Cells
Cell Size and Shape
The shapes of cells are quite varied with some, such as neurons, being longer than they are wide
and others, such as parenchyma (a common type of plant cell) and erythrocytes (red blood cells)
being equidimensional. Some cells are encased in a rigid wall, which constrains their shape, while
others have a flexible cell membrane (and no rigid cell wall).
Cells range in size from small bacteria (1µm) to large animal and plant cells (100 µm), unfertilized
eggs laid by birds and dinosaurs. These sizes are not visible to the naked eye and so a
microscope is an important tool used by biologists to magnify small objects.
http://learn.genetics.utah.edu/content/begin/cells/scale/
In science we use the metric system for measuring. 1 meter = 100 cm = 1,000 mm = 1,000,000 µm
= 1,000,000,000 nm
1 centimeter (cm) = 1/100 meter = 10 mm
1 millimeter (mm) = 1/1000 meter = 1/10 cm
1 micrometer (µm) = 1/1 000 000 meter = 1/1000 mm
1 nanometer (nm) = 1/1 000 000 000 meter = 1/1 000 000 mm
Microscopes
Types of Microscopes
http://www.udel.edu/biology/ketcham/microscope/scope.html
Resources: Powerpoint Presentation
| Blank Microscope Handout (labeling)
Other Resources
Nanoworld Image Gallery
Microworld Images - SEM, TEM images
MicroscopyU - technical support for microscopy, image galleries
Molecular Expressions - features hundreds of photomicrographs and includes virtual microscopes
Microangela - SEM images of insects that have been colorized, dramatic photos
http://www.xtalent.com.au/gallery/index.php?cat=3
Light Microscope - the models found in most schools, use compound lenses and light to magnify
objects. The lenses bend or refract the light, which makes the object beneath them appear closer.
Dissecting Microscope- this microscope allows for binocular (two eyes) viewing of larger
specimens.
Scanning Electron Microscope - allow scientists to view a universe too small to be seen with a light
microscope. SEMs don’t use light waves; they use electrons to magnify objects up to two million
times.
Transmission Electron Microscope - also uses electrons, but instead of scanning the surface (as
with SEM's) electrons are passed through very thin specimens.
Parts of the Light Microscope
http://www.udel.edu/biology/ketcham/microscope/scope.html
THE LIGHT MICROSCOPE.
The common light microscope used in the laboratory is called a compound microscope because
it contains two types of lenses that function to magnify an object. The lens closest to the eye is
called the ocular, while the lens closest to the object is called the objective. Most microscopes
have on their base an apparatus called a condenser, which condenses light rays to a strong
beam. A diaphragm located on the condenser controls the amount of light coming through it. Both
coarse and fine adjustments are found on the light microscope.
To magnify an object, light is projected through an opening in the stage, where it hits the object
and then enters the objective. An image is created, and this image becomes an object for the
ocular lens, which remagnifies the image. Thus, the total magnification possible with the
microscope is the magnification achieved by the objective multiplied by the magnification achieved
by the ocular lens.
A compound light microscope often contains four objective lenses: the scanning lens (4X), the lowpower lens (10X), the high-power lens (40 X), and the oil-immersion lens (100 X). With an ocular
lens that magnifies 10 times, the total magnifications possible will be 40 X with the scanning lens,
100 X with the low-power lens, 400 X with the high-power lens, and 1000 X with the oil-immersion
lens. Most microscopes are parfocal. This term means that the microscope remains in focus when
one switches from one objective to the next objective.
The ability to see clearly two items as separate objects under the microscope is called the
resolution of the microscope. The resolution is determined in part by the wavelength of the light
used for observing. Visible light has a wavelength of about 550 nm, while ultraviolet light has a
wavelength of about 400 nm or less. The resolution of a microscope increases as the wavelength
decreases, so ultraviolet light allows one to detect objects not seen with visible light. The resolving
power of a lens refers to the size of the smallest object that can be seen with that lens. The
resolving power is based on the wavelength of the light used and the numerical aperture of the
lens. The numerical aperture (NA) refers to the widest cone of light that can enter the lens; the NA
is engraved on the side of the objective lens.
