdarga, Lotje, MJD86, Madhero88, Magnus Manske, Majorclanger, MarcoTolo, Marshman, Materialscientist, MauriceJFox3, Mav,
Meeples, Megaman en m, Mentifisto, Merrimoles, Michael Daly, Microscopist, Mikael Häggström, Mike2vil, Mintleaf, Monedula, Mpe, Mr.Z-man, Ncross35, Nineteenninetyfour, Nmedard,
Nonagonal Spider, Numbo3, Okedem, Oliver12, Omicronpersei8, Overney, Oxymoron83, Pb30, Peter G Werner, Peter XX, Peterlewis, PhilKnight, Philopp, Piano non troppo, Pichote,
Plastikspork, Psyche825, Puchiko, Pxma, Quigabyte, Quinsareth, RJHall, Rcej, Res2216firestar, Rettetast, Rigadoun, RockMFR, RokasT, Sagaci, Sangak, Sarindam7, Scarian, Scotto001,
Sebhaase, Sentausa, Shadowjams, Slooj, Snigbrook, Squidonius, Srleffler, Stephenchou0722, Sunray, Superweapons, Svance, Tamasflex, Tameeria, Tellyaddict, Thelb4, Topory, Trabelsiismail,
Velela, W0lfie, WLU, Webmaster5000, WikHead, WingkeeLEE, Yamamoto Ichiro, Yosri, Zaphraud, Zephyr2k, Zephyris, Zzuuzz, 375 anonymous edits
Image Sources, Licenses and Contributors
Image:Stelluti bees1630.jpg Source: http://en.wikipedia.org/w/index.php?title=File:Stelluti_bees1630.jpg License: unknown Contributors: Francesco Stelluti
Image:Optical microscope nikon alphaphot.jpg Source: http://en.wikipedia.org/w/index.php?title=File:Optical_microscope_nikon_alphaphot.jpg License: Public Domain Contributors:
Image:Microscope-optical path.svg Source: http://en.wikipedia.org/w/index.php?title=File:Microscope-optical_path.svg License: GNU Free Documentation License Contributors: Adjustit,
Image:Optical stereo microscope nikon smz10.jpg Source: http://en.wikipedia.org/w/index.php?title=File:Optical_stereo_microscope_nikon_smz10.jpg License: Public Domain Contributors:
Image:Stereomic.png Source: http://en.wikipedia.org/w/index.php?title=File:Stereomic.png License: Creative Commons Attribution-Sharealike 3.0 Contributors: User:Tamasflex
Image:manusingmicroscope.jpg Source: http://en.wikipedia.org/w/index.php?title=File:Manusingmicroscope.jpg License: Public Domain Contributors: Dietzel65, Maksim
Image:2008Computex DnI Award AnMo Dino-Lite Digital Microscope.jpg Source:
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Image:Microscope And Digital Camera.JPG Source: http://en.wikipedia.org/w/index.php?title=File:Microscope_And_Digital_Camera.JPG License: GNU Free Documentation License
Creative Commons Attribution-Share Alike 3.0 Unported
http:/ / creativecommons. org/ licenses/ by-sa/ 3. 0/
Learning unit 6
By the end of this unit, you will be able to:
demonstrate O the correct set-up and use of a binocular
microscope with artificial and with natural light;
demonstrate O the correct use of the x10 paired oculars and x100
oil immersion objective;*
operate O the mechanical stage correctly;
name O correctly 10 component parts of the microscope;
describe O the correct way in which to maintain a microscope in
good working order;
describe O two ways of storing a microscope correctly; and
demonstrate O the correct way of packing a microscope for long-
* Or x7 oculars if they are used in the programme
For efficient malaria microscopy, learn
to use the microscope correctly; know
its limitations and how to keep it in
good working condition.
Monocular microscopes have a single eyepiece (ocular). They are most useful when
no power supply is available. Daylight provides a bright microscopic field for mon-
ocular microscopes. Binocular microscopes, with two eyepieces, have replaced
monocular ones, as they are more comfortable to use, but daylight provides poor
illumination for these microscopes.
