tools of the biologist. history anton von leeuwenhoekanton von leeuwenhoek born in holland 1632...
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Tools of the Biologist
HistoryHistory
• Anton Von LeeuwenhoekAnton 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)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 –Simple microscope – Hand lens (magnifying glass)
• 3 – 40 times magnification
2.2. Compound Light MicroscopeCompound 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 magnificationTotal 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 DiaphragmThe Diaphragm• Use the Diaphragm to
adjust the amount of light
• Image of pollen grain under good brightness (left) and poor brightness (right)
FocusingFocusing
• 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
MagnificationField of View InversionWorking DistanceDepth of FieldResolution
Magnification
• Need light and lens
• Image formation
• Convex lens
Field of View
Inversion
Microscope Image
Original Object
Working Distance
Depth of Field/Focus
ResolutionResolution
• 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. Use only the assigned microscope2. Carry & place the scope properly (3cm from edge of table)3. Do not let the cords dangle or get into the sinks4. Clean lens only with lens paper. NO FINGERS!5. Do not reuse the same spot on your lens paper6. Start on low (4x) power when you start your observations7. Always focus (move the stage) away from the slide8. Use the coarse adjustment first then the fine adjustment9. Be careful when switching to high (40x) power to se that there is
enough clearance between the objective and the slide10.Do not use the coarse adjustment knob on high (40x) power11.When you are done with the scope, turn off the light switch12.Always put scope away with cord wrapped around it, cover on & the
low power objective in place13.Put scopes away with the numbers facing out into the proper slot14.Clean and dry all slides and cover slips before putting them away
Making a Wet Mount
The Letter “e”Normal View
40X
400X
100X
Crossed Threads Total Magnification
Gold Thread
Blue Thread
Diameter = 3.75 mm
or 3750 um
1mm 1mm1mm 1mm
Field of View
Specimen = 4/3750um
Length of Specimen =937.5um
Calculating Fields of ViewCalculating 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:Low Power Field of View (um) = Medium Power MagMedium Power Field of View (um) Low Power Mag
For High Power:Low Power Field of View (um = High Power MagHigh Power Field of View (um) Low Power Mag
Low Power Field of ViewMedium Power Field of View
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 imagesCompound Microscope images
Paramecium
Vorticella Daphnia
Amoeba
Diatom
Hydra budding
Since most of the specimens we observe will be clear, what could be done to enhance the image we view
through the scope?
1. Adjust the diaphragm to allow less light to come through
2. Use a Stain to make transparent specimens visible. Ie. Iodine, methyl blue
3. Specimens must be sliced very thin. Use a Microtome to make thin slices
Microtome
Electron MicroscopesElectron 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 typesTransmission Electron Microscope (TEM)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
Staphylococcus aureus
E. coli bacteria
Herpes simplex viruses
Scanning Electron Microscope Scanning Electron Microscope (SEM)(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
Radiolarian
Diatom
Tick
Side 2: 02255
Limitations of Electron MicroscopesLimitations 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