2.3 More microscopy

Question 1

What happens during light microscopy?

Answer 1

In electron microscopy, a beam of electrons with a wavelength of less than 1nm is used to illuminate the specimen. The electrons are fired from a cathode and focused, by magnets rather than glass lenses, on to a screen or photographic plate.

Question 2

In electron microscopy, why can more of the cell ultrastructure be seen?

Answer 2

Fast-travelling electrons have a wavelength about 125 000 times smaller than that of the central part of the visible light spectrum. This accounts for an electron microscope’s much better resolution compared with an optical microscope. They can produce images with magnifications of up to x500 000 and still have clear resolution.

Question 3

What are 4 disadvantages of electron microscopes?

Answer 3

• They are very expensive pieces
• They can only be used inside a carefully controlled environment in a dedicated space
• Specimens can be damaged by the electron beam
• Because the preparation process is very complex, there is a problem with artefacts (structures that are produced due to the preparation process).
• The metallic salt stains used for staining specimens may be potentially hazardous to the user.

Question 4

Why do samples have to be prepared specifically for the use of electron microscopes?

Answer 4

The inside of an electron microscope is a vacuum to ensure the electron beams travel in straight lines. Because of this, samples need to be processed in a specific way.

Question 5

How are specimens prepared for the use of electron microscopes?

Answer 5

Specimen preparation involves fixation using chemicals or freezing, staining with heavy metals and dehydration with solvents. Samples for a TEM will then be set in resin and may be stained again. Samples for a SEM may be fractured to expose the inside and will then need to be coated with heavy metals.

Question 6

Describe how electron microscopes work

Answer 6

• Electron microscopes use a beam of fast-travelling electrons with a wavelength of about 0.004 nm. This means that they have much greater resolution than optical microscopes and can be used to give clear and highly magnified images.
• The electrons are fired from a cathode and focused, by magnets rather than glass lenses, on to a screen or photographic plate.

Question 7

What are the two different types of electron microscopes?

Answer 7

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

Question 8

Which type of microscope has the best (highest) resolution?

Answer 8

Transmission electron microscope

Question 9

Describe the 3 step process by which transmission electron microscopes work

Answer 9

1) The specimen has to be chemically fixed by being dehydrated and stained.
2) The beam of electrons passes through the specimen, which is stained with metal salts. Some electrons pass through and are focused on the screen or photographic plate.
3) The electrons form a 2D black-and-white (grey scale) image. When photographed this is called an electron micrograph. Transmission electron microscopes can produce a magnification of up to 2 million times.

Question 10

Describe how scanning electron microscopes work

Answer 10

The specimen has to be placed in a vacuum and is often coated with a fine film of metal. Electrons do not pass through the specimen, which is whole, but cause secondary electrons to “bounce off” the specimen’s surface and be focused on to a screen. This gives a 3D image with a magnification from x15 up to x200 000. The image is black and white, but computer software programmes can add false colour.

Question 11

Both types of electron microscope: (2)

Answer 11

• are large and very expensive
• need a great deal of skill and training to use

Question 12

What is an artefact in microscopy?

Answer 12

An artefact is a visible structural detail caused by processing the specimen and not a feature of the specimen. Artefacts appear in both light and electron microscopy. The bubbles that get trapped under the cover slip as you prepare a slide for light microscopy are artefacts.

Question 13

Changes in the ultrastructure of cells are inevitable during the processing that the samples must undergo. Name 3 examples of these changes

Answer 13

• Loss of continuity in membranes
• Distortion of organelles
• Empty spaces in the cytoplasm of cells

Question 14

What was the name given to invaginations [inward foldings] of cell membranes that were observed using electron microscopes after bacterial specimens had been chemically fixed?

Answer 14

Mesosome

Question 15

Originally, scientists believed that mesosomes were a normal structure found within prokaryotes. What did they believe their function was?

Answer 15

The large surface area of the folded membrane was considered to be an important site for the process of oxidative phosphorylation.

Question 16

How did scientists find out that mesosomes were not part of the prokaryotes naturally?

Answer 16

When specimens were fixed by the more recently developed, non-chemical technique called cryofixation, mesosomes were no longer visible

Question 17

How is fluorescent microscopy different from optical microscopy?

Answer 17

Conventional optical microscopes use visible light to illuminate specimens and a lens to produce a magnified image. In fluorescent microscopes a higher light intensity is used to illuminate a specimen that has been treated with a fluorescent chemical (a fluorescent “dye”). Fluorescence is the absorption and re-radiation of light. Light of a longer wavelength and lower energy is emitted and used to produce a magnified image.

Question 18

Describe how laser scanning confocal microscopes work (5)

Answer 18

• A laser scanning confocal microscope moves a single spot of focused light across a specimen (point illumination). This causes fluorescence from the components labelled with a “dye”. The emitted light from the specimen is filtered through a pinhole aperture. Only light radiated from very close to the focal plane (the distance that gives the sharpest focus) is detected.
• Light emitted from other parts of the specimen would reduce the resolution and cause blurring. This unwanted radiation does not pass through the pinhole and is not detected. A laser is used instead of light to get higher intensities, which improves the illumination.
• As very thin sections of specimen are examined and light from elsewhere is removed, very high resolution images can be obtained.
• The beamsplitter is a dichroic mirror, which only reflects one wavelength (from the laser) but allows other wavelengths (produced by the sample) to pass through.
• The positions of the two pinholes means the light waves from the laser (illuminating the sample) follow the same path as the light waves radiated when the sample fluoresces. This means they will both have the same focal plane, hence the term confocal.

