Observing Cells

Cell Structure

Define a light microscope.

A microscope that uses a beam of light and glass lenses to magnify specimens, usually up to about ×1500.

What does a light microscope use to produce a magnified image?

Visible light and glass lenses.

Define magnification.

A measure of how much larger the image is than the real object.

Define resolution (resolving power).

A measure of how close together two objects can be while still being seen as separate objects.

What is the difference between magnification and resolution?

Magnification describes how much larger an image appears, whereas resolution describes the ability to distinguish two close points as separate.

Why is resolution important in microscopy?

Higher resolution allows smaller structures that are close together to be distinguished as separate.

Approximately what is the resolution of the human eye?

About 0.1 mm or 100 µm.

Approximately what is the resolution of a light microscope?

About 0.2 µm.

Why does a light microscope have limited resolution?

Visible light has a relatively long wavelength, limiting how close two points can be while still being distinguished.

What are the main lenses in a light microscope?

The objective lens and the eyepiece lens.

What does the objective lens do?

It produces a magnified image of the specimen.

What does the eyepiece lens do?

It magnifies the image produced by the objective lens.

How is the total magnification of a light microscope calculated?

Magnification of objective lens × magnification of eyepiece lens.

If the objective lens is ×40 and the eyepiece lens is ×10, what is the total magnification?

×400.

What is the equation linking image size, actual size and magnification?

Image size = actual size × magnification.

How do you calculate actual size?

Actual size = image size ÷ magnification.

How do you calculate magnification?

Magnification = image size ÷ actual size.

A cell image is 10 mm across at ×400 magnification. What is its actual size?

0.025 mm, or 25 µm.

Why must image size and actual size use the same units when calculating magnification?

To ensure the calculated magnification is correct.

How many micrometres are in 1 millimetre?

1000 µm.

How many nanometres are in 1 micrometre?

1000 nm.

How many nanometres are in 1 millimetre?

1,000,000 nm.

How do you convert millimetres to micrometres?

Multiply by 1000.

How do you convert micrometres to millimetres?

Divide by 1000.

Can living specimens be viewed using a light microscope?

Yes, living cells, tissues and small organisms can be viewed directly.

How are many specimens prepared for light microscopy?

They are preserved, sectioned into thin slices, stained and mounted on a slide.

Why are specimens sectioned before light microscopy?

To produce thin slices that allow light to pass through the specimen.

Why are biological specimens stained?

To increase contrast and make particular cells or cell structures easier to distinguish.

What does haematoxylin stain?

Nuclei of plant and animal cells purple, blue or brown.

What does methylene blue stain?

Nuclei of animal cells blue.

What does acetocarmine stain?

Chromosomes in dividing plant and animal cells.

What does iodine stain in plant cells?

Starch-containing material blue-black.

Give an advantage of light microscopes involving living material.

They can be used to observe living cells, tissues and organisms.

Why is viewing living material with a light microscope useful?

It allows prepared specimens to be compared with living tissue and allows biological processes to be observed.

Give an economic advantage of light microscopes.

They are relatively cheap compared with electron microscopes.

Where are light microscopes commonly available?

Schools, universities, hospitals, industrial laboratories and research laboratories.

Give a practical advantage of light microscopes.

They are relatively light and portable, so they can be used in many locations.

What is a disadvantage of light microscopes?

They have limited magnification and resolution compared with electron microscopes.

What are artefacts in microscopy?

Structures or features produced during specimen preparation that are not naturally present in the living tissue.

How can artefacts be produced?

Processes such as preserving, staining and sectioning specimens can alter the tissue.

Why can artefacts cause problems when interpreting micrographs?

They may be mistaken for genuine cell structures.

Define a graticule.

A scale placed in the eyepiece of a microscope that can be used to measure specimens accurately.

How is a graticule used?

It is calibrated for the magnification being used and then used to measure cells or structures.

Why must a graticule be calibrated?

The value represented by each division changes with magnification.

Define an electron microscope.

A microscope that uses a beam of electrons and electromagnetic or electrostatic lenses to produce highly magnified images.

What does an electron microscope use instead of light?

A beam of electrons.

What lenses are used in an electron microscope?

