Introduction to the

Microscope
It is expected that you should
. be able to :
 Describe the history of microscopes from
simplest type to the modern complex types of
microscopes
 State types of microscopes
 State and distinguish the different types of
compound microscopes
 State and differentiate different type light
microscopes
 State and differentiate different types of
Electron microscope
 Why do we use a microscope?
Many things are smaller than we can see with
the naked eye.
To accurately study Biology we need a tool to
help us see these tiny things.
 Sizes of living things

The tallest living thing was probably a tree

(Eucalyptus regnans) which was found in
Australia in 1872. This was over 154m in
height and this did not include the roots.
From that maximum the sizes of living things
go down a long way to the minimum size of
viruses (about 10nm).
The human eye can at best resolve (see)
objects that are as small as 100µm in
diameter.
 We cannot see smaller objects because a
limit is imposed by the structure of our eyes.
 Magnifying lenses, made of rock crystal are
said to have been used by the ancient
Chinese, centuries before Christ, but glass
lenses only became regularly available in
Europe in the 16th Century.
Better lenses of this type were developed,
giving greater magnification, but their
usefulness was limited because any lens made
of single piece of glass produces coloured
fringes around the edges of the images of
objects under magnification.
A mounting lens of this sort is called a SIMPLE
MICROSCOPE.
One way of improving the quality of the
.
magnifying lens is to mount two or three
lenses, made of different sorts of glass,
together in one mount.
In this way the problem of colour fringes can
be overcome.
Lenses mounted together in this way can be
made to give magnifying power of up to about
30X.
Circa 1000AD –
The first vision aid
was invented
(inventor unknown)
called a reading
stone. It was a glass sphere that
magnified when laid on top of reading
materials.
Circa 1284 –
Italian, Salvino D'Armate is credited
with inventing the first wearable eye
glasses.
1590 – Two Dutch eye glass makers,
Zaccharias Janssen and son Hans Janssen
experimented with multiple lenses
placed in a tube. The Janssens observed
that viewed objects in front of the tube
appeared greatly enlarged, creating both
the forerunner of the compound
microscope and the telescope.
In 1609 – Galileo Galilei developed a
compound microscope with a convex and
concave lens.
In 1625 - The name microscope was coined by
Giovanni Faber for Galileo Galilei’s compound
microscope.
In the 1660s – the extensive use of
microscopes in research began in Italy,
Holland and England.
1665 – English
physicist, Robert
Hooke looked at a
sliver of cork
through a
microscope lens and
noticed some
"pores" or "cells" in
it.
1674 – Anton van Leeuwenhoek built a
simple microscope with only one lens to
examine blood, yeast, insects and many
other tiny objects. Leeuwenhoek was the
first person to describe bacteria, and he
invented new methods for grinding and
polishing microscope lenses that allowed
for curvatures providing magnifications of
up to 270 diameters, the best available
lenses at that time.
October 9th 1676 – Antonie Van
Leeuewenhoek, the father of microscopy,
reported the discovery of micro-organisms.
Van Leeuewnhoek also made the single lens
microscope and in 1670 developed a method
for grinding very small glass lenses, the lenses
were so thin (in order of millimetres in
diameter) they could magnify several hundred
times.
In the late 17th century – a Dutchman,
Christiaan Huygens, developed a simple 2 lens
ocular system that was achromatically
corrected (a lens that is designed to limit the
effects of chromatic and spherical aberration).
Achromatic lenses are corrected to bring two
wavelengths, typically red and blue, into focus
in the same plane, this was a big improvement
in microscopes.
18th century – Technical innovations
improved microscopes, leading to
microscopy becoming popular among
scientists. Lenses combining two types
of glass reduced the "chromatic effect"
the disturbing halos resulting from
differences in refraction of light.
1830 – Joseph Jackson Lister reduced
spherical aberration or the "chromatic
effect" by showing that several weak
lenses used together at certain distances
gave good magnification without
blurring the image. This was the
prototype for the compound microscope.
1872 – Ernst Abbe, then research
director of the Zeiss Optical Works,
wrote a mathematical formula called the
"Abbe Sine Condition". His formula
provided calculations that allowed for
the maximum resolution in microscopes
possible.
• In 1893 – August Kohler developed a key
technique for sample illumination. The Kohler
illumination is central to modern light
microscopy.
1903 – Richard
Zsigmondy developed
the ultramicroscope
that could study
objects below the
wavelength of light.
He won the Nobel
Prize in Chemistry in
1925.
1932 – Frits Zernike
invented the phase-
contrast microscope
that allowed for the
study of colorless and
transparent biological
materials for which he
won the Nobel Prize
in Physics in 1953.
1931 – Ernst Ruska co-invented the
electron microscope for which he won the
Nobel Prize in Physics in 1986. An
electron microscope depends on electrons
rather than light to view an object,
electrons are speeded up in a vacuum
until their wavelength is extremely short,
only one hundred-thousandth that of
white light. Electron microscopes make
1931 – Ernst
Ruska

