MICROSCOPY Unit Two Part II

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Microscopes are devices that produce magnified images of structures that are too small to see with the unaided eye

Humans cannot see objects much smaller than 0.1mm in size.

Development began in 1500’s and by the 1800’s most microscopes had combinations of lenses that provided clear images. Anton Van Leeuwenhoek, discovered microscopic

organisms in pond water (he called them “wee beasties”)

Robert Hooke was able to recognize the cell as the basic unit of life.

MICROSCOPES

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Two Types of compound light microscopes

Traditional compound light microscope

Also just called a compound light microscope

Stereoscope

Also called dissecting microscope

COMPOUND LIGHT MICROSCOPES

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Compound

The image is focused by two sets of lenses

Ocular lens – located in the eyepiece; the lens system

closest to the eye.

Objective lenses - located near the specimen

The image is magnified by both sets of lenses.

Light

The image is formed through the transmission of

light.

COMPOUND LIGHT MICROSCOPES

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Use transmitted light to form image

Light is transmitted through very thin specimen

Translucent – will let light through

Specimen must be prepared and thin

enough to let light through

Can purchase prepared slides

Image is formed through a series of lenses

Lenses are parfocal - the image remains

focused when switching from one objective

lens to the next with little refocusing required!

TRADITIONAL COMPOUND LIGHT

MICROSCOPES

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View through 2 lenses at any one time

Ocular lens

Found inside eyepiece

Magnification power is written on side of eyepiece

Objective lenses

Can have 2-4 different lenses

Magnification power is listed on side of lenses

Use rotating nosepiece to change objective lenses!!!

The image is magnified by both sets of lenses

TRADITIONAL COMPOUND LIGHT

MICROSCOPES

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Staining

Thin objects are often colorless

Need to add color to be able to see the object

Different stains for different objects

Handling slides

Always hold by edges or sides to avoid

fingerprints

Cover slip – used to cover or protect object

being viewed.

TRADITIONAL COMPOUND LIGHT

MICROSCOPES

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2 dimensional image produced

Image produced is upside down and reversed

from left to right due to orientation of mirrors

inside microscope

Can view

living or dead organisms

whole small organisms

parts of larger specimens

natural or man-made objects

TRADITIONAL COMPOUND LIGHT

MICROSCOPES

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Magnification

Degree to which a microscope can enlarge an

image compared to the objects real size

Today the strongest compound light microscopes

are able to magnify objects up to 2,500X

Our classroom compound light microscopes have

ocular lenses with a magnification power of 10x

Our classroom compound light microscopes have

objective lenses with a power of 4x, 10x, and 40x

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TRADITIONAL COMPOUND LIGHT

MICROSCOPE

Total Magnification

The total amount an object is magnified when

viewed through a compound light microscope

Total magnification is a combination of the power of

the ocular lens and the power of the objective lens

being used

Total magnification = ocular lens power X objective

lens power

TRADITIONAL COMPOUND LIGHT

MICROSCOPES

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Total Magnification examples: If looking through the ocular lens and using the

scanning power objective: 10 x 4 = 40x

If looking through the ocular lens and using the low power objective: 10 x 10 = 100x

If looking through the ocular lens and using the high power objective: 10 x 40 = 400x

What you are seeing is 400 times larger than it is in real life!!!

TRADITIONAL COMPOUND LIGHT

MICROSCOPES

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Resolution, or resolving power, is a measure

of the clarity of an image; the measurement of

how close two points can be and still be

distinguished as separate.

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TRADITIONAL COMPOUND LIGHT

MICROSCOPE

Objective lenses

Scanning, Low power, High power, Oil immersion

Parfocal capability – ability of the image to remain in focus as move from one objective lens to another with little refocusing!

Resolving power – the measurement of how close two points can be and still be distinguished as separate.

Field of view – the area visible through the microscope. As the power of the objective increases, the sixe of the field of view decreases.

Focal plane – the portion of the specimen on the slide that is in focus at any moment

Depth of focus (field) – the vertical distance that can be sharply focused on a specimen. As the power of magnification increases, the depth of field/focus decreases.

COMPOUND LIGHT MICROSCOPES

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Also called dissecting microscopes

Use reflected light for form images

Light is directed down on the

specimen and reflected back into the

objectives and eyepieces.

Specimen does NOT have to be thin

Image orientation is same as

object

STEREOSCOPES

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Paired objectives and ocular lenses

Each eye sees image separately

Aligned so you only see one 3-D image

Objective lenses usually 1x-4x

Ours are 2x and 4x

Ocular lenses are usually 10x or 15x

Ours are 10x

STEREOSCOPES

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Advantages

Able to see small details on large specimens

Helpful for dissections of small but visible

organisms

No preparation needed, object easily manipulated

Overall object is easily recognizable

STEREOSCOPES

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Developed in the 1950’s

Focuses beams of electrons to produce an

image on a computer screen

Originally black and white (can use computer

to add color)

Can magnify specimens more than 200,000

times their actual size

A few can even see individual atoms!

ELECTRON MICROSCOPES

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Used to view objects 1000x smaller than can

be viewed with compound light microscope

No living organisms because the specimens

being studied have to be in a vacuum.

Two main types of electron microscopes

Scanning electron microscope (SEM)

Transmission electron microscope (TEM)

ELECTRON MICROSCOPES

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Scanning electron microscopes (SEM)

Scans the surface with a beam of electrons

Surface usually coated with thin layer of metal

that deflects the electrons

Computer forms 3D image from measurements

of the deflected electrons.

SCANNING ELECTRON MICROSCOPES

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Transmission electron

microscope (TEM)

Transmits electrons through

a thin slice of a specimen

Creates a 2 dimensional

image similar to that of a light

microscope but with a much

higher magnification

TRANSMISSION ELECTRON MICROSCOPES

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X-rays

Formed by x-rays which pass through soft tissues

such as skin and muscle, but are absorbed by

bones and teeth

Good for looking at the skeleton but not muscle,

cartilage, ligaments, or organs

OTHER IMAGING TECHNOLOGY

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Magnetic Resonance

Imaging

MRI

Strong magnetic field used

to produce a cross-section

image of a part of the body

Functional MRI and show

which areas of the brain are

active while a person is

doing a task.

OTHER IMAGING TECHNOLOGY

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