Microscopy

Question 1

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

Answer 1

1. Obtain thin section of tissue - EG using ultratome or maceration
2. Place plant tissue in a drop of water
3. Stain tissue on a slide to make structures visible.
4. Add coverslip using mounted needle at 45° to avoid trapping air bubbles

Question 2

How do light microscopes work

Answer 2

1. Lenses focus rays of light and magnify the view of a thin slice of specimen
2. Different structures absorb different amounts and wavelengths of light
3. Reflected light is transmitted to the observer via the objective lens and eyepiece

Question 3

Describe how a transmission electron microscope (TEM) works

Answer 3

1. Pass a high energy beam of electrons through a thin slice of specimen.
2. More dense structures Turks- appear darker since they absorb more electrons
3. Focus image onto fluorescent screen or photographic plate using magnetic lenses

Question 4

Describe how a scanning electron microscope (SEM) works

Answer 4

1. Focus beam of electrons onto a specimen’s surface using electromagnetic lenses.
2. 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

1. Focus laser beam onto a small area on a sample’s surface using objective lenses.
2. Fluorophores in the sample emit photons
3. Photomultiplier tube amplifies the signal onto a detector. An image is produced pixel by pixel in the correct order

Question 6

How should field of view on microscopy be recorded

Answer 6

Draw diagram with a sharp pencil.
No sketchy lines or shading
Include a scale bar
Annotate visible strutures

Question 7

Actual size = ?

Answer 7

Actual size = image size / magnification

Question 8

Define magnification

Answer 8

Factor by which the image is larger than the actual specimen

Question 9

Define Resolution

Answer 9

Smallest separation distance at which 2 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 the structures
Facilitates absorption of wavelengths of light to produce image.

Differential staining: contrast between heavily and lightly stained areas distinguishes structures

Question 11

Magnification and resolution of a compound light microscopes

Answer 11

Magnification = x2000
Resolution= 200nm

Question 12

Magnification and resolution of a TEM

Answer 12

Magnification = x500,000

Resolution = 0.5nm

Question 13

Magnification and resolution of an SEM

Answer 13

Magnification = x500,000

Resolution = 3 - 10nm

Question 14

How to use an eyepiece graticule and stage micrometer to measure the size of a structure

Answer 14

1. Place micrometer on stage to a calibrate eyepiece graticule
2. Line up scales on the graticule and micrometer. Count how many graticule divisions are in the 100μm on the micrometer.
3. Length of 1 eyepiece division = 100μm/ number of divisions
4. Use calibrated va,use to calculate actual length of structures