it possible to
view objects
as small as
the diameter
of an atom.
In 1932 – Fritz Zernike invented the phase-
contrast microscope that enabled the study of
colourless and transparent biological
materials.
In 1953 he won a Nobe Prize in physics for this
invention.
1981 – Gerd Binnig and Heinrich Rohrer
invented the scanning tunneling
microscope that gives three-dimensional
images of objects down to the atomic
level. Binnig and Rohrer won the Nobel
Prize in Physics in 1986. The powerful
scanning tunneling microscope is the
strongest microscope to date.
Microscope

•Care
• Parts & functions
• use and
focusing
• Light microscope
•Simple microscopes
•Stereo Microscope- Dissection Microscope
•mounted lens
•Compound Microscope
•Binocular Compound Microscope
•Monocular compound microscopes
 Compound light microscopes
– bright-field microscope
– dark-field microscope
– phase-contrast microscope
– fluorescence microscopes
• Electron microscopes
– Scanning Electron Microscope
– Transmission Electron Microscope
– (REM) Reflection Electron Microscope
– (STEM) Scanning Transmission Electron
Microscope
– (LVEM) Low Voltage Electron Microscope
– (STM) scanning tunnelling microscope
 Light microscope
Simple microscopes
 .
Compound Simple Dissecting
Compound microscopes are light
illuminated. The image seen with this type
of microscope is two dimensional. This
microscope is the most commonly used.
You can view individual cells, even living
ones. It has high magnification. However,
it has a low resolution.
• THE COMPOUND MICROSCOPE (CM) was first
.
designed in 1590, by using two sets of lenses
mounted at opposite ends of a tube; it is
possible to obtain much higher magnification.
One lens system (the OBJECTIVE) is close to
the object being examined and forms an
image high in the tube. This image again is
magnified by a lens high in the tube (the
EYEPIECE OR OCULAR LENS).
• BY 1850 the CM had been perfected as far as
.
magnification and resolution were concerned.
Subsequent improvement to the LIGHT
MICROSCOPES (CMs using light visible to the
eye) have made them easier to use but have
not increased their RESOLVING POWER (Ability
to distinguish as separate objects, those
objects which are less than certain distance
apart). This limit is set by the wavelength of
visible light. Even if the magnification is
increased, no more detail is seen.
 .
Since the wavelength of light is the limiting
factor, attempts were made to construct
microscopes using “light” of shorter
wavelength.
This gave rise to utra-violet light microscopes
and eventually to the birth ELECTRON
MICROSCOPE (EM) IN 1940.
 Paulownia Wood c.s.
200x

