CH 4 Microscopy

Microscopy

Use of light or electrons to magnify objects

1 meter

1,000,000 micrometer

1,000,000 micrometer

= 1, 000,000,000 nanometers

Relation of electromagnetic radiation to microscopy

electrons act as waves with wavelengths dependent on the voltage of an electron beam

• and using radiation of smaller wavelengths results in enhanced microscopy

Resolution

The ability to distinguish between two objects that are close together

Wavelength affects quality of resolution how?

Shorter wavelengths produce better quality of resolution

How image altered when seen through a microscope?

inverted, reversed, and enlarged

Reflection

the wave bounces off a surface.

Refraction

the wave bends as it passes from one material to another.

Transmission

the wave passes completely through a material.

Absorption

the wave's energy is captured and converted into heat.

Immersion Oil Use

Light travels through immersion oil at same uniform speed as glass.

• Prevents light from refracting or bending

• Increases numerical apeture

• Increases resolution due to more light rays

Ocular Lens

the lenses closest to the eyes, can be monocular or binocular, magnify 10X

• Remagnifies image formed by objective lens

Condenser Lens

Focuses light through specimen

Contrast

Refers to the different degrees of intensity between 2 objects or an object and it’s background

• Determines resolution

• Staining is used to provide better contrast in microscopy studies

Two factors that determine resolving power (resolution)

the wavelength of the electromagnetic radiation and the numerical aperture of the lens

Numerical aperture

the ability of a lens to gather light

Bright-field Microscope

background is illuminated, can be simple (singular lens) or compound (multiple lens)

• Used to observe dead stained specimen or live naturally colored specimen

Dark-field Microscope

Pale specimen made to appear light against a dark background

• They prevent light from directly entering the objective lens, instead light rays are reflected inside of the condenser and surpass the lens

Phase Microscopy

use the alignment or misalignment of light waves to achieve the desired contrast between a living specimen and its background

• Used to examine living organisms or specimens that would be damaged or altered by attaching them to slides or staining

• two types = phase-contrast and differential interference contrast

Phase-Contrast Microscope

produce sharply defined images in which fine structures can be seen within living cells such as cilia and flagella

Differential Interference Contrast Microscope

significantly increase contrast and give an image a dramatic three dimensional and shadowed appearance as if light were striking the specimen from one side, can even produce unnatural colors to increase contrast

Transmission Electron Microscope

Generates a beam of electrons that pass through specimen and produces an image on a fluorescent screen

• MUST vacuum thick specimen

• Can’t be used on living organisms

Transmission Electron Microscope Use

To observe internal ultrastructural detail of cells and viruses or small bacteria

• Produces 2D image of ultrastructural cells

Scanning Electron Microscope

uses magnetic fields within a vacuum tube to manipulate a beam of electrons, rapidly focusing back and forth across a specimens metal coated surface

• whole specimens can be observed because sectioning is not required, but can only view external structure of dead organisms

Scanning Electron Microscope Use

Observe surfaces structure details

• Realistic 3D images of surface of microbes