The microscope you will use during training and back at your home base is called a
compound binocular microscope. Optimal malaria microscopy is done with micro-
scopes fitted with x10 paired eyepieces and an x100 oil immersion objective.1
To ensure the high standards of illumination required for routine binocular ma-
laria microscopy, it is essential to have a good, reliable source of artificial light. If a
constant supply of electricity is not available, a generator can be used. Delivering
1 Some programmes prefer ×7-paired oculars but they are not easy to obtain. The ×7 ocular covers more blood per
field and is therefore considered by some workers to be more sensitive.
Basic MALARIA MICROSCOPY
even small generators and fuel to remote clinics can be difficult, however, and high
running costs make this method unacceptable. Cheaper, easier sources of artificial
light for microscopy are light-emitting diodes (LED), a form of electrolumines-
cence that can be derived from small, low-voltage batteries. The batteries can be
charged by a small solar panel mounted on a pole or the roof of the laboratory. A
range of these products is available on the market. Most are affordable, easy to use
and require minimal maintenance. Your facilitator will discuss this subject further,
depending on how important it is to you and the programme.
The LED light illustrated here can run for a minimum of 200 hours on four standard 1.5-volt batteries.
Parts of the compound binocular microscope
The main parts of a typical compound binocular microscope are shown above.
1 and 2. Main tube and body tube
Collectively called the microscope head, the main tube and body tube are designed
to slope towards the user and are called an ‘inclined head’. Polished glass prisms
Learning Unit 6. The microscope
inside the body tube of the inclined head bend the light so that the image reaches
the user’s eyes through the paired oculars.
3. Revolving nosepiece
Three or four objective lenses of different magnifications screw into the nosepiece.
The nosepiece revolves to place a different objective over the specimen, in line with
the eyepieces, which increases or decreases magnification of the specimen.
4. Objective lenses
All the parts of the microscope are important, but the objective lenses must be
treated with particular care. An objective consists of two or more lenses kept in
place by a special glue or cement. Solvents such as alcohol, xylol and acetone can
dissolve the cement holding the lens in place and should not be used to clean the
objectives or any other part of the microscope.
An objective is referred to by its magnifying power, which is usually marked on the
side of the body. Each microscope usually has a x10, a x40 and a x100 objective. The
x100 is called the ‘oil immersion objective’ and can be identified by a distinctive
black, red or white ring.
When you examine an objective lens, you will notice that the size of the front lens
decreases with the magnifying power. The working distance between the front lens
and the focused specimen on the stage changes with the magnification. Thus, the
higher the objective’s magnifying power, the shorter the working distance. Care
must therefore be taken not to damage the specimen with the objective lens.
Although there may be small variations according to the manufacturer, the work-
ing distance for each objective is approximately:
x10 15.98 mm
x40 4.31 mm
x100 1.81 mm (oil immersion)
The microscope must be used with care, as specimens, slides and even the ob-
jective lens can easily be damaged by rough manipulation or when objectives are
5. Mechanical stage
The mechanical stage holds the slide secure while allowing specimens to be moved
smoothly. A scale fitted to two sides shows the specimen’s position and subsequent
movement during examination. This scale is called the Vernier scale. You will use
this scale to trace portions of the blood film that should be re-examined or shown
to others. In modern binocular microscopes, the stage moves when the specimen is
focused. In older microscopes, the body and tube move during focusing.
21!Y 51!Y 211!Y
Objectives, showing working distance between
front lens and specimen
Basic MALARIA MICROSCOPY
6. Substage condenser, iris diaphragm and filter holder
The substage condenser consists of a number of lenses that centre the light from
the source or mirror onto a central spot on the microscopic field. The substage con-
denser can be raised or lowered to give maximum or minimum illumination.
Inside the condenser is the iris diaphragm, which is used to control the amount of
light passing through the condenser. It consists of a number of thin, interlocking
metal leaves, which are adjusted by moving a small lever.
Beneath the iris diaphragm is the filter holder, in which a frosted blue-glass filter is
placed when electricity is the light source. This makes the microscopic field appear
white rather than yellow.
The procedure for setting the correct illumination of the microscope, i.e. Köhler
illumination, is important for optimum resolution and contrast, ensuring an even-
ly illuminated field, removing glare and reducing heating of the specimen, as de-
scribed in the enclosed CD-ROM.
Modern microscopes have a fixed illuminator, in which a built-in prism mirror
brings light to the microscopic field. Others have a removable illuminator, which
can be replaced by a mirror when electricity is not available.