Frog’s blood
1,000x
A dissection microscope is light
illuminated. The image that appears is
three dimensional. It is used for dissection
to get a better look at the
larger specimen. You cannot
see individual cells because
it has a low magnification.
(also called stereo
microscope)
• One way of improving.the quality of the
magnifying lens is to mount two or three
lenses, made of different sorts of glass,
together in one mount.
• Lenses mounted together in this way can be
made to give magnifying power of up to about
30X but anything over 20X becomes difficult
to handle. Some DISSECTING MICROSCOPES,
have lenses made up of several pieces of glass
mounted together.
One lens- DMS .System of lenses - DMS
Head of a moth pupa
60x

Sunflower with moth

pupa in the stem
10x
• The EM uses a beam of . electrons as “light”
and electromagnets as “lenses”. Electrons will
not penetrate through air so there must be a
vacuum inside the microscope. Since electrons
are invisible to the eye, the image is projected
on a TV screen or is captured on photographic
film. The specimen must be ultrathin inorder
for the electrons to penetrate a section of the
specimen. With EMs objects as small as atoms
can be “seen
• Types of EMs.
• TRANSIMISION EM (TEM)
• THE SCANNING EM (SEM)
• SCANNING TRANSMISSION EM (STEM
SEM use electron illumination. The
image is seen in 3-D. It has high
magnification and high resolution. The
specimen is coated in gold
and the electrons bounce
off to give you and exterior
view of the specimen. The
pictures are in black and
white.
 pigeon blood

cockroach antenna
TEM is electron illuminated. This gives a
2-D view. Thin slices of specimen are
obtained. The electron beams pass
through this. It has
high magnification
and high resolution.
 bacillus bacteria
dividing

mitochondrion
• It expected that you should be able among others to :
.
1. state the parts of a microscope
2. Mention the name /label the part of a given a microscope or
drawing of a microscope
3 state the function of each part of a microscope
4 Explain the steps you would follow to view a microscopic
specimen under the microscope.
5 explain how you would take care of a microscope
7 State the precautions you would observe to ensure proper
and safe use of a microscope
8 use the microscope safely and successfully .
 Know the following Microscope parts
and their function.
• Eyepiece • Fine adjustment knob
• Ocular lens • Coarse adjustment knob
• Nosepiece • Stage manipulator knobs
• Objective lens • Condenser
• Stage • Light source
• Stage clip • Iris diaphragm knob
• Light switch • Cord holder
• Light intensity knob • Microscope body
 Ocular lens
Body Tube

Revolving Nosepiece
Arm
Objective Lens

Stage
Stage
Clips Coarse adjustment knob
Diaphragm
Fine adjustment knob
Light

Base
• .
 .
• .
Ocular lens

magnifies; where you

look through to see the
image of your specimen.
They are usually 10X or
15X power. Our
microscopes have an ocular
lens power of 10x.
 supports the tube and
connects it to the
arm base
 the flat platform
where you place
your slides
stage
 moves stage (or body
tube) up and down

coarse adjustment knob

 small, round knob on
the side of the
microscope used to
fine-tune the focus of
your specimen
fine adjustment knob
after using the coarse
adjustment knob
 the bottom of the
microscope, used for
support

base
 body tube

connects the eyepiece

to the objective
lenses
the part that holds two
or more objective lenses
revolving nosepiece
and can be rotated to
easily change power
 Adds to the magnification
Usually you will find 3 or
4 objective lenses on a
microscope. They almost
objective lens
always consist of 4X, 10X,
40X and 100X
powers. When coupled
with a 10X (most common)
eyepiece lens, we get total
magnifications of 40X (4X
times 10X), 100X , 400X
and 1000X.
The shortest objective lenses
lens is the lowest power, the
longest one is the lens with
the greatest power. Lenses
are color coded.
The high power objective
lenses are retractable (i.e.
40XR). This means that if
they hit a slide, the end of
the lens will objective lenses
push in (spring loaded)
thereby protecting the lens
and the slide.
Stage clips hold the slides in
place. If your microscope
has a mechanical stage, you
will be able to move the
slide around by turning two
knobs. One stage clips
moves it left and right, the
other moves it up and down.
 controls the amount of light
going through the specimen
Many microscopes have a
rotating disk under the
stage. This diaphragm has
different sized holes and is
diaphragm
used to vary the intensity
and size of the cone of light
that is projected upward into
the slide. There is no set rule
regarding which setting to use
for a particular power. Rather,
the setting is a function of the
transparency of the specimen,
the degree of contrastdiaphragm
you desire and the particular
objective lens in use.
makes the specimen
easier to see

