Tools of the Biologist
HistoryHistory
•Anton Von Leeuwenhoek•Anton Von LeeuwenhoekBorn in Holland 1632
•First to observe livingbacteria & drew them.
•Also looked at protists,sperm, blood
•1st simple scope
•Made over 500"microscopes"
•Robert Hooke (1665)•Robert Hooke (1665)
•Used compoundscope to examinecork.
•Coined the term “cell”referring to the manylittle boxes. Actuallysaw dead plant cells.
Types of Microscopes
Simple microscope –1.Simple microscope –Hand lens (magnifyingglass)
•3 – 40 timesmagnification
2.Compound Light Microscope2.Compound Light Microscope
The type we use in our labs
•Most commonly used microscope
»Uses light and lenses to magnify & view thespecimen
»Has two sets of lenses – Ocular (eye piece) &Objective (near the object being viewed)
»Total magnification»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 bythis
Stage and clips – Holds slides in place
Adjustments – Coarse & Fine. Focuses image
Diaphragm – Controls the amount of lightcoming through the stage
Light – Electric light source
Base – Bottom of scope. One hand goesunderneath
The DiaphragmThe Diaphragm
•Use the Diaphragm toadjust the amount oflight
•Image of pollen grainunder good brightness(left) and poorbrightness (right)
FocusingFocusing
•Use the Adjustmentknobs to focus theimage
•Coarse adjustmentbrings the image intonear focus
•Fine adjustment(smaller knob) bringsit into fine focus
•Use fine adjustmentunder 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
Microscope Image
Original Object
Working Distance
Depth of Field/Focus
ResolutionResolution
•Ability to clearlydistinguish two objectsthat are close together.
•Image of pollen grain withgood resolution (left) andpoor resolution (right)
• Resolving power of ourscope = 0.2um
Rules for using the Microscope
1.Use only the assigned microscope
2.Carry & place the scope properly (3cm from edge of table)
3.Do not let the cords dangle or get into the sinks
4.Clean lens only with lens paper. NO FINGERS!
5.Do not reuse the same spot on your lens paper
6.Start on low (4x) power when you start your observations
7.Always focus (move the stage) away from the slide
8.Use the coarse adjustment first then the fine adjustment
9.Be careful when switching to high (40x) power to se that there isenough 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 & thelow 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”
Normal View
40X
400X
100X
Crossed Threads
TotalMagnification
Gold Thread
Blue Thread
![MCAN02069_0000[1]](data/images/img32.png)
Diameter = 3.75 mm
or 3750 um
1mm
1mm
1mm
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 willno longer use the ruler: GIVE BACK THE RULER
For Medium Power:
Low Power Field of View (um) = Medium Power Mag
Medium Power Field of View (um) Low Power Mag
For High Power:
Low Power Field of View (um = High Power Mag
High Power Field of View (um) Low Power Mag
Low Power Field of View
Medium PowerField of View
3. Binocular (Has twooculars)
Gives a 3D image.
•Also called aDissecting scope orStereo 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 viewthrough the scope?
1.Adjust the diaphragm toallow less light to comethrough
2.Use a Stain to maketransparent specimensvisible. Ie. Iodine,methyl blue
3.Specimens must besliced very thin. Use aMicrotome to make thinslices
Microtome
Electron MicroscopesElectron Microscopes
1.Useselectromagnets andstreams of electronsto view a specimen
2.Limit of Resolutionis 1000x finer thanlight microscope
3.200,000 –1,000,000xmagnification
Two types
Transmission Electron Microscope (TEM)Transmission Electron Microscope (TEM)1931 (Germany)
Image is seen on a fluorescent screen
•Specimen must be thinly sliced and coated withAu or Ag.
•Gives a 2D image of specimen
•Specimen must be dead
Staphylococcus aureus
E. coli bacteria
Herpes simplex viruses
1.Gives a 3D image
2.Electrons scan aroundspecimen
3.Shows only the outside ofthe specimen
4.Gives very clear surfacedetails
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 (Mountingchamber is vacuum sealed)
4.Specimens must be dead
5.Black and white images only! Any coloryou 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