The substage mirror is used to direct light from the light source to the microscope
field. It has two sides: one plane (flat) and the other concave. The flat surface is used
with the substage condenser. The concave side is used without the substage con-
denser, as the curved surface itself acts as a condenser.
8. Base or foot
To avoid movement or wobbling, the solid base, or foot, of the microscope must
rest on a firm, flat surface. The shape of the foot may vary. Most have a threaded
hole in the underside of the base to receive a securing screw that keeps the micro-
scope rigid in the box during transport.
9. Ocular, or eyepiece
The top of the main tube of modern microscopes is fitted with a binocular head, i.e
with two oculars, one for each eye. Monocular microscopes are seldom used today
in national malaria control programmes.
The ocular fits into the upper end of the main tube, and the microscopist looks
through it when using the microscope. The magnifying power of each ocular is
marked on it. The ‘magnifying power’ is the number of times by which it will mag-
nify the image produced by the objective. For example, with oculars of x10 and
an oil immersion objective of x100, the total magnification of the specimen would
be 10 x 100 = 1000 diameters. The magnification is actually a little more, but 1000
diameters is accurate enough for our purposes.
Oculars are available in a range of powers, from x5 to x25 or even x30. In ma-
laria microscopy, a range of x6 to x10 is used routinely. One large programme has
used x5 oculars for many years. Today, x10 is probably the most commonly used.
Programmes are strongly advised to use oculars between x7 and x10 for routine
Oculars fitted to binocular microscopes are called paired oculars. The mark-
ing ‘x10P’ on the rim of a x10 ocular indicates that it is one of a paired set of
10. Arm or limb
The arm forms a rigid support for the main tube and stage of the microscope. It is
robust and can be used as a handle for carrying the microscope. When carrying a
microscope in this way, always support the base of the microscope with the other
11 and 12. Coarse and fine adjustments
The two adjustment systems, coarse and fine, are used to focus on the specimen
being examined. The coarse adjustment is used for rapid, relatively large vertical
focusing movements, while the fine adjustment is for the more precise focusing
required with higher-powered objectives. In modern microscopes, the coarse and
fine adjustments raise and lower the mechanical stage. In older microscopes, the
main tube is raised to focus.
Usually, a specimen is first examined with the coarse adjustment and then exam-
ined in detail with the fine adjustment.
The coarse adjustment is used differently when the oil immersion objective is used,
as will be explained in a later learning unit.
Use of the microscope
In the practical sessions, you will use and become familiar with all the features of
the microscope. Early on, you will see the image of the specimen becoming larger
as the magnification is increased. This takes place when you change objectives.
You will also examine everyday objects and see how different they look under the
microscope. These exercises are designed to help you learn to adjust the illumina-
tion correctly and to use the substage condenser and iris diaphragm. You will also
practise using the mechanical stage and Vernier scale.
The light source
A good source of artificial light is needed to examine specimens properly. Light
that is either too bright or too dim will interfere with malaria microscopy.
When the oil immersion objective of a binocular microscope is used routinely, elec-
tric light from a mains supply or a generator should be used. Battery-operated LED
light sources are a useful alternative when electric light is not available and should
be directed towards the mirror. Artificial LED light travels through the mirror on
a path from the source as follows:
source mirror substage condenser and diaphragm specimen objective oculars
When artificial light is used, a frosted blue filter must be placed between the source
and the substage condenser. The flat side of the mirror is used.
Learning Unit 6. The microscope
Basic MALARIA MICROSCOPY
Daylight should be used only in an emergency. When daylight is the light source,
the concave mirror should be used without the substage condenser. It is danger-
ous to point the mirror directly at the sun when obtaining illumination, as serious
damage can be caused to the eyes.
Obtaining even illumination
Using x10 paired oculars and an x10 objective:
Place the slide on the mechanical stage, with the specimen over the central 1.
opening in the stage.
Focus on the specimen using the coarse adjustment.2.
Make sure that the iris diaphragm is wide open, and raise the substage con-3.
denser until the microscopic field is brightest.
Remove the eyepieces and, looking down the tube, adjust the mirror (if it is be-4.
ing used) until the objective lens is fully illuminated.
Replace the eyepieces. Use the fine adjustment to sharpen the focus on the 5.
Remove the eyepieces again, and slowly close the iris diaphragm until the aper-6.
ture of the objective is two-thirds visible. The specimen will appear clearer, with
Replace the eyepieces, and revolve the nosepiece to select the objective you want 7.
to use. Each time you change the objective, you must refocus.
