Transcript microscopes

Tools of the
Biologist
The Incredible World of Microscopes video
History
• Anton Von
Leeuwenhoek Born in
Holland 1632
• First to observe living
bacteria & drew them.
• Also looked at protists,
sperm, blood
• Used a 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 – 1000
times
» Total magnification = Ocular (10X) x Objective
(40X)
Ocular – Eye piece
Neck – Supports the eyepiece.
Objectives – 4 – 100x 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)
• Image of pollen grain
with good contrast
(left) and poor
contrast (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
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 (6cm 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
Wet Mount
Clean slide
Put coverslip at a right angle
into the side of the water
Add drop of water to
specimen
View specimen
Making a Wet Mount Slide
• 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:
• 2. Place ONE drop of water directly over the specimen. If you
put too much water over the specimen, then the coverslip will
float on top of the water, making it harder to draw the
specimens as they float past the field of view!
•
3. Place the coverslip at a 45 degree angle (approximately),
with one edge touching the water drop, and let go.
The Letter “e”
40x
400x
Threads
100x
Beware of Air Bubbles!!
Compound Microscope images
Diatom
Paramecium
Amoeba
Vorticella
Hydra budding
Daphnia
3. Binocular (Has two
oculars)
Gives a 3D image.
•
Also called a
Dissecting scope
or Stereo scope
Monocular (1 ocular)
Light Microscope
•
2D image
Since most of the specimens we observe will be clear
to near 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
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
Medium Power
For Medium Power:
Low Power Field of View
Field of View
Low Power Field of View (um) =
Medium Power Field of View (um)
For High Power:
Low Power Field of View (um =
High Power Field of View (um)
Medium Power Mag
Low Power Mag
High Power Mag
Low Power Mag
Field of View
1mm
1mm
1mm
Diameter = 3.75 mm
or 3750 um
1mm
4 cells fit across field
Specimen = 3750um
4
Length of Specimen =937.5um
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)
Side 1 2286-99
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
These have all had color added to them!!!
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
2256 - 2285
Images
Weevil
Tick
Diatom
Radiolarian
• Gold coater - $1,950 used
• Transmission Electron Microscopes
(TEM):$90,000 - $2,000,000
• UsedScanning Electron Microscopes
(SEM):$45,000 - $200,000 Used
Limitations of Electron Microscopes
1. Specimens must be very thin (TEM)
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