OCR Biology A Level Biology Microscopy

Give 3 reasons we use light microscopes

More readily available/cheaper than other types of microscope, can be used in the field, can view live or dead specimens

Why would you stain a sample?

Stains make structures visible and increase the contrast as different components take up different stains to different degrees.

How would you prepare a specimen for a dry mount?

Cut the specimen into very thin slices with a sharp blade (sectioning)

Give 4 examples of specimens which could be viewed under a dry mount

Any 4 of- hair, pollen, insect parts, muscle tissue, plant leaves or plant stems

How would you prepare a wet mount?

Suspend the specimen in a liquid such as immersive oil or water. Then, lower the cover slip onto the liquid droplet at an angle.

Why do we use wet mounts?

So that aquatic (or other) organisms can be viewed alive and whole.

Give 2 examples of specimens which could be viewed under a wet mount

Any 2 from- Small insects, aquatic plants, algae

How would one produce a smear slide?

By using the edge of another slide to create an even coating of a liquid on a slide

Give 2 examples of specimens which could be viewed using a smear slide

Blood samples or spinal fluid.

Why would a squash slide be used rather than a wet mount?

We would use it if the specimen did not need to be alive or needed to be thinner for better visibility

How would one prepare a squash slide?

Prepare a wet mount, then gently press down the cover slide onto the droplet

Give 2 examples of specimens which could be viewed using squash slides

Any 2 from- Fungi, soft fruit, parts of plants (i.e root tips)

What is magnification?

How much larger or smaller an image is than the thing being viewed

What is resolution?

The smallest distance between two individual objects that can be seen separately

What is the magnification of an electron microscope?

Up to 500,000 times

What is the resolution of a TEM?

0.5nm

What is the resolution of an SEM?

3-10nm

What is the resolution of a light microscope and why is it limited to this?

200 nm because this is the wavelength of visible light.

What is the magnification of a light microscope?

Up to 1500 times

How does a Transmission Electron Microscope work?

A beam of electrons is transmitted through the specimen, then focused to produce an image in a manner similar to light microscopy.

Why does an electron microscope have a better resolution than a light microscope?

Because the electron beam has a smaller wavelength than light.

How does a Scanning Electron Microscope work?

A beam of electrons is passed across the surface of the specimen, and the reflected electrons collected.

What sort of images do SEMs produce?

Very detailed images of the surface of specimens

What is the magnification of an SEM?

Up to 500,000 times

What is the resolution of an SEM?

3-10nm

Why is the inside of an electron microscope a vacuum?

So that the electrons are not deflected and the electron beam stays in a straight line

How are samples prepared for electron microscopes?

Set in resin (for TEM), fractured (for SEM), fixed with chemicals, frozen, stained with heavy metals, dehydrated with solvents.

What is an artifact?

A visible structural feature which is produced whilst a specimen is being prepared.

What are some examples of artifacts in electron microscopy?

Distortion of organelles, loss of continuity in the cell membrane, empty spaces in the cytoplasm.

Why are artifacts a problem?

Scientists cannot always tell what is an artifact and what isn't.

What is point illumination?

When a single spot of focused light is moved across a specimen during laser scanning confocal microscopy.

What is fluorescence?

The absorption and re-radiation of light

How does Laser Scanning Confocal Microscopy work?

Point illumination is done. This causes fluorescence in parts of a sample labeled with dye. This light is then filtered through a pinhole aperture, and only light from very close to the focal plane will pass through.

What is used to split the beam in Laser Scanning Confocal Microscopy?

A dichroic mirror

What is the resolution of Laser Scanning Confocal Microscopy?

200nm+

Explain the term 'confocal' in laser scanning confocal microscopy.

The position of the pinholes (laser out and light in) means the light from the laser and the light from the fluorescing sample is on the same focal plane

Why is a laser used in laser scanning confocal microscopy?

Because it provides better illumination

How can the usual 2D image from a laser scanning confocal microscope be made 3D?

By shining the laser from different focal planes

Why is laser scanning confocal microscopy used?

It is non-invasive, specimens can be live and it has a very high resolution

What things is laser scanning confocal microscopy used for?

Diagnosing human eye disorders, development of new drugs.

What is a stage micrometer?

A tiny scaled measuring instrument, to be viewed under a microscope

What is an eyepiece graticule?

An arbitrary scale on a microscope lens, which must be calculated for each objective lens

What is the equation to work out the size of each eyepiece division?

Eyepiece division (micrometers)= Number of micrometers on stage micrometer/eyepiece divisions

What is a fluorescent tag?

A protein which produces fluorescent light

Why would a fluorescent tag be used?

It can be attached to genes coding for certain proteins via genetic engineering (different tags can produce different colours). This allows the distribution of different proteins to be monitored.

How does atomic force microscopy work?

A very sharp probe on a cantilever is brought very close to the surface of a specimen. This causes the cantilever to move very slightly, and these deflections are measured using a laser.

What is the resolution of atomic force microscopy?

0.1nm

What is a use of AFM?

Identifying new drugs

What is Super Resolved Fluorescent Microscopy?

1. Converting many very small images into 1 very detailed image.
2. Superimposing many images with normal resolution into 1 very high resolution image.

What is an advantage of super resolved fluorescence microscopy?

Can image individual DNA strands

What is stimulated emission deflection?

Where a 2nd laser is used is super resolved fluorescence microscopy to negate fluorescence in all but a molecular sized area

What are 2 common staining chemicals?

Eosin and Methylene Blue