Microscopes

Question 1

What is a microscope?

Answer 1

An instrument employing lenses to produce a magnified image and fine detail of objects too small to see with the naked eye

Question 2

What is magnification?

Answer 2

How many times the size of an image is larger than the object itself (it is indicated by a number)

Question 3

What is resolution?

Answer 3

The degree to which it is possible to distinguish between two separate objects which are close to each other (it is indicated by a length)

Question 4

What are the two categories of microscopes?

Answer 4

Optical and electron microscopes

Question 5

What are the key points about the resolution of a optical microscope?

Answer 5

• Resolution of an object/feature in a microscope is limited to half the wavelength of the wave employed
• Optical microscopes can resolve objects up to 200nm or 0.2µm

Question 6

What is the range of wavelengths used by optical microscopes?

Answer 6

450-600nm

Question 7

What is the limit of magnification of an optical microscope?

Answer 7

x1500-2000

Question 8

How does an optical microscope work?

Answer 8

• Objective lens produces a magnified image which is magnified again by the eyepiece lens => object is magnified twice
• Light is shon through the specimen/object to be viewed

Question 9

How does an electron microscope work?

Answer 9

• A beam of electrons with a wavelength of less than 1nm is used to illuminate the specimen/object
• The lenses are magnets

Question 10

How do electrons in electron microscopes produce an image?

Answer 10

Electrons produce an image when focused on a fluorescent screen

Question 11

What is the limit of magnification and resolution in an electron microscope?

Answer 11

• The limit of magnification is x500,000
• The limit of resolution is 0.2nm

Question 12

What are the two types of electron microscopes?

Answer 12

• Transmission electron microscopes (TEM)
• Scanning electron microscopes (SEM)

Question 13

How does a TEM work?

Answer 13

• Tissue/cell is inflitrated with plastic resin (=> very hard) and is cut into very thin slices using a diamon knife
• A beam of electrons is passed through exceedingly thin slices of the specimen/material and produces an image on a flourescent screen or photographic film
• This allows for the interior of the cells to be seen

Question 14

How does a SEM work?

Answer 14

• The specimen is shadowed with a layer of heavy metal and covered with carbon
• It is scanned by a fine electron beam which is scattered from the surface of a specimen and the transmitted to a detector
• This allows the surface of specimens to be seen
• Except when freeze-fracture (the cell is frozen in liquid nitrogen at -70 degrees celcius) is being used where the inside of the cell is exposed

Question 15

What is the range of magnification and resolution of a TEM?

Answer 15

Magnification: x500,000 - 2,000,000
Resolution: 0.2 - 0.5nm

Question 16

What is the range of magnification and resolution of a SEM?

Answer 16

Magnification: x100,000 - 500,000
Resolution: 3 - 10nm

Question 17

What is the difference between a TEM and SEM?

Answer 17

TEM:
- Higher resolution than SEM
- Higher magnifcation than SEM
- Colour of image is black and white
- Can view interior of cells
- 2D image formed

SEM:
- Lower resolution than TEM
- Lower magnifcation than TEM
- Colour of image is black and white
- Can view exterior of cells (=> can only view interior through freeze fracturing)
- 3D image formed

Question 18

What are the advantages of a light microscope?

Answer 18

• Small and portable
• Natural colour of the material can be observed
• Cheap to purchase
• Material rarely disorted by preparation
• Living material may be viewed
• Preparation of material is relatively quick and simple, requires only a little expertise
• Unaffected by magnetic fields
• Doesn’t require a vacuum
• Cheap to operate

Question 19

What are the disadvantages of a light microscope?

Answer 19

• The depth of field is restricted
• Magnifies objects up to 1500x
• Can resolve objects 0.2µm apart

Question 20

What are the advantages of the electron microscope?

Answer 20

• Possible to see a greater depth of field (SEM 3D image)
• Magnifies objects up to 500,000x
• Can resolve objects up to 0.001µm apart

Question 21

What are the disadvantages of the electron microscope?

