Microscopy Flashcards
What size does an object have to be in order to be visualized?
1/2 the wavelength of the electromagnetic radiation being shown on it
What is resolution?
resolution refers to the ability to distinguish two points from each other. Smaller resolution = better
What is the wavelength of a light microscope?
0.2 um
What is the wavelength of electron microscopy?
2 nm
How can you improve resolution?
- change the illumination source (electrons)
- increase the intensity of the illumination
- replace the medium light travel through (prevent refraction)
What oil is used for 100x objective?
immersion oil
What is contrast?
how distinct is the specimen from the background
How can you improve contrast?
- Positive stain (specimen)
- Negative stain (background)
What are the different types of light microscopy?
- Bright field
- Darkfield
- phase contrast
- Interference
- Fluorescence
- “Superresolution” fluorescence
- Confocal scanning laser
How does brightfield work?
Uses light, diaphragm, and condenser to condense light through slide.
How does darkfield work?
Uses a special type of condenser to bend light away from objective. If light hits specimen, it will be bent up into lens
What is phase contrast?
Uses phase-shifting of refracted light (caused by photon drag) to enhance contrast. refractive index differences are amplified
What is interference microscope?
uses 2 beams of 90 degree polarized light to detect 3D details in the sample. light is separated by prism and recombine after specimen.
What is fluorescence microscope?
Uses excitation wavelength as illumination light, can see emission wavelength of fluorescently-labeled specimen
What are fluorophores?
chemicals that emit visible light when they are illuminated by a shorter wavelength of light (usually UV)
What is superresolution fluorescence?
uses very brief excitation of low-intensity fluorophore stain, followed by computer averaging, to enhance resolution of light microscope
Can you use multiple fluorophores?
Yes, they are linked to different antibodies
What is confocal scanning laser microscope?
uses an aperture to shut out light from all but one focal plane of the object. Computer builds up 3-d object plane by plane
Types of electron microscopy?
- TEM
- SEM
- electron cyrotomography
What is TEM?
transmission electron microscopy; electron beam passes through the specimen, just like a beam of light. uses dye that absorbs electrons
What is SEM?
Scanning electron microscopy; detects electrons and x-rays scattered from a surface, dramatic 3D views
What is electron cryotomography?
rotate object; capture images at different angles. use software to compile a 3d shape
What is atomic force microscopy?
electric field is monitored at the tip of a nanotube probe. Field varies as probe is pushed over a surface. Can see atoms
What is a simple stain?
shows outline of bacteria; basic (+) dye attaches to (-) phospholipid head groups?
What is differential stain?
more than one stain is used - cell stain differently based on call properties. Includes gram stain and acid-fast stain
What is a gram stain?
shows thickness of peptidoglycan; more than one stain is used
What are gram stain results?
pink = thin (gram -)
purple = thick (gram +)
What is acid-fast stain?
used for bacteria with waxy cell walls; differentiates based on waxy coating impervious to acidified alcohol
What are acid-fast stain results?
red = acid-fast (waxy)
blue = not
What is negative stain?
stains the background, not the cell