If the user is to see objects clearly, sufficient light must enter the objective lens. With modern
microscopes, entry to the objective is not a problem for scanning, low-power, and high-power
lenses. However, the oil-immersion lens is exceedingly narrow, and most light misses it. Therefore,
the object is seen poorly and without resolution. To increase the resolution with the oil-immersion
lens, a drop of immersion oil is placed between the lens and the glass slide. Immersion oil has the
same light-bending ability (index of refraction) as the glass slide, so it keeps light in a straight line
as it passes through the glass slide to the oil and on to the glass of the objective, the oil-immersion
lens. With the increased amount of light entering the objective, the resolution of the object
increases, and one can observe objects as small as bacteria. Resolution is important in other
types of microscopy as well.
There are several concepts fundamental to microscopy.
Magnification: is the ratio of enlargement (or reduction) between the specimen and its image
(either printed photograph or the virtual image seen through the eyepiece). To calculate
magnification we multiply the power of each lens through which the light from the specimen
passes. For example: if the light passes through two lenses (an objective lens and an ocular lens)
we multiply the 10X ocular value by the value of the objective lens (say it is 4X): 10 X 4=40, or 40X
magnification.
Magnification of LMs is generally limited by the properties of the glass used to make microscope
lenses and the physical properties of their light sources. The generally accepted maximum
magnifications in biological uses are between 1000X and 1250X. Only thin specimens (or thin
slices of a specimen) can be viewed with this type of microscope as light is transmitted through the
specimen.
To view relatively large objects at lower magnifications we utilize the dissecting microscope
Common uses of this microscope include examination of prepared microscope slides at low
magnification, dissection (hence the name) of flowers or animal organs, and examinations of the
surface of objects such as coins.
Resolution: is the ability to distinguish between two objects (or points). The closer the two objects
are, the easier it is to distinguish recognize the distance between them. What microscopes do is to
bring small objects "closer" to the observer by increasing the magnification of the sample. Since
the sample is the same distance from the viewer, a "virtual image" is formed as the light (or
electron beam) passes through the magnifying lenses. (LM) were the first to be developed, and still
the most commonly used ones. The best resolution of light microscopes (LM) is 0.2 µm.
Working distance: is the distance between the specimen and the magnifying lens.
Depth of field: is a measure of the amount of a specimen that can be in focus.
How to Use the Microscope
General Procedures
1.
2.
3.
4.
5.
6.
EVERYTHING on a good quality microscope is unbelievably expensive, so be careful.
Make sure all backpacks and junk are out of the aisles.
Always carry microscopes by the arm and set them flat on your desk.
Hold a microscope firmly by the stand, only. Never grab it by the eyepiece holder, for example.
Plug your microscope in to the plug socket.
Remove the cover, plug the microscope in, and place the excess cord on the table! If you let
the excess cord dangle over the edge, your knee could get caught on it, and the next sound
you hear will be a very expensive crash.
7. Always start and end with the Scanning Objective. Do not remove slides with the high power
objective into place - this will scratch the lens!
8. Hold the plug (not the cable) when unplugging the illuminator.
9. Since bulbs are expensive, and have a limited life, turn the illuminator off when you are done.
10. Always wrap electric cords and returning them to the cabinet.
11. Always make sure the stage and lenses are clean before putting away the microscope.
12. NEVER use a paper towel, your shirt, or any material other than good quality lens tissue or a
cotton swab (must be 100% natural cotton) to clean an optical surface. Be gentle! You may use
an appropriate lens cleaner or distilled water to help remove dried material. Organic solvents
may separate or damage the lens elements or coatings.
13. Focus smoothly; don't try to speed through the focusing process or force anything. For example
if you encounter increased resistance when focusing then you've probably reached a limit and
you are going in the wrong direction.
14. Microscopes should be stored with the Scanning Objective clicked into place.
