Imaging and Studying Cells

Question 1

Define ultrastructure

Answer 1

Level of detail obtainable with the EM

Question 2

Define magnification

Answer 2

The ration of an objects image size to its real size

Question 3

Define resolution

Answer 3

The measure of the clarity of the image or the minimum distance of two distinguishable points

Question 4

Define contrast

Answer 4

Visible differences in brightness or colour between parts of the sample

Question 5

How does the bright field microscope work?

Answer 5

Light passing through specimen (specimen must be partially transparent)

Question 6

What is the resolution of light microscope?

Answer 6

0.2 microns

Question 7

How does a fluorescent light microscope work?

Answer 7

Fluorescent substances absorb short wavelengths and emit longer wavelengths emitting visible light

Question 8

How does a confocal light microscope work?

Answer 8

Uses lasers for optical sectioning. Regions with a narrow depth are focussed

Question 9

Outline the main steps for light microscope sample preparation

Answer 9

Small transparent specimens can be used

Tissues must be sectioned. Fixation occurs using chemical fixation. Water removed by dehydration and clearing. Embedding; the specimen is infiltrated with molten wax. Cut into thin sections. Wax is removed and tissue is stained.

Question 10

How does a advanced light microscope work?

Answer 10

Light phase shifts to generate contrast

Question 11

What is deconvolution microscopy?

Answer 11

Algorithms remove out of focus light and sharpens the image and improves resolution.

Question 12

How does super resolution microscopy work?

Answer 12

Gathers light from individual fluorescent molecules and records their position.

Question 13

Why does the electron microscope have a higher resolution?

Answer 13

Electrons have a very short wavelengths (resolution is 100xs bigger)

Question 14

Why do electron microscopes require a vacuum?

Answer 14

Electrons have poor penetrating power

Question 15

Describe how a transmission electron microscope works

Answer 15

Electron beam passes through specimen
Image focussed by magnetic field
Electron image is converted into visible image by a fluorescent screen which is viewed through a glass window
Photographs can be taken

Question 16

Describe the process of transmission electron microscope sample preparation

Answer 16

Whole mount examined
Fixation in Glutaraldehyde then in Osmium tetroxide
Dehydration in an ethanol series
Specimen embedded in plastic resins
Cut 
Heavy metal stain

Question 17

Which microscope always gives 3D appearance?

Answer 17

SEM- scanning electron microscope

Question 18

Describe the process of scanning electron microscope sample preparation

Answer 18

Sample fixed and dried
Fixation in Glutaraldehyde then in Osmium tetroxide
Dehydration by ethanol replacing water
Critical point drying to remove ethanol and minimise shrinking
Coat in thin layer of gold

Question 19

Why is SEM specimen coated with gold?

Answer 19

To prevent electron beam damage

Question 20

Why is TEM specimen stained with heavy metals such as lead?

Answer 20

To improve contrast

Question 21

What is the purpose of cell fractionation?

Answer 21

Allow major organelles to be individually separated out. They’re homogenised to release organelles.

Question 22

Define differential centrifugation

Answer 22

Isolates cell components on the basis of size and density by using increasing durations and g forces