Electromagnetic or electrostatic lenses.

Why do electron microscopes have a higher resolution than light microscopes?

Electrons have a much shorter wavelength than visible light.

Approximately what resolution can an electron microscope achieve according to the textbook?

Less than 1 nm, or about 0.0001 µm.

Approximately how much better can electron microscope resolution be than light microscope resolution?

About 1000 times better.

Why does a shorter wavelength improve resolution?

It allows smaller structures and points closer together to be distinguished.

Why must specimens in an electron microscope be placed in a vacuum?

Air molecules would scatter the electrons and blur the image.

Can living specimens be observed using an electron microscope?

No, specimens must be placed in a vacuum and undergo preparation that kills them.

How are specimens prepared for electron microscopy?

They may undergo chemical preservation, freeze-drying, freeze-fracturing, dehydration, embedding, sectioning and mounting on a metal grid.

Why are heavy metal ions such as lead and uranium used in electron microscopy?

They increase electron scattering and therefore improve contrast.

Do heavy metal stains in electron microscopy identify tissues by colour?

No, they are mainly used to improve electron scattering and image contrast.

How are electron microscope images viewed?

They are displayed on a monitor or computer screen.

What colour are electron microscope images naturally?

They are naturally monochrome.

Why are some electron micrographs brightly coloured?

Colour may be added digitally afterwards to make structures easier to identify.

Is the colour in a colourised electron micrograph the specimen's natural colour?

No, the colour is added digitally after the image is produced.

What is a major advantage of electron microscopes?

They have much greater magnification and resolution than light microscopes.

Why have electron microscopes improved our understanding of cells?

They allow very small details of cell ultrastructure to be observed.

What is cell ultrastructure?

The detailed internal structure of a cell that can be observed at very high resolution.

What is a disadvantage of electron microscopes involving specimens?

Specimens must be dead and placed in a vacuum.

Why can electron microscope preparation produce artefacts?

The extensive preparation can alter or damage cell structures.

Why can electron microscopes not be used easily in the field?

They are large, expensive and require controlled conditions and a vacuum.

What environmental conditions are required for an electron microscope?

A carefully controlled temperature and pressure and an internal vacuum.

What is an economic disadvantage of electron microscopes?

They are extremely expensive.

What is a transmission electron microscope (TEM)?

An electron microscope in which electrons pass through a very thin specimen to produce a high-resolution two-dimensional image.

What type of image does a TEM produce?

A two-dimensional image.

What is a major advantage of a TEM?

It has very high magnification and resolution and can show detailed internal cell structures.

Why must TEM specimens be very thin?

Electrons must be able to pass through the specimen.

Define a transmission electron micrograph (TEM).

A micrograph produced by a transmission electron microscope in which electrons pass through a thin specimen to produce a 2D image.

What is a scanning electron microscope (SEM)?

An electron microscope that scans the surface of a specimen with electrons to produce a three-dimensional image.

What type of image does an SEM produce?

A three-dimensional image showing the specimen's surface.

Does an SEM generally have higher or lower magnification than a TEM?

Lower magnification than a TEM.

What is a major advantage of an SEM?

It produces detailed three-dimensional images of specimen surfaces.

Define a scanning electron micrograph (SEM).

A micrograph produced by a scanning electron microscope that shows a three-dimensional view of a specimen's surface.

What is the main difference between TEM and SEM?

TEM shows detailed internal structures in a 2D image, whereas SEM shows the surface of a specimen in a 3D image.

Which microscope is best for observing the internal ultrastructure of a mitochondrion?

A transmission electron microscope (TEM).

Which microscope is best for producing a 3D image of the surface of an insect?

A scanning electron microscope (SEM).

Which microscope would be most suitable for observing living cells?

A light microscope.

Which microscope provides the greatest detail of cell ultrastructure?

A transmission electron microscope.

Why is high magnification alone not enough to show more cell detail?

If resolution is too low, increasing magnification only produces a larger blurred image without revealing additional detail.

What is meant by useful magnification?

Magnification that reveals additional detail because the microscope has sufficient resolution.

Why can an image become larger without becoming more detailed?

Magnification can increase image size even when resolution is not high enough to distinguish additional structures.