If the intensity of the light from the substage lamp is constant, the illumination 8.
can be adjusted by increasing or decreasing the aperture of the iris diaphragm.
In some microscopes, it is possible to adjust the intensity of the light from the
Using the oil immersion objective
When preparing the microscope for oil immersion microscopy:
Arrange the illumination as described above, then observe the next steps from 1.
the side of the microscope.
Using the coarse adjustment, rack the stage down, away from the objective 2.
Place the slide on the microscope stage, with the blood film uppermost.3.
Making sure that there will be sufficient space between the stage and the x100 4.
objective, revolve the nosepiece until the x100 objective is over the specimen.
Place one or two drops of immersion oil on the area of the blood film to be 5.
Using the coarse adjustment, move the stage until the objective lens is in contact 6.
with the immersion oil. Raise the stage slightly, making sure that the lens and
oil remain in contact.
Looking down the eyepieces, focus on the specimen with the fine adjustment. 7.
Make sure that the lens does not touch the slide. Correct the illumination by
adjusting the iris diaphragm.
Immersion oil is used between the microscope slide and the objective lens to re-
duce scattering of transmitted light. The oil must reproduce the optical properties
of the glass used for the lenses and must therefore have a refractive index of 1.515,
which is approximately 1.5 times the refractive index of water.
Commercially available immersion oils can be cleaned off the objective lens with a
soft cotton cloth. Do not use this cloth to clean other lenses. Immersion oil on blood
films can be gently washed away with the solvent recommended by the manufac-
turers, or the slides can be placed face down for a while on clean, white absorbent
tissue paper that soaks up the oil. Some workers wipe the oil off films with absor-
bent tissue, but this method is rough and is not recommended. Another method is
to roll examined slides in white tissue paper (toilet paper will do), with one layer of
tissue paper between each slide. After a few days, when the paper has absorbed the
oil, the slides can be removed from the paper. Coloured tissue should not be used
as it is often acidic and will de-stain blood films.
Care of the microscope
Provided normal care and common sense are exercised, your microscope will re-
main in good condition for many years.
Removing dust and grease
During the day, when the microscope is not in use, it should be kept covered with
a clean cloth or plastic cover to protect the lenses from settling dust. Overnight, or
if the microscope will remain unused for a long time, it should be placed inside its
box, with the door tightly closed. To protect the objective lenses, the x10 objective
should be rotated to line up with the ocular.
Oil from eyelashes, facial skin and fingers is easily deposited on lenses and oculars
during use. These parts should be cleaned carefully with lens tissue or a soft cotton
Oil immersion objectives must be cleaned immediately after use. If not, the oil
will thicken and harden over time, and the objective will become useless. To avoid
further transfer of oils, never use contaminated cloths to clean other objectives,
oculars or the mirror.
Preventing fungal growth
In warm, humid climates, fungal growths are easily established on lenses and
prisms. Fungal growth causes problems and can become so bad that a microscope
cannot be used. In such cases, the affected surfaces might have to be cleaned and
repolished—a job usually done by the manufacturer, which takes time and can be
Fungus cannot grow on glass surfaces when the atmosphere is dry. Therefore, it O
is important to store the microscope in dry conditions when not in use. One of
the following methods should be used.
Keep the microscope in a ‘warm cupboard’, which has a tightly fitting door and O
two or more, constantly burning 25-watt bulbs, depending on the size of the
Learning Unit 6. The microscope
Basic MALARIA MICROSCOPY
cupboard. The temperature inside the cupboard should be a constant 30–35 °C,
with low humidity.
Keep all lenses and prism heads in an airtight box or desiccator containing O
active silica gel, which is a ‘desiccant’ and absorbs water vapour from the air.
Self-indicating silica gel is blue when active and becomes pink as it absorbs
water vapour. When it is bright pink, it can be reactivated by heating; it is ready
to use again (after cooling) when it has become bright blue.
If possible, keep the microscope in a continuously air-conditioned room. Rooms O
that are air-conditioned only during the working day are not suitable.
Transporting the microscope
When transporting the microscope between laboratories or to the field, it is im-
portant to ensure that it is properly secured inside its box. The best way to do this
is by screwing the securing device through the hole in the bottom of the box into
the base or foot of the microscope. When this is done correctly, the microscope
remains rigid in its box on even the roughest road.