15. Be careful with the equipment, and be sure to leave the lab in the same condition it was in
when you arrived.
Focusing Specimens
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
Magnification Ocular lens
Total Magnification
Scanning
4x
10x
40x
Low Power
10x
10x
100x
High Power
40x
10x
400x
1. Place the slide on the microscope stage, with the specimen directly over the center of the
glass circle on the stage (directly over the light).
2. Always start with the scanning objective. Lower the objective lens to the lowest point, then
focus using first the coarse knob, then the fine focus knob. The specimen will be in focus when
the scanning objective is close to the lowest point, so start there and focus by slowly raising
the lens. Use the Coarse Knob to focus, the image may be small at this magnification, but you
won't be able to find it on the higher powers without this first step If you can’t get it at all into
focus using the coarse knob, then switch to the fine focus knob.
3. Adjust the Diaphragm as you look through the Eyepiece, and you will see that MORE detail is
visible with a greater depth of field, when you allow in LESS light! Too much light will give the
specimen a washed-out appearance.
4. Once you've focused on Scanning, switch to Low Power. Use the Fine Knob to refocus. Again,
if you haven't focused on this level, you will not be able to move to the next level.
5. Once you've focused on Low Power, switch to viewing field, High Power. Use the Fine Knob to
refocus.
6. If you have a thick slide, or a slide without a cover, do NOT use the high power objective. At
this point, ONLY use the Fine Adjustment Knob to focus specimens.
7. If the specimen is too light or too dark, try adjusting the diaphragm.
8. If the image is too dark then adjust the diaphragm; make sure your light is on.
9. If there are spots in the field of view that stay in the same place even when you move the slide,
then your lens is dirty. Rotate the ocular lens. If the spot rotates, then the ocular lens is dirty.
Use lens paper, and only lens paper to carefully clean the ocular lens. If the spot does not
rotate, clean the objective lens.
10. If you can't see anything under high power. Remember the steps, if you can't focus under
scanning and then low power, you won't be able to focus anything under high power.
11. If only half of the viewing field is lit and looks like there's a half-moon in there, you probably
don't have your objective fully clicked into place.
Practice Using the Microscope
Obtain a slide of colored threads and view them under the scanning and low power. Use the
focusing procedure described above.
1) Can you tell which thread is above the other?
View the threads under high power (400X or 300X). Use the fine focus to focus to determine the
order of the threads from top to bottom. As you rotate the fine focus, different strands will go out of
focus while others will become more sharply focused. This procedure will therefore enable you to
determine the order of the threads.
2) Are all of the threads in focus at the same time?
3) What is the order (from top to bottom)?
"Depth of field" refers to the thickness of the plane of focus. With a large depth of field, all of the
threads can be in focused at the same time. With a smaller or narrower depth of field, only one
thread or a part of one thread can be focused, everything else will be out of focus. In order to view
the other threads, you must focus downward to view the ones underneath and upward to view the
ones that are above.
4a)
What happens to the depth of field when you increase to a higher magnification (increases,
decreases, or remains the same)?
4b)
Explain how the slide with threads could be used to answer the question above.
Obtain a slide of the letter e and view it under the scanning objective. Move the slide to the left.
5)
What way did the image move when the slide was moved to the left?
6)
How does the orientation of the image compare to the image on the slide?
Determining the Size of a Specimen
It is useful to know the size of the field of view so that you can estimate the size of objects. If the
field of view is 2 mm and an object is about one half that big, then the object length is ½ X 2 mm =
1 mm.
We can measure the diameter of the field of view under low power using a ruler. You cannot use a
ruler under high power because it is too big.
Example #1
Suppose you measure the low-power field of view with a ruler and it is 2 mm.
If high power is 10X more magnification than the low power, the field of view will be 1/10 as big.
The field of view under high power will be 2 mm X 1/10 = 0.2 mm.
