Microscopes Flashcards
How does a light microscope work?
Compound ones have two lenses (objective and eyepiece). Using both allows for higher magnification, resolution and reduced aberration
Describe the process of creating a dry mount?
Solid specimens are sectioned, add cover slip
E.g. hair, dust, muscle tissue
Describe the process of creating a wet mount?
Suspended in liquid (H20, oil), angled a cover slip
Describe the process of creating a squash slide? (3)
- Wet mount
- Pressed down cover slip.
For soft, spongey samples.
Describe the process of creating a blood smear slide?
Edge of the slide is used to smear the sample. Hold at 45º Even thin coating. Cover slip on top.
E.g. blood smears.
Why do cells show low contrast and how to solve this?
Most cells do not absorb a lot of light. Use a stain
What is resolution?
The shortest distance between two objects that are still seen as separate objects
What is resolution limited by?
By the diffraction of light. Light waves spread as they pass close to specimens. As these objects are close to each other, the light reflected can overlap so they can no longer be seen as separate entities
Why do electron microscopes have high resolving powers than a light microscope?
They use electron beams which have a 1000x shorter wavelength, still diffract but won’t overlap so the details aren’t lost.
Describe the process of calibration?
1) Put the stage micrometer in place and the eyepiece graticule into the eyepiece
2) Get the scale on the micrometer in clear focus
3) Align the micrometer scale with the scale in the eyepiece. Take a reading from both scales
4) Use these readings to calculate the calibration factor for the objective lense (1 grat.div.=no.µm/no. grat.div.)
5) Remove stage micrometer and place prepared slide on the stage.
Describe the stains and the method of gram positive/negative bacteria staining?
Crystal violet & methylene blue (charged dyes).
Bacteria separation; crystal violet on specimen, then iodine to fix the dye, alcohol wash. Gram+ will retain colour. Gram- has thinner cell walls so loses stain. (stained with safranin dye - counterstain)
What wavelength do electron microscopes have?
0.004nm (1000x shorter than light)
Features of Scanning Electron Microscopes (SEM) [6]
e- beams deflected by air; expensive; dead samples; requires training and preparation; >0.1nm resolution; detailed, 3D images
Features of Transmission Electron Microscope (TEM)
Focus beam of e- transmitted through specimen; 2D image; highest resolution (0.0002µm); Specimen must be thinly sliced; angle cut affects how they appear
How do Laser Scanning Microscopes work? (4)
- Moves a single spot of focused light across
- causes fluorescence from dyed components
- Emitted light is filtered
- only light radiated from very close to the focal plain is detected