light
 Microscope Part Function
1- Ocular lens (eyepiece) Lens through which you view
magnified specimen
2- Revolving nosepiece Movable mount for selecting
objective lens best suited for
extent magnification desired.
3- Objective lens. Lens on revolving nosepiece
which accomplishes the initial
magnification of the specimen.
4- Stage Flat work surface upon which
the slide is placed. Some
microscopes have a mechanical
stage which is used for precise
movement of a slide via control
knobs geared to the stage.
5- Disc diaphragm Regulates the intensity of light
passing through the stage
aperture and specimen.
6- Light Source A lamp beneath the disc
diaphragm, provides a constant
light source.
7- Stage clips Fit over ends of a slide to hold it
in place. A microscope having a
mechanical stage has a Lever
which is opened laterally (never
lifted)To secure the side surfaces
of a slide.
8- Coarse adjustment knob Gives gross movement of the
nosepiece for initial focus
effort.
9- Fine adjustment knob Gives refined movement of
the nosepiece for finishing
focus.
10- Base Supports the microscope.
 THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE

FEATURE LIGHT ELECTRON

Electromagnetic
MICROSCOPE
Visible light
MICROSCOPE
Electrons
spectrum used 760nm (red) – app. 4nm
390nm Colours Monochrome
Maximum app. 200nm
visible 0.2nm
resolving power Fine detail
Maximum
magnification x1000 – x1500 x500 000
Radiation Tungsten or quartz High voltage (50kV)
source halogen lamp tungsten lamp
Lenses Glass Magnets
Interior Air-filled Vacuum
Focussing Human eye (retina), fluorescent (TV) screen,
screen photographic film photographic film
© 2007 Paul Billiet ODWS
 THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE

ELECTRON
FEATURE LIGHT MICROSCOPE
MICROSCOPE
Preparation of Temporary mounts Tissues must be
specimens living or dead dehydrated
= dead
Fixation Alcohol OsO4 or KMnO4
Embedding Wax Resin
Sectioning Hand or microtome Microtome only.
slices  20 000nm Slices  50nm
Whole cells visible Parts of cells visible
Stains Water soluble dyes Heavy metals

Support Glass slide Copper grid

© 2007 Paul Billiet ODWS
 .

• .
 .

• .
 .

• .
BRIGHTFIELD ILLUMINATION:
No Stain
BRIGHTFIELD ILLUMINATION:
With Stain
DARKFIELD ILLUMINATION
DARKFIELD ILLUMINATION
Brightfield vs Darkfield
PHASE CONTRAST MICROSCOPY
DIFFERENTIAL INTERFERENCE
CONTRAST
 Fluorescence Microscopy
• Uses UV light.
• Fluorescent
substances absorb
UV light and emit
visible light.
• Cells may be
stained with
fluorescent dyes
(fluorochromes).
Figure 3.6b
FLUORESCENCE MICROSCOPY
FLUORESCENCE MICROSCOPY
Transmission Electron Microscope
Transmission Electron Microscope
Transmission Electron Microscope:
Inside of a Plant Cell
Scanning Electron Microscope
Scanning Electron Microscope:
Flea
Scanning Electron Microscope:
Pollen
 COMPARISON OF MICROSCOPES
BRIGHTFIELD Dark objects are visible against a bright background.
Light reflected off the specimen does not enter the objective lens
Not for looking at live cells
Maximum resolution is 0.2µm and maximum magnification is
2000x
Stains are used on specimens
DARKFIELD Light objects are visible against dark background
Used for live cells, cilia, flagella
Especially good for spirochetes
Uses special condenser with an opaque disc that eliminates all light
in the center
PHASE- No staying required
CONTRAST Accentuates diffraction of the light that passes through a specimen
Good for live cells; good contrast
Most sensitive; cilia shows up
Not three-dimensional
DIFFERENTIAL Uses two beams of light
INTERFERENCE Shows three dimensions
CONTRAST Has a prism to get different colors
Good for live cells (unstained)
Best resolution
 COMPARISON OF MICROSCOPES