Example #2
Suppose that the low power diameter (LPD) is 2.5 mm (2500 um).
LPM (low power magnification) = 100X (10X objective and 10X eyepiece)
HPM = 400X (40X objective)
What is the HPD? In other words, if 100X is 2500um wide, how wide is 400X?
Answer: It is 1/4 as wide. = 2500 X 100/400
Place a small transparent ruler on the stage of your microscope and measure the diameter of the
field of view using the scanning (4X) objective.
1) Record the diameter in millimeters.
2) Convert this number to micrometers.
3) Record the total magnification when using the scanning objective.
4) Record the total magnification when using the high power objective.
5) Calculate the diameter of the field of view under high power by using the formula below.
Making a Wet Mount
1. Gather a thin slice/piece of whatever your specimen is. If your specimen is too thick, then the
coverslip will wobble on top of the sample like a see-saw, and you will not be able to view it
under High Power.
2. Place ONE drop of water directly over the specimen. If you put too much water, then the
coverslip will float on top of the water, making it hard to draw the specimen, because they
might actually float away. (Plus too much water is messy)
3. Place the coverslip at a 45 degree angle (approximately) with one edge touching the water
drop and then gently let go. Performed correctly the coverslip will perfectly fall over the
specimen.
How to Stain a Slide
1. Place one drop of stain (iodine, methylene blue….there are many kinds) on the edge of the
coverslip.
2. Place the flat edge of a piece of paper towel on the opposite side of the coverlip. The paper
towel will draw the water out from under the coverslip, and the cohesion of water will draw the
stain under the slide.
3. As soon as the stain has covered the area containing the specimen, you are finished. The
stain does not need to be under the entire coverslip. If the stain does not cover as needed, get
a new piece of paper towel and add more stain until it does.
4. Be sure to wipe off the excess stain with a paper towel
Drawing Specimens
1. Use pencil - you can erase and shade areas
2. All drawings should include clear and proper labels (and be large enough to view details).
Drawings should be labeled with the specimen name and magnification.
3. Labels should be written on the outside of the circle. The circle indicates the viewing field as
seen through the eyepiece, specimens should be drawn to scale - ie..if your specimen takes up the
whole viewing field, make sure your drawing reflects that.
Tips on Making Good Drawings:
1. All drawings to be done in PENCIL! Drawings in PEN are UNACCEPTABLE! You can erase
pencil!
2. Draw solid, continuous lines. DO NOT SKETCH!
3. DO NOT SHADE.
4. Each Drawing must be 1/2 page in size, and must include clear, proper labels! In the upper left
hand corner of the page include the specimen name as written on the slide label. In the upper
right hand corner, include the magnification (100x or 430x).
Cleanup
1. Store microscopes with the scanning objective in place.
2. Wrap cords and cover microscopes.
3. Wash slides and the coverslips and return them to the correct places!.
4. All slides must be put away in the proper trays! Students will not leave until all materials have
been put way properly. You are a team!
REVISION
Fill in the missing values.
Given: Ocular lens
20x
Objective lenses 15x, 30x, 40x
What is the magnification at: low power?
________
Medium power? ________
High power?
________
Calculate the field of view under low power and high power if, under medium power, the field
measured 4.5mm.
Low power __________
High power __________
Name 3 types of microscopes and the maximum magnification power of each.
Name of microscope
Maximum magnification
a)
b)
c)
Name two microscopes used in the classroom, list their main functions, and briefly describe how
they differ.
__________________________________________
__________________________________________
Field of view - fill in the missing values
ocular lens 10x
objective lenses 4x, 10x, 40x
Magnification
Low power
Medium power
High power
ocular lens 15x
objective lenses 10x, 25x, 90x
Magnification
Low power
Medium power
High power
Field of view
10mm
Field of view
3.8mm
1. Identify the microscope parts: Place the correct number in the appropriate space.
_____ Low power objective
____ Light source
_____ Medium power objective
_____ Slide clips
_____ On/Off switch
_____ Nosepiece
_____ Ocular lens
_____ Body tube
_____ Diaphragm
_____ Fine focus
_____ Arm
_____ Stage
_____ Base
_____ Coarse focus
_____ High power objective
PREPARATION OF SLIDE SAMPLES

