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"

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

• 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