FLUORESCENCE Uses ultraviolet light

Stained cells with fluorescent dye; energizes electrons and creates
visible light
No live cells
Quick diagnosis of TB and syphilis
TRANSMISSION Get flat images
ELECTRON Have vacuum pumps to allow electrons to float better
Stain with heavy metal salts
Shows sections of cell, revealing organelles
Requires an ultramicrotome
Best resolution of all microscopes
SCANNING Surface view only
ELECTRON Needs a vacuum
No live cells
Three-dimensional view
SCANNING Physical probe scans the specimen
PROBE Raster scan: image is cut up into pixels and transmitted to computer
Not limited by diffraction
Slower in acquiring images
Maximum image size is smaller
• Start with
• 1. Lowest objective
2 Next lower objective
3 in the order up to the highest objective
Normally the 3x, 4x 10 x, 15, 20, 30 and 40 are
used
However the light microscope normally have
up to 100x objective
•
The proper way to focus a microscope is
to start with the lowest power objective
lens first and while looking from the
side, crank the lens down as close to the
specimen as possible without touching
it. Now, look through the eyepiece lens
and focus upward only until the image
is sharp. If you can't get it in focus,
repeat the process again.
Once the image is sharp with the low
power lens, you should be able to
simply click in the next power lens and
do minor adjustments with the focus
knob.
If your microscope has a fine focus
adjustment, turning it a bit should be all
that's necessary. Continue with
subsequent objective lenses and fine
Rotate to 40x objective, locate desired
portion of specimen in the center of the
field. Refocus very carefully so that the
specimen is focused as sharply as
possible. (Do not
alter focus for the
Following steps )
Partially rotate so that 40x and 100x
objectives straddle the specimen.
Place a small drop of oil on the slide in
the center of the lighted area. (Take care
not to dribble on the stage.)
Put the small drop
of oil directly over
the area of the
specimen to be
Examined.
Rotate so that the 100x oil immersion
objective touches the oil and clicks into
place.
Focus only with fine focus. Hopefully,
the specimen will come into focus easily.
Do not change focus dramatically.
Clean up!: When you have finished for
the day, wipe the 100x oil immersion
objective carefully with lens paper to
remove all oil. Wipe oil from the slide
thoroughly with a Kimwipe. Cleanse
stage should any oil have spilled on it.
Recap the immersion oil container
securely, replace in drawer.
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 Wet Mounts:

Poorly
Done:

Nicely
Done:
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 The proper way to carry your
microscope.
• Always carry the
microscope with two
hands, holding it close
to your body.
 Improper carrying.
• Carrying the microscope
like this could result in
your ruining a $1500.00
piece of equipment.

• This will not score you

brownie points with
your instructor!
 .
• Only use lens paper for cleaning
• Do not force knobs
• Keep objects clear of desk and cords
• When you are finished with your "scope",
rotate the nosepiece so that it's on the low
power objective, roll the stage down to lowest
level, rubber band the cord, then replace the
dust cover
 Proper usage
• At the lab table, unwrap only
as much cord as you need, the
rest should remain stored
around the cord wrap.
• The microscope should
always be stored with
the 4X (red) lens in
place and the stage in
its lowest position.
 • .
• Always store your .
microscope in its
numbered position,
with the dust cover in
place.
 How to focus your image
• Place the slide on the stage, held in place by the stage
clip.

• ALWAYS start with the 4X lens. Focus your image.

• Move to the 10X lens, focus.

• NEVER use the coarse focus higher than 4X.

