Microscopy

Question 1

How does a light microscope work?

Answer 1

There are two lenses;
objective lens- placed near the specimen
eyepiece lens- the specimen is viewed through this
The objective lens provides a magnified image, which is magnified again by the eyepiece lens - this allows for a much higher magnification
Illumination is provided by light underneath the sample

Question 2

Describe a dry mount

Answer 2

Solid samples are cut into thin slices (sectioning)

The specimen is placed in the middle of the slide and a cover slip is placed on top

Question 3

Describe a wet mount

Answer 3

Specimens are suspended in a liquid. A cover slip is placed on top, at an angle (to prevent air bubbles forming)

Question 4

Describe a squash slide

Answer 4

A wet mount is prepared then two slides are used to press down the cover slip

Question 5

Describe a smear slide

Answer 5

The edge of a slide is used to smear a sample, to create a thin coating on the slide. A cover slip is then placed on the sample.

Question 6

Why is staining useful?

Answer 6

Stains increase contrast as different components of a cell take up stains to different degrees. Increase in contrast- components become more visible and easy to view.
No staining- low contrast- cells do not absorb a lot of light- harder to view components of a cell

Question 7

What is negative staining?

Answer 7

Negatively charged dyes are repelled from the negatively charged cytosol of the cells- therefore they stay out of the cells and leave them unstained. This makes the cell stand out against the stained background.

Question 8

What is differential staining?

Answer 8

Used to distinguish between two types of organisms/ different organelles which would otherwise be hard to identify.

Question 9

What is a gram stain technique?

Answer 9

Separates bacteria into two groups- gram positive and gram negative.
Crystal violet, then iodine (fixes the dye)
Gram positive retain the crystal violet- appearing purple
Gram negative lose the crystal violet as they have thinner cell walls- they are stained with a counterstain and appear red

Question 10

What is an acid fast technique?

Answer 10

Differentiate species of myobacterium from other bacteria

Question 11

What is magnification?

Answer 11

How much bigger the image is than the actual specimen

Question 12

What is resolution?

Answer 12

The ability to distinguish between two points that are close together.

Question 13

What is the formula for magnification?

Answer 13

Magnification=image size/real size

Question 14

How do you calibrate a light microscope?

Answer 14

Line up the stage micrometer with the eyepiece graticule

Question 15

Why is the resolution better in an electron microscope?

Answer 15

More detail of cell ultrastructure can be seen because electrons have a shorter wavelength than light

Question 16

What are the limitations of electron microscopy?

Answer 16

• Expensive

- Specimens can be damaged with the electron beam

Question 17

Transmission electron microscopes

Answer 17

A beam of electrons is transmitted through a specimen and focused to produce an image.
Best resolution.

Question 18

Scanning electron microscopes

Answer 18

A beam of electrons is sent across the surface of a specimen and reflected electrons are collected. Resolution is not as good as a TEM but 3D images are produced.

Question 19

What are the differences between light and electron microscopes?

Answer 19

Electron microscope:

• is expensive to buy/operate
• is large/needs to be installed
• has complex sample preparation (distorts/ creates artefacts)
• requires a vacuum
• black and white image produced
• specimen must be dead

Question 20

What is an artefact?

Answer 20

Visible structural detail caused by processing the specimen- not a feature of the specimen. Eg air bubble trapped when you put the cover slip on the slide.

Question 21

What is a laser scanning confocal microscope?

Answer 21

It moves a single spot of focused light across a specimen. This causes fluorescence from the components with a dye. The emitted light from the fluorescence is filtered through a pinhole aperture

Question 22

Why does all light not pass through the aperture and be detected?

Answer 22

Light emitted from other parts of the specimen would cause blurring and reduce the resolution

Question 23

Why is a laser used in LSCF?

Answer 23

To obtain higher intensities and improve the illumination

Question 24

Why are fluorescent tags used in LSCF?

Answer 24

To target specific features and get more precision.

Question 25

Advantage OF LSCF?

Answer 25

Can be used on 3D specimen

High resolution

Question 26

What is atomic force microscopy?

Answer 26

Feels the surface of a specimen with a mechanical probe to generate a 3D image of its surface.
Adv- specimen can be viewed in normal cell conditions, even living (fixation/staining not required)
High resolution- atomic level