Biological Tools

Microscopes

Introduction• Most widely used tool in biology• Allows us to see things we can’t

see with the naked eye.• The science of Microbiology

started with the invention of microscopes

Magnification: An increase in an objects apparent size mag·ni·fi·ca·tion (m g n -f -k sh n) n.

1. The act of magnifying or the state of being magnified.2. a. The process of enlarging the size of something, as an optical image.

b. Something that has been magnified; an enlarged representation, image, or model.

3. The ratio of the size of an image to the size of an object.

Resolution : The power to show details clearly. The fineness of

detail that can be distinguished in an image

VS

As a general rule, magnification and resolution are inversely related. That is as magnification increases, resolution decreases.

Simple Light Scopes• Simple Light Scopes use a single

piece of curved glass (lens) to produce a larger image of a specimen

– Refraction = bending of light causes distortion/enlargement

– Most common example = Magnifying Glass

– Again, as magnification increases, resolution decreases

Compound Light Scopes• Contains two lenses:

1. Ocular Lens (Eyepiece)

2. Objective LensTotal Magnification = Ocular x Objective

Light Scopes have limits – can reach about 2,000X magnification

X

Parts/Usage of Microscopes

Parts/Usage of Microscopes

• Eyepiece (Ocular Lens)• Usually contains a 10X

lens

• Arm: curved or solid back of the microscope

• Helps support the weight,

• This is where you hold the scope when you are carrying it!

• Body Tube: Proper space between eyepiece and objective lenses.

• Allows for proper resolution (clarity)

• (Revolving) Nosepiece: rotating plate that attaches multiple objective lenses.

• Allows you change objective powers.

• Objective Lenses: 2nd set of lenses closest to the specimen.

• Many scopes have multiple lenses with different powers (4X, 10X, 40X)

• Lower power – shorter lenses• Higher powers – longer

lenses

• Stage: platform to hold slides

– Some have stage clips to hold the slide

– Some have mechanical stages

• Course Adjustment Knob: The larger of the two focusing knobs

• Used for focusing in lower powers – makes large scale adjustment by moving the stage up and down.

• Never use under high power objectives !

• Fine Adjustment Knob: The smaller of the two focusing knobs

• Used for focusing in higher powers – makes microscopic adjustments. (you won’t be able to see the stage move)

• Light Source: Located under the stage.

• Designed to run a beam of light through the specimen to be magnified

• Old scopes used mirrors to reflect light up through the stage.

• Diaphragm: rotating disk on the underside of stage (hard to find)

• Controls how much light passes through specimen– Thin transparent

specimens require little light

– Thick colorful specimens require a lot of light

• Base: bottom of scope

• Used to support the weight.

• Also have one hand underneath when carrying !

Proper Care• Always use two hands when carrying• Keep the scope close to your belly !• Keep scopes away from the table edge• Never drape the cord where someone can walk• Avoid moving the scope around on the desk (use the

revolving eyepiece)

Specimens• Specimens should be rather thin – light must be

able to pass through them.• Stains or chemicals are sometimes used to

highlight or darken certain structures for easy viewing

UNSTAINED CHEEK CELLS

STAINED CHEEK CELLS

Other Biological Tools

Stereomicroscope/Dissection Scope• Light Scope• 3-D Images• Low Magnification• Used in dissections, with insects, and small living things/structures

Electron microscopes• Most Powerful scopes• Uses electrons• Resolution no longer a problem with high magnification

• Several Types (TEM,SEM)

Knoll and Ruska in 1947

How do electron scopes work?In an electron microscope…

The light source is replaced by a beam of very fast moving electrons.The specimen usually has to be specially prepared and held inside a vacuum chamber from which the air has been pumped out (because electrons do not travel very far in air). The lenses are replaced by a series of coil-shaped electromagnets through which the electron beam travels. In an ordinary microscope, the glass lenses bend (or refract) the light beams passing through them to produce magnification. In an electron microscope, the coils bend the electron beams the same way.The image is formed as a photograph (called an electron micrograph) or as an image on a computer screen.

Transmission Electron MicroscopeTEM

• Most powerful scope (1,000,000X and up)

• Image projects onto a photographic plate

• Used to see internal cellular structures

• Samples must be treated with heavy metal stains (kills living cells)

TEM Micrographs(Notice they are 1D or flat images)

Scanning Electron MicroscopeSEM

• Produces a 3 dimensional image   • Specimens aren’t sliced but are sprayed with a fine metal coating  

• A beam of electrons is passed over the surface of the metal coating to emit a shower of electrons

• Showered electrons are projected onto a fluorescent screen or photographic plate  

• SEM's produce greatly magnified image of surface details of specimens  

• Can magnify up to 100,000 times • Can not be used to view living specimens  

SEM Micrographs

ANT

Honeybee Head with pollen on itBacteria Cells Light Scope vs. SEM

Leukemia (cancer) Cells on left

Normal Bone marrow cells on right

Microtome• Used to slice extremely thin microscope specimens

• Biopsy placed in wax to slice

Centrifuge• Used to separate materials by high speed spinning - Centrifugation

Parts of a mixture will separate based on size and density

An Apheresis Centrifuge

Apheresis Donation: 1. Blood drawn & centrifuged 2. Plasma and/or platelets removed3. Red Blood Cells returned to donor

Blood Centrifugation

Chromatography• Separation technique of chemicals• Based on dissolving rates (solvents)

Gel Electrophoresis• Used to separate large molecules into smaller pieces

• Primarily Based on charge

• Common with DNA/Forensics