chapter 2 part 2

Question 1

Electron microscopy:

Answer 1

In light microscopy, increased magnification can be achieved easily using the appropriate lenses, but if the image is blurred no more detail will be seen.
Resolution is the limiting factor.
In electron microscopy, a beam of electrons with a wavelength of less than 1 nm is used to illuminate the specimen.
More detail of cell ultrastructure can be seen because electrons have a much smaller wavelength than light waves.
They can produce images with magnifications of up to x500,000 and still have clear resolution.
Electron microscopes have changed the way we understand cells but there are some disadvantages to this technique.

Question 2

Disadvantages of electron microscopes

Answer 2

They are very expensive pieces of equipment and can only be used inside a carefully controlled environment in a dedicated space.
Specimens can also be damaged by the electron beam and because the preparation process is very complex, there is a problem with artefacts (structures that are produced due to the preparation process).
However, as techniques improve a lot of these artefacts can be eliminated.

Question 3

two types of electron microscope:

Answer 3

transmission electron microscope (TEM)

scanning electron microscope (SEM)

Question 4

transmission electron microscope (TEM)

Answer 4

A beam of electrons is transmitted through a specimen and focused to produce an image.
This is similar to light microscopy.
This has the best resolution with a resolving power of 0.5 nm

Question 5

scanning electron microscope (SEM)

Answer 5

a beam of electrons is sent across the surface of a specimen and the reflected electrons are collected.
The resolving power is from 3-10mm, so the resolution is not as good as with transmission electron microscopy but three-dimensional images of surfaces are produced, giving us valuable information about the appearance of different organisms

Question 6

Sample preparation for electron microscopes:

Answer 6

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.
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 7

Why do they need fixation using chemicals or freezing, staining with heavy metals and dehydration with solvents:

Answer 7

Fixation will stabilise the sample and prevents decomposition
dehydration prevents vaporisation of water in vacuum. vaporisation would damage sample
embedding in resin allows thin slices to be obtained
staining with heavy metals creates contrast in electron beams

Question 8

light vs electron

Answer 8

Question 9

Creation of artefacts:

Answer 9

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.
They are the bubbles that get trapped under the cover slip as you prepare a slide for light microscopy
When preparing specimens for electron microscopy, changes in the ultrastructure of cells are inevitable during the processing that the samples must undergo.
They are seen as the loss of continuity in membranes, distortion of organelles and empty spaces in the cytoplasm of cells
Experience enables scientists to distinguish between an artefact and a true structure.

Question 10

Identifying artefacts:

Answer 10

• ‘Mesosome’ was the name given to invaginations (inward foldings) of cell membranes that were observed using electron microscopes after bacterial specimens had been chemically fixed.
• They were thought to be a normal structure, or organelle, found within prokaryotes.
• The large surface area of the folded membrane was considered to be an important site for the process of oxidative phosphorylation.
• However, when specimens were fixed by the more recently developed, non-chemical technique called cryofixation, the mesosomes were no longer visible.
• It is now widely thought that the majority of mesosomes observed are actually artefacts produced by the chemicals used in the fixation process in electron microscopy preparation, which damage bacterial cell membranes.
• However, there are still a number of scientists who believe that some species of bacteria do have mesosomes as part of their normal structure, but this is not the general consensus.

Question 11

More microscopes:

Answer 11

Light microscopy has also continued to develop.
Some of the latest technology produces images that are very different from electron micrographs but are just as useful.
Conventional optical microscopes use visible light to illuminate specimens and a lens to produce a magnified image.

Question 12

fluorescent microscopes:

Answer 12

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 13

A laser scanning confocal microscope:

Answer 13

• 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 spot illuminating the specimen is moved across the specimen and a two dimensional image is produced.
• A three dimensional image can be produced by creating images at different focal planes.

Question 14

Use of Laser scanning confocal microscopy:

Answer 14

Laser scanning confocal microscopy is non-invasive and is currently used in the diagnosis of diseases of the eye and is also being developed for use in endoscopic procedures.
The fact that it can be used to see the distribution of molecules within cells means it is also used in the development of new drugs.
The future uses for advanced optical microscopy include virtual biopsies, particularly in cases of suspected skin cancer.

Question 15

Structure of Laser scanning confocal microscopy:

Answer 15

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 16

Laser scanning confocal microscopy diagram

Answer 16

Question 17

Fluorescent tags:

Answer 17

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.
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 18

Suggest whether fluorescent microscopy has a higher resolution than normal light microscopy.

Answer 18

resolution the same because resolution limited by wavelength of light
fluorescence is light emitted so super resolved fluorescent microscopy has higher resolution

Question 19

Atomic force microscopy:

Answer 19

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.

Question 20

What does Atomic force microscopy consists of:

Answer 20

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.

Question 21

Preparation of Atomic force microscopy?:

Answer 21

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 22

Resolution of Atomic force microscopy:

Answer 22

The resolution of AFM is very high, in the order of 0.1 nm.
Information can be gained at the atomic level, even about the bonds within molecules.

Question 23

Use of Atomic force microscopy:

Answer 23

The pharmaceutical industry in particular 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.
AFM is also being employed to identify new drugs.
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.
The case study below is a good example of the importance of AFM.

Question 24

Atomic force microscopy diagram

Answer 24

Question 25

Case Study: Deep sea molecules:

Answer 25

In 2010, scientists working on a species of bacterium from a mud sample taken from the Mariana Trench - the deepest place on the planet located nearly 11000 metres beneath the Pacific Ocean, 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.
This was the first time this method had been used in this way.
This new approach could lead to much faster identification of unknown compounds and ultimately speed up the process of the development of new medicines.

Question 26

why atomic force microscopy has a greater resolution than traditional light microscopy.

Answer 26

image not formed by light - image is formed by deflections of tip / probe as the tip / probe moves across surface of specimen

Question 27

AFM is capable of producing magnifications equal to or better than electron microscopes. Explain why

Answer 27

higher resolution than electron microscope - magnification depends on resolution

Question 28

Discuss why, despite the comparable magnification, atomic force microscopy could not have resulted in the same advances in the study of cell function as electron microscopy.

Answer 28

(AFM) only scans surface which means it cannot see into cells and we need to see how
organelles are related to understand function

Question 29

Super resolved fluorescence microscopy
part 1

Answer 29

Electron microscopes cannot be used to examine living cells and it was always believed that the maximum resolution for light microscopes was 0.2 um, about half the wavelength of light.
This limits the detail that can be seen in living cells.
In 2014 Eric Betzig, Stefan W. Hell, and William E. Moerner were awarded the Nobel Prize in Chemistry for achieving resolutions greater than 0.2 um 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 30

Super resolved fluorescence microscopy
part 2

Answer 30

Stefan Hell developed stimulated emission depletion [STED) which 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.
Eric Betzig and William E. Moerner independently developed the second principle which 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 um.
It is now 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 a molecular level.

Question 31

Explain why electron microscopes cannot be used to examine living cells.

Answer 31

specimen preparation kills cells (1); detail e.g., fixation (1)

Question 32

Describe how the ability to control the fluorescence of individual molecules helped uncover cell processes.

Answer 32

single molecules can fluoresce (1); multiple images obtained (1); different molecules fluoresce
in each image (1); images superimposed (1); idea of individual molecules seen in relation to each other interacting