Question 19

What are fluorescent tags useful for?

Answer 19

By using antibodies with fluorescent ‘tags’, specific features can be targeted and therefore studied by confocal microscopy with much more precision than when using staining and light microscopy.

Question 20

What is green fluorescent protein (GFP) produced by?

Answer 20

The jellyfish Aequorea victoria

Question 21

Describe the green fluorescent protein (GFP)

Answer 21

• Green fluorescent protein (GFP) is produced by the jellyfish Aequorea victoria.
• The protein emits bright green light when illuminated by ultraviolet light.
• GFP molecules have been engineered to fluoresce different colours, meaning different components of a specimen can be studied at the same time.
• The gene for this protein has been isolated and can be attached, by genetic engineering, to genes coding for proteins under investigation. The fluorescence indicates that a protein is being made and is used to see where it goes within the cell or organism. Bacterial, fungal, plant, and human cells have all been modified to express this gene and fluoresce. The use of these fluorescing proteins provides a non-invasive technique to study the production and distribution of proteins in cells and organisms.

Question 22

What is a benefit of using fluorescent proteins?

Answer 22

The use of fluorescent proteins provides a non-invasive technique to study the production and distribution of proteins in cells and organisms.

Question 23

Describe how atomic force microscopy works (3)

Answer 23

• The atomic force microscope (AFM) gathers information about a specimen by ‘feeling’ its surface with a mechanical probe. These are scanning microscopes that generate three-dimensional images of surfaces.
• An AFM consists of a sharp tip (probe) on a cantilever (a lever supported at one end) that is used to scan the surface of a specimen. When this is brought very close to a surface, forces between the tip and the specimen cause deflections of the cantilever. These deflections are measured using a laser beam reflected from the top of the cantilever into a detector.
• Fixation and staining are not required and specimens can be viewed in almost normal cell conditions without the damage caused during the preparation of specimens for electron microscopy. Living systems can even be examined.

Question 24

What is the resolution of AFM?

Answer 24

0.1nm

Question 25

With atomic force microscopy, what level can information be gained at?

Answer 25

Atomic level

Question 26

What industry uses AFM?

Answer 26

The pharmaceutical industry

Question 27

Describe how the pharmaceutical industry uses AFM

Answer 27

The pharmaceutical industry uses AFM to identify potential drug targets on cellular proteins and DNA. These microscopes can lead to a better understanding of how drugs interact with their target molecule or cell.

Question 28

What is AFM being employed to do?

Answer 28

Identify new drugs

Question 29

Describe how AFM can be used to identify new drugs

Answer 29

Finding and identifying new chemical compounds from the natural world, which may have medical applications, takes a long time, and is expensive. The molecular structures need to be understood before their potential use in medicine is known. Atomic force microscopes can speed up this process, saving money and, potentially, lives.

Question 30

Describe how AFM was used with deep sea molecules to image the molecules at very high, atomic level resolution

Answer 30

• In 2010, scientists working on a species of bacterium from a mud sample taken from the Mariana Trench found that the bacteria produced an unknown chemical compound.
• The chemical composition (the number and type of atoms present) was easily determined. However, the molecular structure, the way in which the atoms were joined together, was not so easy to work out and would have taken months using conventional techniques.
• Using atomic force microscopy the scientists were able to image the molecules at very high, atomic level resolution within one week, giving them the molecular structure they needed.

Question 31

It was always believed that the maximum resolution for light microscopes was 0.2 micrometres, about half the wavelength of light. In 2014, how did the 3 scientists prove this theory wrong?

Answer 31

They achieved resolutions greater than 2 micrometres using light microscopy. Two principles were involved, both forms of super resolution fluorescent microscopy (SRFM).
- One involved building up a very high resolution image by combining many very small images.
- The other involved superimposing many images with normal resolution to create one very high resolution image.

Question 32

Describe how stimulated emission depletion (STED) works

Answer 32

Stimulated emission depletion (STED) involves the use of two lasers which are slightly offset. The first laser scans a specimen causing fluorescence, followed by the second laser which negates the fluorescence from all but a molecular sized area. A picture is built up with a resolution much greater than that produced normally in light microscopy. In this way, individual strands of DNA become visible

Question 33

Describe how the second form of super resolution fluorescent microscopy (SRFM) works

Answer 33

The second principle relies on the ability to control the fluorescence of individual molecules. Specimens are scanned multiple times but each time different molecules are allowed to fluoresce. The images are then superimposed and the resolution of the combined image is at the molecular level, much greater than 0.2 micrometres.

Question 34

What does super resolved fluorescence microscopy allow us to do in the medical field?

Answer 34

It is possible to follow individual molecules during cellular processes. proteins involved in Parkinson’s and Alzheimer’s diseases can be observed interacting and fertilised eggs dividing into embryos can be studied at molecular level.