Read Learning unit 7 in preparation
for the next session.
Kohler!Illumination:!!It(is(a(method(of(illumination(which(involves(optimizing(a(microscope’s(optical(train(to(produce(homogenously(bright(light(free(from(artifacts(and(glare.(In(Kohler(illumination,(four(separate(planes(combine( to( form( conjugate( planes( in( both( the( illumination( and( imageCforming( light( pathways.( The( lamp(filament,(aperture(diaphragm,(back(focal(plane(of(the(objective(lens,(and(the(eye(point(which(is(approximately(one(centimeter(above(the(top(lens(of(the(ocular,(form(the(illumination(conjugate(plane.(The(conjugate(planes(of(the(imaging(light(path(are(the(field(diaphragm,(specimen,(the(fixed(diaphragm(of(the(ocular,(and(the(retinal(plane( of( the( viewer.( In( Kohler( illumination( the( collector( lens( or( field( diaphragm( collects( light( from( the(illumination(source(and( focuses( it(at( the( front( focal(plane(of( the(subCstage(condenser’s(aperture(diaphragm(which,(in(essence,(projects(an(image(of(the(lamp(filament(onto(the(lens.(
#! Text&Box! Comments!
1! Begin:!Microscope! Begin!diagnostic!process!for!a!work!order!for!Microscope.!Maintenance!is!generally!requested!on!a!microscope!when!a!specimen!cannot!be!viewed!clearly!or!at!all.!
2! Does!the!microscope!power!on!when!plugged!in?! When!plugged!in,!the!microscope!should!power!on!completely.!!
3! Troubleshoot!power!supply!(separate!chart).! If!no!power!reaches!the!machine,!there!may!be!problems!with!the!switch,!fuse,!power!supply!components,!or!wiring.!!See!flowchart!on!Power!Supply!and!BTA!skills!on!Power!Supply.!
4! Does!the!light!source!turn!on!and!stay!on?! The!light!source!should!remain!constant!across!the!stage!when!on.!
5! Does!the!light!turn!on!and!flicker?! If!the!light!turns!on!but!does!not!remain!constant,!there!may!be!a!minor!problem!that!can!be!fixed!without!replacing!the!bulb!completely.!
6! Ensure!contact!is!clean!and!secure!any!loose!connection.! The!inside!of!the!lamp!house!or!the!connections!may!be!dirty.!The!connections!should!also!be!secured!firmly.!See!BTA!skills!for!Connections.!
7! Is!resistance!at!each!end!of!the!filament!0!ohms?! This!checks!if!the!filament!in!the!bulb!is!functioning.!
8! Check!internal!circuitry,!dimmer!knob,!or!diaphragm,!if!applicable.! Ensure!that!the!circuitry!that!connects!to!the!light!source!is!intact.!In!addition,!ensure!the!dimmer!knob!is!turned!on!and!that!the!diaphragm!is!open.!
9! Replace!bulb!with!same!type!of!bulb.! If!the!light!source!still!does!not!turn!on,!replace!the!bulb!with!another!of!the!same!type.!If!the!same!type!of!bulb!is!not!available,!a!new!source!can!be!wired!in.!See!BTA!skills!on!Replacement!of!Light!Bulbs!and!Light!Fixtures.!10! Go!to!begin.! Restart!the!diagnostic!process.!
11! Focus!the!microscope!at!the!lowest!objective.! Begin!this!portion!of!the!diagnostic!process!at!the!lowest!objective.!
12! Can!the!specimen!be!viewed!clearly?! If!the!specimen!is!out!of!focus!or!cannot!be!viewed!at!all,!the!objectives!may!need!attention.!
14! Do!the!mechanical!adjustment!knobs!turn!easily?! The!knobs!and!stage!should!be!able!to!move!freely!and!also!maintain!a!steady!position.!The!screws!holding!each!in!place!may!need!some!adjustment.!
16! Ensure!the!microscope!is!covered!to!avoid!dust!settling!on!it.!! When!covering!the!microscope,!put!a!small!amount!of!uncooked!rice!to!prevent!fungal!growth.!Replace!uncooked!rice!weekly.!17! Microscope!is!working!properly.! Return!the!machine!to!the!appropriate!clinical!personnel.!