Fixation: Chemicals preserve material in a life like condition. Does not distort the specimen.

Dehydration: Water removed from the specimen using ethanol. Particularly important for
electron microscopy because water molecules deflect the electron beam which blurs the
image.

Embedding: Supports the tissue in wax or resin so that it can be cut into thin sections.
Sectioning produces very thin slices for mounting. Sections are cut with a microtome or an
ulramicrotome to make them either a few micrometers (light microscopy) or nanometers
(electron microscopy) thick.

Staining: Most biological material is transparent and needs staining to increase the contrast
between different structures. Different stains are used for different types of tissues.
Methylene blue is often used for animal cells, while iodine in KI solution is used for plant
tissues.

Mounting: Mounting on a slide protects the material so that it is suitable for viewing over a
long period.
ELECTRON MICROSCOPES:

Two examples of which are shown below, are more rarely encountered by beginning biology
students. However, the images gathered from these microscopes reveal a greater structure
of the cell, so some familiarity with the strengths and weaknesses of each type is useful.
Instead of using light as an imaging source, a high energy beam of electrons (between five
thousand and one billion electron volts) is focused through electromagnetic lenses (instead
of glass lenses used in the light microscope). The increased resolution results from the
shorter wavelength of the electron beam, increasing resolution in the transmission electron
microscope (TEM) to a theoretical limit of 0.2 nm. The magnifications reached by TEMs are
commonly over 100,000X, depending on the nature of the sample and the operating
condition of the TEM. The other type of electron microscope is the scanning electron
microscope (SEM). It uses a different method of electron capture and displays images on
high resolution television monitors. The resolution and magnification of the SEM are less
than that of the TEM although still orders of magnitude above the LM.
SEM
Comparison between light and electron microscope
Electron Microscopy.
This uses a beam of electrons, rather than electromagnetic radiation, to "illuminate" the specimen.
This may seem strange, but electrons behave like waves and can easily be produced (using a hot
wire), focused (using electromagnets) and detected (using a phosphor screen or photographic
film). A beam of electrons has an effective wavelength of less than 1 nm, so can be used to
resolve small sub-cellular ultrastructure. The development of the electron microscope in the 1930s
revolutionized biology, allowing organelles such as mitochondria, ER and membranes to be seen
in detail for the first time.
The main problem with the electron microscope is that specimens must be fixed in plastic and
viewed in a vacuum, and must therefore be dead. Other problems are that the specimens can be
damaged by the electron beam and they must be stained with an electron-dense chemical (usually
heavy metals like osmium, lead or gold). Initially there was a problem of artifacts (i.e. observed
structures that were due to the preparation process and were not real), but improvements in
technique have eliminated most of these.
There are two kinds of electron microscope. The transmission electron microscope (TEM) works
much like a light microscope, transmitting a beam of electrons through a thin specimen and then
focusing the electrons to form an image on a screen or on film. This is the most common form of
electron microscope and has the best resolution. The scanning electron microscope (SEM) scans
a fine beam of electron onto a specimen and collects the electrons scattered by the surface. This
has poorer resolution, but gives excellent 3-dimentional images of surfaces.
Transmission Electron Microscope (TEM)



Pass a beam of electrons through the
specimen. The electrons that pass through
the specimen are detected on a
fluorescent screen on which the image is
displayed.
Thin sections of specimen are needed for
transmission electron microscopy as the
electrons have to pass through the
specimen for the image to be produced.
This is the most common form of electron
microscope and has the best resolution
Scanning Electron Microscope (SEM)

Pass a beam of electrons over the
surface of the specimen in the form of a
‘scanning’ beam.

Electrons are reflected off the surface of
the specimen as it has been previously
coated in heavy metals.

It is these reflected electron beams that
are focussed of the fluorescent screen in
order to make up the image.

Larger, thicker structures can thus be
seen under the SEM as the electrons do
not have to pass through the sample in
order to form the image. This gives
excellent 3-dimensional images of
surfaces

However the resolution of the SEM is
lower than that of the TEM.
Bacterium (TEM)
(Image provided by: ceiba.cc.ntu.edu.tw)
Four conodont elements stuck on a pin head
(SEM)
(Image provided by Mark Purnell)
Comparison of the light and electron microscope
Light Microscope
Electron Microscope
Cheap to purchase
Expensive to buy
Cheap to operate.
Expensive to produce electron beam.
Small and portable.
Large and requires special rooms.
Simple and easy sample preparation.
Lengthy and complex sample prep.
Material rarely distorted by preparation.
Preparation distorts material.
Vacuum is not required.
Vacuum is required.
Natural colour of sample maintained.
All images in black and white.
Magnifies objects only up to 2000 times
Magnifies over 500 000 times.
Specimens can be living or dead
Specimens are dead, as they must be fixed in plastic
and viewed in a vacuum
Stains are often needed to make the cells
visible
The electron beam can damage specimens and they
must be stained with an electron-dense chemical
(usually heavy metals like osmium, lead or gold).
Specimen Preparation TEM