Microscopy Flashcards
What are the types of light microscopy?
There’s 5
1. bright-field
2. phase contrast
3. DIC (differential interference contrast)
4. Dark field (scattered light)
5. Fluorescence
What type of electron microscopy are there?
There’s 2:
1. Transmission electron microscopy
2. Scanning electron microscopy
What is brightfield?
it is light microscopy that takes advantage of staining
What are the types of staining for brightfield?
- Crystal violet
- Carbol fuchsin
- Methylene blue
- Negative staining
- Flagellar stain
- Endospore stain
- Capsule stain
- Gram staining
What is gram staining and what are the steps to it?
Gram staining works due to the different cell envelopes that the cells have
- there’s gram positive and gram negative cells and gram positive cells are able to retain the stains
the staining is done in 5 steps
1. Fixation
- Helps stick the cell to the glass
2. Crystal violet
- Colors the cell blue
3. Iodine (mordant)
4. Decolorization (ethanol)
- Gram negative cells would be clear
- Gram positive cells would be purple
5. Counterstain with safranin
- Gram negative cell would be pink
- Gram positive cells would be blue-purple
What is an example of a gram positive and gram negative cell
gram positive: Staphylococcus
gram negative: E.coli
Phase contrast
Allows one to see transparent cell (so no staining required)
when light passes through the specimen, the speed is slowed and the phase is delayed and the light ray bends (refraction)
so this causes the light to be out of phase with the other light waves
the lights are half a wavelength out of sync from each other and this is destructive interference so cells appear dark
what is DIC
differential interference contrast
it uses polarized light and allows us to view things while they are still alive
uses gradients in the optical path length and phase shifts of light to make phase objects visible when viewed with a microscope
What is dark field?
allows you to see things that you might not without it
it enhances shapes
it has better light resolution than light microscopes
what is fluorescence
the cells are labeled with fluorophore
Illuminated with light of a shorter wavelength
The fluorophore emits light of a longer wavelength
- And since you cant lose energy it will explode and emit as longer wavelength
So the fluorophore binds to the cell directly and antibody conjugated to the fluoropore so genetically engineered cells to express a fluorescent protein fusion
transmission electron microscopy
Electrons are detected and they are focused by magnets
They spray the cells with heavy metal ions that allow the electron to bounce off
It reveals differences in density
They look 2-dimensional or flat
Scanning electron microscopy
Scattered electrons are detected
Specimen look 3-dimensional
True or false? Scanning electron microscopy produces images that are flatter (more 2 dimensional) than transmission electron microscopy?
False
True or false? electron microscopes are more powerful than light microscopes because they have greater limit of resolution
false
True or false? With a light microscope, you can see the flagella of living bacteria
false, you can only see non-living bacteria
True or false? Darkfield microscopy relies upon a negative stain
false
True or false? fluorescence microscopy detects light that has travelled through the specimen
false
True or false? electrons have a longer wavelength than rays of visible light
false, because electrons have a shorter wavelength than light
True or false? DIC microscopy relies upon polarized light
true
True or false? phase contrast microscopy causes cells to look like bright objects against a grey background
false, it looks darker not brighter
Fill in the blank:
On a compound microscope, the light travels from the bulb, up through two ___ to the specimen on the stage. From their it proceeds to the ____ lens, and then to the ____ lens.
On a compound microscope, the light travels from the bulb, up through two condenser to the specimen on the stage. From their it proceeds to the objective lens, and then to the optic lens.
The term describing light bending as it passes through different material is
refraction
Fill in the blank:
During the gram stain procedure, just before adding the counterstain, the gram positive cells are ___ and the gram negative cells are ____
During the gram stain procedure, just before adding the counterstain, the gram positive cells are purple and the gram negative cells are clear/white
Darkfield microscopy is different from brightfield, because of what two features of the light rays?
- lights come from the side and angle, shines obliquely
- light scattering