• Repeat at the 40X lens, focus using the fine focus.
Distance between lens and slide
• Use only the fine focus
knob with the 40X and
100X lens.
 The Oil Immersion Lens
• The oil immersion lens or 100X lens is used
with special optical oil. It makes the image
clear at a higher magnification
• Your instructor will tell you if you need to use
this lens.
• It is important to remove all the oil if you use
the oil immersion lens.
 Cleaning
• Please use only lens paper to clean the lenses,
never paper towels or Kimwipes.

• Never use dry lens paper to clean eyepieces.

Use the cleaning solution provided or breathe
on the surface to be cleaned, then wipe.
Troubleshooting
Occasionally you may have trouble with working your microscope. Here are
some common problems and solutions.

1. Image is too dark!

Adjust the diaphragm, make sure your light is on.

2. There's a spot in my viewing field, even when I move the slide the spot stays
in the same place!
Your lens is dirty. Use lens paper, and only lens paper to carefully clean the
objective and ocular lens. The ocular lens can be removed to clean the
inside. The spot is probably a spec of dust.

3. I can't see anything under high power!

Remember the steps, if you can't focus under low power, you won't be able to
focus anything under high power. Start at scanning lowest power through the
steps again.

4. Only half of my viewing field is lit, it looks like there's a half-moon in there!
You probably don't have your objective fully clicked into place..
 Ten things to consider when storing a
microscope
• These ten items for consideration are not in
any order or importance or sequence.
• These are the minimal steps to take care of
our microscopes, which are excellent when
they are well cared for.
• Think of your use of the microscope as a new
professional skill.
 #1 Remove slides from stage
• #1 Remove slides from stage.
– Be sure to place the prepared slides in their
correct tray. Look at the label of the slide and the
label of the edge of the tray. Viola!
– Wet mounts should be rinsed by you and the
cover slips and slides placed in the labeled
beakers.
 #2 Rheostat set to lowest level
• #2 Rheostat (or power level) is set at zero or
lowest setting.
• Why?
– If the microscope is turned on with the rheostat at
the highest level a surge of energy can blow the
light bulb.
– Bulbs cost $25 each!
#3 Store the microscope in it’s proper
cabinet
• #3 Put the microscope in its proper cabinet.
The number on the bottom of the arm will
have a room and cabinet number (220-52, for
room LMC 220 and cabinet 52).
• Why is this important?
– To keep track of the microscopes, to determine
which are being repaired or borrowed and not
stolen! Always place it in the correct cabinet.
 #4 Cover
• #4 Cover with a plastic bag or cover.
• Why?
– To protect it from dust. These are very expensive
and sensitive pieces of equipment and we want
them to work well for you.
 #5 Clean the Microscope
• #5 Clean the Microscope
• How?
– Lens paper and glass cleaners for the lenses only!
Don’t forget the eyepieces, too. Although oil
should only be used with the 100X objective it
often gets onto the 40X objective lens and stage
as well.
– Kim wipes can be used to clean the stage and
other parts of the microscope.
 #6 Power is off
• #6 Power is off.
• Why is this important?
If someone plugs in the microscope the and
the power is on the surge of electricity can
blow-out the halogen lamp. Each lamp cost
more than $25!
 #7 Lowest power objective
• #7 Lowest power objective in place.
• Why? One reason is to prevent the stage from
accidentally crashing into the objective lens.
The other reason is that when you begin to
use the microscope you must (should) always
start with the lowest magnification. This is the
best technique and prevents lens damage.
 #8 Arm facing out
• #8 Place microscope in cabinet with arm
facing out.
• Why?
– So that the next student can easily remove it from
the cupboard.
– Also the head piece of the microscope can be
loosened and if the microscope is placed in the
cupboard backwards it may be grabbed by the
headpiece and the bottom dropped!
 #9 Wrap power cord
• #9 Wrap power cord around the base of the
microscope.
• Tip. Don’t wrap it too tight! The wire inside
the power cord can break inside the plastic
covering if it is stressed too much.
 #10 Lock it up
• #10 Lock it up.
• Why? We don’t want to lose them, they’re
very expensive.
• Don’t forget to return the key to the front of
the room or lab cart. If you discover you
accidentally pocketed the key – just bring it
back ASAP.
 RESOLUTION
• The ability of the lenses to distinguish two
points.
 Resolving Power

• Resolving power is a measure of lens quality.

