MicroscopMicroscopyyhttp://www.microbehunter.com/wp/wp-content/uploads/2010/04/sem_pollen.jpg

Microscopy• the study of

objects or organisms (e.g., bacteria, protists, cells, etc.) too small to be seen by the naked eye using a tool called a microscope

• an instrument that gives an enlarged image of the object under study– Compound

microscope– Electron microscope– Scanning probe

microscope

Microscope

http://www.kennislink.nl/ , http://www.sciencelearn.org.nz/

Type of microscope

Magnification Generated image Key features

How it works

Optical compound microscope

Up to 1 000x Magnified color view

Samples on glass slides

are sometimes

stained

Visible light and lenses produce a

magnified image of an object

Scanning electron

microscope

Up to 50 000x Sharp 3D black and white image

Surface structures of

a sample

Thin beam of electrons scanned across the

surface of the sample; reflected electrons make

up image

Transmission electron

microscope

Up to 10 000 000x

Sharp 2D black and white image

Details inside cells and

nanoparticles (1 micrometer thick samples)

Beam of electrons passed through sample,

image is magnified, focused by a lens and

captured by a computer

Scanning probe

microscope

Up to 90 000 000x

3D black and white computer image of atoms on surface of

sample

Looks at individual

atoms

A probe with a tip only an atom wide

scans across a sample’s surface

Compound light microscope

• Magnification– Enlargement of the image of

the specimen– Total magnification =

objective lens magnification * ocular lens magnification

• Resolution– ability to see a gap

separating two dots in an image that, to the naked eye, are not separated.

– Influenced by frequency of light waves and quality of the lens

• Contrast– Condenser and diaphragm

modify size and intensity of a light beam

http://www.cls.zju.edu.cn, http://science.howstuffworks.com

Using the microscope1. Carrying the

microscope properly

2. Mounting the slide3. Viewing the

specimen*images are backward and inverted*FOV gets darker as magnification increases

4. Preparing the microscope for storage

Illuminating the field of view

Brighter Darker

Mirror Correct angle, concave side

Wrong angle, plane side

Iris diaphragm Fully open Partially closed

Objective Lower magnification

Higher magnification

Condenser Closer to stage

Farther from stage

Preparing samples for viewing

http://www.biosci.ohio-state.edu, http://www.doctortee.com/dsu/tiftickjian/bio100/cell-lab.html

Staining cells for better visibility1.Place several drops of stain on one edge of the cover slip2.The process of diffusion will allow the stain to go under the cover slip and stain the specimen 3.(tissue paper may be placed on the opposite edge of the cover slip to help the stain diffuse under the cover slip)

Microscope Math

• Theoretical magnification= ocular X objective

• Estimating the diameter of the field of view(LPO diam)(LPO mag) = (HPO diam)(HPO mag)

• Estimating cell size= diameter / # of cells spanning the diameter

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