microscopy

Question 1

outline how a student could prepare a temporary mount of tissue for a light microscope

Answer 1

1. obtain a thin section of tissue
2. place plant tissue in a drop of water
3. stain the tissue to make structures visible
4. place tissue on a slide, add a coverslip using a a mounted needle at a 45 degree angle to avoid trapping air bubbles

Question 2

describe how light microscopes work

Answer 2

• lenses focus rays of light and magnify the view of a thin slice of specimen
• different structures absorb different amounts and wavelengths of light
• reflected light is transmitted to the observer via the objective lens and the eyepiece

Question 3

describe how a transmission electron microscope (TEM) works

Answer 3

• pass a high energy beam of electrons through a thin slice of specimen
• more dense structures appear darker since they absorb more electrons
• focus image onto fluorescent screen or photographic plate using magnetic lenses

Question 4

describe how a scanning electron microscope (SEM) works

Answer 4

• focus a beam of electrons onto a specimen’s surface using electromagnetic lenses
• reflected electrons hit a collecting device and are amplified to produce an image on a photographic plate

Question 5

describe how a laser scanning confocal microscope works

Answer 5

• focus a laser beam onto a small area on a sample’s surface using objective lenses
• fluorophores in the sample emit photons
• photomultiplier tube amplifies the signal onto a detector, an image is produced pixel by pixel in the correct order

Question 6

how should the field of view in microscopy be recorded?

Answer 6

• draw a diagram with a sharp pencil
• do not use sketchy lines or shading
• label visible structures with label lines (with no arrowhead) drawn with a ruler
• include a scale bar/magnification

Question 7

state the equation of magnification

Answer 7

magnification = image size ÷ actual size

Question 8

define magnification

Answer 8

the number of times larger an image appears compared to the real size of the specimen

Question 9

define resolution

Answer 9

the smallest separation distance at which two separate structures can be distinguished from one another

Question 10

why do samples need to be stained for light microscopes?

Answer 10

• coloured dye binds to structures
• different colours absorb different wavelengths of light to produce the image
• differential staining causes a contrast between heavily and lightly stained area to be able to distinguish different structures

Question 11

state the magnification and resolution of a compound light optical microscope

Answer 11

mag. = x2000
resolution = 200nm

Question 12

state the magnification and resolution of a TEM

Answer 12

mag. = x500000
resolution = 0.5nm

Question 13

state the magnification and resolution of an SEM

Answer 13

mag. = x500000
resolution = 3-10nm

Question 14

explain how to use an eyepiece graticule and stage micrometre to measure the size of a structure

Answer 14

1. place the micrometre on the stage of the microscope in order to calibrate the eyepiece graticule
2. line up scales on graticule and micrometre. count how many graticule divisions are in 100μm on the micrometre
3. length of one eyepiece division = 100μm (number of micrometre divisions) ÷ number of graticule divisions
4. use calibrated values to calculate actual length of structures