Chapter 2 Microscopy

Question 1

Why is staining used?

Answer 1

Increases contrast of the cells/organelles/image
Allows internal structures e.g organelles to be more easily seen, also helping distinguish between them
Can help distinguish between different types of cells
Effectively increases clarity of the image

Question 2

What is staining?

Answer 2

Add a chemical which is absorbed by certain cells/organelles of a sample, increasing contrast

Question 3

How should samples be generally prepared?

Answer 3

Using a sharp blade, cut the sample very thinly to allow the light to pass through the specimen, increasing resolution
Place on a slide with forceps
Use some sort of mount ideally- wet mount or smear or squash, to allow more cells to be seen

Question 4

How should a stain be added?

Answer 4

Place the very thin sample on the slide with forceps
Add one or two drops of the stain to the edge of the sample
Use blotting paper to remove excess stain
Lower the cover slip at an angle with an angled needle

Question 5

What is the purpose of a wet mount? What is the purpose of squashing?

Answer 5

Wet- prevents dehydration/distortion of the tissue
Squash- allows light to penetrate more easily

Question 6

What is the resolution, magnification, image and use of TEMs?

Answer 6

Around 0.2nm x500000
2D, black and white image with a very high resolution
Used to find details of cell ultrastructure

Question 7

What is the resolution, magnification, image and use of SEMs?

Answer 7

Around 3-10nm x100000
3D, black white images
Used to determine information of cell surface, as 3D

Question 8

What is the resolution, magnification, image and use of light microscopes?

Answer 8

Around 200nm x2000
2D, coloured images
Info about live samples, cells

Question 9

How do TEMs work? What are some disadvantages of them?

Answer 9

Beam of electron through sample
Can produce artefacts, require complex sample preparation, including very, very thin samples

Question 10

What is an artefact?

Answer 10

Distortions in the image caused by the sample preparation, mostly with electron microscopes, can be air bubbles with light

Question 11

How does SEMs work? What are some advantages of them?

Answer 11

Beam of electrons send across the surface of a specimen, electrons detected to form a 3D image

Very expensive, lower resolution than TEM

Question 12

What are the general advantages/disadvantages of light microscopes?

Answer 12

Cheap, Portable, Easy to prepare samples, Alive or dead sample, natural colour, no vacuum needed

But lower resolution

Question 13

What is the process of electron sample preperation?

Answer 13

Chemical Fixation
Cryofixation
Dehydration, ethanol to preserve
Embedding in resin
Sectioning
Staining with heavy metals
Mounting

Question 14

What the types of slide preperation of light microscopes?

Answer 14

Dry Mount- cover slip parallel
Wet Mount- in liquid e.g oil, cover slip angled
Squash- Wet then squash
Smear- with cover slip

Question 15

What is fluorescence?

Answer 15

The absorption and re-emittance of of light at lower energy and higher wavelength, so magnified

Question 16

How does a LSCM work?

Answer 16

Laser Scanning Confocal Microscope

Dye in sample
Point illumination- moving a laser across the specimen
Light emitted as fluorescence, only light closest to focal plane allowed through pinhole aperture to increase resolution, remove background noise

Question 17

What are advantages of LSCM?

Answer 17

Very high resolution
Depth selectivity

Question 18

Define magnification

Answer 18

How many times larger an image is compared to the real size

Question 19

Define resolution

Answer 19

The ability to see two points which are close together as two separate entities

Question 20

What must be increased to see greater detail?

Answer 20

Magnification and Resolution

Question 21

What happens with light that limits resolution?

Answer 21

Tendency for light to spread when passing through a specimen, diffraction
If distances between points closer than 0.5 wavelength of light, cannot be seen individually, as waves overlap
Wavelength of electrons lower, so small distances between points able to be seen

Question 22

What is the equation with microscopes?

Answer 22

Image Size= Real Size x Magnification

Question 23

What is an eyepiece graticule?

Answer 23

Glass disc on microscope with arbitrary units, used with a stage micrometer to measure the length of samples

Question 24

What is a stage micrometer?

Answer 24

A microscope slide containing a total distance of 1mm, with 100 divisions. Each division is 10 micrometres
Used to calibrate at each magnification

Question 25

How can a microscope be calibrated?

Answer 25

Align stage micrometer with eyepiece graticule
Divide to get the value of one EPU
Multiple any measurement of sample at this magnification by the value of one EPU

Question 26

How can you tell an image is from an TEM?

Answer 26

High resolution and magnification
As very small organelles such as ribosomes can be seen, and more detail of organelles such as nuclear pores