Light microscopy

Question 1

What is the smallest measurement visible to the naked eye?

Answer 1

0.2mm

Question 2

What are the 2 steps to which a classic compound microscope magnifies?

Answer 2

1. Objective lens produces enlarged image of object in ‘real’ image plane
2. Real image magnified by ocular lens on eyepiece = produces virtual image

Question 3

Define magnification

Answer 3

Ratio of size of image to size of object

Question 4

How do you calculate the total magnification?

Answer 4

Power of objective x power of eyepiece

Question 5

Define resolution

Answer 5

Clarity of image, illumination & quality of the optics

Question 6

Define contrast

Answer 6

Contrast between lightest & darkest areas of the sample

Question 7

What is the numerical aperture of a lens?

Answer 7

The size of the cone of light coming from each point on the specimen

Question 8

Who 1st named cells?

Answer 8

Robert Hooke

Question 9

What did Antony van Leeuwenhoek do?

Answer 9

improved production of lenses & observes cell types e.g. RBCs

Question 10

What did Zeiss do?

Answer 10

Made lenses with resolution at the theoretical limits

Question 11

What do all microscopes have?

Answer 11

Light source
Stage
Objectives 
Lens
Eyepiece

Question 12

What does the objective lens do?

Answer 12

Collect a cone of light rays to create an image

Question 13

What does the condenser lens do?

Answer 13

Focuses cone of light rays onto each point of specimen

Question 14

What are the 5 types of light microscopy?

Answer 14

> Bright field
> Phase contrast 
> DIC = differential interference contrast 
> Fluorescence
> Confocal

Question 15

What do bright field microscopes produce?

Answer 15

Relatively poor images compared w/ Phase contrast & DIC

Question 16

What do phase contrast microscopes produce?

Answer 16

High contrast images

- can see organelles clearly

Question 17

How are phase contrast images produced?

Answer 17

Diffracted & un-diffracted rays give rise to change in brightness

Question 18

What do DIC microscopes produce?

Answer 18

Images w/ depth

Question 19

How does a DIC microscope work?

Answer 19

Uses polarised light

• -> through beam splitter prism
• -> through specimen
• -> through-combining prism

Question 20

How are the different colours on DIC images produced?

Answer 20

1 beam diffracted by specimen
–> recombined by prism
–> interfere
= generates contrast from black to white

Beams not diffracted
–> don’t interfere when recombined
=pale grey

Question 21

Briefly, how can cellular components, cell types or cell viability be detected?

Answer 21

1. add dyes
2. chromogenic enzyme substrates
3. detect whether dyes have been included or excluded

Question 22

Which dyes interact w/ phenolic compounds?

Answer 22

Histo- or cyto-chemical dyes

Question 23

How can enzyme substrates be used in microscopy?

Answer 23

Specific chemical bind to enzymes and give rise to coloured products that can reveal sites of activity

e.g. proteases

Question 24

How can living or dead cells be identified?

Answer 24

Dead = dye not retained
(damaged vacuolar membrane)

Alive = vacuoles take up dye

Question 25

What is fluorescence?

Answer 25

Property of absorbing light of a particular wavelength & then emitting light of a diff colour & wavelength

Question 26

What is the difference between the path light takes in bright field vs epifluorescence microscopy?

Answer 26

Epi:
Light comes from side
Goes through filter
Monochromatic green light reflects off mirror onto specimen

Bright field:
Single beam of light from below the specimen

Question 27

What are the autofluorescence examples?

Answer 27

Lignin

Avenacin
anti-fungal compound in epidermis of oat roots

Question 28

How do confocal microscopes work?

Answer 28

They focus on 1 plane of a 3D specimen at a time

Question 29

What can confocal microscopes be used for?

Answer 29

> Looking at co-localisation
Intra-cellular studies
On thick specimens
3D studies