Light microscopes

Question 1

How it works:

Answer 1

• Has 2 lenses - objective lens placed near the specimen, and an eyepiece lens through which the specimen is viewed
• Objective lens produces magnified image (x4, x10, x40) which is magnified again by eyepiece (x10)
• This configuration allows for higher magnification and reduced chromatic aberration
• Illumination provided by a light or mirror under the sample

Question 2

Sample preparation:

Answer 2

• Dry mount: Solid specimens are viewed whole/cut into very thin slices (sectioning). Specimen is placed on the centre of slide and cover slip in placed on the sample.
• Wet mount: Specimens suspended in liquid. Cover slip is placed on from an angle.
• Squash slides: squashing sample between 2 slides
• Smear slides: Use edge of slide to smear the sample and create a thin coating

Question 3

Use of staining:

Answer 3

• Sample is illuminated from below by white light and observed from above (brightfield microscopy). Whole sample is illuminated at once (wide-field microscopy).
• Imaged have low contrast as most cells don’t absorb lot of light. Resolution is limited by wavelength of light.
• Cytosol of cells are often transparent.
• Stains INCREASE CONTRAST as different components within the cell take up stains to different degrees based on charges of dyes as cytosol is negative
• Allows organelles to be identified (Differential staining)

Question 4

Using a graticule:

Answer 4

• Eyepiece graticule is a disc marked with a fine scale of 1-100. Has no units.
• Stage micrometre is a slide with very accurate scale in micrometres engraved on it : 100 divisions = 1mm so 1 division = 10um
• Magnification factor = num. of micrometres/ num. of graticule divisons