Tools of the Tools of the BiologistBiologist

The Incredible World of Microscopes video

HistoryHistory

• Anton Von Anton Von LeeuwenhoekLeeuwenhoek 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"

http://en.wikipedia.org/wiki/Image:Leeuwenhoek_Eschenholz.jpg

• Robert Hooke (1665)Robert Hooke (1665) • Used compound

scope to examine cork.

• Coined the term “cellcell” 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 – 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 DiaphragmThe 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)

FocusingFocusing

• Use the Adjustment Adjustment knobs knobs to focus the image

• Coarse adjustment brings the image into near focus

• Fine adjustment (smaller knob) brings it into fine focus

ResolutionResolution

• Ability to clearly Ability to clearly distinguish two distinguish two objects that are objects that are close togetherclose together.

• Image of pollen grain with good resolution (left) and poor resolution (right)

• Resolving power of our scope = 0.2um

Rules for using the Microscope1. Use only the assigned microscope2. Carry & place the scope properly (6cm 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

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 imagesCompound Microscope images

Paramecium

Vorticella Daphnia

Amoeba

Diatom

Hydra budding

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. 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

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

Diameter = 3.75 mmor 3750 um

1mm 1mm1mm 1mm

Field of View

4 cells fit across fieldSpecimen = 3750um 4

Length of Specimen =937.5um

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) 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

Staphylococcus aureus

E. coli bacteria

Herpes simplex viruses

These have all had color added to them!!!

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

Radiolarian

Diatom

Tick

• 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 MicroscopesLimitations 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