Ch. 3 Observing Microorganisms through a Microscope Flashcards
Microorganisms are measured in
micrometers and nanometers
Types of light microscopy
– Compound light microscopy
– Darkfield microscopy
– Phase-contrast microscopy
– Differential interference contrast (DIC) microscopy
– Fluorescence microscopy
– Confocal microscopy
Compound microscope
image from the objective lens is magnified again by the ocular lens
Total magnification =
objective lens x ocular lens
Resolution
the ability of the lens to distinguish two points
refractive index
a measure of the light-bending ability of a medium
Brightfield illumination
– Dark objects are visible against a bright background
– Light reflected off the specimen does not enter the
objective lens
Darkfield Microscopy
- Light objects are visible against a dark background
- Opaque disk placed in condenser
- Only light reflected off the specimen enters the objective
lens
Phase-Contrast Microscopy
- Allows examination of living organisms and internal cell
structures - Brings together two sets of light rays, direct rays, and
diffracted rays to form an image
Differential Interference Contrast Microscopy
- Similar to phase-contrast
- Uses two light beams and prisms to split light beams,
giving more contrast and color to the specimen
Fluorescence Microscopy
- Uses UV (short wavelength) light
- Fluorescent substances absorb UV light and emit longer
wavelength (visible) light - Cells may be stained with fluorescent dyes
(fluorochromes) if they do not naturally fluoresce
Confocal Microscopy
- Cells are stained with fluorochrome dyes
- Short-wavelength (blue) light is used to excite a single
plane of a specimen - Each plane in a specimen is illuminated and a three-
dimensional image is constructed with a computer - Can examine layers of cells to a depth of 100 μm
Two-Photon Microscopy
- Cells are stained with fluorochrome dyes
- Two photons of long-wavelength (red) light are used to
excite the dyes - Can study living cells up to 1 millimeter deep
Super-Resolution Light Microscopy
- Uses two laser beams
– One wavelength stimulates fluorescent molecules to
glow
– Second wavelength cancels out all fluorescence
except for that in one nm - A computer scans the specimen nm by nm, then puts the
images together
Scanning Acoustic Microscopy
- Measures sound waves that are reflected back from a
specimen - Used to study cells attached to surfaces
- Resolution of 1 μm
Electron Microscopy
- Uses electrons instead of light
- The shorter wavelength of electrons gives greater
resolution - Used for images too small to be seen with light
microscopes, such as viruses
Transmission Electron Microscopy
- A beam of electrons passes through ultrathin sections of
a specimen, then through an electromagnetic lens, then
focused on a projector lens - Specimens may be stained with heavy-metal salts for
contrast - Magnifies objects 10,000 to 10,000,000x; resolution of 10
pm
Scanning Electron Microscopy
- An electron gun produces a beam of electrons that scans
the surface of an entire specimen - Secondary electrons emitted from the specimen produce
a three-dimensional image - Magnifies objects 1,000 to 500,000x; resolution of 10 nm
Scanning Tunneling Microscopy
- Uses a tungsten probe to scan a specimen and reveal
details of its surface - Resolution of 1/100 of an atom
Atomic Force Microscopy
- Uses a metal-and-diamond probe placed onto a
specimen; movements are recorded - Produces three-dimensional images at near atomic detail
Staining
coloring microorganisms with a dye that emphasizes certain structures
Smear
a thin film of a material containing microorganisms spread over a slide
Fixed
Microorganisms are attached to the slide, which kills the microorganisms
Chromophore
colored stain consisting of a positive and negative ion
