EXAM 1: LO 6 Flashcards
Microscopy
the use of light or electrons to magnify objects
Wavelength
the distance between two corresponding parts of a wave
Magnification
the apparent increase in the size of an object
Resolution
the ability to distinguish between two objects that are close together
Contrast
the difference in intensity between two objects, or between an object and its background
Optical sectioning
the process of creating a 3D reconstruction of a specimen from images captured at different focal planes.
Explain Wavelength of radiation
- (NON IONIZING) Radiation = traveling energetic particles of energy
- Disruption of wavelength is what allows us to see an image
- The smaller the wavelength, the smaller the object can be that disrupts it
- Smaller the wavelength, the higher the resolving power (ability to distinguish between two objects)
Explain magnification
- Enlargement of appearance
- Includes the refractive index: a measure of how greatly a substance slows the velocity of light
What is refractive index?
a measure of how greatly a substance slows the velocity of light
What is resolution?
What is the trend that’s associated with it?
- Distinguishing between objects/points
- The shortest distance between two point that can still be distinguished by observer as separate entities
- Increasing resolution = increasing the amount of information into lens = better image & ability to distinguish between 2 objects
What is contrast?
- Important in determining resolution
- Staining increases contrast
- Use of light that is in phase increases contrast
- Difference in intensity between two objects, or between an object and background
Explain how the use of oil affects microscopy
- Oil is there to reduce change in density as light travels through the air (keeps resolution high and produces sharper image as magnification increases
- Oil immersion lens increases resolution & numerical aperture
Bright field microscopy
- Light passes through the specimen into the objective lens
- Can have 1 or 2 ocular lenses
- Most have a condenser lens (direct light through the specimen → increases the intensity of light)
Dark field microscopy
- Best for observing pale objects (light is inverted) → the specimen appears light against dark background
- Only light rays scattered by the specimen enter the objective lens
- Increases contrast and enables observation of more details
- For a pale specimen, you would have to kill the organism to stain it to see it with a bright field.
- For a dark field, you wouldn’t need to kill the organism
Phase contrast microscopes
- Used to examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining
- Light rays “in phase” produce a brighter image. Light rays “out of phase” produce a darker image
- Contrast is created because light waves are out of phase
Fluorescent Microscope
- Direct UV light source at the specimen
- Specimen radiates energy back as a longer, visible wavelength
- UV light increases resolution and contrast
- Some cells are naturally fluorescent others must be stained
- Used in immunofluorescence to identify pathogens and to make visible a variety of proteins
Confocal microscopy
- Uses fluorescent dyes
- Uses UV lasers to illuminate fluorescent chemicals in a single plane
- Resolution increased because emitted light passes through pinhole aperture
- Allows for optical sectioning
- Computer constructs 3D composite images from digitized images
Discuss the similarities and differences between light microscopy, confocal microscopy, and electron microscopy
- Dependent on the wavelength and resolving power
- Electron microscopes differ from light microscopes in that they produce an image of a specimen by using a beam of electrons rather than a beam of light.
What makes electron microscopy special?
- Electrons have much a shorter wavelength than visible light, and this allows electron microscopes to produce higher-resolution images than standard light microscopes.
What are the 2 types of electron microscopy?
Transmission Electron Microscope (TEM)
Scanning Electron Microscope (SEM)
Transmission Electron Microscope (TEM)
- Can not be used to study living organisms
- Preparation can cause artifacts
- Electrons scatter when they pass through thin sections of a specimen
- Transmitted electrons are under vacuum which reduces scatter and are used to produce a clear image
- Denser regions in the specimen, scatter more electrons and appear darker
Scanning Electron Microscope (SEM)
- Uses electron beam to scatter electrons from the surface of a specimen to create detailed image → Produces a realistic 3D image of a specimen’s surface features
- No sectioning required
- The whole organism can be viewed
- No internal structures
Staining for Electron Microscopy
- Chemicals containing heavy metals used for TEM
- Stains may bind molecules in specimens or the background
List the general principles of microscopy
1) Wavelength of radiation
2) Magnification
3) Resolution
4) Contrast
