Tools of the Biologist
History
• Anton Von Leeuwenhoek
Born in Holland 1632
• First to observe living
bacteria & drew them.
• Also looked at protists,
sperm, blood
• 1st simple scope
• Made over 500
"microscopes"
• Robert Hooke (1665)
• Used compound
scope to examine
cork.
• Coined the term
“cell” referring to the
many little boxes.
Actually saw dead
plant cells.
Types of Microscopes
1. Simple microscope –
Hand lens (magnifying
glass)
• 3 – 40 times
magnification
2. Compound Light Microscope
The type we use in our labs
•
Most commonly used microscope
» Uses light and lenses to magnify & view the
specimen
» Has two sets of lenses – Ocular (eye piece) &
Objective (near the object being viewed)
» Total magnification on our scopes = 40-400 times
» Total magnification = Ocular (10X) x Objective
(40X)
Ocular – Eye piece 10x
Body Tube – Supports the eyepiece.
Nosepiece – rotates objectives
Objectives – 40 – 400x total magnification
Arm – Supports neck and objectives. Carry by
this
Stage and clips – Holds slides in place
Adjustments – Coarse & Fine. Focuses image
Diaphragm – Controls the amount of light
coming through the stage
Light – Electric light source
Base – Bottom of scope. One hand goes
underneath
The Diaphragm
• Use the Diaphragm to
adjust the amount of
light
• Image of pollen grain
under good brightness
(left) and poor
brightness (right)
Focusing
• Use the Adjustment
knobs to focus the
image
• Coarse adjustment
brings the image into
near focus
• Fine adjustment
(smaller knob) brings
it into fine focus
• Use fine adjustment
under 40x
Microscope Principles
 Magnification
 Field of View
 Inversion
 Working Distance
 Depth of Field
 Resolution
Magnification
• Need light and lens
• Image formation
• Convex lens
Field of View
Inversion
Original Object
Microscope Image
Working Distance
Depth of Field/Focus
Resolution
• Ability to clearly
distinguish two objects
that are close together.
• Image of pollen grain with
good resolution (left) and
poor resolution (right)
•
Resolving power of our
scope = 0.2um
Rules for using the Microscope
1.
2.
3.
4.
5.
6.
7.
8.
9.
Use only the assigned microscope
Carry & place the scope properly (3cm from edge of table)
Do not let the cords dangle or get into the sinks
Clean lens only with lens paper. NO FINGERS!
Do not reuse the same spot on your lens paper
Start on low (4x) power when you start your observations
Always focus (move the stage) away from the slide
Use the coarse adjustment first then the fine adjustment
Be careful when switching to high (40x) power to se that there is
enough clearance between the objective and the slide
10.Do not use the coarse adjustment knob on high (40x) power
11.When you are done with the scope, turn off the light switch
12.Always put scope away with cord wrapped around it, cover on & the
low power objective in place
13.Put scopes away with the numbers facing out into the proper slot
14.Clean and dry all slides and cover slips before putting them away
Making a Wet Mount
The Letter “e”
40X
Normal View
400X
100X
Crossed Threads
Total
Magnification
Blue Thread
Gold Thread
Field of View
1mm
1mm
1mm
Diameter = 3.75 mm
or 3750 um
1mm
Specimen = 4/3750um
Length of Specimen =937.5um
Calculating Fields of View
Once you have your field of view for Low Power, you will
no longer use the ruler: GIVE BACK THE RULER
For Medium Power:
Medium Power
Low Power
Field
of View
Low Power Field of View
(um)
= View
Medium Power Mag
Field of
Medium Power Field of View (um)
Low Power Mag
For High Power:
Low Power Field of View (um =
High Power Field of View (um)
High Power Mag
Low Power Mag
3. Binocular (Has two
oculars)
Gives a 3D image.
•
Also called a
Dissecting scope or
Stereo scope
Monocular (1 ocular)
Light Microscope
•
2D image
Compound Microscope images
Diatom
Paramecium
Amoeba
Vorticella
Hydra budding
Daphnia
Since most of the specimens we observe will be clear,
what could be done to enhance the image we view
through the scope?
1.
2.
3.
Adjust the diaphragm to
allow less light to come
through
Use a Stain to make
transparent specimens
visible. Ie. Iodine,
methyl blue
Specimens must be
sliced very thin. Use a
Microtome to make thin
slices
Microtome
Electron Microscopes
1. Uses
electromagnets and
streams of electrons
to view a specimen
2. Limit of Resolution
is 1000x finer than
light microscope
3. 200,000 –
1,000,000x
magnification
Two types
Transmission Electron Microscope (TEM)
1931 (Germany)
Image is seen on a fluorescent screen
•
•
•
Specimen must be thinly sliced and coated with
Au or Ag.
Gives a 2D image of specimen
Specimen must be dead
Herpes simplex viruses
Staphylococcus aureus
E. coli bacteria
Scanning Electron Microscope
(SEM) – 1935 (Germany)
1. Gives a 3D image
2. Electrons scan around
specimen
3. Shows only the outside of
the specimen
4. Gives very clear surface
details
Images
Weevil
Diatom
Side 2: 02255
Tick
Radiolarian
Limitations of Electron Microscopes
1. Specimens must be very thin
2. Specimens must be stained or coated
3. Specimens must be dried out (Mounting
chamber is vacuum sealed)
4. Specimens must be dead
5. Black and white images only! Any color
you may see is added in
• Gold coater - $1,950 used
• Transmission Electron Microscopes
(TEM):$90,000 - $2,000,000
• UsedScanning Electron Microscopes
(SEM):$45,000 - $200,000 Used