Quality lenses have a high resolving power,
which is the capacity to deliver a clear image
in fine detail. If a lens has a high magnifying
power but a low resolving power it is of little
value. Although the image may be large, it will
not be clear enough to show fine detail.
• Another factor that reduces resolving power is
the cleanliness of the lenses. Dirt, water, or oil
on the lens may scatter light and reduce the
effective resolving power of th microscope.
Therefore, lenses should always be kept clean.
Use only the recommended paper to clean the
highly polished lens’ surfaces.
 RESOLVING POWER
• The distance between two closely adjacent
objects where the objects still appear separate
and distinct. The shorter the distance (the
smaller the number), the better the resolving
power (the sharper the image).
 RESOLVING POWER
• To calculate the resolving power (to see how
close two objects can be so you can still see
them):
• D = distance (smaller number is better)
• 0.61 = a constant number, does not change
• NAobj = numerical aperture of the objective (larger
number causes D to decrease, which is better)
• λ = (lambda): the wavelength (nm) of the light going
through the microscope. Convert nm to mm by dividing
by 1000000 (six zeros)
D = 0.61 λ / NAobj
 Resolving Power

2 objects 2 objects not Same 2 objects resolved with

resolved resolved better optical instrument
Resolving power limits for several
optical instruments:
Optical Instrument Resolving Power
0.2 millimeters
Human eye
(mm)
0.25 micrometers
Light microscope
(µm)
Scanning electron microscope 5-10 nanometers
(SEM) (nm)
Transmission electron microscope 0.5 nanometers
(TEM) (nm)
 PRISM
• This is a triangular device that breaks up light
into its various wavelengths so you can see all
the colors of the rainbow (the visible
spectrum).
• The visible spectrum of colors starts with
violet (350nm), and goes on to indigo, blue,
green (550nm), yellow, orange, red (700nm).
 Sample Problem
• When we want to observe the color green
(550nm) under an oil-immersion objective
lens of a microscope, where the NAobj is 1.25,
the resolution is as follows:
• D = 0.61 λ / NAobj
• D = (0.61)(0.000550) / 1.25
• D = 0.0002684 mm  convert this to
microns (µm) by multiplying by 1000
• D = 0.27 µm
 Sample Problem

• The NAobj for the high-dry (400x) lens is 0.65

• What is the resolving power (D) of this
objective when viewing a wavelength of
550nm?
• D = 0.61 λ / NAobj
• D = (0.61)(0.000550) / 0.65
• D = 5.1615mm
• D = 0.52µm
 Magnification
• The compound microscope combines the
magnifying powers of the ocular lens with the
magnifying powers of the objective lens.
• The magnifying power of each lens is marked on
its tubular housing.
• Simply multiply the magnification value that is
marked on the ocular lens housing times the
value marked on the objective lens housing to
determine how many times your specimen is
enlarged.
• Microscopes often have two additional
objective lenses, namely a high-power lens
and an oil-immersion lens. The latter will be
the longest and have the highest
magnification. The oil-immersion lens will be
used only after you have had special training
in oil-immersion technique.
Magnification

Your microscope has 3 magnifications: Scanning, Low and High.

Each objective will have written the magnification. In addition to
this, the ocular lens (eyepiece) has a magnification. The total
magnification is the ocular x objective
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• Suppose you make a drawing viewed under
LP(4X) and you have estimated that 9 cells are
fitting across the field of view and the length
of your drawing is 10cm. What will be the
magnification of your drawing ?
• Remember formula for mag.
• Magn. = size of the drawing/actual size of the
specimen.