Answer 21

• Very large and must be operated in special rooms
• All images in black and white
• Expensive to purchase
• Preparation of material may distort it
• A high vacuum is required
• Living material can’t be viewed
• Preparation of material is length, requires a considerable expertise ad complex equipment
• Affected by magnetic fields
• Expensive to operate: requires up to 100,000V to produce an electron beam
• Can produce artifacts

Question 22

What are the different ways that samples and specimens can be prepared?

Answer 22

• Dry mount
• Wet mount
• Squash slides
• Smear slides

Question 23

How is a dry mount prepared?

Answer 23

• Solid specimens are viewed whole or cut into very thin slices with a sharp blade, this is called sectioning
• The specimen is placed on the centre of the slide and a cover slip is placed over the sample
• A cover slip is needed to keep the specimen flat and to maintain the same focal length throughout

Question 24

How is a wet mount prepared?

Answer 24

• Specimen is placed on the slide
• Drops of a stain/water is put on specimen
• A cover slip is lowered at an angle using a mounted needle to minimise trapping air bubbles

Question 25

How is a smear slide prepared?

Answer 25

- A spreader slide is pulled backwards across another slide and used to smear the sample - The spreader slide is pushed forwards in one smooth motion with constant downward pressure - This creates a thin, even coating on the slide - A cover slip is placed over the sample

Question 26

How is a squash slide prepared?

Answer 26

- A wet mount is first prepared, then a lens tissue is used to gently press down the cover slip - Depending on the material, potential damage to a cover slip can be avoided by squashing the sample between two microscope slides

Question 27

What are the stages of preparing a permanent slide?

Answer 27

Fixing: chemicals like formaldehyde are used to preserve specimens in as near-natural a state as possible with minimum distortion Dehydration: specimens are dehydrated (water I removed) with alcohols (to kill bacteria and prevent any from growing) Clearing: removing the dehydration alcohol to leave specimen transparent Embedding: placing it in a mould with wax or resin to form a hard block Sectioning: using a microtome to produce thin slices to form a block Staining: treating the thin slices with chemical agents (stain) to "dye" different structures different colours Mounting: the specimens are then secured to a microscope slide and a cover slip placed on top

Question 28

What are the advantages of staining?

Answer 28

- Provides contrast between components of cells e.g. organelles - Provides contrast between a structure (e.g. cell) and its background - Allows different components (e.g. organelles) of cells to be identified

Question 29

Why does a stain give a different colour with different cellular structures?

Answer 29

Cellular structures are composed of different molecules which interact differently with the specific stain molecules e.g. a positively charged stain will interact with a negatively charged molecule such as DNA

Question 30

What is differential staining?

Answer 30

- This is staining that uses more than one chemical stain - This makes differences between cells or between different structures within cells more visible

Question 31

What is the gram stain technique used for?

Answer 31

It is used to separate bacteria into two groups, Gram-positive bacteria and Gram-negative bacteria

Question 32

What are the steps of the Gram stain technique?

Answer 32

- **Crystal violet** is first applied to a bacterial specimen on a slide - **Iodine** is added to fix the dye - The slide is then washed with **alcohol** - Gram-positive bacteria retains the crystal violet stain and will appear blue or purple under a microscope - Gram-negative bacteria have a thinner cell wall and so therefore lose the stain - Gram-negative bacteria is then stained with **Safranin dye** which is called a counter stain - These bacteria will appear red

Question 33

What is an artificat?

Answer 33

A visible structural detail that is due to the processing of the specimen and not a natural feature of the specimen

Question 34

What are some examples of positively charged dyes?

Answer 34

Crystal violet and methylene blue

Question 35

What are some examples of negatively charged dyes?

Answer 35

Nigrosin and congo red

Question 36

What is diffraction?

Answer 36

The tendency of light waves to spread as they pass close to physical structures such as those present in the specimen being studied