2.1.1: Cells and Microscopy Flashcards
(92 cards)
Describe the four ways in which a sample can be prepared (light microscope examination)
- Dry mount
- Wet mount
- Squash slides
- Smear slides
How light microscope works:
1) Light passes from bulb
2) Through condenser lens which focuses rays
3) Through specimen
4) Then focused through one of the 4 objective lenses to view specimen at different magnification
5) Light passes into eyepiece –> specimen can be viewed
Why must the cover slip by placed on a wet mount at an angle?
• To avoid trapping air bubbles
Why should the refractive index of the medium for a wet mount be close to that of glass?
- To avoid excess refraction of light
* Thereby keep image clear
Why is staining necessary?
- Increase the contrast between different components of cells
- Thereby make them easier to identify
What is a simple stain?
- A process that involves using one solution of a single dye.
- Can make it possible to discern the shapes of different organisms like bacteria.
What is differential staining?
- A process that involves using more than one chemical stain.
- Can help distinguish between different microorganisms or provide contrast between different organelles in an organism.
What is resolution?
The ability to see individual objects as separate entities. Allows more detail to be seen.
Points closer than…
1/2 the wavelength of light cannot be seen as separate entities.
What is magnification?
The degree to which the size of the image is larger than the size of the object itself.
How does Laser Scanning Confocal Microscopy work?
1) Laser excitation source emits rays of light of certain wavelength
2) Light passes through illumination pinhole
3) Light reflected by dichromatic mirror
4) Reflected light refracted through the objective lens
5) Light focused on single focal plane of the specimen (illumination point)
6) Light causes fluorescence from components stained with a fluorescent antibody tag
7) Light emitted back through objective lens, passed through dichromatic mirror
8) Light enters detector through confocal pinhole
Why does the fluorescent light emitted pass through the dichromatic mirror?
Because this light has a different wavelength to the light emitted by the laser excitation source.
What does the confocal pinhole do?
Prevents out of focus light from a different focal plane being detected.
What does confocal mean?
Both the light waves from the laser and radiated when the sample fluoresces have the same focal plane and both follow the same paths.
What is the beamsplitter?
The dichromatic mirror, which reflects light of the wavelength emitted by the laser but lets light of other wavelengths pass through.
How can a 3D image be produced from LSCM?
- 2D image slices layered on top of each other.
* Reconstructed into 3D model by computer.
How can antibodies be used for fluorescence microscopy?
- Antibodies specific to a particular molecule are dyed.
* They then attach to this feature in the cell.
DNA in eukaryotic cells is…
- linear
- contained in nucleus
- wrapped around histomes
DNA in prokaryotic cells is:
- circular
- ”naked” = not wrapped around histomes
- exists in nucleoid region or in plasmids
Organelles in prokaryotic cells are:
Not membrane bound
Organelles in eukaryotic cells are…
Membrane bound
Size of eukaryotic cell:
10-40 µm (micrometers)
Size of prokaryotic cells:
0.5-5 µm (micrometers)
Cell walls in a prokaryotic cell:
- always present
* made of peptidoglycan